Rapamycin regulates the proliferation of Huh7, a hepatocellular carcinoma cell line, by up-regulating p53 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Sora; Jeon, Ji-Sook; Ahn, Curie

Rapamycin, a specific inhibitor of mTOR used extensively as an immunosuppressant, has been expanded recently to cancer therapy, because the mTOR signal is known to be up-regulated in various cancer cells including hepatocellular carcinoma (HCC) cells. In spite of extensive efforts to employ mTOR inhibitors as anti-HCC therapy, they have not yet been approved by the FDA. Because of the heterogeneity and complexity of molecular signaling in HCC, suitable biomarkers should be identified or discovered to improve clinical efficacy of mTOR-specific inhibitors to HCC cells. In this study, the effect of rapamycin was investigated on two different HCC cell lines,more » Huh7 cells and HepG2 cells. Rapamycin was found to inhibit the proliferation of Huh7 cells but not of HepG2 cells. Moreover, it was found that rapamycin can up-regulate p53 at the protein level, but not affect its transcript. To understand the critical role of p53 in the rapamycin effect, knock-down experiments were performed using small-interfering RNAs (siRNAs). The anti-proliferative effect of rapamycin on Huh7 cells clearly disappeared after blocking p53 production with siRNA, which indicates that p53 is a critical factor in the anti-proliferative effect of rapamycin in HCC cells. The over-expression system of p53 was also employed to mimic the effect of rapamycin and found that cell proliferation was clearly down-regulated by p53 over-expression. Finally, we found that the extracellular signal-regulated kinase 1/2 (ERK1/2) signal was regulated by p53 whose expression was induced by rapamycin. Overall, this study demonstrates that rapamycin inhibited the proliferation of Huh7 cells by up-regulating the expression of p53 and down-regulating the ERK1/2 signal, indicating that p53 is a useful biomarker for anti-cancer therapy using the specific inhibitor of mTOR signal, rapamycin, against hepatocellular carcinoma cells. - Highlights: • Rapamycin inhibits the proliferation of hepatocellular carcinoma cells depending on the expression of p53. • Rapamycin up-regulates p53 at the protein level, but not affect its transcript. • The up-regulation of p53 expression by rapamycin inhibits ERK signal.« less

Characterization of CD133+ parenchymal cells in the liver: histology and culture.

PubMed

Yoshikawa, Seiichi; Zen, Yoh; Fujii, Takahiko; Sato, Yasunori; Ohta, Tetsuo; Aoyagi, Yutaka; Nakanuma, Yasuni

2009-10-21

To reveal the characteristics of CD133(+) cells in the liver. This study examined the histological characteristics of CD133(+) cells in non-neoplastic and neoplastic liver tissues by immunostaining, and also analyzed the biological characteristics of CD133(+) cells derived from human hepatocellular carcinoma (HCC) or cholangiocarcinoma cell lines. Immunostaining revealed constant expression of CD133 in non-neoplastic and neoplastic biliary epithelium, and these cells had the immunophenotype CD133(+)/CK19(+)/HepPar-1(-). A small number of CD133(+)/CK19(-)/HepPar-1(+) cells were also identified in HCC and combined hepatocellular and cholangiocarcinoma. In addition, small ductal structures, resembling the canal of Hering, partly surrounded by hepatocytes were positive for CD133. CD133 expression was observed in three HCC (HuH7, PLC5 and HepG2) and two cholangiocarcinoma cell lines (HuCCT1 and CCKS1). Fluorescence-activated cell sorting (FACS) revealed that CD133(+) and CD133(-) cells derived from HuH7 and HuCCT1 cells similarly produced CD133(+) and CD133(-) cells during subculture. To examine the relationship between CD133(+) cells and the side population (SP) phenotype, FACS was performed using Hoechst 33342 and a monoclonal antibody against CD133. The ratios of CD133(+)/CD133(-) cells were almost identical in the SP and non-SP in HuH7. In addition, four different cellular populations (SP/CD133(+), SP/CD133(-), non-SP/CD133(+), and non-SP/CD133(-)) could similarly produce CD133(+) and CD133(-) cells during subculture. This study revealed that CD133 could be a biliary and progenitor cell marker in vivo. However, CD133 alone is not sufficient to detect tumor-initiating cells in cell lines.

Methylsulfonylmethane suppresses hepatic tumor development through activation of apoptosis

PubMed Central

Kim, Joo-Hyun; Shin, Hye-Jun; Ha, Hye-Lin; Park, Young-Ho; Kwon, Tae-Ho; Jung, Mi-Ra; Moon, Hyung-Bae; Cho, Eun-Sang; Son, Hwa-Young; Yu, Dae-Yeul

2014-01-01

AIM: To investigate the effect of methylsulfonylmethane (MSM), recently reported to have anti-cancer effects, in liver cancer cells and transgenic mice. METHODS: Three liver cancer cell lines, HepG2, Huh7-Mock and Huh7-H-rasG12V, were used. Cell growth was measured by Cell Counting Kit-8 and soft agar assay. Western blot analysis was used to detect caspases, poly (ADP-ribose) polymerase (PARP), and B-cell lymphoma 2 (Bcl-2) expressions. For in vivo study, we administered MSM to H-ras12V transgenic mice for 3 mo. RESULTS: MSM decreased the growth of HepG2, Huh7-Mock and Huh7-H-rasG12V cells in a dose-dependent manner. That was correlated with significantly increased apoptosis and reduced cell numbers in MSM treated cells. Cleaved caspase-8, cleaved caspase-3 and cleaved PARP were remarkably increased in the liver cancer cells treated with 500 mmol/L of MSM; however, Bcl-2 was slightly decreased in 500 mmol/L. Liver tumor development was greatly inhibited in the H-ras12V transgenic mice treated with MSM, compared to control, by showing reduced tumor size and number. Cleaved PARP was significantly increased in non-tumor treated with MSM compared to control. CONCLUSION: Liver injury was also significantly attenuated in the mice treated with MSM. Taken together, all the results suggest that MSM has anti-cancer effects through inducing apoptosis in liver cancer. PMID:24575169

Very-Low-Density Lipoprotein (VLDL)-Producing and Hepatitis C Virus-Replicating HepG2 Cells Secrete No More Lipoviroparticles than VLDL-Deficient Huh7.5 Cells

PubMed Central

Jammart, Baptiste; Michelet, Maud; Pécheur, Eve-Isabelle; Parent, Romain; Bartosch, Birke; Zoulim, Fabien

2013-01-01

In the plasma samples of hepatitis C virus (HCV)-infected patients, lipoviroparticles (LVPs), defined as (very-) low-density viral particles immunoprecipitated with anti-β-lipoproteins antibodies are observed. This HCV-lipoprotein association has major implications with respect to our understanding of HCV assembly, secretion, and entry. However, cell culture-grown HCV (HCVcc) virions produced in Huh7 cells, which are deficient for very-low-density lipoprotein (VLDL) secretion, are only associated with and dependent on apolipoprotein E (apoE), not apolipoprotein B (apoB), for assembly and infectivity. In contrast to Huh7, HepG2 cells can be stimulated to produce VLDL by both oleic acid treatment and inhibition of the MEK/extracellular signal-regulated kinase (ERK) pathway but are not permissive for persistent HCV replication. Here, we developed a new HCV cell culture model to study the interaction between HCV and lipoproteins, based on engineered HepG2 cells stably replicating a blasticidin-tagged HCV JFH1 strain (JB). Control Huh7.5-JB as well as HepG2-JB cell lines persistently replicated viral RNA and expressed viral proteins with a subcellular colocalization of double-stranded RNA (dsRNA), core, gpE2, and NS5A compatible with virion assembly. The intracellular RNA replication level was increased in HepG2-JB cells upon dimethyl sulfoxide (DMSO) treatment, MEK/ERK inhibition, and NS5A overexpression to a level similar to that observed in Huh7.5-JB cells. Both cell culture systems produced infectious virions, which were surprisingly biophysically and biochemically similar. They floated at similar densities on gradients, contained mainly apoE but not apoB, and were not neutralized by anti-apoB antibodies. This suggests that there is no correlation between the ability of cells to simultaneously replicate HCV as well as secrete VLDL and their capacity to produce LVPs. PMID:23427158

Very-low-density lipoprotein (VLDL)-producing and hepatitis C virus-replicating HepG2 cells secrete no more lipoviroparticles than VLDL-deficient Huh7.5 cells.

PubMed

Jammart, Baptiste; Michelet, Maud; Pécheur, Eve-Isabelle; Parent, Romain; Bartosch, Birke; Zoulim, Fabien; Durantel, David

2013-05-01

In the plasma samples of hepatitis C virus (HCV)-infected patients, lipoviroparticles (LVPs), defined as (very-) low-density viral particles immunoprecipitated with anti-β-lipoproteins antibodies are observed. This HCV-lipoprotein association has major implications with respect to our understanding of HCV assembly, secretion, and entry. However, cell culture-grown HCV (HCVcc) virions produced in Huh7 cells, which are deficient for very-low-density lipoprotein (VLDL) secretion, are only associated with and dependent on apolipoprotein E (apoE), not apolipoprotein B (apoB), for assembly and infectivity. In contrast to Huh7, HepG2 cells can be stimulated to produce VLDL by both oleic acid treatment and inhibition of the MEK/extracellular signal-regulated kinase (ERK) pathway but are not permissive for persistent HCV replication. Here, we developed a new HCV cell culture model to study the interaction between HCV and lipoproteins, based on engineered HepG2 cells stably replicating a blasticidin-tagged HCV JFH1 strain (JB). Control Huh7.5-JB as well as HepG2-JB cell lines persistently replicated viral RNA and expressed viral proteins with a subcellular colocalization of double-stranded RNA (dsRNA), core, gpE2, and NS5A compatible with virion assembly. The intracellular RNA replication level was increased in HepG2-JB cells upon dimethyl sulfoxide (DMSO) treatment, MEK/ERK inhibition, and NS5A overexpression to a level similar to that observed in Huh7.5-JB cells. Both cell culture systems produced infectious virions, which were surprisingly biophysically and biochemically similar. They floated at similar densities on gradients, contained mainly apoE but not apoB, and were not neutralized by anti-apoB antibodies. This suggests that there is no correlation between the ability of cells to simultaneously replicate HCV as well as secrete VLDL and their capacity to produce LVPs.

A novel matrine derivate inhibits differentiated human hepatoma cells and hepatic cancer stem-like cells by suppressing PI3K/AKT signaling pathways

PubMed Central

Liu, Ying; Qi, Yang; Bai, Zhi-hui; Ni, Chen-xu; Ren, Qi-hui; Xu, Wei-heng; Xu, Jing; Hu, Hong-gang; Qiu, Lei; Li, Jian-zhong; He, Zhi-gao; Zhang, Jun-ping

2017-01-01

Matrine is an alkaloid extracted from a Chinese herb Sophora flavescens Ait, which has shown chemopreventive potential against various cancers. In this study, we evaluated the anticancer efficacy of a novel derivative of matrine, (6aS, 10S, 11aR, 11bR, 11cS)-10- methylamino-dodecahydro- 3a,7a-diazabenzo (de) (MASM), against human hepatocellular carcinoma (HCC) cells and their corresponding sphere cells in vitro and in vivo. Human HCC cell lines (Hep3B and Huh7) were treated with MASM. Cell proliferation was assessed using CCK8 and colony assays; cell apoptosis and cell cycle distributions were examined with flow cytometry. The expression of cell markers and signaling molecules was detected using Western blot and qRT-PCR analyses. A sphere culture technique was used to enrich cancer stem cells (CSC) in Hep3B and Huh7 cells. The in vivo antitumor efficacy of MASM was evaluated in Huh7 cell xenograft model in BALB/c nude mice, which were administered MASM (10 mg·kg−1·d−1, ig) for 3 weeks. After the treatment was completed, tumor were excised and weighed. A portion of tumor tissue was enzymatically dissociated to obtain a single cell suspension for the spheroid formation assays. MASM (2, 10, 20 μmol/L) dose-dependently inhibited the proliferation of HCC cells, and induced apoptosis, which correlated with a reduction in Bcl-2 expression and an increase in PARP cleavage. MASM also induced cell cycle arrest in G0/G1 phase, which was accompanied by increased p27 and decreased Cyclin D1 expression. Interestingly, MASM (2, 10, and 20 μmol/L) drastically reduced the EpCAM+/CD133+ cell numbers, suppressed the sphere formation, inhibited the expression of stem cell marker genes and promoted the expression of mature hepatocyte markers in the Hep3B and Huh7 spheroids. Additionally, MASM dose-dependently suppressed the PI3K/AKT/mTOR and AKT/GSK3β/β-catenin signaling pathways in Hep3B and Huh7 cells. In Huh7 xenograft bearing nude mice, MASM administration significantly inhibited Huh7 xenograft tumor growth and markedly reduced the number of surviving cancer stem-like cells in the tumors. MASM administration also reduced the expression of stem cell markers while increasing the expression of mature hepatocyte markers in the tumor tissues. The novel derivative of matrine, MASM, markedly suppresses HCC tumor growth through multiple mechanisms, and it may be a promising candidate drug for the treatment of hepatocellular carcinoma. PMID:27773936

The fibrous form of intracellular inclusion bodies in recombinant variant fibrinogen-producing cells is specific to the hepatic fibrinogen storage disease-inducible variant fibrinogen.

PubMed

Arai, Shinpei; Ogiwara, Naoko; Mukai, Saki; Takezawa, Yuka; Sugano, Mitsutoshi; Honda, Takayuki; Okumura, Nobuo

2017-06-01

Fibrinogen storage disease (FSD) is a rare disorder that is characterized by the accumulation of fibrinogen in hepatocytes and induces liver injury. Six mutations in the γC domain (γG284R, γT314P, γD316N, the deletion of γG346-Q350, γG366S, and γR375W) have been identified for FSD. Our group previously established γ375W fibrinogen-producing Chinese hamster ovary (CHO) cells and observed aberrant large granular and fibrous forms of intracellular inclusion bodies. The aim of this study was to investigate whether fibrous intracellular inclusion bodies are specific to FSD-inducible variant fibrinogen. Thirteen expression vectors encoding the variant γ-chain were stably or transiently transfected into CHO cells expressing normal fibrinogen Aα- and Bβ-chains or HuH-7 cells, which were then immunofluorescently stained. Six CHO and HuH-7 cell lines that transiently produced FSD-inducible variant fibrinogen presented the fibrous (3.2-22.7 and 2.1-24.5%, respectively) and large granular (5.4-25.5 and 7.7-23.9%) forms of intracellular inclusion bodies. Seven CHO and HuH-7 cell lines that transiently produced FSD-non-inducible variant fibrinogen only exhibit the large granular form. These results demonstrate that transiently transfected variant fibrinogen-producing CHO cells and inclusion bodies of the fibrous form may be useful in non-invasive screening for FSD risk factors for FSD before its onset.

[1-9-NαC]-crourorb A1 isolated from Croton urucurana latex induces G2/M cell cycle arrest and apoptosis in human hepatocarcinoma cells.

PubMed

de Matos Cândido-Bacani, Priscila; Ezan, Frédéric; de Oliveira Figueiredo, Patrícia; Matos, Maria de Fátima Cepa; Rodrigues Garcez, Fernanda; Silva Garcez, Walmir; Baffet, Georges

2017-05-05

[1-9-NαC]-crourorb A1 is a cyclic peptide isolated from Croton urucurana Baillon latex, found in midwestern Brazil, that has been shown to exert cytotoxic effects against a panel of cancer cell lines. However, the underlying mechanisms responsible for the crourorb A1-induced cytotoxicity in cancer cells remain unknown. In this study, the effects of crourorb A1 on the viability, apoptosis, cell cycle and migration of Huh-7 (human hepatocarcinoma) cells were investigated. We evaluated the viability of Huh-7 cells treated with crourorb A1 in 2D and 3D collagen cultures and found that cells in 3D culture exhibited increased resistance to crourorb A1 compared to cells in 2D culture (IC 50 : 62μg/ml versus 35.75μg/ml). Crourorb A1 treatment decreases the viability of Huh-7 cells in a dose- and time-dependent manner and is associated with the induction of apoptosis, in the absence of necrotic cells, through the activation of caspase-3/7 and increased expression of the pro-apoptotic proteins Bak, Bid, Bax, Puma, Bim, and Bad. The effects of crourorb A1 are also associated with G2/M phase cell cycle arrest and increases in cyclin-dependent kinase (CDK1) and cyclin B1 expression. A significant reduction in Huh-7 cell migration induced by crourorb A1 was also observed in the presence of mitomycin C. Finally, we showed that the JNK/MAP pathway, but not ERK signaling, is involved in crourorb A1-induced hepatocarcinoma cell mortality. Copyright © 2017 Elsevier B.V. All rights reserved.

A dimeric urea of the bisabolene sesquiterpene from the Okinawan marine sponge Axinyssa sp. inhibits protein tyrosine phosphatase 1B activity in Huh-7 human hepatoma cells.

PubMed

Abdjul, Delfly B; Kanno, Syu-Ichi; Yamazaki, Hiroyuki; Ukai, Kazuyo; Namikoshi, Michio

2016-01-15

Protein tyrosine phosphatase 1B (PTP1B) plays an important role as a negative regulator of the insulin and leptin signaling pathways. Therefore, this enzyme is regarded as an attractive therapeutic target for the treatment of type 2 diabetes and obesity. Our screening program for PTP1B inhibitors led to the isolation of four sesquiterpenes and sterol: N,N'-bis[(6R,7S)-7-amino-7,8-dihydro-α-bisabolen-7-yl]urea (1), (6R,7S)-7-amino-7,8-dihydro-α-bisabolene (2), (1R,6S,7S,10S)-10-isothiocyanato-4-amorphene (3), axinisothiocyanate J (4), and axinysterol (5) from the marine sponge Axinyssa sp. collected at Iriomote Island. Of these, compound 1 was the most potent inhibitor of PTP1B activity (IC50=1.9μM) without cytotoxicity at 50μM in two human cancer cell lines, hepatoma Huh-7 and bladder carcinoma EJ-1 cells. Compound 1 also moderately enhanced the insulin-stimulated phosphorylation levels of Akt in Huh-7 cells. Therefore, compound 1 has potential as a new type of anti-diabetic drug candidate possessing PTP1B inhibitory activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

RNA-seq reveals distinctive RNA profiles of small extracellular vesicles from different human liver cancer cell lines.

PubMed

Berardocco, Martina; Radeghieri, Annalisa; Busatto, Sara; Gallorini, Marialucia; Raggi, Chiara; Gissi, Clarissa; D'Agnano, Igea; Bergese, Paolo; Felsani, Armando; Berardi, Anna C

2017-10-10

Liver cancer (LC) is one of the most common cancers and represents the third highest cause of cancer-related deaths worldwide. Extracellular vesicle (EVs) cargoes, which are selectively enriched in RNA, offer great promise for the diagnosis, prognosis and treatment of LC. Our study analyzed the RNA cargoes of EVs derived from 4 liver-cancer cell lines: HuH7, Hep3B, HepG2 (hepato-cellular carcinoma) and HuH6 (hepatoblastoma), generating two different sets of sequencing libraries for each. One library was size-selected for small RNAs and the other targeted the whole transcriptome. Here are reported genome wide data of the expression level of coding and non-coding transcripts, microRNAs, isomiRs and snoRNAs providing the first comprehensive overview of the extracellular-vesicle RNA cargo released from LC cell lines. The EV-RNA expression profiles of the four liver cancer cell lines share a similar background, but cell-specific features clearly emerge showing the marked heterogeneity of the EV-cargo among the individual cell lines, evident both for the coding and non-coding RNA species.

PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation

PubMed Central

Gedaly, Roberto; Galuppo, Roberto; Musgrave, Yolanda; Angulo, Paul; Hundley, Jonathan; Shah, Malay; Daily, Michael F.; Chen, Changguo; Cohen, Donald A.; Spear, Brett T.; Evers, B. Mark

2015-01-01

Background Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells). Materials and methods Flow cytometry was performed on the resulting tumors to identify the LCSC markers CD133, CD44, CD24, and aldehyde dehydrogenase 1. Methylthiazol tetrazolium assay was used to assess cellular proliferation. Finally, a Western blot assay was used to evaluate for inhibition of specific enzymes in these two signaling pathways. Results Using flow cytometry, we found that LCSC contain 64.4% CD133 + cells, 83.2% CD44 + cells, and 96.4% CD24 + cells. PKI-587 and sorafenib caused inhibiton of LCSC and HCC cell proliferation. PLC cells were more sensitive to PKI-587 than LCSC or Huh7 (P < 0.001). Interestingly, HuH7 cells were more sensitive to sorafenib than LCSC or PLC cells. Additionally, combination therapy with PKI-587 and sorafenib caused significantly more inhibition than monotherapy in HuH7, PLC, and LCSC. Using the methylthiazol tetrazolium assay, we found that the LCSC proliferation was inhibited with sorafenib monotherapy 39% at 5 μM (P < 0.001; n = 12) and 67% by PKI-587 at 0.1 μM (P = 0.002, n = 12) compared with control. The combination of PKI-587 and sorafenib, however, synergistically inhibited LCSC proliferation by 86% (P = 0.002; n = 12). Conclusions LCSC (CD133+, CD44+, CD24+) were able to develop very aggressive tumors with low cell concentrations at 4 to 6 wk. Cells CD133+, CD44+, CD24+ demonstrated at least moderate resistance to therapy in vitro. The combination of PKI-587 and sorafenib was better than either drug alone at inhibiting of LCSC and on HCC cell proliferation. PMID:23769634

PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation.

PubMed

Gedaly, Roberto; Galuppo, Roberto; Musgrave, Yolanda; Angulo, Paul; Hundley, Jonathan; Shah, Malay; Daily, Michael F; Chen, Changguo; Cohen, Donald A; Spear, Brett T; Evers, B Mark

2013-11-01

Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells). Flow cytometry was performed on the resulting tumors to identify the LCSC markers CD133, CD44, CD24, and aldehyde dehydrogenase 1. Methylthiazol tetrazolium assay was used to assess cellular proliferation. Finally, a Western blot assay was used to evaluate for inhibition of specific enzymes in these two signaling pathways. Using flow cytometry, we found that LCSC contain 64.4% CD133 + cells, 83.2% CD44 + cells, and 96.4% CD24 + cells. PKI-587 and sorafenib caused inhibiton of LCSC and HCC cell proliferation. PLC cells were more sensitive to PKI-587 than LCSC or Huh7 (P < 0.001). Interestingly, HuH7 cells were more sensitive to sorafenib than LCSC or PLC cells. Additionally, combination therapy with PKI-587 and sorafenib caused significantly more inhibition than monotherapy in HuH7, PLC, and LCSC. Using the methylthiazol tetrazolium assay, we found that the LCSC proliferation was inhibited with sorafenib monotherapy 39% at 5 μM (P < 0.001; n = 12) and 67% by PKI-587 at 0.1 μM (P = 0.002, n = 12) compared with control. The combination of PKI-587 and sorafenib, however, synergistically inhibited LCSC proliferation by 86% (P = 0.002; n = 12). LCSC (CD133+, CD44+, CD24+) were able to develop very aggressive tumors with low cell concentrations at 4 to 6 wk. Cells CD133+, CD44+, CD24+, which demonstrated at least moderate resistance to therapy in vitro. The combination of PKI-587 and sorafenib was better than either drug alone at inhibiting of LCSC and on HCC cell proliferation. Copyright © 2013 Elsevier Inc. All rights reserved.

Comparison of protein expression between human livers and the hepatic cell lines HepG2, Hep3B, and Huh7 using SWATH and MRM-HR proteomics: Focusing on drug-metabolizing enzymes.

PubMed

Shi, Jian; Wang, Xinwen; Lyu, Lingyun; Jiang, Hui; Zhu, Hao-Jie

2018-04-01

Human hepatic cell lines are widely used as an in vitro model for the study of drug metabolism and liver toxicity. However, the validity of this model is still a subject of debate because the expressions of various proteins in the cell lines, including drug-metabolizing enzymes (DMEs), can differ significantly from those in human livers. In the present study, we first conducted an untargeted proteomics analysis of the microsomes of the cell lines HepG2, Hep3B, and Huh7, and compared them to human livers using a sequential window acquisition of all theoretical mass spectra (SWATH) method. Furthermore, high-resolution multiple reaction monitoring (MRM-HR), a targeted proteomic approach, was utilized to compare the expressions of pre-selected DMEs between human livers and the cell lines. In general, the SWATH quantifications were in good agreement with the MRM-HR analysis. Over 3000 protein groups were quantified in the cells and human livers, and the proteome profiles of human livers significantly differed from the cell lines. Among the 101 DMEs quantified with MRM-HR, most were expressed at substantially lower levels in the cell lines. Thus, appropriate caution must be exercised when using these cell lines for the study of hepatic drug metabolism and toxicity. Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Coronaviruses and arteriviruses display striking differences in their cyclophilin A-dependence during replication in cell culture.

PubMed

de Wilde, Adriaan H; Zevenhoven-Dobbe, Jessika C; Beugeling, Corrine; Chatterji, Udayan; de Jong, Danielle; Gallay, Philippe; Szuhai, Karoly; Posthuma, Clara C; Snijder, Eric J

2018-04-01

Cyclophilin A (CypA) is an important host factor in the replication of a variety of RNA viruses. Also the replication of several nidoviruses was reported to depend on CypA, although possibly not to the same extent. These prior studies are difficult to compare, since different nidoviruses, cell lines and experimental set-ups were used. Here, we investigated the CypA dependence of three distantly related nidoviruses that can all replicate in Huh7 cells: the arterivirus equine arteritis virus (EAV), the alphacoronavirus human coronavirus 229E (HCoV-229E), and the betacoronavirus Middle East respiratory syndrome coronavirus (MERS-CoV). The replication of these viruses was compared in the same parental Huh7 cells and in CypA-knockout Huh7 cells generated using CRISPR/Cas9-technology. CypA depletion reduced EAV yields by ~ 3-log, whereas MERS-CoV progeny titers were modestly reduced (3-fold) and HCoV-229E replication was unchanged. This study reveals that the replication of nidoviruses can differ strikingly in its dependence on cellular CypA. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Polo-like kinase 1, a new therapeutic target in hepatocellular carcinoma

PubMed Central

Mok, Wei Chuen; Wasser, Shanthi; Tan, Theresa; Lim, Seng Gee

2012-01-01

AIM: To investigate the role of polo-like kinase 1 (PLK1) as a therapeutic target for hepatocellular carcinoma (HCC). METHODS: PLK1 gene expression was evaluated in HCC tissue and HCC cell lines. Gene knockdown with short-interfering RNA (siRNA) was used to study PLK1 gene and protein expression using real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, and cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium (MTS) and bromodeoxyuridine (BrdU) assays. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and caspase-inhibition assay. Huh-7 cells were transplanted into nude mice and co-cultured with PLK1 siRNA or control siRNA, and tumor progression was compared with controls. RESULTS: RT-PCR showed that PLK1 was overexpressed 12-fold in tumor samples compared with controls, and also was overexpressed in Huh-7 cells. siRNA against PLK1 showed a reduction in PLK1 gene and protein expression of up to 96% in Huh-7 cells, and a reduction in cell proliferation by 68% and 92% in MTS and BrdU cell proliferation assays, respectively. There was a 3-fold increase in apoptosis events, and TUNEL staining and caspase-3 assays suggested that this was caspase-independent. The pan-caspase inhibitor Z-VAD-FMK was unable to rescue the apoptotic cells. Immnofluorescence co-localized endonuclease-G to fragmented chromosomes, implicating it in apoptosis. Huh-7 cells transplanted subcutaneously into nude mice showed tumor regression in siPLK1-treated mice, but not in controls. CONCLUSION: Knockdown of PLK1 overexpression in HCC was shown to be a potential therapeutic target, leading to apoptosis through the endonuclease-G pathway. PMID:22826617

Manipulating the mitochondria activity in human hepatic cell line Huh7 by low-power laser irradiation

PubMed Central

Lynnyk, Anna; Lunova, Mariia; Jirsa, Milan; Egorova, Daria; Kulikov, Andrei; Kubinová, Šárka; Lunov, Oleg; Dejneka, Alexandr

2018-01-01

Low-power laser irradiation of red light has been recognized as a promising tool across a vast variety of biomedical applications. However, deep understanding of the molecular mechanisms behind laser-induced cellular effects remains a significant challenge. Here, we investigated mechanisms involved in the death process in human hepatic cell line Huh7 at a laser irradiation. We decoupled distinct cell death pathways targeted by laser irradiations of different powers. Our data demonstrate that high dose laser irradiation exhibited the highest levels of total reactive oxygen species production, leading to cyclophilin D-related necrosis via the mitochondrial permeability transition. On the contrary, low dose laser irradiation resulted in the nuclear accumulation of superoxide and apoptosis execution. Our findings offer a novel insight into laser-induced cellular responses, and reveal distinct cell death pathways triggered by laser irradiation. The observed link between mitochondria depolarization and triggering ROS could be a fundamental phenomenon in laser-induced cellular responses. PMID:29541521

Synergistic growth inhibition by acyclic retinoid and vitamin K2 in human hepatocellular carcinoma cells.

PubMed

Kanamori, Toh; Shimizu, Masahito; Okuno, Masataka; Matsushima-Nishiwaki, Rie; Tsurumi, Hisashi; Kojima, Soichi; Moriwaki, Hisataka

2007-03-01

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. However, effective chemopreventive and chemotherapeutic agents for this cancer have not yet been developed. In clinical trials acyclic retinoid (ACR) and vitamin K(2) (VK(2)) decreased the recurrence rate of HCC. In the present study we examined the possible combined effects of ACR or another retinoid 9-cis retinoic acid (9cRA) plus VK(2) in the HuH7 human HCC cell line. We found that the combination of 1.0 microM ACR or 1.0 microM 9cRA plus 10 microM VK(2) synergistically inhibited the growth of HuH7 cells without affecting the growth of Hc normal human hepatocytes. The combined treatment with ACR plus VK(2) also acted synergistically to induce apoptosis in HuH7 cells. Treatment with VK(2) alone inhibited phosphorylation of the retinoid X receptor (RXR)alpha protein, which is regarded as a critical factor for liver carcinogenesis, through inhibition of Ras activation and extracellular signal-regulated kinase phosphorylation. Moreover, the inhibition of RXRalpha phosphorylation by VK(2) was enhanced when the cells were cotreated with ACR. The combination of retinoids plus VK(2) markedly increased both the retinoic acid receptor responsive element and retinoid X receptor responsive element promoter activities in HuH7 cells. Our results suggest that retinoids (especially ACR) and VK(2) cooperatively inhibit activation of the Ras/MAPK signaling pathway, subsequently inhibiting the phosphorylation of RXRalpha and the growth of HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.

RNA-seq reveals distinctive RNA profiles of small extracellular vesicles from different human liver cancer cell lines

PubMed Central

Berardocco, Martina; Radeghieri, Annalisa; Busatto, Sara; Gallorini, Marialucia; Raggi, Chiara; Gissi, Clarissa; D’Agnano, Igea; Bergese, Paolo; Felsani, Armando; Berardi, Anna C.

2017-01-01

Liver cancer (LC) is one of the most common cancers and represents the third highest cause of cancer-related deaths worldwide. Extracellular vesicle (EVs) cargoes, which are selectively enriched in RNA, offer great promise for the diagnosis, prognosis and treatment of LC. Our study analyzed the RNA cargoes of EVs derived from 4 liver-cancer cell lines: HuH7, Hep3B, HepG2 (hepato-cellular carcinoma) and HuH6 (hepatoblastoma), generating two different sets of sequencing libraries for each. One library was size-selected for small RNAs and the other targeted the whole transcriptome. Here are reported genome wide data of the expression level of coding and non-coding transcripts, microRNAs, isomiRs and snoRNAs providing the first comprehensive overview of the extracellular-vesicle RNA cargo released from LC cell lines. The EV-RNA expression profiles of the four liver cancer cell lines share a similar background, but cell-specific features clearly emerge showing the marked heterogeneity of the EV-cargo among the individual cell lines, evident both for the coding and non-coding RNA species. PMID:29137313

Transforming growth factor-β decreases side population cells in hepatocellular carcinoma in vitro.

PubMed

Kim, Jong Bin; Lee, Seulki; Kim, Hye Ri; Park, Seo-Young; Lee, Minjong; Yoon, Jung-Hwan; Kim, Yoon Jun

2018-06-01

Hepatocellular carcinoma (HCC) can result from hepatitis B or C infection, fibrosis or cirrhosis. Transforming growth factor-β (TGF-β) is one of the main growth factors associated with fibrosis or cirrhosis progression in the liver, but its role is controversial in hepatocarcinogenesis. In the present study, the effect of TGF-β on the HCC Huh-7 and Huh-Bat cell lines was evaluated. To study the effect of TGF-β, Huh-7 and Huh-Bat cells were treated with TGF-β and a TGF-β receptor inhibitor (SB431542). Cell survival, cell cycle, numbers of side population (SP) cells and expression of the cancer stem cell marker cluster of differentiation (CD)133, epithelial-mesenchymal transition markers (E-cadherin, α-smooth muscle actin and vimentin) and TGF-β-regulated proteins [phospho-c-Jun N-terminal kinase (p-JNK), p-c-Jun and p-smad2] were investigated. TGF-β treatment resulted in decreased cell survival with a targeted effect on SP cells. Expression of CD133 and vimentin was upregulated by treatment with the TGF-β receptor antagonist SB431542, but not with TGF-β. By contrast, TGF-β induced accumulation of cells at G0/G1, and upregulated expression of p-JNK, p-c-Jun and p-smad2. However, these effects were blocked when cells were treated with TGF-β plus SB431542, indicating the specificity of the TGF-β effect. The present results indicated that TGF-β has anticancer effects mediated by survival inhibition of cancer stem cells, which may be developed as a novel therapy for HCC.

Cell Spheroids with Enhanced Aggressiveness to Mimic Human Liver Cancer In Vitro and In Vivo.

PubMed

Jung, Hong-Ryul; Kang, Hyun Mi; Ryu, Jea-Woon; Kim, Dae-Soo; Noh, Kyung Hee; Kim, Eun-Su; Lee, Ho-Joon; Chung, Kyung-Sook; Cho, Hyun-Soo; Kim, Nam-Soon; Im, Dong-Soo; Lim, Jung Hwa; Jung, Cho-Rok

2017-09-05

We fabricated a spheroid-forming unit (SFU) for efficient and economic production of cell spheroids. We optimized the protocol for generating large and homogenous liver cancer cell spheroids using Huh7 hepatocellular carcinoma (HCC) cells. The large Huh7 spheroids showed apoptotic and proliferative signals in the centre and at the surface, respectively. In particular, hypoxia-induced factor-1 alpha (HIF-1α) and ERK signal activation were detected in the cell spheroids. To diminish core necrosis and increase the oncogenic character, we co-cultured spheroids with 2% human umbilical vein endothelial cells (HUVECs). HUVECs promoted proliferation and gene expression of HCC-related genes and cancer stem cell markers in the Huh7 spheroidsby activating cytokine signalling, mimicking gene expression in liver cancer. HUVECs induced angiogenesis and vessel maturation in Huh7 spheroids in vivo by activating epithelial-mesenchymal transition and angiogenic pathways. The large Huh7 cell spheroids containing HUVECs survived at higher concentrations of anti-cancer drugs (doxorubicin and sorafenib) than did monolayer cells. Our large cell spheroid provides a useful in vitro HCC model to enable intuitive observation for anti-cancer drug testing.

Rocaglamide overcomes tumor necrosis factor-related apoptosis-inducing ligand resistance in hepatocellular carcinoma cells by attenuating the inhibition of caspase-8 through cellular FLICE-like-inhibitory protein downregulation.

PubMed

Luan, Zhou; He, Ying; He, Fan; Chen, Zhishui

2015-01-01

The enhancement of apoptosis is a therapeutic strategy used in the treatment of cancer. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent. However, hepatocellular carcinoma (HCC) cells exhibit marked resistance to the induction of cell death by TRAIL. The present study investigated whether rocaglamide, a naturally occurring product isolated from the genus Aglaia, is able to sensitize resistant HCC cells to TRAIL-mediated apoptosis. Two HCC cell lines, HepG2 and Huh-7, were treated with rocaglamide and/or TRAIL and the induction of apoptosis and effects on the TRAIL signaling pathway were investigated. The in vivo efficacy of rocaglamide was determined in TRAIL-resistant Huh-7-derived tumor xenografts. Rocaglamide significantly sensitized the TRAIL-resistant HCC cells to apoptosis by TRAIL, which resulted from the rocaglamide-mediated downregulation of cellular FLICE-like inhibitory protein and subsequent caspase-8 activation. Furthermore, rocaglamide markedly inhibited tumor growth from Huh-7 cells propagated in severe combined immunodeficient mice, suggesting that chemosentization also occurred in vivo. These data suggest that rocaglamide acted synergistically with TRAIL against the TRAIL-resistant HCC cells. Thus, it is concluded that rocaglamide as an adjuvant to TRAIL-based therapy may present a promising therapeutic approach for the treatment of HCC.

Influence of P53 on the radiotherapy response of hepatocellular carcinoma

PubMed Central

Gomes, Ana R.; Abrantes, Ana M.; Brito, Ana F.; Laranjo, Mafalda; Casalta-Lopes, João E.; Gonçalves, Ana C.; Sarmento-Ribeiro, Ana B.; Tralhão, José G.

2015-01-01

Background/Aims Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and it has a poor prognosis and few therapeutic options. Radiotherapy is one of the most effective forms of cancer treatment, and P53 protein is one of the key molecules determining how a cell responds to radiotherapy. The aim of this study was to determine the therapeutic efficacy of iodine-131 in three human HCC cell lines. Methods Western blotting was used to measure P53 expression. The effects of radiotherapy with iodine-131 were assessed by using the clonogenic assay to evaluate cell survival. Flow cytometry was carried out to examine the effects of iodine-131 on cell death, oxidative stress, reduced intracellular glutathione expression, the mitochondrial membrane potential, and the cell cycle. Results The P53 protein was not expressed in Hep3B2.1-7 cells, was expressed at normal levels in HepG2 cells, and was overexpressed in HuH7 cells. P53 expression in the HuH7 and HepG2 cell lines increased after internal and external irradiation with iodine-131. Irradiation induced a decrease in cell survival and led to a decrease in cell viability in all of the cell lines studied, accompanied by cell death via late apoptosis/necrosis and necrosis. Irradiation with 131-iodine induced mostly cell-cycle arrest in the G0/G1 phase. Conclusions These results suggest that P53 plays a key role in the radiotherapy response of HCC. PMID:26527121

Effect of Bmi-1-mediated NF-κB signaling pathway on the stem-like properties of CD133+ human liver cancer cells.

PubMed

Ma, De-Qiang; Zhang, Yin-Hua; Ding, De-Ping; Li, Juan; Chen, Lin-Li; Tian, You-You; Ao, Kang-Jian

2018-05-11

To investigate the impact of Bmi-1-mediated NF-κB pathway on the biological characteristics of CD133+ liver cancer stem cells (LCSCs). Flow cytometry was used to isolate CD133+ LCSC cells from Huh7, Hep3B, SK-hep1, and PLC/PRF-5 cells. CD133+ Huh7 cells were divided into Control, Blank, Bmi-1 siRNA, JSH-23 (NF-κB pathway inhibitor), and Bmi-1 + JSH-23 groups. The properties of CD133+ Huh7 cells were detected by the colony-formation and sphere-forming assays. Besides, Transwell assay was applied for the measurement of cell invasion and migration, immunofluorescence staining for the detection of NF-κB p65 nuclear translocation, and qRT-PCR and Western blotting for the determination of SOX2, NANOG, OCT4, Bmi-1, and NF-κB p65 expression. CD133+ Huh-7 cells were chosen as the experiment subjects after flow cytometry. Compared with CD133- Huh-7 cells, the expression of CD133, OCT4, SOX2, NANOG, Bmi-1, and NF-κB p65, the nuclear translocation of NF-κB p65, the number of cell colonies and Sphere formation, as well as the abilities of invasion and migration were observed to be increased in CD133+ Huh-7 cells, which was inhibited after treated with Bmi-1 siRNA or JSH-23, meanwhile, the cell cycle was arrested at the G0/G1 and S phases with apparently enhanced cell apoptosis. Importantly, no significant differences in the biological characteristics of CD133 + Huh-7 cells were found between the Blank group and Bmi-1 + JSH-23 group. Down-regulating Bmi-1 may inhibit the biological properties of CD133+ LCSC by blocking NF-κB signaling pathway, which lays a scientific foundation for the clinical treatment of liver cancer.

Evaluating hepatocellular carcinoma cell lines for tumour samples using within-sample relative expression orderings of genes.

PubMed

Ao, Lu; Guo, You; Song, Xuekun; Guan, Qingzhou; Zheng, Weicheng; Zhang, Jiahui; Huang, Haiyan; Zou, Yi; Guo, Zheng; Wang, Xianlong

2017-11-01

Concerns are raised about the representativeness of cell lines for tumours due to the culture environment and misidentification. Liver is a major metastatic destination of many cancers, which might further confuse the origin of hepatocellular carcinoma cell lines. Therefore, it is of crucial importance to understand how well they can represent hepatocellular carcinoma. The HCC-specific gene pairs with highly stable relative expression orderings in more than 99% of hepatocellular carcinoma but with reversed relative expression orderings in at least 99% of one of the six types of cancer, colorectal carcinoma, breast carcinoma, non-small-cell lung cancer, gastric carcinoma, pancreatic carcinoma and ovarian carcinoma, were identified. With the simple majority rule, the HCC-specific relative expression orderings from comparisons with colorectal carcinoma and breast carcinoma could exactly discriminate primary hepatocellular carcinoma samples from both primary colorectal carcinoma and breast carcinoma samples. Especially, they correctly classified more than 90% of liver metastatic samples from colorectal carcinoma and breast carcinoma to their original tumours. Finally, using these HCC-specific relative expression orderings from comparisons with six cancer types, we identified eight of 24 hepatocellular carcinoma cell lines in the Cancer Cell Line Encyclopedia (Huh-7, Huh-1, HepG2, Hep3B, JHH-5, JHH-7, C3A and Alexander cells) that are highly representative of hepatocellular carcinoma. Evaluated with a REOs-based prognostic signature for hepatocellular carcinoma, all these eight cell lines showed the same metastatic properties of the high-risk metastatic hepatocellular carcinoma tissues. Caution should be taken for using hepatocellular carcinoma cell lines. Our results should be helpful to select proper hepatocellular carcinoma cell lines for biological experiments. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Hepatitis C virus utilizes VLDLR as a novel entry pathway.

PubMed

Ujino, Saneyuki; Nishitsuji, Hironori; Hishiki, Takayuki; Sugiyama, Kazuo; Takaku, Hiroshi; Shimotohno, Kunitada

2016-01-05

Various host factors are involved in the cellular entry of hepatitis C virus (HCV). In addition to the factors previously reported, we discovered that the very-low-density lipoprotein receptor (VLDLR) mediates HCV entry independent of CD81. Culturing Huh7.5 cells under hypoxic conditions significantly increased HCV entry as a result of the expression of VLDLR, which was not expressed under normoxic conditions in this cell line. Ectopic VLDLR expression conferred susceptibility to HCV entry of CD81-deficient Huh7.5 cells. Additionally, VLDLR-mediated HCV entry was not affected by the knockdown of cellular factors known to act as HCV receptors or HCV entry factors. Because VLDLR is expressed in primary human hepatocytes, our results suggest that VLDLR functions in vivo as an HCV receptor independent of canonical CD81-mediated HCV entry.

The silencing of Pokemon attenuates the proliferation of hepatocellular carcinoma cells in vitro and in vivo by inhibiting the PI3K/Akt pathway.

PubMed

Lin, Chan-Chan; Zhou, Jing-Ping; Liu, Yun-Peng; Liu, Jing-Jing; Yang, Xiao-Ning; Jazag, Amarsanaa; Zhang, Zhi-Ping; Guleng, Bayasi; Ren, Jian-Lin

2012-01-01

Pokemon (POK erythroid myeloid ontogenic factor), which belongs to the POK protein family, is also called LRF, OCZF and FBI-1. As a transcriptional repressor, Pokemon assumes a critical function in cellular differentiation and oncogenesis. Our study identified an oncogenic role for Pokemon in human hepatocellular carcinoma (HCC). We successfully established human HepG2 and Huh-7 cell lines in which Pokemon was stably knocked down. We demonstrated that Pokemon silencing inhibited cell proliferation and migration. Pokemon knockdown inhibited the PI3K/Akt and c-Raf/MEK/ERK pathways and modulated the expression of various cell cycle regulators in HepG2 and Huh-7 cells. Therefore, Pokemon may also be involved in cell cycle progression in these cells. We confirmed that Pokemon silencing suppresses hepatocellular carcinoma growth in tumor xenograft mice. These results suggest that Pokemon promotes cell proliferation and migration in hepatocellular carcinoma and accelerates tumor development in an Akt- and ERK-signaling-dependent manner.

CXCR4 expression affects overall survival of HCC patients whereas CXCR7 expression does not

PubMed Central

Neve Polimeno, Maria; Ierano, Caterina; D'Alterio, Crescenzo; Simona Losito, Nunzia; Napolitano, Maria; Portella, Luigi; Scognamiglio, Giosuè; Tatangelo, Fabiana; Maria Trotta, Anna; Curley, Steven; Costantini, Susan; Liuzzi, Raffaele; Izzo, Francesco; Scala, Stefania

2015-01-01

Hepatocellular carcinoma (HCC) is a heterogeneous disease with a poor prognosis and limited markers for predicting patient survival. Because chemokines and chemokine receptors play numerous and integral roles in HCC disease progression, the CXCR4–CXCL12–CXCR7 axis was studied in HCC patients. CXCR4 and CXCR7 expression was analyzed by immunohistochemistry in 86 HCC patients (training cohort) and validated in 42 unrelated HCC patients (validation cohort). CXCR4 levels were low in 22.1% of patients, intermediate in 30.2%, and high in 47.7%, whereas CXCR7 levels were low in 9.3% of patients, intermediate in 44.2% and high in 46.5% of the patients in the training cohort. When correlated to patient outcome, only CXCR4 affected overall survival (P=0.03). CXCR4–CXCL12–CXCR7 mRNA levels were examined in 33/86 patients. Interestingly, the common CXCR4–CXCR7 ligand CXCL12 was expressed at significantly lower levels in tumor tissues compared to adjacent normal liver (P=0.032). The expression and function of CXCR4 and CXCR7 was also analyzed in several human HCC cell lines. CXCR4 was expressed in Huh7, Hep3B, SNU398, SNU449 and SNU475 cells, whereas CXCR7 was expressed in HepG2, Huh7, SNU449 and SNU475 cells. Huh7, SNU449 and SNU475 cells migrated toward CXCL12, and this migration was inhibited by AMD3100/anti-CXCR4 and by CCX771/anti-CXCR7. Moreover, SNU449 and Huh7 cells exhibited matrix invasion in the presence of CXCL12 and CXCL11, a ligand exclusive to CXCR7. In conclusion, CXCR4 affects the prognosis of HCC patients but CXCR7 does not. Therefore, the CXCR4–CXCL12–CXCR7 axis plays a role in the interaction of HCC with the surrounding normal tissue and represents a suitable therapeutic target. PMID:25363530

External beam radiation therapy enhances mesenchymal stem cell-mediated sodium iodide symporter gene delivery.

PubMed

Schug, Christina; Sievert, Wolfgang; Urnauer, Sarah; Müller, Andrea Maria; Schmohl, Kathrin Alexandra; Wechselberger, Alexandra; Schwenk, Nathalie; Lauber, Kirsten; Schwaiger, Markus; Multhoff, Gabriele; Wagner, Ernst; Nelson, Peter J; Spitzweg, Christine

2018-05-04

The tumor-homing properties of mesenchymal stem cells (MSC) have led to their development as delivery vehicles for the targeted delivery of therapeutic genes such as the sodium iodide symporter (NIS) to solid tumors. External beam radiation therapy (EBRT) may represent an ideal setting for the application of engineered MSC-based gene therapy as tumor irradiation may enhance MSC recruitment into irradiated tumors through the increased production of select factors linked to MSC migration. In the present study, the irradiation of human liver cancer cells (HuH7) (1-10 Gy) showed a strong dose-dependent increase in steady state mRNA levels of CXCL8, CXCL12/SDF-1, FGF2, PDGFβ, TGFβ1, TSP-1 and VEGF (0-48 h), which was verified for most factors at the protein level (after 48 h). Radiation effects on directed MSC migration was tested in vitro using a live cell tracking migration assay and supernatants from control and irradiated HuH7 cells. A robust increase in mean forward migration index (yFMI), mean center of mass (yCoM) and mean directionality of MSCs towards supernatants was seen from irradiated as compared to nonirradiated tumor cells. Transferability of this effect to other tumor sources was demonstrated using the human breast adenocarcinoma cell line (MDA-MB-231), which showed a similar behavior to radiation as seen with HuH7 cells in qPCR and migration assay. To evaluate this in a more physiologic in vivo setting, subcutaneously growing HuH7 xenograft tumors were irradiated with 0, 2 or 5 Gy followed by CMV-NIS-MSC application 24 h later. Tumoral iodide uptake was monitored using ¹²³I-scintigraphy. The results showed increased tumor-specific dose-dependent accumulation of radioiodide in irradiated tumors. Our results demonstrate that EBRT enhances the migratory capacity of MSCs and may thus increase the therapeutic efficacy of MSC-mediated NIS radionuclide therapy.

Expression of CD133 confers malignant potential by regulating metalloproteinases in human hepatocellular carcinoma.

PubMed

Kohga, Keisuke; Tatsumi, Tomohide; Takehara, Tetsuo; Tsunematsu, Hinako; Shimizu, Satoshi; Yamamoto, Masashi; Sasakawa, Akira; Miyagi, Takuya; Hayashi, Norio

2010-06-01

Although CD133 expression is identified as a cancer stem cell marker of hepatocellular carcinoma (HCC), the detailed characteristics of HCC cells expressing CD133 remain unclear. We examined the malignant characteristics of CD133-expressing HCC cells. CD133-expressing cells could be detected with low frequency in 5 HCC tissues. We derived two different HCC cell lines by (1) transfection of CD133 siRNA in PLC/PRF/5 cells in (CD133si-PLC/PRF/5), and (2) by a magnetic cell sorting method that allowed to divide Huh7 cells into two CD133 positive (+) and negative (-) groups. CD133 knockdown in PLC/PRF/5 cells resulted in a decrease of the mRNA and protein expressions of matrix metalloproteinase (MMP)-2 and a disintegrin and metalloproteinase (ADAM)9. We next examined the malignant characteristics related to decreasing MMP-2 and ADAM9 in HCC cells. In CD133si-PLC/PRF/5 cells and CD133- Huh7 cells, invasiveness and vascular endothelial growth factor (VEGF) production, which are both related to the activity of MMP-2, were inhibited compared CD133-expressing HCC cells. We previously demonstrated that ADAM9 protease plays critical roles in the shedding of MHC class I-related chain A (MICA) which regulates the sensitivity of tumor cells to natural killer cells (NK). Decreasing ADAM9 expression in CD133si-PLC/PRF/5 cells and CD133- Huh7 cells resulted in an increase in membrane-bound MICA and a decrease in soluble MICA production. Both CD133si-PLC/PRF/5 cells and CD133- Huh7 cells were susceptible to NK activity, depending on the expression levels of membrane-bound MICA, but CD133-expressing HCC cells were not. These results demonstrate that CD133 expression in HCC cells confers malignant potential which may contribute to the survival of HCC cells. Copyright 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

The dual targeting of insulin and insulin-like growth factor 1 receptor enhances the mTOR inhibitor-mediated antitumor efficacy in hepatocellular carcinoma

PubMed Central

Pivonello, Claudia; Negri, Mariarosaria; De Martino, Maria Cristina; Napolitano, Maria; de Angelis, Cristina; Provvisiero, Donatella Paola; Cuomo, Gaia; Auriemma, Renata Simona; Simeoli, Chiara; Izzo, Francesco; Colao, Annamaria; Hofland, Leo J.; Pivonello, Rosario

2016-01-01

Deregulation of mTOR and IGF pathways is frequent in hepatocellular carcinoma (HCC), thus mTOR and IGF1R represent suitable therapeutic targets in HCC. The aim of this study was to evaluate the effects of mTOR inhibitors (mTORi) and OSI-906, blocker of IGF1R/IR, on HCC cell proliferation, viability, migration and invasion, and alpha-fetoprotein (α-FP) secretion. In HepG2 and HuH-7 we evaluated, the expression of mTOR and IGF pathway components; the effects of Sirolimus, Everolimus, Temsirolimus and OSI-906 on cell proliferation; the effects of Sirolimus, OSI-906, and their combination, on cell secretion, proliferation, viability, cell cycle, apoptosis, invasion and migration. Moreover, intracellular mechanisms underlying these cell functions were evaluated in both cell lines. Our results show that HepG2 and HuH-7 present with the same mRNA expression profile with high levels of IGF2. OSI-906 inhibited cell proliferation at high concentration, while mTORi suppressed cell proliferation in a dose-time dependent manner in both cell lines. The co-treatment showed an additive inhibitory effect on cell proliferation and viability. This effect was not related to induction of apoptosis, but to G0/G1 phase block. Moreover, the co-treatment prevented the Sirolimus-induced AKT activation as escape mechanism. Both agents demonstrated to be differently effective in inhibiting α-FP secretion. Sirolimus, OSI-906, and their combination, blocked cell migration and invasion in HuH-7. These findings indicate that, co-targeting of IGF1R/IR and mTOR pathways could be a novel therapeutic approach in the management of HCC, in order to maximize antitumoral effect and to prevent the early development of resistance mechanisms. PMID:26756219

Comparative analysis of TGF-β/Smad signaling dependent cytostasis in human hepatocellular carcinoma cell lines.

PubMed

Dzieran, Johanna; Fabian, Jasmin; Feng, Teng; Coulouarn, Cédric; Ilkavets, Iryna; Kyselova, Anastasia; Breuhahn, Kai; Dooley, Steven; Meindl-Beinker, Nadja M

2013-01-01

Hepatocellular carcinoma (HCC) is a major public health problem due to increased incidence, late diagnosis and limited treatment options. TGF-β is known to provide cytostatic signals during early stages of liver damage and regeneration, but exerts tumor promoting effects in onset and progression of liver cancer. To understand the mechanistic background of such a switch, we systematically correlated loss of cytostatic TGF-β effects with strength and dynamics of its downstream signaling in 10 HCC cell lines. We demonstrate that TGF-β inhibits proliferation and induces apoptosis in cell lines with low endogenous levels of TGF-β and Smad7 and strong transcriptional Smad3 activity (PLC/PRF/5, HepG2, Hep3B, HuH7), previously characterized to express early TGF-β signatures correlated with better outcome in HCC patients. TGF-β dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-β and Smad7 and showing significantly reduced Smad3 signaling. Of those, HLE and HLF exhibit late TGF-β signatures, which is associated with bad prognosis in HCC patients. RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7. HCC-M and HCC-T represent a third group of cell lines lacking cytostatic TGF-β signaling despite strong and prolonged Smad3 phosphorylation and low Smad7 and TGF-β expression. Inhibitory linker phosphorylation, as in HCC-T, may disrupt C-terminally phosphorylated Smad3 function. In summary, we assort 10 HCC cell lines in at least two clusters with respect to TGF-β sensitivity. Cell lines responsive to the TGF-β cytostatic program, which recapitulate early stage of liver carcinogenesis exhibit transcriptional Smad3 activity. Those with disturbed TGF-β/Smad3 signaling are insensitive to TGF-β dependent cytostasis and might represent late stage of the disease. Regulation of this switch remains complex and cell line specific. These features may be relevant to discriminate stage dependent TGF-β functions for the design of efficient TGF-β directed therapy in liver cancer.

HBV life cycle is restricted in mouse hepatocytes expressing human NTCP.

PubMed

Li, Hanjie; Zhuang, Qiuyu; Wang, Yuze; Zhang, Tianying; Zhao, Jinghua; Zhang, Yali; Zhang, Junfang; Lin, Yi; Yuan, Quan; Xia, Ningshao; Han, Jiahuai

2014-03-01

Recent studies have revealed that human sodium taurocholate cotransporting polypeptide (SLC10A1 or NTCP) is a functional cellular receptor for hepatitis B virus (HBV). However, whether human NTCP can support HBV infection in mouse hepatocyte cell lines has not been clarified. Because an HBV-permissible mouse model would be helpful for the study of HBV pathogenesis, it is necessary to investigate whether human NTCP supports the susceptibility of mouse hepatocyte cell lines to HBV. The results show that exogenous human NTCP expression can render non-susceptible HepG2 (human), Huh7 (human), Hepa1-6 (mouse), AML-12 (mouse) cell lines and primary mouse hepatocyte (PMH) cells susceptible to hepatitis D virus (HDV) which employs HBV envelope proteins. However, human NTCP could only introduce HBV susceptibility in human-derived HepG2 and Huh7 cells, but not in mouse-derived Hepa1-6, AML-12 or PMH cells. These data suggest that although human NTCP is a functional receptor that mediates HBV infection in human cells, it cannot support HBV infection in mouse hepatocytes. Our study indicated that the restriction of HBV in mouse hepatocytes likely occurs after viral entry but prior to viral transcription. We have excluded the role of mouse hepatocyte nuclear factors in the restriction of the HBV life cycle and showed that knockdown or inhibition of Sting, TBK1, IRF3 or IRF7, the components of the anti-viral signaling pathways, had no effect on HBV infection in mouse hepatocytes. Therefore, murine restriction factors that limit HBV infection need to be identified before a HBV-permissible mouse line can be created.

Occludin-Knockout Human Hepatic Huh7.5.1-8-Derived Cells Are Completely Resistant to Hepatitis C Virus Infection.

PubMed

Shirasago, Yoshitaka; Shimizu, Yoshimi; Tanida, Isei; Suzuki, Tetsuro; Suzuki, Ryosuke; Sugiyama, Kazuo; Wakita, Takaji; Hanada, Kentaro; Yagi, Kiyohito; Kondoh, Masuo; Fukasawa, Masayoshi

2016-05-01

It is well known that occludin (OCLN) is involved in hepatitis C virus (HCV) entry into hepatocytes, but there has been no conclusive evidence that OCLN is essential for HCV infection. In this study, we first established an OCLN-knockout cell line derived from human hepatic Huh7.5.1-8 cells using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system, in which two independent targeting plasmids expressing single-guide RNAs were used. One established cell clone, named OKH-4, had the OCLN gene truncated in the N-terminal region, and a complete defect of the OCLN protein was shown using immunoblot analysis. Infection of OKH-4 cells with various genotypes of HCV was abolished, and exogenous expression of the OCLN protein in OKH-4 cells completely reversed permissiveness to HCV infection. In addition, using a co-culture system of HCV-infected Huh7.5.1-8 cells with OKH-4 cells, we showed that OCLN is also critical for cell-to-cell HCV transmission. Thus, we concluded that OCLN is essential for HCV infection of human hepatic cells. Further experiments using HCV genomic RNA-transfected OKH-4 cells or HCV subgenomic replicon-harboring OKH-4 cells suggested that OCLN is mainly involved in the entry step of the HCV life cycle. It was also demonstrated that the second extracellular loop of OCLN, especially the two cysteine residues, is critical for HCV infection of hepatic cells. OKH-4 cells may be a useful tool for understanding not only the entire mechanism of HCV entry, but also the biological functions of OCLN.

The Silencing of Pokemon Attenuates the Proliferation of Hepatocellular Carcinoma Cells In Vitro and In Vivo by Inhibiting the PI3K/Akt Pathway

PubMed Central

Liu, Yun-Peng; Liu, Jing-Jing; Yang, Xiao-Ning; Jazag, Amarsanaa; Zhang, Zhi-Ping; Guleng, Bayasi; Ren, Jian-Lin

2012-01-01

Pokemon (POK erythroid myeloid ontogenic factor), which belongs to the POK protein family, is also called LRF, OCZF and FBI-1. As a transcriptional repressor, Pokemon assumes a critical function in cellular differentiation and oncogenesis. Our study identified an oncogenic role for Pokemon in human hepatocellular carcinoma (HCC). We successfully established human HepG2 and Huh-7 cell lines in which Pokemon was stably knocked down. We demonstrated that Pokemon silencing inhibited cell proliferation and migration. Pokemon knockdown inhibited the PI3K/Akt and c-Raf/MEK/ERK pathways and modulated the expression of various cell cycle regulators in HepG2 and Huh-7 cells. Therefore, Pokemon may also be involved in cell cycle progression in these cells. We confirmed that Pokemon silencing suppresses hepatocellular carcinoma growth in tumor xenograft mice. These results suggest that Pokemon promotes cell proliferation and migration in hepatocellular carcinoma and accelerates tumor development in an Akt- and ERK-signaling-dependent manner. PMID:23300578

Prognostic relevance of miR-137 in patients with hepatocellular carcinoma.

PubMed

Sakabe, Tomohiko; Azumi, Junya; Umekita, Yoshihisa; Toriguchi, Kan; Hatano, Etsuro; Hirooka, Yasuaki; Shiota, Goshi

2017-02-01

Cancer stem cells (CSCs) play a pivotal role in progression, metastasis and recurrence of cancer. Therefore, it is clinically useful to identify the relevant CSC marker that is associated with prognosis of hepatocellular carcinoma (HCC) and clarify its genetic and biological characteristics. Expression of four CSC markers, CD13, EpCAM, CD44 and CD44v9, was examined in 99 HCC patients. Biological and cDNA/miRNA microarray data were compared among CD44-positive/-negative HCC cells and normal hepatic cells. The significance of the representative miRNAs was examined with regard to prognosis of additional 110 HCC patients. CD44-positive HuH7 cells proliferated faster and showed a greater sphere forming ability than CD44-negative HuH7 cells. CD44-positive HuH7 cells exhibited higher expression of specific genes involved in resistance to reactive oxygen species, anticancer drugs and tumour invasion than CD44-negative HCC cells. Higher expression of six miRNAs was observed in CD44-positive HuH7 cells, CD44-negative HuH7 cells, and human normal hepatic cells in that order. Of the six miRNAs, miR-137 was closely associated with overall and cancer-specific survivals, as well as with invasion of hepatic vein, hepatic artery, portal vein and bile duct, and alpha-foetoprotein in additional 110 HCC patients. miR-137 may serve as a prognostic marker in patients with HCC and may be a potential target for the elimination of liver CSCs. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

PKI-587 and sorafenib targeting PI3K/AKT/mTOR and Ras/Raf/MAPK pathways synergistically inhibit HCC cell proliferation.

PubMed

Gedaly, Roberto; Angulo, Paul; Hundley, Jonathan; Daily, Michael F; Chen, Changguo; Evers, B Mark

2012-08-01

Deregulated Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study aimed to test the inhibitory effects of PKI-587 and sorafenib as single agents or in combination on HCC (Huh7 cell line) proliferation. (3)H-thymidine incorporation and MTT assay were used to assess Huh7 cell proliferation. Phosphorylation of the key enzymes in the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways was detected by Western blot. We found that PKI-587 is a more potent PI3K/mTOR inhibitor than PI-103. Combination of PKI-587 and sorafenib was a more effective inhibitor of Huh7 proliferation than the combination of PI-103 and sorafenib. Combination of PKI-587 and sorafenib synergistically inhibited epidermal growth factor (EGF)-stimulated Huh7 proliferation compared with monodrug therapy. EGF increased phosphorylation of Ras/Raf downstream signaling proteins MEK and ERK; EGF-stimulated activation was inhibited by sorafenib. However, sorafenib, as a single agent, increased AKT (Ser473) phosphorylation. EGF-stimulated AKT (ser473) activation was inhibited by PKI-587. PKI-587 is a potent inhibitor of AKT (Ser473), mTOR (Ser2448), and S6K (Thr389) phosphorylation; in contrast, rapamycin stimulated mTOR complex 2 substrate AKT(Ser473) phosphorylation although it inhibited mTOR complex 1 substrate S6K phosphorylation. PKI-587, as a single agent, stimulated MEK and ERK phosphorylation. However, when PKI-587 and sorafenib were used in combination, they inhibited all the tested kinases in the Ras/Raf /MAPK and PI3K/AKT/mTOR pathways. The combination of PKI-587 and sorafenib has the advantage over monodrug therapy on inhibition of HCC cell proliferation by blocking both PI3K/AKT/mTOR and Ras/Raf/MAPK signaling pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

Melatonin-induced increase in sensitivity of human hepatocellular carcinoma cells to sorafenib is associated with reactive oxygen species production and mitophagy

PubMed Central

Prieto-Domínguez, Néstor; Ordóñez, Raquel; Fernández, Anna; Méndez-Blanco, Carolina; Baulies, Anna; Garcia-Ruiz, Carmen; Fernández-Checa, José C.; Mauriz, José L.; González-Gallego, Javier

2016-01-01

Effects of sorafenib in hepatocellular carcinoma (HCC) are frequently transient due to tumor-acquired resistance, a phenotype that could be targeted by other molecules to reduce this adaptive response. Because melatonin is known to exert antitumor effects in HCC cells, this study investigated whether and how melatonin reduces resistance to sorafenib. Susceptibility to sorafenib (10 nM to 50 μM) in the presence of melatonin (1 and 2 mM) was assessed in HCC cell lines HepG2, HuH7 and Hep3B. Cell viability was reduced by sorafenib from 1 μM in HepG2 or HuH7 cells, and 2.5 μM in Hep3B cells. Co-administration of melatonin and sorafenib exhibited a synergistic cytotoxic effect on HepG2 and HuH7 cells, while Hep3B cells displayed susceptibility to doses of sorafenib that had no effect when administrated alone. Co-administration of 2.5 μM sorafenib and 1 mM melatonin induced apoptosis in Hep3B cells, increasing PARP hydrolysis and BAX expression. We also observed an early colocalization of mitochondria with lysosomes, correlating with the expression of mitophagy markers PINK1 and Parkin and a reduction of mitofusin-2 and mtDNA compared with sorafenib administration alone. Moreover, increased reactive oxygen species production and mitochondrial membrane depolarization were elicited by drug combination, suggesting their contribution to mitophagy induction. Interestingly, Parkin silencing by siRNA to impair mitophagy significantly reduced cell killing, PARP cleavage and BAX expression. These results demonstrate that the pro-oxidant capacity of melatonin and its impact on mitochondria stability and turnover via mitophagy increase sensitivity to the cytotoxic effect of sorafenib. PMID:27484637

Isolation and Characterization of Highly Replicable Hepatitis C Virus Genotype 1a Strain HCV-RMT

PubMed Central

Arai, Masaaki; Tokunaga, Yuko; Takagi, Asako; Tobita, Yoshimi; Hirata, Yuichi; Ishida, Yuji; Tateno, Chise; Kohara, Michinori

2013-01-01

Multiple genotype 1a clones have been reported, including the very first hepatitis C virus (HCV) clone called H77. The replication ability of some of these clones has been confirmed in vitro and in vivo, although this ability is somehow compromised. We now report a newly isolated genotype 1a clone, designated HCV-RMT, which has the ability to replicate efficiently in patients, chimeric mice with humanized liver, and cultured cells. An authentic subgenomic replicon cell line was established from the HCV-RMT sequence with spontaneous introduction of three adaptive mutations, which were later confirmed to be responsible for efficient replication in HuH-7 cells as both subgenomic replicon RNA and viral genome RNA. Following transfection, the HCV-RMT RNA genome with three adaptive mutations was maintained for more than 2 months in HuH-7 cells. One clone selected from the transfected cells had a high copy number, and its supernatant could infect naïve HuH-7 cells. Direct injection of wild-type HCV-RMT RNA into the liver of chimeric mice with humanized liver resulted in vigorous replication, similar to inoculation with the parental patient’s serum. A study of virus replication using HCV-RMT derivatives with various combinations of adaptive mutations revealed a clear inversely proportional relationship between in vitro and in vivo replication abilities. Thus, we suggest that HCV-RMT and its derivatives are important tools for HCV genotype 1a research and for determining the mechanism of HCV replication in vitro and in vivo. PMID:24358200

Isolation and characterization of highly replicable hepatitis C virus genotype 1a strain HCV-RMT.

PubMed

Arai, Masaaki; Tokunaga, Yuko; Takagi, Asako; Tobita, Yoshimi; Hirata, Yuichi; Ishida, Yuji; Tateno, Chise; Kohara, Michinori

2013-01-01

Multiple genotype 1a clones have been reported, including the very first hepatitis C virus (HCV) clone called H77. The replication ability of some of these clones has been confirmed in vitro and in vivo, although this ability is somehow compromised. We now report a newly isolated genotype 1a clone, designated HCV-RMT, which has the ability to replicate efficiently in patients, chimeric mice with humanized liver, and cultured cells. An authentic subgenomic replicon cell line was established from the HCV-RMT sequence with spontaneous introduction of three adaptive mutations, which were later confirmed to be responsible for efficient replication in HuH-7 cells as both subgenomic replicon RNA and viral genome RNA. Following transfection, the HCV-RMT RNA genome with three adaptive mutations was maintained for more than 2 months in HuH-7 cells. One clone selected from the transfected cells had a high copy number, and its supernatant could infect naïve HuH-7 cells. Direct injection of wild-type HCV-RMT RNA into the liver of chimeric mice with humanized liver resulted in vigorous replication, similar to inoculation with the parental patient's serum. A study of virus replication using HCV-RMT derivatives with various combinations of adaptive mutations revealed a clear inversely proportional relationship between in vitro and in vivo replication abilities. Thus, we suggest that HCV-RMT and its derivatives are important tools for HCV genotype 1a research and for determining the mechanism of HCV replication in vitro and in vivo.

Identification of a Calcium Signalling Pathway of S-[6]-Gingerol in HuH-7 Cells.

PubMed

Li, Xiao-Hong; McGrath, Kristine C Y; Tran, Van H; Li, Yi-Ming; Mandadi, Sravan; Duke, Colin C; Heather, Alison K; Roufogalis, Basil D

2013-01-01

Calcium signals in hepatocytes control cell growth, proliferation, and death. Members of the transient receptor potential (TRP) cation channel superfamily are candidate calcium influx channels. NF κ B activation strictly depends on calcium influx and often induces antiapoptotic genes favouring cell survival. Previously, we reported that S-[6]-gingerol is an efficacious agonist of the transient receptor potential cation channel subfamily V member 1 (TRPV1) in neurones. In this study, we tested the effect of S-[6]-gingerol on HuH-7 cells using the Fluo-4 calcium assay, RT-qPCR, transient cell transfection, and luciferase measurements. We found that S-[6]-gingerol induced a transient rise in [Ca(2+)] i in HuH-7 cells. The increase in [Ca(2+)] i induced by S-[6]-gingerol was abolished by preincubation with EGTA and was also inhibited by the TRPV1 channel antagonist capsazepine. Expression of TRPV1 in HuH-7 cells was confirmed by mRNA analysis as well as a test for increase of [Ca(2+)] i by TRPV1 agonist capsaicin and its inhibition by capsazepine. We found that S-[6]-gingerol induced rapid NF κ B activation through TRPV1 in HuH-7 cells. Furthermore, S-[6]-gingerol-induced NF κ B activation was dependent on the calcium gradient and TRPV1. The rapid NF κ B activation by S-[6]-gingerol was associated with an increase in mRNA levels of NF κ B-target genes: cIAP-2, XIAP, and Bcl-2 that encode antiapoptotic proteins.

GATA4 promotes hepatoblastoma cell proliferation by altering expression of miR125b and DKK3.

PubMed

Pei, Yihua; Yao, Qin; Yuan, Sibo; Xie, Bozhen; Liu, Yan; Ye, Chunsheng; Zhuo, Huiqin

2016-11-22

GATA4 is a zinc finger DNA-binding protein that plays an important role in mammalian liver development. However, the effects of GATA4 in hepatoblastoma (HB), a common liver cancer in pediatric patients, remain largely unknown. In this study, we demonstrate that GATA4 promotes growth and survival in the Huh6 human hepatoblastoma cell line. GATA4 expression was high in Huh6 cells, and its knockdown decreased expression of Dickkopf-related protein 3 (DKK3), a gene that may contribute to premature or undifferentiated phenotypes in HB. GATA4 also directly bound to the promoter regions of the miRNA miR125b and inhibited its expression in Huh6 cells. DKK3 was a direct target of miR125b in Huh6 cells. Inhibition of miR125b or overexpression of DKK3 promoted proliferation, survival, migration, and invasion in Huh6 cells. This is the first report to demonstrate that GATA4 promotes oncogenesis by inhibiting miR125b-dependent suppression of DKK3 expression. This GATA4/miR125b/DKK3 axis may be a major regulator of growth, migration, invasion, and survival in hepatoma cells, and is therefore a potential therapeutic target or biomarker for progression in HB patients.

Inhibition of mitogen-activated protein kinase Erk1/2 promotes protein degradation of ATP binding cassette transporters A1 and G1 in CHO and HuH7 cells.

PubMed

Mulay, Vishwaroop; Wood, Peta; Manetsch, Melanie; Darabi, Masoud; Cairns, Rose; Hoque, Monira; Chan, Karen Cecilia; Reverter, Meritxell; Alvarez-Guaita, Anna; Rye, Kerry-Anne; Rentero, Carles; Heeren, Joerg; Enrich, Carlos; Grewal, Thomas

2013-01-01

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

A microRNA-7/growth arrest specific 6/TYRO3 axis regulates the growth and invasiveness of sorafenib-resistant cells in human hepatocellular carcinoma.

PubMed

Kabir, Tasnuva D; Ganda, Clarissa; Brown, Rikki M; Beveridge, Dianne J; Richardson, Kirsty L; Chaturvedi, Vishal; Candy, Patrick; Epis, Michael; Wintle, Larissa; Kalinowski, Felicity; Kopp, Christina; Stuart, Lisa M; Yeoh, George C; George, Jacob; Leedman, Peter J

2018-01-01

Sorafenib remains the only approved drug for treating patients with advanced hepatocellular carcinoma (HCC). However, the therapeutic effect of sorafenib is transient, and patients invariably develop sorafenib resistance (SR). Recently, TYRO3, a member of the TYRO3-AXL-MER family of receptor tyrosine kinases, was identified as being aberrantly expressed in a significant proportion of HCC; however, its role in SR is unknown. In this study, we generated two functionally distinct sorafenib-resistant human Huh-7 HCC cell lines in order to identify new mechanisms to abrogate acquired SR as well as new potential therapeutic targets in HCC. Initially, we investigated the effects of a microRNA (miR), miR-7-5p (miR-7), in both in vitro and in vivo preclinical models of human HCC and identified miR-7 as a potent tumor suppressor of human HCC. We identified TYRO3 as a new functional target of miR-7, which regulates proliferation, migration, and invasion of Huh-7 cells through the phosphoinositide 3-kinase/protein kinase B pathway and is markedly elevated with acquisition of SR. Furthermore, miR-7 effectively silenced TYRO3 expression in both sorafenib-sensitive and sorafenib-resistant Huh-7 cells, inhibiting TYRO3/growth arrest specific 6-mediated cancer cell migration and invasion. We identified a mechanism for acquiring SR in HCC that is through the aberrant expression of the TYRO3/phosphoinositide 3-kinase/protein kinase B signal transduction pathway, and that can be overcome by miR-7 overexpression. Taken together, these data suggest a potential role for miR-7 as an RNA-based therapeutic to treat refractory and drug-resistant HCC. (Hepatology 2018;67:216-231). © 2017 by the American Association for the Study of Liver Diseases.

Proteomics reveal energy metabolism and mitogen-activated protein kinase signal transduction perturbation in human Borna disease virus Hu-H1-infected oligodendroglial cells.

PubMed

Liu, X; Yang, Y; Zhao, M; Bode, L; Zhang, L; Pan, J; Lv, L; Zhan, Y; Liu, S; Zhang, L; Wang, X; Huang, R; Zhou, J; Xie, P

2014-05-30

Borna disease virus (BDV) is a neurotropic, non-cytolytic RNA virus which replicates in the cell nucleus targeting mainly hippocampal neurons, but also astroglial and oligodendroglial cells in the brain. BDV is associated with a large spectrum of neuropsychiatric pathologies in animals. Its relationship to human neuropsychiatric illness still remains controversial. We could recently demonstrate that human BDV strain Hu-H1 promoted apoptosis and inhibited cell proliferation in a human oligodendroglial cell line (OL cells) whereas laboratory BDV strain V acted contrariwise. Here, differential protein expression between BDV Hu-H1-infected OL cells and non-infected OL cells was assessed through a proteomics approach, using two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry. A total of 63 differential host proteins were identified in BDV Hu-H1-infected OL cells compared to non-infected OL cells. We found that most changes referred to alterations related to the pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, the tricarboxylic acid (TCA) cycle, and glycolysis /gluconeogenesis. By manual querying, two differential proteins were found to be associated with mitogen-activated protein kinase (MAPK) signal transduction. Five key signaling proteins of this pathway (i.e., p-Raf, p-MEK, p-ERK1/2, p-RSK, and p-MSK) were selected for Western blotting validation. p-ERK1/2 and p-RSK were found to be significantly up-regulated, and p-MSK was found to be significantly down-regulated in BDV Hu-H1-infected OL cells compared to non-infected OL cell. Although BDV Hu-H1 constitutively activated the ERK-RSK pathway, host cell proliferation and nuclear translocation of activated pERK in BDV Hu-H1-infected OL cells were impaired. These findings indicate that BDV Hu-H1 infection of human oligodendroglial cells significantly perturbs host energy metabolism, activates the downstream ERK-RSK complex of the Raf/MEK/ERK signaling cascade, and disturbs host cell proliferation possibly through impaired nuclear translocation of pERK, a finding which warrants further research. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Near-infrared fluorescence imaging and photodynamic therapy with indocyanine green lactosome has antineoplastic effects for hepatocellular carcinoma.

PubMed

Tsuda, Takumi; Kaibori, Masaki; Hishikawa, Hidehiko; Nakatake, Richi; Okumura, Tadayoshi; Ozeki, Eiichi; Hara, Isao; Morimoto, Yuji; Yoshii, Kengo; Kon, Masanori

2017-01-01

Anticancer agents and operating procedures have been developed for hepatocellular carcinoma (HCC) patients, but their prognosis remains poor. It is necessary to develop novel diagnostic and therapeutic strategies for HCC to improve its prognosis. Lactosome is a core-shell-type polymeric micelle, and enclosing labeling or anticancer agents into this micelle enables drug delivery. In this study, we investigated the diagnostic and therapeutic efficacies of indocyanine green (ICG)-loaded lactosome for near-infrared fluorescence (NIF) imaging and photodynamic therapy (PDT) for HCC. The human HCC cell line HuH-7 was treated with ICG or ICG-lactosome, followed by PDT, and the cell viabilities were measured (in vitro PDT efficiency). For NIF imaging, HuH-7 cells were subcutaneously transplanted into BALB/c nude mice, followed by intravenous administration of ICG or ICG-lactosome. The transplanted animals were treated with PDT, and the antineoplastic effects were analyzed (in vivo PDT efficiency). PDT had toxic effects on HuH-7 cells treated with ICG-lactosome, but not ICG alone. NIF imaging revealed that the fluorescence of tumor areas in ICG-lactosome-treated animals was higher than that of contralateral regions at 24 h after injection and thereafter. PDT exerted immediate and continuous phototoxic effects in the transplanted mice treated with ICG-lactosome. Our results demonstrate that ICG-lactosome accumulated in xenograft tumors, and that PDT had antineoplastic effects on these malignant implants. NIF imaging and PDT with ICG-lactosome could be useful diagnostic and/or therapeutic strategies for HCC.

Novel Combination of Sorafenib and Celecoxib Provides Synergistic Anti-Proliferative and Pro-Apoptotic Effects in Human Liver Cancer Cells

PubMed Central

Lampiasi, Nadia; Cusimano, Antonella; Azzolina, Antonina; McCubrey, James A.; Montalto, Giuseppe

2013-01-01

Molecular targeted therapy has shown promise as a treatment for advanced hepatocellular carcinoma (HCC). Sorafenib, a multikinase inhibitor, recently received FDA approval for the treatment of advanced HCC. However, although sorafenib is well tolerated, concern for its safety has been expressed. Celecoxib (Celebrex®) is a selective cyclooxygenase-2 (COX-2) inhibitor which exhibits antitumor effects in human HCC cells. The present study examined the interaction between celecoxib and sorafenib in two human liver tumor cell lines HepG2 and Huh7. Our data showed that each inhibitor alone reduced cell growth and the combination of celecoxib with sorafenib synergistically inhibited cell growth and increased apoptosis. To better understand the molecular mechanisms underlying the synergistic antitumor activity of the combination, we investigated the expression profile of the combination-treated liver cancer cell lines using microarray analysis. Combination treatment significantly altered expression levels of 1,986 and 2,483 transcripts in HepG2 and Huh7 cells, respectively. Genes functionally involved in cell death, signal transduction and regulation of transcription were predominantly up-regulated, while genes implicated in metabolism, cell-cycle control and DNA replication and repair were mainly down-regulated upon treatment. However, combination-treated HCC cell lines displayed specificity in the expression and activity of crucial factors involved in hepatocarcinogenesis. The altered expression of some of these genes was confirmed by semi-quantitative and quantitative RT-PCR and by Western blotting. Many novel genes emerged from our transcriptomic analyses, and further functional analyses may determine whether these genes can serve as potential molecular targets for more effective anti-HCC strategies. PMID:23776502

SUMOylation of HSP27 by small ubiquitin-like modifier 2/3 promotes proliferation and invasion of hepatocellular carcinoma cells.

PubMed

Ge, Haize; Du, Juan; Xu, Jingman; Meng, Xiangliang; Tian, Jinchuan; Yang, Jie; Liang, Huimin

2017-08-03

Primary hepatocellular carcinoma (PHC) is a major health problem worldwide and is one of the 10 most commonly diagnosed cancers in China. Heat shock protein 27 (HSP27) were found to be overexpressed in a wide range of malignancies including PHC, however, post-translational modification of HSP27 still needs exploration in PHC. Recently, SUMOylation, an important post-translational modification associating with the development of many kinds of cancers has been intensively studied. In the current study, mRNA and protein level of HSP27 in archived tumor samples representing various pathological characteristics of PHC were examined, and modification of HSP27 by SUMO2/3 was investigated. HSP27 were expressed abundantly in patients' tumor tissues, and found to be associated with pathological progression. Besides, HSP27 was also elevated significantly in liver cancer cell lines Huh7 and HepG2 compared with human hepatocyte cells L02. Furthermore, knockdown of HSP27 was found to be associated with the decreased proliferation and invasion ability in Huh7 and HepG2 cells. Immunofluorescence assay showed that HSP27 and SUMO2/3 were co-localized in the subcellular, and co-immunoprecipitation verified the interaction between HSP27 and SUMO2/3. Overexpression of SUMO2/3 upregulated the HSP27 protein level and promotes Huh7 and HepG2 cell proliferation and invasion, and vice versa when the SUMO2/3 was knockdown. Taken together, increased protein level of HSP27 through SUMO2/3-mediated SUMOylation plays crucial roles in the progression of PHC, and this finding may shed light on developing potential therapeutic targets for PHC.

Antiviral Effect of Sub Fraction Cassia alata Leaves Extract to Dengue Virus Serotype-2 strain New Guinea C in Human Cell Line Huh-7 it-1

NASA Astrophysics Data System (ADS)

Angelina, Marissa; Hanafi, Muhammad; Suyatna, Franciscus D.; Mirawati S., T.; Ratnasari, Shirley; Ernawati Dewi, Beti

2017-12-01

Dengue virus (DENV) is one of the most common viral infections found Indonesia and tropical regions, and no specific antiviral for DENV. Indonesia has several of herbal medicine that were not explored of their potency as antiviral DENV. This study was done to evaluate the activity and toxicity of 4 derived fractions: Hexane (CA1), ethyl acetate (CA2), buthanol (CA3 ) and water (CA4) of Cassia alata leaf extract (CA) as an antiviral drug to DENV. The DENV was treated with various concentration of extract and added to Huh-7 it-1. The decrease of virus titer was determined by Focus assay. The toxicity of extract was measured by MTT assay. In our previous study, we found that CA on Huh-7 cells showed IC50, CC50 and SI values of <10 μg/mL, 323.45 μg/mL, and more than 32.3, respectively. For the fractions, CA3 showed best antiviral activity among other, with IC50, CC50 and SI of <10 μg/mL, 645.8 μg/mL, and more than 64.5, respectively. CA and CA3 were proven to possess antiviral activity that is potent when tested against DENV-2. Future study was needed to explore the inhibition mechanism and compound of CA that have potency as antiviral drug to DENV.

Simultaneous imaging of temporal changes of NF-κB activity and viable tumor cells in Huh7/NF-κB-tk-luc2/rfp tumor-bearing mice.

PubMed

Wang, Wei-Hsun; Chiang, I-Tsang; Liu, Yu-Chang; Hsu, Fei-Ting; Chen, Hong-Wen; Chen, Chuan-Lin; Lee, Yi-Jang; Lin, Wuu-Jyh; Hwang, Jeng-Jong

2013-01-01

Few studies have reported that the effect of sorafenib on advanced human hepatocellular carcinoma (HCC) is taking place via the inhibition of NF-κB signal transduction. Here we constructed a human HCC Huh7 stable clone with NF-κB-responsive element to drive dual reporter genes, herpes simplex virus thymidine kinase (tk) and firefly luciferase (luc2), and co-transfected with a third red fluorescent protein (rfp) gene, renamed as Huh7/NF-κB-tk-luc2/rfp cells, and combined with bioluminescent imaging (BLI) and red fluorescent protein imaging (RFPI) to monitor the effect of sorafenib on NF-κB activation and tumor inhibition. The results show that sorafenib could suppress the NF-κB-DNA binding activity, and the expression of downstream effector proteins. Notably, the relative photon fluxes obtained from RFPI and BLI, which represent the viable tumor cells and cells with NF-κB activation, decreased after sorafenib treatment by 50 to 65%, and 87.5 to >90%, respectively, suggesting that NF-κB activation is suppressed in viable HCC cells by sorafenib. Simultaneous molecular imaging of the temporal change of NF-κB activity and of viable cells in the same Huh7/NF-κB-tk-luc2/rfp tumors of the animal may reflect the real status of NF-κB activity and the viable tumor cells at the time of imaging.

High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources

PubMed Central

Haraszti, Reka A.; Didiot, Marie-Cecile; Sapp, Ellen; Leszyk, John; Shaffer, Scott A.; Rockwell, Hannah E.; Gao, Fei; Narain, Niven R.; DiFiglia, Marian; Kiebish, Michael A.; Aronin, Neil; Khvorova, Anastasia

2016-01-01

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types. PMID:27863537

Anti-LRP/LR Specific Antibody IgG1-iS18 Impedes Adhesion and Invasion of Liver Cancer Cells

PubMed Central

Chetty, Carryn; Khumalo, Thandokuhle; Da Costa Dias, Bianca; Reusch, Uwe; Knackmuss, Stefan; Little, Melvyn; Weiss, Stefan F. T.

2014-01-01

Two key events, namely adhesion and invasion, are pivotal to the occurrence of metastasis. Importantly, the 37 kDa/67 kDa laminin receptor (LRP/LR) has been implicated in enhancing these two events thus facilitating cancer progression. In the current study, the role of LRP/LR in the adhesion and invasion of liver cancer (HUH-7) and leukaemia (K562) cells was investigated. Flow cytometry revealed that the HUH-7 cells displayed significantly higher cell surface LRP/LR levels compared to the poorly-invasive breast cancer (MCF-7) control cells, whilst the K562 cells displayed significantly lower cell surface LRP/LR levels in comparison to the MCF-7 control cells. However, Western blotting and densitometric analysis revealed that all three tumorigenic cell lines did not differ significantly with regards to total LRP/LR levels. Furthermore, treatment of liver cancer cells with anti-LRP/LR specific antibody IgG1-iS18 (0.2 mg/ml) significantly reduced the adhesive potential of cells to laminin-1 and the invasive potential of cells through the ECM-like Matrigel, whilst leukaemia cells showed no significant differences in both instances. Additionally, Pearson's correlation coefficients suggested direct proportionality between cell surface LRP/LR levels and the adhesive and invasive potential of liver cancer and leukaemia cells. These findings suggest the potential use of anti-LRP/LR specific antibody IgG1-iS18 as an alternative therapeutic tool for metastatic liver cancer through impediment of the LRP/LR- laminin-1 interaction. PMID:24798101

Composite fatty acid ether amides suppress growth of liver cancer cells in vitro and in an in vivo allograft mouse model.

PubMed

Cao, Mengde; Prima, Victor; Nelson, David; Svetlov, Stanislav

2013-06-01

The heterogeneity of liver cancer, in particular hepatocellular carcinoma (HCC), portrays the requirement of multiple targets for both its treatment and prevention. Multifaceted agents, minimally or non-toxic for normal hepatocytes, are required to address the molecular diversity of HCC, including the resistance of putative liver cancer stem cells to chemotherapy. We designed and synthesized two fatty acid ethers of isopropylamino propanol, C16:0-AIP-1 and C18:1-AIP-2 (jointly named AIPs), and evaluated their anti-proliferative effects on the human HCC cell line Huh7 and the murine hepatoma cell line BNL 1MEA.7R.1, both in vitro and in an in vivo allograft mouse model. We found that AIP-1 and AIP-2 inhibited proliferation and caused cell death in both Huh7 and BNL 1MEA.7R.1 cells. Importantly, AIP-1 and AIP-2 were found to block the activation of putative liver cancer stem cells as manifested by suppression of clonal 'carcinosphere' development in growth factor-free and anchorage-free medium. The AIPs exhibited a relatively low toxicity against normal human or rat hepatocytes in primary cultures. In addition, we found that the AIPs utilized multifaceted pathways that mediate both autophagy and apoptosis in HCC, including the inhibition of AKTs and CAMK-1. In immune-competent mice, the AIPs significantly reduced BNL 1MEA.7R.1 cell-driven tumor allograft development, with a higher efficiency than sorafenib. A combination of AIP-1 + AIP-2 was most effective in reducing the tumor allograft incidence. AIPs represent a novel class of simple fatty acid derivatives that are effective against liver tumors via diverse pathways. They show a low toxicity towards normal hepatocytes. The addition of AIPs may represent a new avenue towards the management of chronic liver injury and, ultimately, the prevention and treatment of HCC.

The ubiquitin ligase TRIM25 inhibits hepatocellular carcinoma progression by targeting metastasis associated 1 protein.

PubMed

Zang, Hong-Liang; Ren, Sheng-Nan; Cao, Hong; Tian, Xiao-Feng

2017-10-01

Metastasis associated 1 protein (MTA1) is one of the prime facilitators of metastatic progression in all solid tumors including hepatocellular carcinoma (HCC). However, the underlying regulatory mechanism of MTA1 expression in HCC is not clear. In this study, we evaluated MTA1 transcript and protein expression in HCC and normal hepatic cell lines. The results revealed that MTA1 protein expression had a significantly increase in HCC cell line, HuH6, compared with that in normal hepatic cell line, THLE-2. Determination of protein half-life using cycloheximide (CHX) treatment did not reveal any statistically significant difference in protein turn-over rates between THLE-2 (3.3 ± 0.25 h) and HuH6 (3.6 ± 0.15 h) cell lines. MTA1 protein level was stabilized in THLE-2 cells after treatment with MG-132 to levels similar to those observed in HuH6 cells. Mass spectrometric analysis of FLAG immunoprecipitates of FLAG-MTA1 transfected THLE-2 cells after MG-132 treated revealed candidate ubiquitin ligases that were interacting with MTA1. RNAi-mediated silencing of each prospective ubiquitin ligase in THLE-2 cells indicated that knockdown of TRIM25 resulted in stabilization of MTA1 protein, indicating TRIM25 as a putative E3 ligase for MTA1. Coimmunoprecipitation of FLAG-tagged MTA1, but not IgG, in MG-132 treated and untreated THLE-2 cells cotransfected with either FLAG-MTA1 or Myc-TRIM25 revealed robust polyubiquitinated MTA1, confirming that the TRIM25 is the ubiquitin ligase for MTA1 degradation. Overexpression of TRIM25 in HuH6 and RNAi mediated silencing of TRIM25 in THLE-2 cells inhibited and increased the cell migration and invasion, respectively. Analysis of The Cancer Genome Atlas data for assessment of TRIM25 transcript level and MTA1 protein expression in 25 HCC patients confirmed an inverse correlation between the expression of TRIM25 and MTA1. Cumulatively, our data reveal a novel mechanism of post-translational to regulate MTA1 expression in normal hepatic cells, which is repressed in HCC. © 2017 IUBMB Life, 69(10):795-801, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

Assessment of in vitro cyto/genotoxicity of sequentially treated electroplating effluent on the human hepatocarcinoma HuH-7 cell line.

PubMed

Naik, Umesh Chandra; Das, Mihir Tanay; Sauran, Swati; Thakur, Indu Shekhar

2014-03-01

The present study compares in vitro toxicity of electroplating effluent after the batch treatment process with that obtained after the sequential treatment process. Activated charcoal prepared from sugarcane bagasse through chemical carbonization, and tolerant indigenous bacteria, Bacillus sp. strain IST105, were used individually and sequentially for the treatment of electroplating effluent. The sequential treatment involving activated charcoal followed by bacterial treatment removed 99% of Cr(VI) compared with the batch processes, which removed 40% (charcoal) and 75% (bacteria), respectively. Post-treatment in vitro cyto/genotoxicity was evaluated by the MTT test and the comet assay in human HuH-7 hepatocarcinoma cells. The sequentially treated sample showed an increase in LC50 value with a 6-fold decrease in comet-assay DNA migration compared with that of untreated samples. A significant decrease in DNA migration and an increase in LC50 value of treated effluent proved the higher effectiveness of the sequential treatment process over the individual batch processes. Copyright © 2014 Elsevier B.V. All rights reserved.

Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties.

PubMed

Chiba, Tetsuhiro; Kita, Kaoru; Zheng, Yun-Wen; Yokosuka, Osamu; Saisho, Hiromitsu; Iwama, Atsushi; Nakauchi, Hiromitsu; Taniguchi, Hideki

2006-07-01

Recent advances in stem cell biology enable us to identify cancer stem cells in solid tumors as well as putative stem cells in normal solid organs. In this study, we applied side population (SP) cell analysis and sorting to established hepatocellular carcinoma (HCC) cell lines to detect subpopulations that function as cancer stem cells and to elucidate their roles in tumorigenesis. Among four cell lines analyzed, SP cells were detected in Huh7 (0.25%) and PLC/PRF/5 cells (0.80%), but not in HepG2 and Huh6 cells. SP cells demonstrated high proliferative potential and anti-apoptotic properties compared with those of non-SP cells. Immunocytochemistry examination showed that SP fractions contain a large number of cells presenting characteristics of both hepatocyte and cholangiocyte lineages. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft transplant experiments showed that only 1 x 10(3) SP cells were sufficient for tumor formation, whereas an injection of 1 x 10(6) non-SP cells did not initiate tumors. Re-analysis of SP cell-derived tumors showed that SP cells generated both SP and non-SP cells and tumor-initiating potential was maintained only in SP cells in serial transplantation. Microarray analysis discriminated a differential gene expression profile between SP and non-SP cells, and several so-called "stemness genes" were upregulated in SP cells in HCC cells. In conclusion, we propose that a minority population, detected as SP cells in HCC cells, possess extreme tumorigenic potential and provide heterogeneity to the cancer stem cell system characterized by distinct hierarchy.

Protective Effects of Moringa oleifera on HBV Genotypes C and H Transiently Transfected Huh7 Cells.

PubMed

Feustel, Sina; Ayón-Pérez, Fabiola; Sandoval-Rodriguez, Ana; Rodríguez-Echevarría, Roberto; Contreras-Salinas, Homero; Armendáriz-Borunda, Juan; Sánchez-Orozco, L V

2017-01-01

Chronic hepatitis B infection treatment implicates a long-lasting treatment. M. oleifera extracts contain compounds with antiviral, antioxidant, and antifibrotic properties. In this study, the effect of M. oleifera was evaluated in Huh7 cells expressing either HBV genotypes C or H for the antiviral, antifibrotic, anti-inflammatory, and antioxidative responses. Huh7 cells were treated with an aqueous extract of M. oleifera (leaves) at doses of 0, 30, 45, or 60 μ g/mL. The replicative virus and TGF-β1 , CTGF , CAT , IFN-β1 , and pgRNA expressions were measured by real time. HBsAg and IL-6 titers were determined by ELISA. CTGF , TGF-β1 , IFN-β1 , and pgRNA expressions decreased with M. oleifera treatment irrespective of the HBV genotype. HBsAg secretion in the supernatant of transfected Huh7 cells with both HBV genotypes was decreased regardless of the dose of M. oleifera . Similar effect was observed in proinflammatory cytokine IL-6, which had a tendency to decrease at 24 hours of treatment. Transfection with both HBV genotypes strongly decreased CAT expression, which is retrieved with M. oleifera treatment. M. oleifera treatment reduced fibrosis markers, IL-6, and HBsAg secretion in HBV genotypes C and H. However, at the level of replication, only HBV-DNA genotype C was slightly reduced with this treatment.

Role of HCV Core gene of genotype 1a and 3a and host gene Cox-2 in HCV-induced pathogenesis

PubMed Central

2011-01-01

Background Hepatitis C virus (HCV) Core protein is thought to trigger activation of multiple signaling pathways and play a significant role in the alteration of cellular gene expression responsible for HCV pathogenesis leading to hepatocellular carcinoma (HCC). However, the exact molecular mechanism of HCV genome specific pathogenesis remains unclear. We examined the in vitro effects of HCV Core protein of HCV genotype 3a and 1a on the cellular genes involved in oxidative stress and angiogenesis. We also studied the ability of HCV Core and Cox-2 siRNA either alone or in combination to inhibit viral replication and cell proliferation in HCV serum infected Huh-7 cells. Results Over expression of Core gene of HCV 3a genotype showed stronger effect in regulating RNA and protein levels of Cox-2, iNOS, VEGF, p-Akt as compared to HCV-1a Core in hepatocellular carcinoma cell line Huh-7 accompanied by enhanced PGE2 release and cell proliferation. We also observed higher expression levels of above genes in HCV 3a patient's blood and biopsy samples. Interestingly, the Core and Cox-2-specific siRNAs down regulated the Core 3a-enhanced expression of Cox-2, iNOS, VEGF, p-Akt. Furthermore, the combined siRNA treatment also showed a dramatic reduction in viral titer and expression of these genes in HCV serum-infected Huh-7 cells. Taken together, these results demonstrated a differential response by HCV 3a genotype in HCV-induced pathogenesis, which may be due to Core and host factor Cox-2 individually or in combination. Conclusions Collectively, these studies not only suggest a genotype-specific interaction between key players of HCV pathogenesis but also may represent combined viral and host gene silencing as a potential therapeutic strategy. PMID:21457561

Nanocurcumin-Mediated Down-Regulation of Telomerase Via Stimulating TGFβ1 Signaling Pathway in Hepatocellular Carcinoma Cells

PubMed

Shariati, Molood; Hajigholami, Samira; Veisi Malekshahi, Ziba; Entezari, Maliheh; Bodaghabadi, Narges; Sadeghizadeh, Majid

2017-10-10

Curcumin, extracted from turmeric, represents enormous potential to serve as an anticancer agent. Telomerase is viewed as a prominent molecular target of curcumin, and Transforming growth factor-β1 (TGFβ1) has proven to be a major inhibitory signaling pathway for telomerase activity. In the current study, we aimed to explore suppressive effects of nanocurcumin on telomerase expression through TGFβ1 pathway in a hepatocellular carcinoma cell line (Huh7). MTT assay was used to determine the effect of nonocurcumin on viability of Huh7 cells. RT-PCR was used to analyze the gene expression patterns. MTT assay revealed that nanocurcumin acts in a dose- and time-dependent manner to diminish the cell viability. RT-PCR analysis indicated that nanocurcumin results in augmentation of TGFβ1 72 hours post treatment and leads to the reduction of telomerase expression 48 and 72 hours post exposure. Also, up-regulation of Smad3 and E2F1 and down-regulation of Smad7 confirmed the effect of nanocurcumin on intermediate components of TGFβ1 pathway. Furthermore, transfection of the proximal promoter of telomerase triggered a significant reduction in luciferase activity. The data from the present study lead us to develop a deeper understanding of the mechanisms underlying nanocurcumin-mediated regulation of telomerase expression, thereby presenting a new perspective to the landscape of using nanocurcumin as a cancer-oriented therapeutic agent.

In vitro antitumor efficacy of berberine: solid lipid nanoparticles against human HepG2, Huh7 and EC9706 cancer cell lines

NASA Astrophysics Data System (ADS)

Meng, Xiang-Ping; Wang, Xiao; Wang, Huai-ling; Chen, Tong-sheng; Wang, Yi-fei; Wang, Zhi-ping

2016-03-01

Hepatocarcinoma and esophageal squamous cell carcinomas threaten human life badly. It is a current issue to seek the effective natural remedy from plant to treat cancer due to the resistance of the advanced hepatocarcinoma and esophageal carcinoma to chemotherapy. Berberine (Ber), an isoquinoline derivative alkaloid, has a wide range of pharmacological properties and is considered to have anti-hepatocarcinoma and antiesophageal carcinoma effects. However its low oral bioavailability restricts its wide application. In this report, Ber loaded solid lipid nanoparticles (Ber-SLN) was prepared by hot melting and then high pressure homogenization technique. The in vitro anti-hepatocarcinoma and antiesophageal carcinoma effects of Ber-SLN relative to efficacy of bulk Ber were evaluated. The particle size and zeta potential of Ber-SLN were 154.3 ± 4.1 nm and -11.7 ± 1.8 mV, respectively. MTT assay showed that Ber-SLN effectively inhibited the proliferation of human HepG2 and Huh7 and EC9706 cells, and the corresponding IC50 value was 10.6 μg/ml, 5.1 μg/ml, and 7.3 μg/ml (18.3μg/ml, 6.5μg/ml, and 12.4μg/ml μg/ml of bulk Ber solution), respectively. These results suggest that the delivery of Ber-SLN is a promising approach for treating tumors.

PD and PDT for hepatoblastoma? Preclinical considerations

NASA Astrophysics Data System (ADS)

Stepp, Herbert; Bergmann, Florian; Johansson, Ann; Heide, Michael; Metzger, Roman; Rolle, Udo; Till, Holger

2011-07-01

Objective: Provide preclinical data on the feasibility of 5-aminolevulinic acid (5-ALA) -based photodetection (PD) and Photodynamic Therapy (PDT) of early childhood tumors. Methods: Hepatoblastoma (HuH6), neuroblastoma (MHH-NB11) and N1-fibroblast cell lines were tested for their relative capacities to synthesize Protoporphyrin IX (PpIX) from 5-ALA and for their susceptibility to PDT in vitro. HuH6-cells were also inoculated in the peritoneum of rats. The pharmacokinetics of porphyrin accumulation was measured in 9 rats by laparoscopic spectroscopy. 5-ALA was applied by i.p. injection of 500 mg/kg bw. In another 21 animals, tumors (n=20), liver (n=5) and peritoneum (n=4) were treated by PDT laparoscopically. 48 h after irradiation, animals were again incubated with 5-ALA and then sacrificed and tissues were removed for further investigation. Results: Both tumor cell lines showed higher levels of porphyrin fluorescence than the fibroblasts. Cell viability testing proved the HuH6 cells to be most susceptible to PDT. Pharmacokinetic measurements of PpIX in xenografted tumors showed a peak at 80-200 min after i.p. injection of 5-ALA. Irradiation resulted in pronounced photobleaching at all irradiated sites and necrosis of tumor and liver tissue, whereas peritoneum appeared to remain unaffected. Necrosis induced by PDT could be seen in fluorescence microscopy due to the lack of porphyrin synthesis in necrotic tissue after the re-incubation with 5-ALA.

Wee1 Kinase Inhibitor AZD1775 Radiosensitizes Hepatocellular Carcinoma Regardless of TP53 Mutational Status Through Induction of Replication Stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cuneo, Kyle C., E-mail: kcuneo@umich.edu; Morgan, Meredith A.; Davis, Mary A.

2016-06-01

Purpose: Wee1 kinase inhibitors are effective radiosensitizers in cells lacking a G{sub 1} checkpoint. In this study we examined the potential effect of Wee1 kinase inhibition on inducing replication stress in hepatocellular carcinoma (HCC). Methods and Materials: Five independent datasets from the Oncomine database comparing gene expression in HCC compared to normal tissue were combined and specific markers associated with Wee1 sensitivity were analyzed. We then performed a series of in vitro experiments to study the effect of Wee1 inhibition on irradiated HCC cell lines with varying p53 mutational status. Clonogenic survival assays and flow cytometry using anti-γH2AX and phospho-histone H3more » antibodies with propidium iodide were performed to study the effect of AZD1775 on survival, cell cycle, and DNA repair. Additionally, nucleoside enriched medium was used to examine the effect of altering nucleotide pools on Wee1 targeted radiation sensitization. Results: Our analysis of the Oncomine database found high levels of CDK1 and other cell cycle regulators indicative of Wee1 sensitivity in HCC. In our in vitro experiments, treatment with AZD1775 radiosensitized and chemosensitized Hep3B, Huh7, and HepG2 cell lines and was associated with delayed resolution of γH2AX foci and the induction of pan-nuclear γH2AX staining. Wee1 inhibition attenuated radiation-induced G{sub 2} arrest in the Hep3B (TP53 null) and Huh7 (TP53 mutant) cell lines but not in the TP53 wild-type cell line HepG2. Supplementation with nucleosides reversed the radiation-sensitizing effect of AZD1775 and reduced the amount of cells with pan-nuclear γH2AX staining after radiation. Conclusions: Radiation sensitization with Wee1 inhibition occurs in cells regardless of their p53 mutational status. In this study we show for the first time that replication stress via the overconsumption of nucleotides plays an important role in AZD1775-induced radiation sensitization.« less

Identification of various cell culture models for the study of Zika virus

PubMed Central

Himmelsbach, Kiyoshi; Hildt, Eberhard

2018-01-01

AIM To identify cell culture models supportive for Zika virus (ZIKV) replication. METHODS Various human and non-human cell lines were infected with a defined amount of ZIKV Polynesia strain. Cells were analyzed 48 h post infection for the amount of intracellular and extracellular viral genomes and infectious viral particles by quantitative real-time PCR and virus titration assay. The extent of replication was monitored by immunofluorescence and western blot analysis by using Env and NS1 specific antibodies. Innate immunity was assayed by luciferase reporter assay and immunofluorescence analysis. RESULTS All investigated cell lines except CHO cells supported infection, replication and release of ZIKV. While in infected A549 and Vero cells a pronounced cytopathic effect was observed COS7, 293T and Huh7.5 cells were most resistant. Although the analyzed cell lines released comparable amounts of viral genomes to the supernatant significant differences were found for the number of infectious viral particles. The neuronal cell lines N29.1 and SH-SY5Y released 100 times less infectious viral particles than Vero-, A549- or 293T-cells. However there is no strict correlation between the amount of produced viral particles and the induction of an interferon response in the analyzed cell lines. CONCLUSION The investigated cell lines with their different tissue origins and diverging ZIKV susceptibility display a toolbox for ZIKV research. PMID:29468137

Comparative Analysis of the Lambda-Interferons IL-28A and IL-29 regarding Their Transcriptome and Their Antiviral Properties against Hepatitis C Virus

PubMed Central

Diegelmann, Julia; Beigel, Florian; Zitzmann, Kathrin; Kaul, Artur; Göke, Burkhard; Auernhammer, Christoph J.; Bartenschlager, Ralf; Diepolder, Helmut M.; Brand, Stephan

2010-01-01

Background Specific differences in signaling and antiviral properties between the different Lambda-interferons, a novel group of interferons composed of IL-28A, IL-28B and IL-29, are currently unknown. This is the first study comparatively investigating the transcriptome and the antiviral properties of the Lambda-interferons IL-28A and IL-29. Methodology/Principal Findings Expression studies were performed by microarray analysis, quantitative PCR (qPCR), reporter gene assays and immunoluminometric assays. Signaling was analyzed by Western blot. HCV replication was measured in Huh-7 cells expressing subgenomic HCV replicon. All hepatic cell lines investigated as well as primary hepatocytes expressed both IFN-λ receptor subunits IL-10R2 and IFN-λR1. Both, IL-28A and IL-29 activated STAT1 signaling. As revealed by microarray analysis, similar genes were induced by both cytokines in Huh-7 cells (IL-28A: 117 genes; IL-29: 111 genes), many of them playing a role in antiviral immunity. However, only IL-28A was able to significantly down-regulate gene expression (n = 272 down-regulated genes). Both cytokines significantly decreased HCV replication in Huh-7 cells. In comparison to liver biopsies of patients with non-viral liver disease, liver biopsies of patients with HCV showed significantly increased mRNA expression of IL-28A and IL-29. Moreover, IL-28A serum protein levels were elevated in HCV patients. In a murine model of viral hepatitis, IL-28 expression was significantly increased. Conclusions/Significance IL-28A and IL-29 are up-regulated in HCV patients and are similarly effective in inducing antiviral genes and inhibiting HCV replication. In contrast to IL-29, IL-28A is a potent gene repressor. Both IFN-λs may have therapeutic potential in the treatment of chronic HCV. PMID:21170333

Uptake of DNA by cancer cells without a transfection reagent.

PubMed

Kong, Yanping; Zhang, Xianbo; Zhao, Yongliang; Xue, Yanfang; Zhang, Ye

2017-01-21

Cancer cells exhibit elevated levels of glucose uptake and may obtain pre-formed, diet-derived fatty acids from the bloodstream to boost their rapid growth; they may also use nucleic acid from their microenvironment. The study of processing nucleic acid by cancer cells will help improve the understanding of the metabolism of cancer. DNA is commonly packaged into a viral or lipid particle to be transferred into cells; this process is called transfection in laboratory. Cancer cells are known for having gene mutations and the evolving ability of endocytosis. Their uptake of DNAs might be different from normal cells; they may take in DNAs directly from the environment. In this report, we studied the uptake of DNAs in cancer cells without a transfection reagent. A group of DNA fragments were prepared with PCR and labeled with isotope phosphorous-32 to test their uptake by Huh 7 (liver cancer) and THLE3 (normal liver cells) after incubation overnight by counting radioactivity of the cells' genomic DNA. Multiple cell lines including breast cancer and lung cancer were tested with the same method. DNA molecules were also labeled with fluorescence to test the location in the cells using a kit of "label it fluorescence in situ hybridization (FISH)" from Mirus (USA). The data demonstrated that hepatocellular carcinoma cells possess the ability to take in large DNA fragments directly without a transfection reagent whereas normal liver cells cannot. Huh7 and MDA-MB231 cells displayed a significantly higher Rhodamine density in the cytoplasmic phagosomes and this suggests that the mechanism of uptake of large DNA by cancer cells is likely endocytosis. The efficacy of uptake is related to the DNA's size. Some cell lines of lung cancer and breast cancer also showed similar uptake of DNA. In the present study, we have revealed the evidence that some cancer cells, but not nontumorigenic cells, can take DNA fragments directly from the environment without the aid of the transfecting reagent.

[Comparison of TTR and CMV promoters in vivo and in vitro via a secreted luciferase reporter system].

PubMed

Luo, Shun-Tao; Tian, Wen-Hong; Wang, Gang; Dong, Xiao-Yan; Yang, Li; Wu, Xiao-Bing

2009-11-01

GLuc (Gaussia luciferase) is a secreted luciferase with high sensitivity. In this study, we primarily compared expression character of PTTR with that of PCMV, relied on easy secretion, high sensitivity and simple and fast detection of GLuc. We firstly constructed two plasmids pAAV2-neo-TTR-GLuc and pAAV2-neo-CMV-GLuc. Then, 4 cell lines were transfected with the two plasmids in aid of Lipofectamine 2000, including Huh7 and HepG2, which are derived from liver cells, as well as HEK293 and HeLaS3 cells, which are non-liver cell lines. We monitored the expression of GLuc in the supernatant of these cell cultures at different time points post-transfection. Furthermore, we injected the two plasmids with different doses into BALB/c mice by the means of hydrodynamic delivery and monitored the GLuc expression in vivo with 2.5 microl tail tip blood since 2 h post-injection. The cell assay results suggested that the expression of GLuc driven by CMV promoter was significantly higher than that of GLuc driven by TTR promoter. And, the luciferase activity of GLuc driven by CMV promoter was 50-300 times higher than that of GLuc driven by TTR promoter in HEK293 and HeLaS3 cell lines, but less than 10 times higher than that of GLuc driven by TTR promoter in the HepG2 and Huh7 cell lines, indicating the relative liver-specificity of TTR promoter. In the animal assay, the higher luciferase activity was determined in CMV promoter group than in TTR promoter group at different doses of the two plasmids. But the expression patterns for the two promoters differed obviously. The expression of GLuc driven by CMV promoter reached the maximum 10 hours post-injection and declined rapidly; while the expression of GLuc driven by TTR promoter reached the maximum 48 hours after delivery, and declined very slowly. These results implied that PTTR could keep expression of driven gene in a long time although its expression intensity is lower than PCMV's. Thus, it is more suitable for maintaining longer expression of target genes in liver.

Targeted transplantation of mitochondria to hepatocytes

PubMed Central

Gupta, Nidhi; Wu, Catherine H; Wu, George Y

2016-01-01

Background Mitochondrial defects in hepatocytes can result in liver dysfunction and death. Hepatocytes have cell-surface asialoglycoprotein receptors (AsGRs) which internalize AsGs within endosomes. The aim of this study was to determine whether mitochondria could be targeted to hepatocytes by AsGR-mediated endocytosis. Materials and methods An AsG, AsOR, was linked to polylysine to create a conjugate, AsOR-PL, and complexed with healthy and functional mitochondria (defined by normal morphology, cytochrome c assays, and oxygen-consumption rates). Huh7 (AsGR+) and SK Hep1 (AsGR−) cells were treated with a mitochondrial toxin to form Huh7-Mito− and SK Hep1-Mito− cells, lacking detectable mitochondrial DNA. An endosomolytic peptide, LLO, was coupled to AsOR to form AsOR-LLO. A lysosomal inhibitor, amantadine, was used in mitochondria-uptake studies as a control for nonspecific endosomal release. Results Coincubation of complexed mitochondria and AsOR-LLO with Huh7-Mito− cells increased mitochondrial DNA to >9,700-fold over control at 7 days (P<0.001), and increased mitochondrial oxygen-consumption rates to >90% of control by 10 days. Conclusion Rescue of mitochondria-damaged hepatocytes can be achieved by targeted uptake of normal mitochondria through receptor-mediated endocytosis. PMID:27942238

Amphipathic α-helices in apolipoproteins are crucial to the formation of infectious hepatitis C virus particles.

PubMed

Fukuhara, Takasuke; Wada, Masami; Nakamura, Shota; Ono, Chikako; Shiokawa, Mai; Yamamoto, Satomi; Motomura, Takashi; Okamoto, Toru; Okuzaki, Daisuke; Yamamoto, Masahiro; Saito, Izumu; Wakita, Takaji; Koike, Kazuhiko; Matsuura, Yoshiharu

2014-12-01

Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly.

Amphipathic α-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles

PubMed Central

Nakamura, Shota; Ono, Chikako; Shiokawa, Mai; Yamamoto, Satomi; Motomura, Takashi; Okamoto, Toru; Okuzaki, Daisuke; Yamamoto, Masahiro; Saito, Izumu; Wakita, Takaji; Koike, Kazuhiko; Matsuura, Yoshiharu

2014-01-01

Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly. PMID:25502789

Anti-hepatocarcinoma effects of berberine-nanostructured lipid carriers against human HepG2, Huh7, and EC9706 cancer cell lines

NASA Astrophysics Data System (ADS)

Meng, Xiang-Ping; Fan, Hua; Wang, Yi-fei; Wang, Zhi-ping; Chen, Tong-sheng

2016-10-01

Hepatocarcinoma and esophageal squamous cell carcinomas threaten human life badly. It is a current issue to seek the effective natural remedy from plant to treat cancer due to the resistance of the advanced hepatocarcinoma and esophageal carcinoma to chemotherapy. Berberine (Ber), an isoquinoline derivative alkaloid, has a wide range of pharmacological properties and is considered to have anti-hepatocarcinoma and antiesophageal carcinoma effects. However its low oral bioavailability restricts its wide application. In this report, Ber loaded nanostructured lipid carriers (Ber-NLC) was prepared by hot melting and then high pressure homogenization technique. The in vitro anti-hepatocarcinoma and antiesophageal carcinoma effects of Ber-NLC relative to efficacy of bulk Ber were evaluated. The particle size and zeta potential of Ber-NLC were 189.3 +/- 3.7 nm and -19.3 +/- 1.4 mV, respectively. MTT assay showed that Ber-NLC effectively inhibited the proliferation of human HepG2 and Huh7 and EC9706 cells, and the corresponding IC50 value was 9.1 μg/ml, 4.4 μg/ml, and 6.3 μg/ml (18.3μg/ml, 6.5μg/ml, and 12.4μg/ml μg/ml of bulk Ber solution), respectively. These results suggest that the delivery of Ber-NLC is a promising approach for treating tumors.

Combination of Mitochondrial and Plasma Membrane Citrate Transporter Inhibitors Inhibits De Novo Lipogenesis Pathway and Triggers Apoptosis in Hepatocellular Carcinoma Cells

PubMed Central

Phokrai, Phornpun; Suwankulanan, Somrudee; Phakdeeto, Narinthorn; Phunsomboon, Pattamaphorn; Pekthong, Dumrongsak; Richert, Lysiane; Pongcharoen, Sutatip

2018-01-01

Increased expression levels of both mitochondrial citrate transporter (CTP) and plasma membrane citrate transporter (PMCT) proteins have been found in various cancers. The transported citrates by these two transporter proteins provide acetyl-CoA precursors for the de novo lipogenesis (DNL) pathway to support a high rate of cancer cell viability and development. Inhibition of the DNL pathway promotes cancer cell apoptosis without apparent cytotoxic to normal cells, leading to the representation of selective and powerful targets for cancer therapy. The present study demonstrates that treatments with CTP inhibitor (CTPi), PMCT inhibitor (PMCTi), and the combination of CTPi and PMCTi resulted in decreased cell viability in two hepatocellular carcinoma cell lines (HepG2 and HuH-7). Treatment with citrate transporter inhibitors caused a greater cytotoxic effect in HepG2 cells than in HuH-7 cells. A lower concentration of combined CTPi and PMCTi promotes cytotoxic effect compared with either of a single compound. An increased cell apoptosis and an induced cell cycle arrest in both cell lines were reported after administration of the combined inhibitors. A combination treatment exhibits an enhanced apoptosis through decreased intracellular citrate levels, which consequently cause inhibition of fatty acid production in HepG2 cells. Apoptosis induction through the mitochondrial-dependent pathway was found as a consequence of suppressed carnitine palmitoyl transferase-1 (CPT-1) activity and enhanced ROS generation by combined CTPi and PMCTi treatment. We showed that accumulation of malonyl-CoA did not correlate with decreasing CPT-1 activity. The present study showed that elevated ROS levels served as an inhibition on Bcl-2 activity that is at least in part responsible for apoptosis. Moreover, inhibition of the citrate transporter is selectively cytotoxic to HepG2 cells but not in primary human hepatocytes, supporting citrate-mediating fatty acid synthesis as a promising cancer therapy. PMID:29546056

Attenuation of Proinflammatory Responses by S-[6]-Gingerol via Inhibition of ROS/NF-Kappa B/COX2 Activation in HuH7 Cells.

PubMed

Li, Xiao-Hong; McGrath, Kristine C Y; Tran, Van H; Li, Yi-Ming; Duke, Colin C; Roufogalis, Basil D; Heather, Alison K

2013-01-01

Introduction. Hepatic inflammation underlies the pathogenesis of chronic diseases such as insulin resistance and type 2 diabetes mellitus. S-[6]-Gingerol has been shown to have anti-inflammatory properties. Important inflammatory mediators of interleukins include nuclear factor κ B (NF κ B) and cyclooxygenase 2 (COX2). We now explore the mechanism of anti-inflammatory effects of S-[6]-gingerol in liver cells. Methods. HuH7 cells were stimulated with IL1β to establish an in vitro hepatic inflammatory model. Results. S-[6]-Gingerol attenuated IL1β-induced inflammation and oxidative stress in HuH7 cells, as evidenced by decreasing mRNA levels of inflammatory factor IL6, IL8, and SAA1, suppression of ROS generation, and increasing mRNA levels of DHCR24. In addition, S-[6]-gingerol reduced IL1β-induced COX2 upregulation as well as NF κ B activity. Similar to the protective effects of S-[6]-gingerol, both NS-398 (a selective COX2 inhibitor) and PDTC (a selective NF κ B inhibitor) suppressed mRNA levels of IL6, IL8, and SAA1. Importantly, PDTC attenuated IL1β-induced overexpression of COX2. Of particular note, the protective effect of S-[6]-gingerol against the IL1β-induced inflammatory response was similar to that of BHT, an ROS scavenger. Conclusions. The findings of this study demonstrate that S-[6]-gingerol protects HuH7 cells against IL1β-induced inflammatory insults through inhibition of the ROS/NF κ B/COX2 pathway.

Nanocurcumin-Mediated Down-Regulation of Telomerase Via Stimulating TGFβ1 Signaling Pathway in Hepatocellular Carcinoma Cells

PubMed Central

Shariati, Molood; Hajigholami, Samira; Malekshahi, Ziba Veisi; Entezari, Maliheh; Bodaghabadi, Narges; Sadeghizadeh, Majid

2018-01-01

Background: Curcumin, extracted from turmeric, represents enormous potential to serve as an anticancer agent. Telomerase is viewed as a prominent molecular target of curcumin, and transforming growth factor-β1 (TGFβ1) has proven to be a major inhibitory signaling pathway for telomerase activity. In the current study, we aimed to explore suppressive effects of nanocurcumin on telomerase expression through TGFβ1 pathway in a hepatocellular carcinoma cell line (Huh7). Methods: MTT assay was used to determine the effect of nonocurcumin on viability of Huh7 cells. RT-PCR was used to analyze the gene expression patterns. Results: MTT assay revealed that nanocurcumin acts in a dose- and time-dependent manner to diminish the cell viability. RT-PCR analysis indicated that nanocurcumin results in augmentation of TGFβ1 72 hours post treatment and leads to the reduction of telomerase expression 48 and 72 hours post exposure. Also, up-regulation of Smad3 and E2F1 and down-regulation of Smad7 confirmed the effect of nanocurcumin on intermediate components of TGFβ1 pathway. Furthermore, transfection of the proximal promoter of telomerase triggered a significant reduction in luciferase activity. Conclusion: The data from the present study lead us to develop a deeper understanding of the mechanisms underlying nanocurcumin-mediated regulation of telomerase expression, thereby presenting a new perspective to the landscape of using nanocurcumin as a cancer-oriented therapeutic agent.

Carbohydrate linked organotin(IV) complexes as human topoisomerase Iα inhibitor and their antiproliferative effects against the human carcinoma cell line.

PubMed

Khan, Rais Ahmad; Yadav, Shipra; Hussain, Zahid; Arjmand, Farukh; Tabassum, Sartaj

2014-02-14

Dimethyltin(IV) complexes with ethanolamine (1) and biologically significant N-glycosides (2 and 3) were designed and synthesized. The structural elucidation of complexes 1-3 was done using elemental and spectroscopic methods; in addition, complex 1 was studied by single crystal X-ray diffraction studies. The in vitro DNA binding profile of complexes 2 and 3 was carried out by employing different biophysical methods to ascertain the feasibility of glycosylated complexes. Further, the cleaving ability of 2 and 3 was investigated by the agarose gel electrophoretic mobility assay with supercoiled pBR322 DNA, and demonstrated significantly good nuclease activity. Furthermore, both the complexes exhibited significant inhibitory effects on the catalytic activity of human Topo I at lower concentration than standard drugs. Computer-aided molecular docking techniques were used to ascertain the mode and mechanism of action towards the molecular target DNA and Topo I. The cytotoxicity of 2 and 3 against human hepatoma cancer cells (Huh7) was evaluated, which revealed significant regression in cancerous cells as compared with the standard drug. The antiproliferative activities of 2 and 3 were tested against human hepatoma cancer cells (Huh7), and results showed significantly good activity. Additionally, to validate the remarkable antiproliferative activity of complexes 2 and 3, specific regulatory gene expression (MMP-2 and TGF-β) was obtained by real time PCR.

Farnesylthiosalicylic acid sensitizes hepatocarcinoma cells to artemisinin derivatives

PubMed Central

Wu, Liping; Pang, Yilin; Qin, Guiqi; Xi, Gaina; Wu, Shengnan; Wang, Xiaoping; Chen, Tongsheng

2017-01-01

Dihydroartemisinin (DHA) and artesunate (ARS), two artemisinin derivatives, have efficacious anticancer activities against human hepatocarcinoma (HCC) cells. This study aims to study the anticancer action of the combination treatment of DHA/ARS and farnesylthiosalicylic acid (FTS), a Ras inhibitor, in HCC cells (Huh-7 and HepG2 cell lines). FTS pretreatment significantly enhanced DHA/ARS-induced phosphatidylserine (PS) externalization, Bak/Bax activation, mitochondrial membrane depolarization, cytochrome c release, and caspase-8 and -9 activations, characteristics of the extrinsic and intrinsic apoptosis. Pretreatment with Z-IETD-FMK (caspase-8 inhibitor) potently prevented the cytotoxicity of the combination treatment of DHA/ARS and FTS, and pretreatment with Z-VAD-FMK (pan-caspase inhibitor) significantly inhibited the loss of ΔΨm induced by DHA/ARS treatment or the combination treatment of DHA/ARS and FTS in HCC cells. Furthermore, silencing Bak/Bax modestly but significantly inhibited the cytotoxicity of the combination treatment of DHA/ARS and FTS. Interestingly, pretreatment with an antioxidant N-Acetyle-Cysteine (NAC) significantly prevented the cytotoxicity of the combination treatment of DHA and FTS instead of the combination treatment of ARS and FTS, suggesting that reactive oxygen species (ROS) played a key role in the anticancer action of the combination treatment of DHA and FTS. Similar to FTS, DHA/ARS also significantly prevented Ras activation. Collectively, our data demonstrate that FTS potently sensitizes Huh-7 and HepG2 cells to artemisinin derivatives via accelerating the extrinsic and intrinsic apoptotic pathways. PMID:28182780

CD40 inhibits replication of hepatitis C virus in primary human hepatocytes by c-Jun N terminal kinase activation independent from the interferon pathway.

PubMed

Rau, Sibylle J; Hildt, Eberhard; Himmelsbach, Kiyoshi; Thimme, Robert; Wakita, Takaji; Blum, Hubert E; Fischer, Richard

2013-01-01

CD40, a member of the tumor necrosis factor receptor family, and its ligand, CD40L (CD154), are important regulators of the antiviral immune response. CD40L is up-regulated on lymphocytes and CD40 on hepatocytes during infection with hepatitis C virus (HCV); we investigated the role of CD40 signaling during HCV replication in hepatocytes. Viral replication was studied in primary human hepatocytes (PHH) and Huh7.5 cells using the infectious HCV Japanese fulminate hepatitis 1 isolate (JFH1) culture system, and in coculture with HCV antigen-specific CD8+ T cells. CD40L rapidly and transiently inhibits expression of the HCV nonstructural proteins NS3 and NS5A as well as HCV structural proteins core and E2 in Huh7.5 cells. Similarly, CD40L prevented replication of HCV in PHH, in synergy with interferon (IFN)-alpha. In Huh7.5 cells with replicating HCV, CD40L prevented production of infectious viral particles. When HCV antigen-specific CD8+ T cells were cocultured with HLA-A2-expressing Huh7 cells that had replicating virus, the T cells became activated, up-regulated CD40L, and inhibited HCV replication. Inhibition of CD40L partially prevented the antiviral activity of the CD8+ T cells. The antiviral effect of CD40L required activation of c-Jun N terminal kinases (JNK)1/2, but not induction of apoptosis or the JAK/STAT pathway that is necessary for the antiviral effects of IFNs. CD40 inhibits HCV replication by a novel, innate immune mechanism. This pathway might mediate viral clearance, and disruptions might be involved in the pathogenesis of HCV infection. Copyright © 2012 American Association for the Study of Liver Diseases.

Design of PEI-conjugated bio-reducible polymer for efficient gene delivery.

PubMed

Nam, Joung-Pyo; Kim, Soyoung; Kim, Sung Wan

2018-07-10

The poly(cystaminebis(acrylamide)-diaminohexane) (poly(CBA-DAH)) was designed previously as a bio-reducible efficient gene delivery carrier. However, the high weight ratio required to form the polyplexes between poly(CBA-DAH) with pDNA is still a problem that needs to be addressed. To solve this problem and increase the transfection efficiency, poly(ethylenimine) (PEI, 1.8 kDa) was conjugated to poly(CBA-DAH) via disulfide bond. The PEI conjugated poly(CBA-DAH) (PCDP) can bind with pDNA at a very low weight ratio of 0.5 and above, like PEI 25 kDa, and form the polyplexes with nano-size (102-128 nm) and positive surface charge (27-34 mV). PCDP and PCDP polyplexes had negligible cytotoxicity and indicated similar or better cellular uptake than the comparison groups such as PEI 25 kDa and Lipofectamine® polyplexes. To confirm the transfection efficiency, the plasmid DNA (pDNA) encoded with the luciferase reporter gene (gWiz-Luc) and green fluorescent protein reporter gene (GFP) were used and treated with PCDP into the A549, Huh-7, and Mia PaCa-2 cells. PCDP/pDNA polyplexes showed highest transfection efficiency in all tested cell lines. In the luciferase assay, PCDP polyplexes showed 10.2 times higher gene transfection efficiency than Lipofectamine® polyplexes in mimic in vivo conditions (30% FBS, A549 cells). The VEGF siRNA expressing plasmid (pshVEGF), which is constructed as a therapeutic gene by our previous work, was delivered by PCDP into the cancer cells. The VEGF gene expression of PCDP/pshVEGF polyplexes was dramatically lower than control and the VEGF gene silencing efficiencies of PCDP/pshVEGF (w/w; 10/1) polyplexes were 54% (A549 cells), 77% (Huh-7 cells), and 66% (Mia PaCa-2 cells). In addition, PCDP/pshVEGF had reduced cell viability rates of about 31% (A549 cells), 39% (Huh-7 cells), and 42% (Mia PaCa-2 cells) and showed better results than all comparison groups. In the transfection efficiency and VEGF silencing assay, PCDP polyplexes showed better results than poly(CBA-DAH) at 4-fold lower weight ratio. The data of all experiments demonstrate that the synthesized PCDP could be used for efficient gene delivery and could be widely applied. Published by Elsevier B.V.

DDX60L Is an Interferon-Stimulated Gene Product Restricting Hepatitis C Virus Replication in Cell Culture

PubMed Central

Grünvogel, Oliver; Esser-Nobis, Katharina; Reustle, Anna; Schult, Philipp; Müller, Birthe; Metz, Philippe; Trippler, Martin; Windisch, Marc P.; Frese, Michael; Binder, Marco; Fackler, Oliver; Bartenschlager, Ralf; Ruggieri, Alessia

2015-01-01

ABSTRACT All major types of interferon (IFN) efficiently inhibit hepatitis C virus (HCV) replication in vitro and in vivo. Remarkably, HCV replication is not sensitive to IFN-γ in the hepatoma cell line Huh6, despite an intact signaling pathway. We performed transcriptome analyses between Huh6 and Huh-7 cells to identify effector genes of the IFN-γ response and thereby identified the DExD/H box helicase DEAD box polypeptide 60-like (DDX60L) as a restriction factor of HCV replication. DDX60L and its homolog DEAD box polypeptide 60 (DDX60) were both induced upon viral infection and IFN treatment in primary human hepatocytes. However, exclusively DDX60L knockdown increased HCV replication in Huh-7 cells and rescued HCV replication from type II IFN as well as type I and III IFN treatment, suggesting that DDX60L is an important effector protein of the innate immune response against HCV. In contrast, we found no impact of DDX60L on replication of hepatitis A virus. DDX60L protein was detectable only upon strong ectopic overexpression, displayed a broad cytoplasmic distribution, but caused cytopathic effects under these conditions. DDX60L knockdown did not alter interferon-stimulated gene (ISG) induction after IFN treatment but inhibited HCV replication upon ectopic expression, suggesting that it is a direct effector of the innate immune response. It most likely inhibits viral RNA replication, since we found neither impact of DDX60L on translation or stability of HCV subgenomic replicons nor additional impact on assembly of infectious virus. Similar to DDX60, DDX60L had a moderate impact on RIG-I dependent activation of innate immunity, suggesting additional functions in the sensing of viral RNA. IMPORTANCE Interferons induce a plethora of interferon-stimulated genes (ISGs), which are our first line of defense against viral infections. In addition, IFNs have been used in antiviral therapy, in particular against the human pathogen hepatitis C virus (HCV); still, their mechanism of action is not well understood, since diverse, overlapping sets of antagonistic effector ISGs target viruses with different biologies. Our work identifies DDX60L as a novel factor that inhibits replication of HCV. DDX60L expression is regulated similarly to that of its homolog DDX60, but our data suggest that it has distinct functions, since we found no contribution of DDX60 in combatting HCV replication. The identification of novel components of the innate immune response contributes to a comprehensive understanding of the complex mechanisms governing antiviral defense. PMID:26269178

Ailanthone Inhibits Huh7 Cancer Cell Growth via Cell Cycle Arrest and Apoptosis In Vitro and In Vivo

PubMed Central

Zhuo, Zhenjian; Hu, Jianyang; Yang, Xiaolin; Chen, Minfen; Lei, Xueping; Deng, Lijuan; Yao, Nan; Peng, Qunlong; Chen, Zhesheng; Ye, Wencai; Zhang, Dongmei

2015-01-01

While searching for natural anti-hepatocellular carcinoma (HCC) components in Ailanthus altissima, we discovered that ailanthone had potent antineoplastic activity against HCC. However, the molecular mechanisms underlying the antitumor effect of ailanthone on HCC have not been examined. In this study, the antitumor activity and the underlying mechanisms of ailanthone were evaluated in vitro and in vivo. Mechanistic studies showed that ailanthone induced G0/G1-phase cell cycle arrest, as indicated by decreased expression of cyclins and CDKs and increased expression of p21 and p27. Our results demonstrated that ailanthone triggered DNA damage characterized by activation of the ATM/ATR pathway. Moreover, ailanthone-induced cell death was associated with apoptosis, as evidenced by an increased ratio of cells in the subG1 phase and by PARP cleavage and caspase activation. Ailanthone-induced apoptosis was mitochondrion-mediated and involved the PI3K/AKT signaling pathway in Huh7 cells. In vivo studies demonstrated that ailanthone inhibited the growth and angiogenesis of tumor xenografts without significant secondary adverse effects, indicating its safety for treating HCC. In conclusion, our study is the first to report the efficacy of ailanthone against Huh7 cells and to elucidate its underlying molecular mechanisms. These findings suggest that ailanthone is a potential agent for the treatment of liver cancer. PMID:26525771

Liver-specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis, and hepatocellular carcinoma in mice.

PubMed

Ko, Kwang Suk; Tomasi, Maria Lauda; Iglesias-Ara, Ainhoa; French, Barbara A; French, Samuel W; Ramani, Komal; Lozano, Juan José; Oh, Pilsoo; He, Lina; Stiles, Bangyan L; Li, Tony W H; Yang, Heping; Martínez-Chantar, M Luz; Mato, José M; Lu, Shelly C

2010-12-01

Prohibitin 1 (PHB1) is a highly conserved, ubiquitously expressed protein that participates in diverse processes including mitochondrial chaperone, growth and apoptosis. The role of PHB1 in vivo is unclear and whether it is a tumor suppressor is controversial. Mice lacking methionine adenosyltransferase 1A (MAT1A) have reduced PHB1 expression, impaired mitochondrial function, and spontaneously develop hepatocellular carcinoma (HCC). To see if reduced PHB1 expression contributes to the Mat1a knockout (KO) phenotype, we generated liver-specific Phb1 KO mice. Expression was determined at the messenger RNA and protein levels. PHB1 expression in cells was varied by small interfering RNA or overexpression. At 3 weeks, KO mice exhibit biochemical and histologic liver injury. Immunohistochemistry revealed apoptosis, proliferation, oxidative stress, fibrosis, bile duct epithelial metaplasia, hepatocyte dysplasia, and increased staining for stem cell and preneoplastic markers. Mitochondria are swollen and many have no discernible cristae. Differential gene expression revealed that genes associated with proliferation, malignant transformation, and liver fibrosis are highly up-regulated. From 20 weeks on, KO mice have multiple liver nodules and from 35 to 46 weeks, 38% have multifocal HCC. PHB1 protein levels were higher in normal human hepatocytes compared to human HCC cell lines Huh-7 and HepG2. Knockdown of PHB1 in murine nontransformed AML12 cells (normal mouse hepatocyte cell line) raised cyclin D1 expression, increased E2F transcription factor binding to cyclin D1 promoter, and proliferation. The opposite occurred with PHB1 overexpression. Knockdown or overexpression of PHB1 in Huh-7 cells did not affect proliferation significantly or sensitize cells to sorafenib-induced apoptosis. Hepatocyte-specific PHB1 deficiency results in marked liver injury, oxidative stress, and fibrosis with development of HCC by 8 months. These results support PHB1 as a tumor suppressor in hepatocytes. Copyright © 2010 American Association for the Study of Liver Diseases.

Altered expression and activation of signal transducers and activators of transcription (STATs) in hepatitis C virus infection: in vivo and in vitro studies.

PubMed

Larrea, E; Aldabe, R; Molano, E; Fernandez-Rodriguez, C M; Ametzazurra, A; Civeira, M P; Prieto, J

2006-08-01

Signal transducers and activators of transcription (STATs) play a critical role in antiviral defence. STAT3 is also important in cell protection against inflammatory damage. STAT proteins are activated by interferons and by hepatoprotective cytokines of the interleukin 6 superfamily, including cardiotrophin 1. We analysed the status of STATs in hepatitis C virus (HCV) infected livers and the relationship between expression and activation of STATs and HCV replication in Huh7 cells transfected with HCV genomic replicon. STAT3alpha expression was reduced in HCV infected livers showing an inverse correlation with serum alanine aminotransferase. In patients with HCV infection, nuclear staining for phosphorylated STAT3 was faint in parenchymal cells (although conspicuous in infiltrating leucocytes), in contrast with strong nuclear staining in hepatocytes from control livers. Expression and activation of STAT1 (a factor activated by both interferon (IFN)-alpha and IFN-gamma) were increased in HCV infected livers, particularly in those with high inflammatory activity. Conversely, phosphorylated STAT2 (a factor selectively activated by IFN-alpha) was undetectable in livers with HCV infection, a finding that was associated with marked downregulation of the two functional subunits of the IFN-alpha receptor. HCV replication in Huh7 cells caused STAT3alpha downregulation and blocked STAT3 phosphorylation by either IFN-alpha or cardiotrophin 1. HCV replication in Huh7 cells also inhibited STAT1 and STAT2 activation by IFN-alpha while there was no impairment of STAT1 phosphorylation by the proinflammatory cytokine IFN-gamma. STAT3 is downregulated in HCV infected livers and in Huh7 cells bearing the full length HCV replicon. HCV replication is associated with impaired Jak-STAT signalling by antiviral and cytoprotective cytokines. These effects may favour viral replication while facilitating the progression of liver disease.

A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Ren-Jie

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G{sub 2}/M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosismore » in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G{sub 2}/M cell cycle regulators, ABT-751 induced G{sub 2}/M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria. - Highlights: • An anti-microtubule agent, ABT-751, induces autophagy and apoptosis in Huh-7 cells. • ABT-751 induces early autophagy and delays apoptosis. • ABT-751 induces autophagy via modulations of nuclear TP53 and AKT/MTOR pathways. • ABT-751-induced apoptosis is ROS-, mitochondria- and caspase-dependent. • Inhibition of autophagy enhances ABT-751-induced apoptosis.« less

The metabolic sensors FXRα, PGC-1α, and SIRT1 cooperatively regulate hepatitis B virus transcription.

PubMed

Curtil, Claire; Enache, Liviu S; Radreau, Pauline; Dron, Anne-Gaëlle; Scholtès, Caroline; Deloire, Alexandre; Roche, Didier; Lotteau, Vincent; André, Patrice; Ramière, Christophe

2014-03-01

Hepatitis B virus (HBV) genome transcription is highly dependent on liver-enriched, metabolic nuclear receptors (NRs). Among others, NR farnesoid X receptor α (FXRα) enhances HBV core promoter activity and pregenomic RNA synthesis. Interestingly, two food-withdrawal-induced FXRα modulators, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and deacetylase SIRT1, have been found to be associated with HBV genomes ex vivo. Whereas PGC-1α induction was shown to increase HBV replication, the effect of SIRT1 on HBV transcription remains unknown. Here, we showed that, in hepatocarcinoma-derived Huh-7 cells, combined activation of FXRα by GW4064 and SIRT1 by activator 3 increased HBV core promoter-controlled luciferase expression by 25-fold, compared with a 10-fold increase with GW4064 alone. Using cell lines differentially expressing FXRα in overexpression and silencing experiments, we demonstrated that SIRT1 activated the core promoter in an FXRα- and PGC-1α-dependent manner. Maximal activation (>150-fold) was observed in FXRα- and PGC-1α-overexpressing Huh-7 cells treated with FXRα and SIRT1 activators. Similarly, in cells transfected with full-length HBV genomes, maximal induction (3.5-fold) of core promoter-controlled synthesis of 3.5-kb RNA was observed in the same conditions of transfection and treatments. Thus, we identified a subnetwork of metabolic factors regulating HBV replication, strengthening the hypothesis that transcription of HBV and metabolic genes is similarly controlled.

Inhibition of sirtuins 1 and 2 impairs cell survival and migration and modulates the expression of P-glycoprotein and MRP3 in hepatocellular carcinoma cell lines.

PubMed

Ceballos, María Paula; Decándido, Giulia; Quiroga, Ariel Darío; Comanzo, Carla Gabriela; Livore, Verónica Inés; Lorenzetti, Florencia; Lambertucci, Flavia; Chazarreta-Cifre, Lorena; Banchio, Claudia; Alvarez, María de Luján; Mottino, Aldo Domingo; Carrillo, María Cristina

2018-06-01

Sirtuins (SIRTs) 1 and 2 deacetylases are overexpressed in hepatocellular carcinoma (HCC) and are associated with tumoral progression and multidrug resistance (MDR). In this study we analyzed whether SIRTs 1 and 2 activities blockage was able to affect cellular survival and migration and to modulate p53 and FoxO1 acetylation in HepG2 and Huh7 cells. Moreover, we analyzed ABC transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 3 (MRP3) expression. We used cambinol and EX-527 as SIRTs inhibitors. Both drugs reduced cellular viability, number of colonies and cellular migration and augmented apoptosis. In 3D cultures, SIRTs inhibitors diminished spheroid growth and viability. 3D culture was less sensitive to drugs than 2D culture. The levels of acetylated p53 and FoxO1 increased after treatments. Drugs induced a decrease in ABC transporters mRNA and protein levels in HepG2 cells; however, only EX-527 was able to reduce MRP3 mRNA and protein levels in Huh7 cells. This is the first work demonstrating the regulation of MRP3 by SIRTs. In conclusion, both drugs decreased HCC cells survival and migration, suggesting SIRTs 1 and 2 activities blockage could be beneficial during HCC therapy. Downregulation of the expression of P-gp and MRP3 supports the potential application of SIRTs 1 and 2 inhibitions in combination with conventional chemotherapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Lysophosphatidylcholine acyltransferase 1 is downregulated by hepatitis C virus: impact on production of lipo-viro-particles.

PubMed

Beilstein, Frauke; Lemasson, Matthieu; Pène, Véronique; Rainteau, Dominique; Demignot, Sylvie; Rosenberg, Arielle R

2017-12-01

HCV is intimately linked with the liver lipid metabolism, devoted to the efflux of triacylglycerols stored in lipid droplets (LDs) in the form of triacylglycerol-rich very-low-density lipoproteins (VLDLs): (i) the most infectious HCV particles are those of lowest density due to association with triacylglycerol-rich lipoproteins and (ii) HCV-infected patients frequently develop hepatic steatosis (increased triacylglycerol storage). The recent identification of lysophosphatidylcholine acyltransferase 1 (LPCAT1) as an LD phospholipid-remodelling enzyme prompted us to investigate its role in liver lipid metabolism and HCV infectious cycle. Huh-7.5.1 cells and primary human hepatocytes (PHHs) were infected with JFH1-HCV. LPCAT1 depletion was achieved by RNA interference. Cells were monitored for LPCAT1 expression, lipid metabolism and HCV production and infectivity. The density of viral particles was assessed by isopycnic ultracentrifugation. Upon HCV infection, both Huh-7.5.1 cells and PHH had decreased levels of LPCAT1 transcript and protein, consistent with transcriptional downregulation. LPCAT1 depletion in either naive or infected Huh-7.5.1 cells resulted in altered lipid metabolism characterised by LD remodelling, increased triacylglycerol storage and increased secretion of VLDL. In infected Huh-7.5.1 cells or PHH, LPCAT1 depletion increased production of the viral particles of lowest density and highest infectivity. We have identified LPCAT1 as a modulator of liver lipid metabolism downregulated by HCV, which appears as a viral strategy to increase the triacylglycerol content and hence infectivity of viral particles. Targeting this metabolic pathway may represent an attractive therapeutic approach to reduce both the viral titre and hepatic steatosis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

A tryptophan derivative, ITE, enhances liver cell metabolic functions in vitro

PubMed Central

Zhang, Xiaoqian; Lu, Juan; He, Bin; Tang, Lingling; Liu, Xiaoli; Zhu, Danhua; Cao, Hongcui; Wang, Yingjie; Li, Lanjuan

2017-01-01

Cell encapsulation provides a three-dimensional support by incorporating isolated cells into microcapsules with the goal of simultaneously maintaining cell survival and function, as well as providing active transport for a bioreactor in vitro similarly to that observed in vivo. However, the biotransformation and metabolic functions of the encapsulated cells are not satisfactory for clinical applications. For this purpose, in this study, hepatoma-derived Huh7 cells/C3A cells were treated with 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), an endogenous non-toxic ligand for aryl hydrocarbon receptor, in monolayer cultures and on microspheres. The mRNA and protein levels, as well as the metabolic activities of drug metabolizing enzymes, albumin secretion and urea synthesis were determined. When the Huh7 and C3A cells cultured in a monolayer on two-dimensional surfaces, ITE enhanced the protein levels and the metabolic activities of the major cytochrome P450 (CYP450) enzymes, CYP1A1, CYP1A2, CYP3A4 and CYP1B1, and slightly increased albumin secretion and urea synthesis. Moreover, when cultured on microspheres, ITE also substantially increased the protein levels and metabolic activities of CYP1A1, CYP1A2, CYP3A4 and CYP1B1 in both liver cell lines. On the whole, our findings indicate that ITE enhances the enzymatic activities of major CYP450 enzymes and the metabolic functions of liver cells cultured in monolayer or on microspheres, indicating that it may be utilized to improve the functions of hepatocytes. Thus, it may be used in the future for the treatment of liver diseases. PMID:27959388

A tryptophan derivative, ITE, enhances liver cell metabolic functions in vitro.

PubMed

Zhang, Xiaoqian; Lu, Juan; He, Bin; Tang, Lingling; Liu, Xiaoli; Zhu, Danhua; Cao, Hongcui; Wang, Yingjie; Li, Lanjuan

2017-01-01

Cell encapsulation provides a three-dimensional support by incorporating isolated cells into microcapsules with the goal of simultaneously maintaining cell survival and function, as well as providing active transport for a bioreactor in vitro similarly to that observed in vivo. However, the biotra-nsformation and metabolic functions of the encapsulated cells are not satisfactory for clinical applications. For this purpose, in this study, hepatoma-derived Huh7 cells/C3A cells were treated with 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), an endogenous non-toxic ligand for aryl hydrocarbon receptor, in monolayer cultures and on microspheres. The mRNA and protein levels, as well as the metabolic activities of drug metabolizing enzymes, albumin secretion and urea synthesis were determined. When the Huh7 and C3A cells cultured in a monolayer on two‑dimensional surfaces, ITE enhanced the protein levels and the metabolic activities of the major cytochrome P450 (CYP450) enzymes, CYP1A1, CYP1A2, CYP3A4 and CYP1B1, and slightly increased albumin secretion and urea synthesis. Moreover, when cultured on microspheres, ITE also substantially increased the protein levels and metabolic activities of CYP1A1, CYP1A2, CYP3A4 and CYP1B1 in both liver cell lines. On the whole, our findings indicate that ITE enhances the enzymatic activities of major CYP450 enzymes and the metabolic functions of liver cells cultured in monolayer or on microspheres, indicating that it may be utilized to improve the functions of hepatocytes. Thus, it may be used in the future for the treatment of liver diseases.

Isolation of human monoclonal antibodies to the envelope e2 protein of hepatitis C virus and their characterization.

PubMed

Shimizu, Yohko K; Hijikata, Minako; Oshima, Masamichi; Shimizu, Kazufumi; Alter, Harvey J; Purcell, Robert H; Yoshikura, Hiroshi; Hotta, Hak

2013-01-01

We isolated and characterized two human monoclonal antibodies to the envelope E2 protein of hepatitis C virus (HCV). Lymphoblastoid cell lines stably producing antibodies were obtained by immortalizing peripheral blood mononuclear cells of a patient with chronic hepatitis C using Epstein-Barr virus. Screening for antibody-positive clones was carried out by immunofluorescence with Huh7 cells expressing the E2 protein of HCV strain H (genotype 1a) isolated from the same patient. Isotype of resulting antibodies, #37 and #55, was IgG1/kappa and IgG1/lambda, respectively. Epitope mapping revealed that #37 and #55 recognize conformational epitopes spanning amino acids 429 to 652 and 508 to 607, respectively. By immunofluorescence using virus-infected Huh7.5 cells as targets both antibodies were reactive with all of the nine different HCV genotypes/subtypes tested. The antibodies showed a different pattern of immuno-staining; while #37 gave granular reactions mostly located in the periphery of the nucleus, #55 gave diffuse staining throughout the cytoplasm. Both antibodies were shown by immuno-gold electron microscopy to bind to intact viral particles. In a neutralization assay (focus-forming unit reduction using chimeric infectious HCV containing structural proteins derived from genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, 6a, and 7a), #55 inhibited the infection of all HCV genotypes tested but genotype 7a to a lesser extent. #37 did not neutralize any of these viruses. As a broadly cross-neutralizing human antibody, #55 may be useful for passive immunotherapy of HCV infection.

SGK2 promotes hepatocellular carcinoma progression and mediates GSK-3β/β-catenin signaling in HCC cells.

PubMed

Liu, Junying; Zhang, Guangdong; Lv, Yanping; Zhang, Xiaoyang; Ying, Cui; Yang, Suocheng; Kong, Xin; Yu, Yanzhang

2017-06-01

The phosphoinositide 3-kinase pathway is one of the most commonly altered pathways in human cancers. The serum/glucocorticoid-regulated kinase (SGK) family of serine/threonine kinases consists of three isoforms, SGK1, SGK2, and SGK3. This family of kinases is highly homologous to the AKT kinase family, sharing similar upstream activators and downstream targets. Few studies have investigated the role of SGK2 in hepatocellular carcinoma. Here, we report that SGK2 expression levels were upregulated in hepatocellular carcinoma tissues and human hepatoma cell lines compared to the adjacent normal liver tissues and a normal hepatocyte line, respectively. We found that downregulated SGK2 inhibits cell migration and invasive potential of hepatocellular carcinoma cell lines (SMMC-7721 and Huh-7).We also found that downregulated SGK2 suppressed the expression level of unphosphorylated (activated) glycogen synthase kinase 3 beta. In addition, SGK2 downregulation decreased the dephosphorylation (activation) of β-catenin by preventing its proteasomal degradation in the hepatocellular carcinoma cell lines. These findings suggest that SGK2 promotes hepatocellular carcinoma progression and mediates glycogen synthase kinase 3 beta/β-catenin signaling in hepatocellular carcinoma cells.

[Expression and identification of eukaryotic expression vectors of Brucella melitensis lipoprotein OMP19].

PubMed

He, Zuoping; Luo, Peifang; Hu, Feihuan; Weng, Yunceng; Wang, Wenjing; Li, Chengyao

2016-04-01

To construct eukaryotic expression vectors carrying Brucella melitensis outer membrane protein 19 (OMP19), express them in transfected Huh7.5.1 and JEG-3 cells, and analyze their role in cell apoptosis. Brucella melitensis lipidated OMP19 (L-OMP19) gene and unlipidated OMP19 (U-OMP19) gene were amplified by PCR and inserted into the vector pZeroBack/blunt. The correct L-OMP19 and U-OMP19 genes verified by XbaI and BamHI double digestion and sequencing were cloned into the lentivirus expression vector pHAGE-CMV-MCS-IZsGreen to construct vectors pHAGE-L-OMP19 and pHAGE-U-OMP19, which were separately transfected into 293FT cells, Huh7.5.1 and JEG-3 cells. L-OMP19 and U-OMP19 in the cells were detected by Western blotting and immunofluorescence technique. Flow cytometry combined with annexin V-PE/7-AAD staining was used to detect the cell apoptosis. The lentiviral vectors pHAGE-L-OMP19 and pHAGE-U-OMP19 were constructed correctly and the recombinant lipoproteins L-OMP19 and U-OMP19 expressed in the above cells were well recognized by the specific antibodies against L-OMP19 in Western blotting and immunofluorescence technique. L-OMP19 and U-OMP19 induced JEG-3 cell death, but did not induce the apoptosis of Huh7.5.1 cells. The eukaryotic expression vectors of L-OMP19 and U-OMP19 have been constructed successfully. Recombinant lipoproteins L-OMP19 and U-OMP19 expressed in cells have a good antigenicity, which could be used as experimental materials for the research on the relationship between host cells and lipoproteins in Brucella infection.

CRM1 inhibitory and antiproliferative activities of novel 4'-alkyl substituted klavuzon derivatives.

PubMed

Kanbur, Tuğçe; Kara, Murat; Kutluer, Meltem; Şen, Ayhan; Delman, Murat; Alkan, Aylin; Otaş, Hasan Ozan; Akçok, İsmail; Çağır, Ali

2017-08-15

Klavuzons are 6-(naphthalen-1-yl) substituted 5,6-dihydro-2H-pyran-2-one derivatives showing promising antiproliferative activities in variety of cancer cell lines. In this work, racemic syntheses of nine novel 4'-alkyl substituted klavuzon derivatives were completed in eight steps and anticancer properties of these compounds were evaluated. It is found that size of the substituent has dramatic effect over the potency and selectivity of the cytotoxic activity in cancerous and healthy pancreatic cell lines. The size of the substituent can also effect the CRM1 inhibitory properties of klavuzon derivatives. Strong cytotoxic activity and CRM1 inhibition can be observed only when a small substituent present at 4'-position of naphthalen-1-yl group. However, these substituents makes the molecule more cytotoxic in healthy pancreatic cells rather than cancerous pancreatic cells. Among the tested compounds 1,2,3,4-tetrahydrophenanthren-9-yl substituted lactone was the most cytotoxic compound and its antiproliferative activity was also tested in 3D spheroids generated from HuH-7 cell lines. Copyright © 2017 Elsevier Ltd. All rights reserved.

Direct methylation of FXR by Set7/9, a lysine methyltransferase, regulates the expression of FXR target genes

PubMed Central

Balasubramaniyan, Natarajan; Ananthanarayanan, Meena

2012-01-01

The farnesoid X receptor (FXR) is a ligand (bile acid)-dependent nuclear receptor that regulates target genes involved in every aspect of bile acid homeostasis. Upon binding of ligand, FXR recruits an array of coactivators and associated proteins, some of which have intrinsic enzymatic activity that modify histones or even components of the transcriptional complex. In this study, we show chromatin occupancy by the Set7/9 methyltransferase at the FXR response element (FXRE) and direct methylation of FXR in vivo and in vitro at lysine 206. siRNA depletion of Set7/9 in the Huh-7 liver cell line decreased endogenous mRNAs of the FXR target genes, the short heterodimer partner (SHP) and bile salt export pump (BSEP). Mutation of the methylation site at K206 of FXR to an arginine prevented methylation by Set7/9. A pan-methyllysine antibody recognized the wild-type FXR but not the K206R mutant form. An electromobility shift assay showed that methylation by Set7/9 enhanced binding of FXR/retinoic X receptor-α to the FXRE. Interaction between hinge domain of FXR (containing K206) and Set7/9 was confirmed by coimmunoprecipitation, GST pull down, and mammalian two-hybrid experiments. Set7/9 overexpression in Huh-7 cells significantly enhanced transactivation of the SHP and BSEP promoters in a ligand-dependent fashion by wild-type FXR but not the K206R mutant FXR. A Set7/9 mutant deficient in methyltransferase activity was also not effective in increasing transactivation of the BSEP promoter. These studies demonstrate that posttranslational methylation of FXR by Set7/9 contributes to the transcriptional activation of FXR-target genes. PMID:22345554

Control of temporal activation of hepatitis C virus-induced interferon response by domain 2 of nonstructural protein 5A.

PubMed

Hiet, Marie-Sophie; Bauhofer, Oliver; Zayas, Margarita; Roth, Hanna; Tanaka, Yasuhito; Schirmacher, Peter; Willemsen, Joschka; Grünvogel, Oliver; Bender, Silke; Binder, Marco; Lohmann, Volker; Lotteau, Vincent; Ruggieri, Alessia; Bartenschlager, Ralf

2015-10-01

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a multifunctional protein playing a crucial role in diverse steps of the viral replication cycle and perturbing multiple host cell pathways. We showed previously that removal of a region in domain 2 (D2) of NS5A (mutant NS5A(D2Δ)) is dispensable for viral replication in hepatoma cell lines. By using a mouse model and immune-competent cell systems, we studied the role of D2 in controlling the innate immune response. In vivo replication competence of NS5A(D2Δ) was studied in transgenic mice with human liver xenografts. Results were validated using primary human hepatocytes (PHHs) and mechanistic analyses were conducted in engineered Huh7 hepatoma cells with reconstituted innate signaling pathways. Although the deletion in NS5A removed most of the interferon (IFN) sensitivity determining-region, mutant NS5A(D2Δ) was as sensitive as the wild type to IFN-α and IFN-λ in vitro, but severely attenuated in vivo. This attenuation could be recapitulated in PHHs and was linked to higher activation of the IFN response, concomitant with reduced viral replication and virus production. Importantly, immune-reconstituted Huh7-derived cell lines revealed a sequential activation of the IFN-response via RIG-I (retinoic acid-inducible gene I) and MDA5 (Myeloma differentiation associated factor 5), respectively, that was significantly higher in the case of the mutant lacking most of NS5A D2. Our study reveals an important role of NS5A D2 for suppression of the IFN response that is activated by HCV via RIG-I and MDA5 in a sequential manner. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singaravelu, Ragunath; National Research Council of Canada, Ottawa, Ontario K1A 0R6; Lyn, Rodney K.

Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limitedmore » cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway.« less

Sofosbuvir inhibits hepatitis A virus replication in vitro assessed by a cell-based fluorescent reporter system.

PubMed

Jiang, Wang; Muhammad, Fawad; Ma, Pengjuan; Liu, Xiyu; Long, Gang

2018-06-01

Hepatitis A virus (HAV) infection remains a major cause of acute hepatitis worldwide and even leads to fulminant hepatitis. For screening antivirals against HAV in vitro, we develop a cell-based fluorescent reporter system named Huh-7.5.1-GA, in which HAV infection is visualized by green fluorescence protein (GFP) translocation from the cytosol into the nucleus. The reliability of Huh-7.5.1-GA for antiviral studies is validated by IFN-α, a known inhibitor of HAV replication, which impedes GFP translocation. Utilizing this in-vitro reporter system, we find that sofosbuvir, an FDA approved prodrug for the treatment of chronic hepatitis C, disturbs GFP translocation and inhibits HAV replication efficiently. In addition, we find that inhibition of HAV by sofosbuvir is hepatic-cell dependent, with IC50 (half-maximal inhibitory concentration) being 6.3 μM and 9.9 μM in Huh-7.5.1, quantified separately by RT-qPCR and image-based analysis. Therefore, our reporter system may serve as a high-throughput platform for screening potent antivirals against HAV. Sofosbuvir may be considered for treatment of hepatitis A, especially in re-infected patients who undergo liver transplantation due to HAV-induced liver failure. Copyright © 2018 Elsevier B.V. All rights reserved.

Four Novel Splice-Switch Reporter Cell Lines: Distinct Impact of Oligonucleotide Chemistry and Delivery Vector on Biological Activity.

PubMed

Rocha, Cristina S J; Lundin, Karin E; Behlke, Mark A; Zain, Rula; El Andaloussi, Samir; Smith, C I Edvard

2016-12-01

New advances in oligonucleotide (ON) chemistry emerge continuously, and over the last few years, several aspects of ON delivery have been improved. However, clear knowledge regarding how certain chemistries behave alone, or in combination with various delivery vectors, is limited. Moreover, characterization is frequently limited to a single reporter cell line and, when different cell types are studied, experiments are commonly not carried out under similar conditions, hampering comparative analysis. To address this, we have developed a small "tissue" library of new, stable, pLuc/705 splice-switching reporter cell lines (named HuH7_705, U-2 OS_705, C2C12_705, and Neuro-2a_705). Our data show that, indeed, the cell type used in activity screenings influences the efficiency of ONs of different chemistry (phosphorothioate with locked nucleic acid or 2'-O-methyl with or without N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine). Likewise, the delivery method, Lipofectamine ® 2000, PepFect14 nanoparticles, or "naked" uptake, also demonstrates cell-type-dependent outcomes. Taken together, these cell lines can potentially become useful tools for future in vitro evaluation of new nucleic acid-based oligomers as well as delivery compounds for splice-switching approaches and cell-specific therapies.

Decrease in Dengue virus-2 infection and reduction of cytokine/chemokine production by Uncaria guianensis in human hepatocyte cell line Huh-7.

PubMed

Mello, Cíntia da Silva; Valente, Ligia Maria Marino; Wolff, Thiago; Lima-Junior, Raimundo Sousa; Fialho, Luciana Gomes; Marinho, Cintia Ferreira; Azeredo, Elzinandes Leal; Oliveira-Pinto, Luzia Maria; Pereira, Rita de Cássia Alves; Siani, Antonio Carlos; Kubelka, Claire Fernandes

2017-06-01

Dengue fever may present hemorrhages and cavitary effusions as result of exacerbated immune responses. We investigated hydro-alcoholic extracts from leaves (UGL) and bark (UGB) of the medicinal species Uncaria guinanensis with respect to antiviral effects in Dengue virus (DENV) infection and in immunological parameters associated with in vivo physiopathological features. Chemical profiles from UGB or UGL were compared in thin layer chromatography and 1H nuclear magnetic resonance using flavonoid compounds and a pentacyclic oxindole alkaloid-enriched fraction as references. DENV-2-infected hepatocytes (Huh-7) were treated with extracts. Cell viability, DENV antigens and immunological factors were detected by enzyme-linked immunosorbent assay (ELISA) or flow cytometry. The UGL mainly differed from UGB by selectively containing the flavonoid kaempferitrin. UGB and UGL improved hepatocyte viability. Both extracts reduced intracellular viral antigen and inhibited the secretion of viral non-structural protein (NS1), which is indicative of viral replication. Reduction in secretion of macrophage migration inhibitory factor was achieved by UGB, of interleukin-6 by UGL, and of interleukin-8 by both UGB and UGL. MAIN. The U. guianensis extracts presented, antiviral and immunomodulatory effects for DENV and possibly a hepatocyte-protective activity. Further studies may be performed to consider these products as potential candidates for the development of an herbal product for the future treatment of dengue.

Semisynthesis, cytotoxicity, antiviral activity, and drug interaction liability of 7-O-methylated analogues of flavonolignans from milk thistle.

PubMed

Althagafy, Hanan S; Graf, Tyler N; Sy-Cordero, Arlene A; Gufford, Brandon T; Paine, Mary F; Wagoner, Jessica; Polyak, Stephen J; Croatt, Mitchell P; Oberlies, Nicholas H

2013-07-01

Silymarin, an extract of the seeds of milk thistle (Silybum marianum), is used as an herbal remedy, particularly for hepatoprotection. The main chemical constituents in silymarin are seven flavonolignans. Recent studies explored the non-selective methylation of one flavonolignan, silybin B, and then tested those analogues for cytotoxicity and inhibition of both cytochrome P450 (CYP) 2C9 activity in human liver microsomes and hepatitis C virus infection in a human hepatoma (Huh7.5.1) cell line. In general, enhanced bioactivity was observed with the analogues. To further probe the biological consequences of methylation of the seven major flavonolignans, a series of 7-O-methylflavonolignans were generated. Optimization of the reaction conditions permitted selective methylation at the phenol in the 7-position in the presence of each metabolite's 4-5 other phenolic and/or alcoholic positions without the use of protecting groups. These 7-O-methylated analogues, in parallel with the corresponding parent compounds, were evaluated for cytotoxicity against Huh7.5.1 cells; in all cases the monomethylated analogues were more cytotoxic than the parent compounds. Moreover, parent compounds that were relatively non-toxic and inactive or weak inhibitors of hepatitis C virus infection had enhanced cytotoxicity and anti-HCV activity upon 7-O-methylation. Also, the compounds were tested for inhibition of major drug metabolizing enzymes (CYP2C9, CYP3A4/5, UDP-glucuronsyltransferases) in pooled human liver or intestinal microsomes. Methylation of flavonolignans differentially modified inhibitory potency, with compounds demonstrating both increased and decreased potency depending upon the compound tested and the enzyme system investigated. In total, these data indicated that monomethylation modulates the cytotoxic, antiviral, and drug interaction potential of silymarin flavonolignans. Copyright © 2013 Elsevier Ltd. All rights reserved.

A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells.

PubMed

Wei, Ren-Jie; Lin, Su-Shuan; Wu, Wen-Ren; Chen, Lih-Ren; Li, Chien-Feng; Chen, Han-De; Chou, Chien-Ting; Chen, Ya-Chun; Liang, Shih-Shin; Chien, Shang-Tao; Shiue, Yow-Ling

2016-11-15

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G 2 /M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G 2 /M cell cycle regulators, ABT-751 induced G 2 /M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria. Copyright © 2016 Elsevier Inc. All rights reserved.

Suppression of AMF/PGI-mediated tumorigenic activities by ursolic acid in cultured hepatoma cells and in a mouse model.

PubMed

Shih, Wen-Ling; Yu, Feng-Ling; Chang, Ching-Dong; Liao, Ming-Huei; Wu, Hung-Yi; Lin, Ping-Yuan

2013-10-01

Our previous studies demonstrated that autocrine motility factor/phosphoglucose isomerase (AMF/PGI) possesses tumorigenic activities through the modulation of intracellular signaling. We then investigated the effects of ursolic acid (UA), oleanolic acid (OA), tangeretin, and nobiletin against AMF/PGI-mediated oncogenesis in cultured stable Huh7 and Hep3B cells expressing wild-type or mutated AMF/PGI and in a mouse model in this study. The working concentrations of the tested compounds were lower than their IC10 , which was determined by Brdu incorporation and colony formation assay. Only UA efficiently suppressed the AMF/PGI-induced Huh7 cell migration and MMP-3 secretion. Additionally, UA inhibited the AMF/PGI-mediated protection against TGF-β-induced apoptosis in Hep3B cells, whereas OA, tangeretin, and nobiletin had no effect. In Huh7 cells and tumor tissues, UA disrupted the Src/RhoA/PI 3-kinase signaling and complex formation induced by AMF/PGI. In the Hep3B system, UA dramatically suppressed AMF/PGI-induced anti-apoptotic signaling transmission, including Akt, p85, Bad, and Stat3 phosphorylation. AMF/PGI enhances tumor growth, angiogenesis, and pulmonary metastasis in mice, which is correlated with its enzymatic activity, and critically, UA intraperitoneal injection reduces the tumorigenesis in vivo, enhances apoptosis in tumor tissues and also prolongs mouse survival. Combination of sub-optimal dose of UA and cisplatin, a synergistic tumor cell-killing effects was found. Thus, UA modulates intracellular signaling and might serve as a functional natural compound for preventing or alleviating hepatocellular carcinoma. © 2012 Wiley Periodicals, Inc.

Mineral pitch induces apoptosis and inhibits proliferation via modulating reactive oxygen species in hepatic cancer cells.

PubMed

Pant, Kishor; Gupta, Parul; Damania, Preeti; Yadav, Ajay K; Gupta, Aanchal; Ashraf, Anam; Venugopal, Senthil K

2016-05-27

Mineral Pitch (MP) is a dark brown coloured humic matter originating from high altitude rocks. It is an Ayurvedic medicinal food, commonly used by the people of the Himalayan regions of Nepal and India for various body ailments. The Huh-7 cells were treated with different concentrations of MP for 24 h, and both apoptosis and proliferation was determined by the TUNEL and MTT assays respectively. The formation of ROS and nitric oxide was analysed by DCFH-DA and Griess reagent respectively. The expression of miRNA-21 and miRNA-22 were checked by the real time PCR. Effect of miRNA-22 on proliferation and c-myc was studied by over-expressing miRNA-22 premiRs in Huh-7 cells. We found that MP enhanced anti-cancer effects by inducing apoptosis and inhibiting proliferation. MP induced both ROS and NO, upon neutralizing them, there was a partial recovery of apoptosis and proliferation. MP also induced miRNA-22 expression, while miRNA-21 expression was inhibited. Over-expression of miRNA-22 resulted in a significant inhibition of proliferation. miRNA-22 directly targeted c-myc gene, thereby inhibited proliferation. These results clearly show that MP induces its anti-cancer activity by more than one pathway. The data clearly indicate that MP induced apoptosis via the production of ROS, and inhibited proliferation by inducing miRNA-22 and inhibiting miRNA-21 in Huh-7 cells.

Short-term starvation is a strategy to unravel the cellular capacity of oxidizing specific exogenous/endogenous substrates in mitochondria.

PubMed

Zeidler, Julianna D; Fernandes-Siqueira, Lorena O; Carvalho, Ana S; Cararo-Lopes, Eduardo; Dias, Matheus H; Ketzer, Luisa A; Galina, Antonio; Da Poian, Andrea T

2017-08-25

Mitochondrial oxidation of nutrients is tightly regulated in response to the cellular environment and changes in energy demands. In vitro studies evaluating the mitochondrial capacity of oxidizing different substrates are important for understanding metabolic shifts in physiological adaptations and pathological conditions, but may be influenced by the nutrients present in the culture medium or by the utilization of endogenous stores. One such influence is exemplified by the Crabtree effect (the glucose-mediated inhibition of mitochondrial respiration) as most in vitro experiments are performed in glucose-containing media. Here, using high-resolution respirometry, we evaluated the oxidation of endogenous or exogenous substrates by cell lines harboring different metabolic profiles. We found that a 1-h deprivation of the main energetic nutrients is an appropriate strategy to abolish interference of endogenous or undesirable exogenous substrates with the cellular capacity of oxidizing specific substrates, namely glutamine, pyruvate, glucose, or palmitate, in mitochondria. This approach primed mitochondria to immediately increase their oxygen consumption after the addition of the exogenous nutrients. All starved cells could oxidize exogenous glutamine, whereas the capacity for oxidizing palmitate was limited to human hepatocarcinoma Huh7 cells and to C2C12 mouse myoblasts that differentiated into myotubes. In the presence of exogenous glucose, starvation decreased the Crabtree effect in Huh7 and C2C12 cells and abrogated it in mouse neuroblastoma N2A cells. Interestingly, the fact that the Crabtree effect was observed only for mitochondrial basal respiration but not for the maximum respiratory capacity suggests it is not caused by a direct effect on the electron transport system. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of miR-122-independent propagation of HCV

PubMed Central

Motooka, Daisuke; Nakamura, Shota; Yamamoto, Satomi; Mori, Hiroyuki; Sato, Asuka; Uemura, Kentaro; Fauzyah, Yuzy; Suda, Takahiro; Nishio, Akira; Hmwe, Su Su; Okamoto, Toru; Tatsumi, Tomohide; Takehara, Tetsuo; Chayama, Kazuaki; Wakita, Takaji; Koike, Kazuhiko

2017-01-01

miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV122KO, possessing G28A substitution in the 5’UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocyte-derived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients. PMID:28494029

Negundoside, an iridiod glycoside from leaves of Vitex negundo, protects human liver cells against calcium-mediated toxicity induced by carbon tetrachloride

PubMed Central

Tasduq, Sheikh A; Kaiser, Peerzada J; Gupta, Bishan D; Gupta, Vijay K; Johri, Rakesh K

2008-01-01

AIM: To evaluate the protective effect of 2'-p-hydroxybenzoylmussaenosidic acid [negundoside (NG), against carbon tetrachloride (CCl4)-induced toxicity in HuH-7 cells. METHODS: CCl4 is a well characterized hepatotoxin, and inducer of cytochrome P450 2E1 (CYP2E1)-mediated oxidative stress. In addition, lipid peroxidation and accumulation of intracellular calcium are important steps in the pathway involved in CCl4 toxicity. Liver cells (HuH-7) were treated with CCl4, and the mechanism of the cytoprotective effect of NG was assessed. Silymarin, a known hepatoprotective drug, was used as control. RESULTS: NG protected HuH-7 cells against CCl4 toxicity and loss of viability without modulating CYP2E1 activity. Prevention of CCl4 toxicity was associated with a reduction in oxidative damage as reflected by decreased generation of reactive oxygen species (ROS), a decrease in lipid peroxidation and accumulation of intracellular Ca2+ levels and maintenance of intracellular glutathione homeostasis. Decreased mitochondrial membrane potential (MMP), induction of caspases mediated DNA fragmentation and cell cycle arrest, as a result of CCl4 treatment, were also blocked by NG. The protection afforded by NG seemed to be mediated by activation of cyclic adenosine monophosphate (cAMP) synthesis and inhibition of phospholipases (cPLA2). CONCLUSION: NG exerts a protective effect on CYP2E1-dependent CCl4 toxicity via inhibition of lipid peroxidation, followed by an improved intracellular calcium homeostasis and inhibition of Ca2+-dependent proteases. PMID:18595136

NOTCH2 signaling confers immature morphology and aggressiveness in human hepatocellular carcinoma cells

PubMed Central

HAYASHI, YOSHIHIRO; OSANAI, MAKOTO; LEE, GANG-HONG

2015-01-01

The NOTCH family of membranous receptors plays key roles during development and carcinogenesis. Since NOTCH2, yet not NOTCH1 has been shown essential for murine hepatogenesis, NOTCH2 rather than NOTCH1 may be more relevant to human hepatocarcinogenesis; however, no previous studies have supported this hypothesis. We therefore assessed the role of NOTCH2 in human hepatocellular carcinoma (HCC) by immunohistochemistry and cell culture. Immunohistochemically, 19% of primary HCCs showed nuclear staining for NOTCH2, indicating activated NOTCH2 signaling. NOTCH2-positive HCCs were on average in more advanced clinical stages, and exhibited more immature cellular morphology, i.e. higher nuclear-cytoplasmic ratios and nuclear densities. Such features were not evident in NOTCH1-positive HCCs. In human HCC cell lines, abundant NOTCH2 expression was associated with anaplasia, represented by loss of E-cadherin. When NOTCH2 signaling was stably downregulated in HLF cells, an anaplastic HCC cell line, the cells were attenuated in potential for in vitro invasiveness and migration, as well as in vivo tumorigenicity accompanied by histological maturation. Generally, inverse results were obtained for a differentiated HCC cell line, Huh7, manipulated to overexpress activated NOTCH2. These findings suggested that the NOTCH2 signaling may confer aggressive behavior and immature morphology in human HCC cells. PMID:26252838

NOTCH2 signaling confers immature morphology and aggressiveness in human hepatocellular carcinoma cells.

PubMed

Hayashi, Yoshihiro; Osanai, Makoto; Lee, Gang-Hong

2015-10-01

The NOTCH family of membranous receptors plays key roles during development and carcinogenesis. Since NOTCH2, yet not NOTCH1 has been shown essential for murine hepatogenesis, NOTCH2 rather than NOTCH1 may be more relevant to human hepatocarcinogenesis; however, no previous studies have supported this hypothesis. We therefore assessed the role of NOTCH2 in human hepatocellular carcinoma (HCC) by immunohistochemistry and cell culture. Immunohistochemically, 19% of primary HCCs showed nuclear staining for NOTCH2, indicating activated NOTCH2 signaling. NOTCH2-positive HCCs were on average in more advanced clinical stages, and exhibited more immature cellular morphology, i.e. higher nuclear-cytoplasmic ratios and nuclear densities. Such features were not evident in NOTCH1‑positive HCCs. In human HCC cell lines, abundant NOTCH2 expression was associated with anaplasia, represented by loss of E-cadherin. When NOTCH2 signaling was stably downregulated in HLF cells, an anaplastic HCC cell line, the cells were attenuated in potential for in vitro invasiveness and migration, as well as in vivo tumorigenicity accompanied by histological maturation. Generally, inverse results were obtained for a differentiated HCC cell line, Huh7, manipulated to overexpress activated NOTCH2. These findings suggested that the NOTCH2 signaling may confer aggressive behavior and immature morphology in human HCC cells.

Multi-Leu PACE4 Inhibitor Retention within Cells Is PACE4 Dependent and a Prerequisite for Antiproliferative Activity

PubMed Central

Ly, Kévin; Levesque, Christine; Kwiatkowska, Anna; Ait-Mohand, Samia; Desjardins, Roxane; Guérin, Brigitte; Day, Robert

2015-01-01

The overexpression as well as the critical implication of the proprotein convertase PACE4 in prostate cancer progression has been previously reported and supported the development of peptide inhibitors. The multi-Leu peptide, a PACE4-specific inhibitor, was further generated and its capability to be uptaken by tumor xenograft was demonstrated with regard to its PACE4 expression status. To investigate whether the uptake of this inhibitor was directly dependent of PACE4 levels, uptake and efflux from cancer cells were evaluated and correlations were established with PACE4 contents on both wild type and PACE4-knockdown cell lines. PACE4-knockdown associated growth deficiencies were established on the knockdown HepG2, Huh7, and HT1080 cells as well as the antiproliferative effects of the multi-Leu peptide supporting the growth capabilities of PACE4 in cancer cells. PMID:26114115

Infection of Hepatocytes With HCV Increases Cell Surface Levels of Heparan Sulfate Proteoglycans, Uptake of Cholesterol and Lipoprotein, and Virus Entry by Up-regulating SMAD6 and SMAD7.

PubMed

Zhang, Fang; Sodroski, Catherine; Cha, Helen; Li, Qisheng; Liang, T Jake

2017-01-01

The signaling molecule and transcriptional regulator SMAD6, which inhibits the transforming growth factor β signaling pathway, is required for infection of hepatocytes by hepatitis C virus (HCV). We investigated the mechanisms by which SMAD6 and another inhibitory SMAD (SMAD7) promote HCV infection in human hepatoma cells and hepatocytes. We infected Huh7 and Huh7.5.1 cells and primary human hepatocytes with Japanese fulminant hepatitis-1 (JFH1) HCV cell culture system (HCVcc). We then measured HCV binding, intracellular levels of HCV RNA, and expression of target genes. We examined HCV entry in HepG2/microRNA (miR) 122/CD81 cells, which support entry and replication of HCV, were transfected these cells with small interfering RNAs targeting inhibitory SMADs to analyze gene expression profiles. Uptake of labeled low-density lipoprotein (LDL) and cholesterol was measured. Cell surface proteins were quantified by flow cytometry. We obtained liver biopsy samples from 69 patients with chronic HCV infection and 19 uninfected individuals (controls) and measured levels of syndecan 1 (SDC1), SMAD7, and SMAD6 messenger RNAs (mRNAs). Small interfering RNA knockdown of SMAD6 blocked the binding and infection of hepatoma cell lines and primary human hepatocytes by HCV, whereas SMAD6 overexpression increased HCV infection. We found levels of mRNAs encoding heparan sulfate proteoglycans (HSPGs), particularly SDC1 mRNA, and cell surface levels of heparan sulfate to be reduced in cells after SMAD6 knockdown. SMAD6 knockdown also reduced transcription of genes encoding lipoprotein and cholesterol uptake receptors, including the LDL receptor (LDLR), the very LDLR, and the scavenger receptor class B member 1 in hepatocytes; knockdown of SMAD6 also inhibited cell uptake of cholesterol and lipoprotein. Overexpression of SMAD6 increased the expression of these genes. Similar effects were observed with knockdown and overexpression of SMAD7. In addition, HCV infection of cells increased the expression of SMAD6, which required the activity of nuclear factor-κB, but not transforming growth factor β. Liver tissues from patients with chronic HCV infection had significantly higher levels of SMAD6, SMAD7, and HSPG mRNAs than controls. In studies of hepatoma cell lines and primary human hepatocytes, we found that infection with HCV leads to activation of nuclear factor-κB, resulting in increased expression of SMAD6 and SMAD7. Up-regulation of SMAD6 and SMAD7 induces the expression of HSPGs, such as SDC1, as well as LDLR, very LDLR, and the scavenger receptor class B member 1, which promote HCV entry and propagation, as well as cellular uptake of cholesterol and lipoprotein. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Hepatitis C virus envelope components alter localization of hepatocyte tight junction-associated proteins and promote occludin retention in the endoplasmic reticulum.

PubMed

Benedicto, Ignacio; Molina-Jiménez, Francisca; Barreiro, Olga; Maldonado-Rodríguez, Alejandra; Prieto, Jesús; Moreno-Otero, Ricardo; Aldabe, Rafael; López-Cabrera, Manuel; Majano, Pedro L

2008-10-01

Hepatocyte tight junctions (TJ) play key roles in characteristic liver functions, including bile formation and secretion. Infection by hepatitis C virus (HCV) may cause alterations of the liver architecture and disruption of the bile duct, which ultimately can lead to cholestasis. Herein, we employed the HCV replicon system to analyze the effect of HCV on TJ organization. TJ-associated proteins occludin, claudin-1, and Zonula Occludens protein-1 (ZO-1) disappeared from their normal localization at the border of adjacent cells in Huh7 clones harboring genomic but not subgenomic replicons expressing only the nonstructural proteins. Furthermore, cells containing genomic replicons showed a cytoplasmic accumulation of occludin in the endoplasmic reticulum (ER). TJ-associated function, measured as FITC-dextran paracellular permeability, of genomic replicon-containing cells, was also altered. Interestingly, clearance of the HCV replicon by interferon-alpha (IFN-alpha) treatment and by short hairpin RNA (shRNA) significantly restored the localization of TJ-associated proteins. Transient expression of all HCV structural proteins, but not core protein alone, altered the localization of TJ-associated proteins in Huh7 cells and in clones with subgenomic replicons. Confocal analysis showed that accumulation of occludin in the ER partially co-localized with HCV envelope glycoprotein E2. E2/occludin association was further confirmed by co-immunoprecipitation and pull-down assays. Additionally, using a cell culture model of HCV infection, we observed the cytoplasmic dot-like accumulation of occludin in infected Huh7 cells. We propose that HCV structural proteins, most likely those of the viral envelope, promote alterations of TJ-associated proteins, which may provide new insights for HCV-related pathogenesis.

Interferon-α Silencing by Small Interference RNA Increases Adenovirus Transduction and Transgene Expression in Huh7 Cells.

PubMed

Sobrevilla-Navarro, Ana Alondra; Sandoval-Rodríguez, Ana; García-Bañuelos, Jesús Javier; Armendariz-Borunda, Juan; Salazar-Montes, Adriana María

2018-04-01

Adenoviruses are the most common vectors used in clinical trials of gene therapy. In 2017, 21.2% of clinical trials used rAds as vectors. Systemic administration of rAds results in high tropism in the liver. Interferon types α and β are the major antiviral cytokines which orchestrate the host's immune response against rAd, limiting therapeutic gene expression and preventing subsequent vector administration. siRNA is small double-strand RNAs that temporally inhibit the expression of a specific gene. The aim is to evaluate the effect of IFN-α blocking by a specific siRNA on Ad-GFP transduction and on transgene expression in Huh7 cells in culture. Huh7 cells were cultured in DMEM and transfected with 70 nM of siRNA-IFN-α. Six hours later, the cells were exposed to 1 × 10 9  vp/ml of rAd-GFP for 24 h. Expression of IFN-α, TNF-α and the PKR gene was determined by RT-qPCR. Percentage of transduction was analyzed by flow cytometry and by qPCR. GFP expression was determined by western blot. 70 nM of siRNA-IFN-α inhibited 96% of IFN-α and 65% of TNF-α gene expression compared to an irrelevant siRNA. Percentage of transduction and transgene expression increased in these cells compared to an irrelevant siRNA. Inhibition of IFN-α expression by siRNA-IFN-α enabled a higher level of transduction and transgene expression GFP, highlighting the role of IFN-α in the elimination of adenovirus in transduced cells and thus suggesting that its inhibition could be an important strategy for gene therapy in clinical trials using adenovirus as a vector directed to liver diseases.

Independent, parallel pathways to CXCL10 induction in HCV-infected hepatocytes.

PubMed

Brownell, Jessica; Wagoner, Jessica; Lovelace, Erica S; Thirstrup, Derek; Mohar, Isaac; Smith, Wesley; Giugliano, Silvia; Li, Kui; Crispe, I Nicholas; Rosen, Hugo R; Polyak, Stephen J

2013-10-01

The pro-inflammatory chemokine CXCL10 is induced by HCV infection in vitro and in vivo, and is associated with outcome of IFN (interferon)-based therapy. We studied how hepatocyte sensing of early HCV infection via TLR3 (Toll-like receptor 3) and RIG-I (retinoic acid inducible gene I) led to expression of CXCL10. CXCL10, type I IFN, and type III IFN mRNAs and proteins were measured in PHH (primary human hepatocytes) and hepatocyte lines harboring functional or non-functional TLR3 and RIG-I pathways following HCV infection or exposure to receptor-specific stimuli. HuH7 human hepatoma cells expressing both TLR3 and RIG-I produced maximal CXCL10 during early HCV infection. Neutralization of type I and type III IFNs had no impact on virus-induced CXCL10 expression in TLR3+/RIG-I+ HuH7 cells, but reduced CXCL10 expression in PHH. PHH cultures were positive for monocyte, macrophage, and dendritic cell mRNAs. Immunodepletion of non-parenchymal cells (NPCs) eliminated marker expression in PHH cultures, which then showed no IFN requirement for CXCL10 induction during HCV infection. Immunofluorescence studies also revealed a positive correlation between intracellular HCV Core and CXCL10 protein expression (r(2) = 0.88, p ≤ 0.001). While CXCL10 induction in hepatocytes during the initial phase of HCV infection is independent of hepatocyte-derived type I and type III IFNs, NPC-derived IFNs contribute to CXCL10 induction during HCV infection in PHH cultures. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

GC–MS-Based Metabonomic Profiling Displayed Differing Effects of Borna Disease Virus Natural Strain Hu-H1 and Laboratory Strain V Infection in Rat Cortical Neurons

PubMed Central

Liu, Siwen; Bode, Liv; Zhang, Lujun; He, Peng; Huang, Rongzhong; Sun, Lin; Chen, Shigang; Zhang, Hong; Guo, Yujie; Zhou, Jingjing; Fu, Yuying; Zhu, Dan; Xie, Peng

2015-01-01

Borna disease virus (BDV) persists in the central nervous systems of a wide variety of vertebrates and causes behavioral disorders. Previous studies have revealed that metabolic perturbations are associated with BDV infection. However, the pathophysiological effects of different viral strains remain largely unknown. Rat cortical neurons infected with human strain BDV Hu-H1, laboratory BDV Strain V, and non-infected control (CON) cells were cultured in vitro. At day 12 post-infection, a gas chromatography coupled with mass spectrometry (GC–MS) metabonomic approach was used to differentiate the metabonomic profiles of 35 independent intracellular samples from Hu-H1-infected cells (n = 12), Strain V-infected cells (n = 12), and CON cells (n = 11). Partial least squares discriminant analysis (PLS-DA) was performed to demonstrate discrimination between the three groups. Further statistical testing determined which individual metabolites displayed significant differences between groups. PLS-DA demonstrated that the whole metabolic pattern enabled statistical discrimination between groups. We identified 31 differential metabolites in the Hu-H1 and CON groups (21 decreased and 10 increased in Hu-H1 relative to CON), 35 differential metabolites in the Strain V and CON groups (30 decreased and 5 increased in Strain V relative to CON), and 21 differential metabolites in the Hu-H1 and Strain V groups (8 decreased and 13 increased in Hu-H1 relative to Strain V). Comparative metabonomic profiling revealed divergent perturbations in key energy and amino acid metabolites between natural strain Hu-H1 and laboratory Strain V of BDV. The two BDV strains differentially alter metabolic pathways of rat cortical neurons in vitro. Their systematic classification provides a valuable template for improved BDV strain definition in future studies. PMID:26287181

Anticancer potential of Hericium erinaceus extracts against human gastrointestinal cancers.

PubMed

Li, Guang; Yu, Kai; Li, Fushuang; Xu, Kangping; Li, Jing; He, Shujin; Cao, Shousong; Tan, Guishan

2014-04-28

Hericium is a genus of mushrooms (fungus) in the Hericiaceae family. Hericium erinaceus (HE) has been used for the treatment of digestive diseases for over 2000 years in China. HE possesses many beneficial functions such as anticancer, antiulcer, antiinflammation and antimicrobial effects, immunomodulation and other activities. The aim of the studies was to evaluate the anticancer efficacy of two extracts (HTJ5 and HTJ5A) from the culture broth of HE against three gastrointestinal cancers such as liver, colorectal and gastric cancers in both of in vitro of cancer cell lines and in vivo of tumor xenografts and discover the active compounds. Two HE extracts (HTJ5 and HTJ5A) were used for the studies. For the study of chemical constituents, the HTJ5 and HTJ5A were separated using a combination of macroporous resin with silica gel, HW-40 and LH-20 chromatography then purified by semipreparative high-performance liquid chromatography (HPLC) and determined by nuclear magnetic resonance (NMR) spectra. For the in vitro cytotoxicity studies, HepG2 and Huh-7 liver, HT-29 colon, and NCI-87 gastric cancer cell lines were used and MTT assay was performed to determine the in vitro cytotoxicity. For in vivo antitumor efficacy and toxicity studies, tumor xenograft models of SCID mice bearing liver cancer HepG2 and Huh-7, colon cancer HT-29 and gastric cancer NCI-87 subcutaneously were used and the mice were treated with the vehicle control, HTJ5 and HTJ5A orally (500 and 1000 mg/kg/day) and compared to 5-fluorouraci (5-FU) at the maximum tolerated dose (MTD, 25-30 mg/kg/day) intraperitoneally daily for 5 days when the tumors reached about 180-200 mg (mm(3)). Tumor volumes and body weight were measured daily during the first 10 days and 2-3 times a week thereafter to assess the tumor growth inhibition, tumor doubling time, partial and complete tumor response and toxicity. Twenty-two compounds were obtained from the fractions of HTJ5/HTJ5A including seven cycli dipeptides, five indole, pyrimidines, amino acids and derivative, three flavones, one anthraquinone, and six small aromatic compounds. HTJ5 and HTJ5A exhibited concentration-dependent cytotoxicity in vitro against liver cancer HepG2 and Huh-7, colon cancer HT-29, and gastric cancer NCI-87 cells with the IC50 in 2.50±0.25 and 2.00±0.25, 0.80±0.08 and 1.50±0.28, 1.25±0.06 and 1.25±0.05, and 5.00±0.22 and 4.50±0.14 mg/ml; respectively. For in vivo tumor xenograft studies, HTJ5 and HTJ5A showed significantly antitumor efficacy against all four xenograft models of HepG2, Huh-7, HT-29 and NCI-87 without toxicity to the host. Furthermore, HTJ5 and HTJ5A are more effective than that of 5-FU against the four tumors with less toxicity. HE extracts (HTJ5 and HTJ5A) are active against liver cancer HepG2 and Huh-7, colon cancer HT-29 and gastric cancer NCI-87 cells in vitro and tumor xenografts bearing in SCID mice in vivo. They are more effective and less toxic compared to 5-FU in all four in vivo tumor models. The compounds have the potential for development into anticancer agents for the treatment of gastrointestinal cancer used alone and/or in combination with clinical used chemotherapeutic drugs. However, further studies are required to find out the active chemical constituents and understand the mechanism of action associated with the super in vivo anticancer efficacy. In addition, future studies are needed to confirm our preliminary results of in vivo synergistic antitumor efficacy in animal models of tumor xenografts with the combination of HE extracts and clinical used anticancer drugs such as 5-FU, cisplatin and doxurubicin for the treatment of gastrointestinal cancers. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

The E3 ligase for metastasis associated 1 protein, TRIM25, is targeted by microRNA-873 in hepatocellular carcinoma.

PubMed

Li, Yu-Hui; Zhong, Ming; Zang, Hong-Liang; Tian, Xiao-Feng

2018-07-01

Tumor metastasis accounts for 90% of all cancer-related deaths. Epithelial to mesenchymal transition (EMT) considered to be centrally important in acquired resistance to chemotherapy and in progression of tumors to secondary organs. One of the important mediators of metastatic progression in hepatocellular carcinoma (HCC) is the metastasis associated protein 1 (MTA-1). We have earlier shown that in the context of HCC and normal liver cell lines, MTA-1 protein is actively stabilized in HCC cell lines and actively degraded in normal liver cells. We have also shown that TRIM25 is the E3 ligase that interacts with and degrades MTA-1 protein. The identity of the factor regulating expression of TRIM25 in normal liver cells and HCC is unknown. In the current work we elucidate that microRNA (miR)- 873 targets TRIM25 in HCC cells. Both metagenomic analysis and quantification of miR-873 and TRIM25 in 25 HCC patients revealed an inverse correlation between the two in HCC patients with high miR-873 and low TRIM25 expression, respectively. The expression pattern was mimicked in the normal liver cells THLE-2 and the HCC cell line, HuH6. In vitro luciferase reporter assays confirmed TRIM25 as the target of miR-873. Transient transfection of HuH6 cells with an anti-miR-873 antagomir significantly decreased both transwell motility in these cells. Furthermore, in in vivo xenograft assays treatment with anti-miR-873 antagomir significantly decreased hepatic nodules formation. Cumulatively, our data indicate that suppression of TRIM25 expression by high levels of miR-873 dictates MTA1 protein upregulation in HCC. Copyright © 2018 Elsevier Inc. All rights reserved.

Positive regulation of spondin 2 by thyroid hormone is associated with cell migration and invasion.

PubMed

Liao, Chen-Hsin; Yeh, Shih-Chi; Huang, Ya-Hui; Chen, Ruey-Nan; Tsai, Ming-Ming; Chen, Wei-Jan; Chi, Hsiang-Cheng; Tai, Pei-Ju; Liao, Chia-Jung; Wu, Sheng-Ming; Cheng, Wan-Li; Pai, Li-Mei; Lin, Kwang-Huei

2010-03-01

The thyroid hormone 3,3',5-triiodo-L-thyronine (T(3)) regulates growth, development, and differentiation processes in animals. These activities are mediated by the nuclear thyroid hormone receptors (TRs). Microarray analyses were performed previously to study the mechanism of regulation triggered by T(3) treatment in hepatoma cell lines. The results showed that spondin 2 was regulated positively by T(3). However, the underlying mechanism and the physiological role of T(3) in the regulation of spondin 2 are not clear. To verify the microarray results, spondin 2 was further investigated using semi-quantitative reverse transcription-PCR and western blotting. After 48 h of T(3) treatment in the HepG2-TR alpha 1#1 cell line, spondin 2 mRNA and protein levels increased by 3.9- to 5.7-fold. Similar results were observed in thyroidectomized rats. To localize the regulatory region in spondin 2, we performed serial deletions of the promoter and chromatin immunoprecipitation assays. The T(3) response element on the spondin 2 promoter was localized in the -1104/-1034 or -984/-925 regions. To explore the effect of spondin 2 on cellular function, spondin 2 knockdown cell lines were established from Huh7 cells. Knockdown cells had higher migration ability and invasiveness compared with control cells. Conversely, spondin 2 overexpression in J7 cells led to lower migration ability and invasiveness compared with control cells. Furthermore, this study demonstrated that spondin 2 overexpression in some types of hepatocellular carcinomas is TR dependent. Together, these experimental findings suggest that spondin 2, which is regulated by T(3), has an important role in cell invasion, cell migration, and tumor progression.

Identification of a unique hepatocellular carcinoma line, Li-7, with CD13(+) cancer stem cells hierarchy and population change upon its differentiation during culture and effects of sorafenib.

PubMed

Yamada, Takeshi; Abei, Masato; Danjoh, Inaho; Shirota, Ryoko; Yamashita, Taro; Hyodo, Ichinosuke; Nakamura, Yukio

2015-04-11

Cancer stem cell (CSC) research has highlighted the necessity of developing drugs targeting CSCs. We investigated a hepatocellular carcinoma (HCC) cell line that not only has CSC hierarchy but also shows phenotypic changes (population changes) upon differentiation of CSC during culture and can be used for screening drugs targeting CSC. Based on a hypothesis that the CSC proportion should decrease upon its differentiation into progenitors (population change), we tested HCC cell lines (HuH-7, Li-7, PLC/PRF/5, HLF, HLE) before and after 2 months culture for several markers (CD13, EpCAM, CD133, CD44, CD90, CD24, CD166). Tumorigenicity was tested using nude mice. To evaluate the CSC hierarchy, we investigated reconstructivity, proliferation, ALDH activity, spheroid formation, chemosensitivity and microarray analysis of the cell populations sorted by FACS. Only Li-7 cells showed a population change during culture: the proportion of CD13 positive cells decreased, while that of CD166 positive cells increased. The high tumorigenicity of the Li-7 was lost after the population change. CD13(+)/CD166(-) cells showed slow growth and reconstructed the bulk Li-7 populations composed of CD13(+)/CD166(-), CD13(-)/CD166(-) and CD13(-)/CD166(+) fractions, whereas CD13(-)/CD166(+) cells showed rapid growth but could not reproduce any other population. CD13(+)/CD166(-) cells showed high ALDH activity, spheroid forming ability and resistance to 5-fluorouracil. Microarray analysis demonstrated higher expression of stemness-related genes in CD166(-) than CD166(+) fraction. These results indicated a hierarchy in Li-7 cells, in which CD13(+)/CD166(-) and CD13(-)/CD166(+) cells serve as slow growing CSCs and rapid growing progenitors, respectively. Sorafenib selectively targeted the CD166(-) fraction, including CD13(+) CSCs, which exhibited higher mRNA expression for FGF3 and FGF4, candidate biomarkers for sorafenib. 5-fluorouracil followed by sorafenib inhibited the growth of bulk Li-7 cells more effectively than the reverse sequence or either alone. We identified a unique HCC line, Li-7, which not only shows heterogeneity for a CD13(+) CSC hierarchy, but also undergoes a "population change" upon CSC differentiation. Sorafenib targeted the CSC in vitro, supporting the use of this model for screening drugs targeting the CSC. This type of "heterogeneous, unstable" cell line may prove more useful in the CSC era than conventional "homogeneous, stable" cell lines.

Primary biliary acids inhibit hepatitis D virus (HDV) entry into human hepatoma cells expressing the sodium-taurocholate cotransporting polypeptide (NTCP).

PubMed

Veloso Alves Pereira, Isabel; Buchmann, Bettina; Sandmann, Lisa; Sprinzl, Kathrin; Schlaphoff, Verena; Döhner, Katinka; Vondran, Florian; Sarrazin, Christoph; Manns, Michael P; Pinto Marques Souza de Oliveira, Cláudia; Sodeik, Beate; Ciesek, Sandra; von Hahn, Thomas

2015-01-01

The sodium-taurocholate cotransporting polypeptide (NTCP) is both a key bile acid (BA) transporter mediating uptake of BA into hepatocytes and an essential receptor for hepatitis B virus (HBV) and hepatitis D virus (HDV). In this study we aimed to characterize to what extent and through what mechanism BA affect HDV cell entry. HuH-7 cells stably expressing NTCP (HuH-7/NTCP) and primary human hepatocytes (PHH) were infected with in vitro generated HDV particles. Infectivity in the absence or presence of compounds was assessed using immunofluorescence staining for HDV antigen, standard 50% tissue culture infectious dose (TCID50) assays and quantitative PCR. Addition of primary conjugated and unconjugated BA resulted in a dose dependent reduction in the number of infected cells while secondary, tertiary and synthetic BA had a lesser effect. This effect was observed both in HuH-7/NTCP and in PHH. Other replication cycle steps such as replication and particle assembly and release were unaffected. Moreover, inhibitory BA competed with a fragment from the large HBV envelope protein for binding to NTCP-expressing cells. Conversely, the sodium/BA-cotransporter function of NTCP seemed not to be required for HDV infection since infection was similar in the presence or absence of a sodium gradient across the plasma membrane. When chenodeoxycolic acid (15 mg per kg body weight) was administered to three chronically HDV infected individuals over a period of up to 16 days there was no change in serum HDV RNA. Primary BA inhibit NTCP-mediated HDV entry into hepatocytes suggesting that modulation of the BA pool may affect HDV infection of hepatocytes.

Nodularin induces tumor necrosis factor-alpha and mitogen-activated protein kinases (MAPK) and leads to induction of endoplasmic reticulum stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meili, Nicole; Christen, Verena

Nodularin is produced by the cyanobacterium Nodularia spumigena. It is of concern due to hepatotoxicity in humans and animals. Here we investigated unexplored molecular mechanisms by transcription analysis in human liver cells, focusing on induction of pro-inflammatory cytokines, the tumor necrosis factor α (TNF-α), endoplasmic reticulum (ER) stress and components of the activator protein-1 complex in human hepatoma cells (Huh7) exposed to non-cytotoxic (0.1 and 1 μM) and toxic concentrations (5 μM) for 24, 48, and 72 h. Transcripts of TNF-α and ER stress marker genes were strongly induced at 1 and 5 μM at all time-points. TNF-α led tomore » induction of mitogen-activated protein kinases (MAPK), as demonstrated by induction of CJUN and CFOS, which form the AP-1 complex. Human primary liver cells reacted more sensitive than Huh7 cells. They showed higher cytotoxicity and induction of TNF-α and ER stress at 2.5 nM, while HepG2 cells were insensitive up to 10 μM due to low expression of organic anion transporting polypeptides. Furthermore, nodularin led to induction of TNF-α protein, and CCAAT/enhancer-binding protein-homologous (CHOP) protein. Our data indicate that nodularin induces inflammation and ER stress and leads to activation of MAPK in liver cells. All of these activated pathways, which were analysed here for the first time in detail, may contribute to the hepatotoxic, and tumorigenic action of nodularin. - Highlights: • Toxicity of nodularin and its mechanisms of action are poorly understood. • We investigated mechanisms of nodularin toxicity in human liver cell lines and human hepatocytes. • We identified several pathways involved in nodularin toxicity. • Nodularin induces TNF-α, MAPK pathway and ER stress • These activated pathways may contribute to the hepatotoxic and tumorigenic action of nodularin.« less

The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mylonis, Ilias; Lakka, Achillia; Tsakalof, Andreas

Research highlights: {yields} Kaempferol inhibits HIF-1 activity in hepatocarcinoma cells; {yields} Kaempferol causes cytoplasmic mislocalization of HIF-1{alpha} by impairing the MAPK pathway. {yields} Viability of hepatocarcinoma cells under hypoxia is reduced by kaempferol. -- Abstract: Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1{alpha} subunit, an important target of anti-cancer therapy, is observed in many cancers includingmore » HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1{alpha} as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC{sub 50} = 5.16 {mu}M). The mechanism of this inhibition did not involve suppression of HIF-1{alpha} protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC{sub 50} = 4.75 {mu}M). Exposure of Huh7 cells to 10 {mu}{Mu} kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10 {mu}M) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.« less

Identification of nucleotides in the 5'UTR and amino acids substitutions that are essential for the infectivity of 5'UTR-NS5A recombinant of hepatitis C virus genotype 1b (strain Con1).

PubMed

Li, Jinqian; Feng, Shengjun; Liu, Xi; Guo, Mingzhe; Chen, Mingxiao; Chen, Yiyi; Rong, Liang; Xia, Jinyu; Zhou, Yuanping; Zhong, Jin; Li, Yi-Ping

2018-05-01

Genotype 1b strain Con1 represents an important reference in the study of hepatitis C virus (HCV). Here, we aimed to develop an advanced infectious Con1 recombinant. We found that previously identified mutations A1226G/F1464L/A1672S/Q1773H permitted culture adaption of Con1 Core-NS5A (C-5A) recombinant containing 5'UTR and NS5B-3'UTR from JFH1 (genotype 2a), thus acquired additional mutations L725H/F886L/D2415G. C-5A containing all seven mutations (C-5A_7m) replicated efficiently in Huh7.5 and Huh7.5.1 cells and had an increased infectivity in SEC14L2-expressing Huh7.5.1 cells. Incorporation of Con1 NS5B was deleterious to C-5A_7m, however Con1 5'UTR was permissive but attenuated the virus. Nucleotides G1, A4, and G35 primarily accounted for the viral attenuation without affecting RNA translation. C-5A_7m was inhibited dose-dependently by simeprevir and daclatasvir, and substitutions at A4, A29, A34, and G35 conferred resistance to miR-122 antagonism. The novel Con1 5'UTR-NS5A recombinant, adaptive mutations, and critical nucleotides described here will facilitate future studies of HCV culture systems and virus-host interaction. Copyright © 2018 Elsevier Inc. All rights reserved.

Isoliensinine, a Bioactive Alkaloid Derived from Embryos of Nelumbo nucifera, Induces Hepatocellular Carcinoma Cell Apoptosis through Suppression of NF-κB Signaling.

PubMed

Shu, Guangwen; Yue, Ling; Zhao, Wenhao; Xu, Chan; Yang, Jing; Wang, Shaobing; Yang, Xinzhou

2015-10-14

Isoliensinine (isolie) is an alkaloid produced by the edible plant Nelumbo nucifera. Here, we unveiled that isolie was able to provoke HepG2, Huh-7, and H22 hepatocellular carcinoma (HCC) cell apoptosis. Isolie decreased NF-κB activity and constitutive phosphorylation of NF-κB p65 subunit at Ser536 in HCC cells. Overexpression of p65 Ser536 phosphorylation mimics abrogated isolie-mediated HCC cell apoptosis. Furthermore, intraperitoneal injection of isolie inhibited the growth of Huh-7 xenografts in nude mice. Additionally, isolie given by both intraperitoneal injection and gavage diminished the proliferation of transplanted H22 cells in Kunming mice. Reduced tumor growth in vivo was associated with inhibited p65 phosphorylation at Ser536 and declined NF-κB activity in tumor tissues. Finally, we revealed that isolie was bioavailable in the blood of mice and exhibited no detectable toxic effects on tumor-bearing mice. Our data provided strong evidence for the anti-HCC effect of isolie.

Arsenite-loaded nanoparticles inhibit PARP-1 to overcome multidrug resistance in hepatocellular carcinoma cells

NASA Astrophysics Data System (ADS)

Liu, Hanyu; Zhang, Zongjun; Chi, Xiaoqin; Zhao, Zhenghuan; Huang, Dengtong; Jin, Jianbin; Gao, Jinhao

2016-08-01

Hepatocellular carcinoma (HCC) is one of the highest incidences in cancers; however, traditional chemotherapy often suffers from low efficiency caused by drug resistance. Herein, we report an arsenite-loaded dual-drug (doxorubicin and arsenic trioxide, i.e., DOX and ATO) nanomedicine system (FeAsOx@SiO2-DOX, Combo NP) with significant drug synergy and pH-triggered drug release for effective treatment of DOX resistant HCC cells (HuH-7/ADM). This nano-formulation Combo NP exhibits the synergistic effect of DNA damage by DOX along with DNA repair interference by ATO, which results in unprecedented killing efficiency on DOX resistant cancer cells. More importantly, we explored the possible mechanism is that the activity of PARP-1 is inhibited by ATO during the treatment of Combo NP, which finally induces apoptosis of HuH-7/ADM cells by poly (ADP-ribosyl) ation suppression and DNA lesions accumulation. This study provides a smart drug delivery strategy to develop a novel synergistic combination therapy for effectively overcome drug- resistant cancer cells.

Expression of Fibroblast Growth Factor 21 and β-Klotho Regulates Hepatic Fibrosis through the Nuclear Factor-κB and c-Jun N-Terminal Kinase Pathways.

PubMed

Lee, Kyong Joo; Jang, Yoon Ok; Cha, Seung-Kuy; Kim, Moon Young; Park, Kyu-Sang; Eom, Young Woo; Baik, Soon Koo

2018-04-27

Fibroblast growth factor (FGF) 21 is associated with hepatic inflammation and fibrosis. However, little is known regarding the effects of inflammation and fibrosis on the β-Klotho and FGF21 pathway in the liver. Enrolled patients had biopsy-confirmed viral or alcoholic hepatitis. FGF19, FGF21 and β-Klotho levels were evaluated using enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blotting. Furthermore, we explored the underlying mechanisms for this process by evaluating nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathway involvement in Huh-7 cells. We observed that the FGF19 and FGF21 serum and mRNA levels in the biopsied liver tissue gradually increased and were correlated with fibrosis stage. Inflammatory markers (interleukin 1β [IL-1β], IL-6, and tumor necrosis factor-α) were positively correlated, while β-Klotho expression was negatively correlated with the degree of fibrosis. In Huh-7 cells, IL-1β increased FGF21 levels and decreased β-Klotho levels. NF-κB and JNK inhibitors abolished the effect of IL-1β on both FGF21 and β-Klotho expression. FGF21 protected IL-1β-induced growth retardation in Huh-7 cells. These results indicate that the inflammatory response during fibrogenesis increases FGF21 levels and suppresses β-Klotho via the NF-κB and JNK pathway. In addition, FGF21 likely protects hepatocytes from hepatic inflammation and fibrosis.

Effects of hepatitis C virus core protein and nonstructural protein 4B on the Wnt/β-catenin pathway.

PubMed

Jiang, Xiao-Hua; Xie, Yu-Tao; Cai, Ya-Ping; Ren, Jing; Ma, Tao

2017-05-25

Hepatitis C virus (HCV) core protein and nonstructural protein 4B (NS4B) are potentially oncogenic. Aberrant activation of the Wnt/β-catenin signaling pathway is closely associated with hepatocarcinogenesis. We investigated the effects of HCV type 1b core protein and NS4B on Wnt/β-catenin signaling in various liver cells, and explored the molecular mechanism underlying HCV-related hepatocarcinogenesis. Compared with the empty vector control, HCV core protein and NS4B demonstrated the following characteristics in the Huh7 cells: significantly enhanced β-catenin/Tcf-dependent transcriptional activity (F = 40.87, P < 0.01); increased nuclear translocation of β-catenin (F = 165.26, P < 0.01); upregulated nuclear β-catenin, cytoplasmic β-catenin, Wnt1, c-myc, and cyclin D1 protein expression (P < 0.01); and promoted proliferation of Huh7 cells (P < 0.01 or P < 0.05). Neither protein enhanced β-catenin/Tcf-dependent transcriptional activity in the LO2 cells (F = 0.65, P > 0.05), but they did significantly enhance Wnt3a-induced β-catenin/Tcf-dependent transcriptional activity (F = 64.25, P < 0.01), and promoted the nuclear translocation of β-catenin (F = 66.54, P < 0.01) and the Wnt3a-induced proliferation of LO2 cells (P < 0.01 or P < 0.05). Moreover, activation of the Wnt/β-catenin signaling pathway was greater with the core protein than with NS4B (P < 0.01 or P < 0.05). HCV core protein and NS4B directly activate the Wnt/β-catenin signaling pathway in Huh7 cells and LO2 cells induced by Wnt3a. These data suggest that HCV core protein and NS4B contribute to HCV-associated hepatocellular carcinogenesis.

HGF/c-Met related activation of β-catenin in hepatoblastoma

PubMed Central

2011-01-01

Background Activation of beta-catenin is a hallmark of hepatoblastoma (HB) and appears to play a crucial role in its pathogenesis. While aberrant accumulation of the beta-catenin is a common event in HB, mutations or deletions in CTNNB1 (beta-catenin gene) do not always account for the high frequency of protein expression. In this study we have investigated alternative activation of beta-catenin by HGF/c-Met signaling in a large cohort of 98 HB patients enrolled in the SIOPEL-3 clinical trial. Methods We performed immunohistochemistry, using antibodies to total beta-catenin and tyrosine654-phosphorylated beta-catenin, which is a good surrogate marker of HGF/c-Met activation. CTNNB1 mutation analysis was also carried out on all samples. We also investigated beta-catenin pathway activation in two liver cancer cell lines, HuH-6 and HuH-7. Results Aberrant beta-catenin expression was seen in the cytoplasm and/or nucleus of 87% of tumour samples. Our results also revealed a large subset of HB, 83%, with cytoplasmic expression of tyrosine654-phosphorylated beta-catenin and 30% showing additional nuclear accumulation. Sequence analysis revealed mutations in 15% of our cohort. Statistical analysis showed an association between nuclear expression of c-Met-activated beta-catenin and wild type CTNNB1 (P-value = 0.015). Analysis of total beta-catenin and Y654-beta-catenin in response to HGF activation in the cell lines, mirrors that observed in our HB tumour cohort. Results We identified a significant subset of hepatoblastoma patients for whom targeting of the c-Met pathway may be a treatment option and also demonstrate distinct mechanisms of beta-catenin activation in HB. PMID:21992464

Inhibition of hepatitis C virus replication through adenosine monophosphate-activated protein kinase-dependent and -independent pathways.

PubMed

Nakashima, Kenji; Takeuchi, Kenji; Chihara, Kazuyasu; Hotta, Hak; Sada, Kiyonao

2011-11-01

Persistent infection with hepatitis C virus (HCV) is closely correlated with type 2 diabetes. In this study, replication of HCV at different glucose concentrations was investigated by using J6/JFH1-derived cell-adapted HCV in Huh-7.5 cells and the mechanism of regulation of HCV replication by AMP-activated protein kinase (AMPK) as an energy sensor of the cell analyzed. Reducing the glucose concentration in the cell culture medium from 4.5 to 1.0 g/L resulted in suppression of HCV replication, along with activation of AMPK. Whereas treatment of cells with AMPK activator 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) suppressed HCV replication, compound C, a specific AMPK inhibitor, prevented AICAR's effect, suggesting that AICAR suppresses the replication of HCV by activating AMPK in Huh-7.5 cells. In contrast, compound C induced further suppression of HCV replication when the cells were cultured in low glucose concentrations or with metformin. These results suggest that low glucose concentrations and metformin have anti-HCV effects independently of AMPK activation. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.

The discovery of IDX21437: Design, synthesis and antiviral evaluation of 2'-α-chloro-2'-β-C-methyl branched uridine pronucleotides as potent liver-targeted HCV polymerase inhibitors.

PubMed

Alexandre, François-René; Badaroux, Eric; Bilello, John P; Bot, Stéphanie; Bouisset, Tony; Brandt, Guillaume; Cappelle, Sylvie; Chapron, Christopher; Chaves, Dominique; Convard, Thierry; Counor, Clément; Da Costa, Daniel; Dukhan, David; Gay, Marion; Gosselin, Gilles; Griffon, Jean-François; Gupta, Kusum; Hernandez-Santiago, Brenda; La Colla, Massimiliano; Lioure, Marie-Pierre; Milhau, Julien; Paparin, Jean-Laurent; Peyronnet, Jérôme; Parsy, Christophe; Pierra Rouvière, Claire; Rahali, Houcine; Rahali, Rachid; Salanson, Aurélien; Seifer, Maria; Serra, Ilaria; Standring, David; Surleraux, Dominique; Dousson, Cyril B

2017-09-15

Herein we describe the discovery of IDX21437 35b, a novel R P d-aminoacid-based phosphoramidate prodrug of 2'-α-chloro-2'-β-C-methyluridine monophosphate. Its corresponding triphosphate 6 is a potent inhibitor of the HCV NS5B RNA-dependent RNA polymerase (RdRp). Despite showing very weak activity in the in vitro Huh-7 cell based HCV replicon assay, 35b demonstrated high levels of active triphosphate 6 in mouse liver and human hepatocytes. A biochemical study revealed that the metabolism of 35b was mainly attributed to carboxyesterase 1 (CES1), an enzyme which is underexpressed in HCV Huh-7-derived replicon cells. Furthermore, due to its metabolic activation, 35b was efficiently processed in liver cells compared to other cell types, including human cardiomyocytes. The selected R P diastereoisomeric configuration of 35b was assigned by X-ray structural determination. 35b is currently in Phase II clinical trials for the treatment of HCV infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

Efficient infectious cell culture systems of the hepatitis C virus (HCV) prototype strains HCV-1 and H77.

PubMed

Li, Yi-Ping; Ramirez, Santseharay; Mikkelsen, Lotte; Bukh, Jens

2015-01-01

The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5'UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3'UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 10(4.0) focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 10(3.5) and 10(4.4) FFU/ml, respectively. Hepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but not in vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developed in vitro infectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, the in vitro infectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The antibacterial peptide from Bombyx mori cecropinXJ induced growth arrest and apoptosis in human hepatocellular carcinoma cells.

PubMed

Xia, Lijie; Wu, Yanling; Ma, J I; Yang, Jianhua; Zhang, Fuchun

2016-07-01

CecropinXJ is a cationic antimicrobial peptide originally isolated from the larvae of Bombyx mori . The anticancer effect of cecropinXJ has been reported in various tumor cells, including leukemia, gastric and esophageal cancer cells. However, the activity of cecropinXJ on hepatocellular carcinoma (HCC) and its underlying mechanism have not been investigated to date. Therefore, the present study investigated the efficacy and associated mechanism of cecropinXJ in Huh-7 cells. Flow cytometric analysis was performed to determine the presence of cell cycle arrested and apoptotic cells. CecropinXJ significantly inhibited the growth of Huh-7 cells in a dose- and time-dependent manner. CecropinXJ treatment for 24 h induced S cell cycle arrest and apoptosis, in addition to loss of the mitochondrial membrane potential, in hepatoma cells. CecropinXJ induced HCC cell apoptosis by activating caspase-3 and poly(ADP-ribose) polymerase. Furthermore, cecropinXJ downregulated the expression of B-cell lymphoma 2 (Bcl-2), while upregulated the expression of Bcl-2-associated death promoter and Bcl-2-associated X protein. In conclusion, the results of the present study suggest that cecropinXJ may be an active anti-HCC agent and provide novel insights into the mechanism of cecropinXJ.

PNPLA3 rs738409 causes steatosis according to viral & IL28B genotypes in hepatitis C.

PubMed

Ampuero, Javier; Del Campo, José A; Rojas, Lourdes; García-Lozano, José R; Solá, Ricard; Andrade, Raúl; Pons, José A; Navarro, José M; Calleja, José L; Buti, María; González-Escribano, María F; Forns, Xavier; Diago, Moisés; García-Samaniego, Javier; Romero-Gómez, Manuel

2014-01-01

Hepatitis C virus (HCV) is associated with a higher prevalence of steatosis compared to the general population. Our aim was to assess the impact of PNPLA3 rs738409 G-allele on steatosis in HCV patients. We included 474 HCV patients treated with peginterferon plus ribavirin. PNPLA3 rs738409 was genotyped and patients were classified according to alleles and genotypes. Steatosis was detected in 46.4% (220/474). Fibrosis was assessed by Scheuer score. Gene expression was analyzed in Huh7.5 and Huh7 cells using Real Time-PCR. PNPLA3 allele-G was associated with steatosis [54.1% (126/233) vs. 39% (94/241)] (p = 0.0001). In HCV-1, allele-G was related to steatosis [50.6% (82/162) vs. 32.3% (53/164)] (p = 0.001), but did not in HCV-3 [61.9% (26/42) vs. 62% (31/50)] (p = 0.993). PNPLA3 allele-G was associated with steatosis in patients with IL28B-CT/TT [57.7% (82/142) vs. 37.1% (56/151)] (p = 0.0001), but did not in IL28B-CC [47.8% (43/90) vs. 42% (37/88)] (p = 0.442). Independent variables associated with steatosis were: PNPLA3 G-allele [O.R. 1.84 (CI95%: 1.06-3.21); p = 0.007], age [O.R. 1.04 (CI95%: 1.01-1.07); p = 0.017], HCV-genotype 3 [O.R. 2.46 (CI95%: 1.30-4.65); p = 0.006], HOMA > 4 [O.R. 2.72 (CI95%: 1.27-5.82); p = 0.010]. Since PNPLA3 RNA could not be detected on PBMC from HCV patients, an in vitro analysis was performed. Huh7.5 cells infected with JFH1 had a decreased PNPLA3 gene expression (fold inhibition = 3.2 ± 0.2), while Huh7 cells presented increased PNPLA3 gene expression (fold induction = 1.5 ± 0.2). PNPLA3 allele-G modulated the development of steatosis, particularly in patients with HCV-1 and IL28B-CT/TT genotype, but was not associated with SVR. Metabolic but not viral steatosis seems to be PNPLA3 regulated. Gene interaction may result in differential PNPLA3 gene expression levels in HCV infection.

Repertoire of virus-derived small RNAs produced by mosquito and mammalian cells in response to dengue virus infection.

PubMed

Schirtzinger, Erin E; Andrade, Christy C; Devitt, Nicholas; Ramaraj, Thiruvarangan; Jacobi, Jennifer L; Schilkey, Faye; Hanley, Kathryn A

2015-02-01

RNA interference (RNAi) is the major defense of many arthropods against arthropod-borne RNA viruses (arboviruses), but the role of RNAi in vertebrate immunity to arboviruses is not clear. RNA viruses can trigger RNAi in vertebrate cells, but the vertebrate interferon response may obscure this interaction. We quantified virus-derived small RNAs (vRNAs) generated by mosquito (U4.4) cells and interferon-deficient (Vero) and interferon-competent (HuH-7) mammalian cells infected with a single isolate of mosquito-borne dengue virus. Mosquito cells produced significantly more vRNAs than mammalian cells, and mosquito cell vRNAs were derived from both the positive- and negative-sense dengue genomes whereas mammalian cell vRNAs were derived primarily from positive-sense genome. Mosquito cell vRNAs were predominantly 21 nucleotides in length whereas mammalian cell vRNAs were between 12 and 36 nucleotides with a modest peak at 24 nucleotides. Hot-spots, regions of the virus genome that generated a disproportionate number of vRNAs, overlapped among the cell lines. Copyright © 2014 Elsevier Inc. All rights reserved.

Novel TRAIL sensitizer Taraxacum officinale F.H. Wigg enhances TRAIL-induced apoptosis in Huh7 cells.

PubMed

Yoon, Ji-Yong; Cho, Hyun-Soo; Lee, Jeong-Ju; Lee, Hyo-Jung; Jun, Soo Young; Lee, Jae-Hye; Song, Hyuk-Hwan; Choi, SangHo; Saloura, Vassiliki; Park, Choon Gil; Kim, Cheol-Hee; Kim, Nam-Soon

2016-04-01

TRAIL (TNF-related apoptosis inducing ligand) is a promising anti-cancer drug target that selectively induces apoptosis in cancer cells. However, many cancer cells are resistant to TRAIL-induced apoptosis. Therefore, reversing TRAIL resistance is an important step for the development of effective TRAIL-based anti-cancer therapies. We previously reported that knockdown of the TOR signaling pathway regulator-like (TIPRL) protein caused TRAIL-induced apoptosis by activation of the MKK7-c-Jun N-terminal Kinase (JNK) pathway through disruption of the MKK7-TIPRL interaction. Here, we identified Taraxacum officinale F.H. Wigg (TO) as a novel TRAIL sensitizer from a set of 500 natural products using an ELISA system and validated its activity by GST pull-down analysis. Furthermore, combination treatment of Huh7 cells with TRAIL and TO resulted in TRAIL-induced apoptosis mediated through inhibition of the MKK7-TIPRL interaction and subsequent activation of MKK7-JNK phosphorylation. Interestingly, HPLC analysis identified chicoric acid as a major component of the TO extract, and combination treatment with chicoric acid and TRAIL induced TRAIL-induced cell apoptosis via JNK activation due to inhibition of the MKK7-TIPRL interaction. Our results suggest that TO plays an important role in TRAIL-induced apoptosis, and further functional studies are warranted to confirm the importance of TO as a novel TRAIL sensitizer for cancer therapy. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Merlin, the product of NF2 gene, is associated with aromatase expression and estrogen formation in human liver tissues and liver cancer cells.

PubMed

Cocciadiferro, Letizia; Miceli, Vitale; Granata, Orazia M; Carruba, Giuseppe

2017-09-01

The product of neurofibromatosis type 2 (NF2) gene, also known as Merlin/neurofibromin 2, homeostatically regulates liver stem cells by controlling abundance and signaling of epidermal growth factor receptor (EGFR), with a mechanism independent of the Hippo pathway. We have reported that locally elevated estrogen formation, driven by abnormally high expression and function of aromatase, may be implicated in development and progression of human hepatocellular carcinoma (HCC) through activation of a rapid signaling pathway mediated by amphiregulin (AREG) and EGFR. We have recently presented a model by which the aromatase-estrogen-amphiregulin-EGFR axis is activated in response to tissue injury and/or inflammatory disease, with its alteration eventually leading to development of major human tumors (liver, breast, prostate) and other chronic diseases (diabetes, obesity, Alzheimer's and heart disease). In this study, we investigated NF2 expression in liver cancer cells and tissues in relation to aromatase expression/function, estrogen receptor (ER) status and amphiregulin. Our data indicate that NF2 expression is associated with aromatase and AREG expression, being elevated in HCC tissues and HepG2 cells, intermediate in cirrhotic tissues and Huh7 cells, and lower in nontumoral liver and HA22T cells. In addition, NF2 expression is inversely related to wild type hERα66 and proportional to the expression of the membrane-associated hERα36 splice variant, as measured by exon-specific RT-PCR analysis, both in vivo and in vitro. Furthermore, incubation with estradiol induced a significant decrease of NF2 expression in both HA22T and Huh7 cells (over 54% and 22%, respectively), while no change could be observed in HepG2 cells, this effect being inversely related to aromatase expression and activity in HCC cell lines. Based on the above combined evidence, we hypothesize that NF2 behaves as a protein sensing tissue damage and aromatase-driven local estrogen formation, eventually leading to regulation of stem cells differentiation and tissue repair. Copyright © 2016 Elsevier Ltd. All rights reserved.

Metabolic profiling reveals that PNPLA3 induces widespread effects on metabolism beyond triacylglycerol remodeling in Huh-7 hepatoma cells

PubMed Central

Min, Hae-Ki; Sookoian, Silvia; Pirola, Carlos J.; Cheng, Jianfeng; Mirshahi, Faridoddin

2014-01-01

PNPLA3 was recently associated with the susceptibility to nonalcoholic fatty liver disease, a common cause of chronic liver disease characterized by abnormal triglyceride accumulation. Although it is established that PNPLA3 has both triacylglycerol lipase and acylglycerol O-acyltransferase activities, is still unknown whether the gene has any additional role in the modulation of the human liver metabolome. To uncover the functional role of PNPLA3 on liver metabolism, we performed high-throughput metabolic profiling of PNPLA3 siRNA-silencing and overexpression of wild-type and mutant Ile148Met variants (isoleucine/methionine substitution at codon 148) in Huh-7 cells. Metabolomic analysis was performed by using GC/MS and LC/MS platforms. Silencing of PNPLA3 was associated with a global perturbation of Huh-7 hepatoma cells that resembled a catabolic response associated with protein breakdown. A significant decrease in amino- and γ-glutamyl-amino acids and dipeptides and a significant increase in cysteine sulfinic acid, myo-inositol, lysolipids, sphingolipids, and polyunsaturated fatty acids were observed. Overexpression of the PNPLA3 Met148 variant mirrored many of the metabolic changes observed during gene silencing, but in the opposite direction. These findings were replicated by the exploration of canonical pathways associated with PNPLA3 silencing and Met148 overexpression. Overexpression of the PNPLA3 Met148 variant was associated with a 1.75-fold increase in lactic acid, suggesting a shift to anaerobic metabolism and mitochondrial dysfunction. Together, these results suggest a critical role of PNPLA3 in the modulation of liver metabolism beyond its classical participation in triacylglycerol remodeling. PMID:24763554

Metabolic profiling reveals that PNPLA3 induces widespread effects on metabolism beyond triacylglycerol remodeling in Huh-7 hepatoma cells.

PubMed

Min, Hae-Ki; Sookoian, Silvia; Pirola, Carlos J; Cheng, Jianfeng; Mirshahi, Faridoddin; Sanyal, Arun J

2014-07-01

PNPLA3 was recently associated with the susceptibility to nonalcoholic fatty liver disease, a common cause of chronic liver disease characterized by abnormal triglyceride accumulation. Although it is established that PNPLA3 has both triacylglycerol lipase and acylglycerol O-acyltransferase activities, is still unknown whether the gene has any additional role in the modulation of the human liver metabolome. To uncover the functional role of PNPLA3 on liver metabolism, we performed high-throughput metabolic profiling of PNPLA3 siRNA-silencing and overexpression of wild-type and mutant Ile148Met variants (isoleucine/methionine substitution at codon 148) in Huh-7 cells. Metabolomic analysis was performed by using GC/MS and LC/MS platforms. Silencing of PNPLA3 was associated with a global perturbation of Huh-7 hepatoma cells that resembled a catabolic response associated with protein breakdown. A significant decrease in amino- and γ-glutamyl-amino acids and dipeptides and a significant increase in cysteine sulfinic acid, myo-inositol, lysolipids, sphingolipids, and polyunsaturated fatty acids were observed. Overexpression of the PNPLA3 Met148 variant mirrored many of the metabolic changes observed during gene silencing, but in the opposite direction. These findings were replicated by the exploration of canonical pathways associated with PNPLA3 silencing and Met148 overexpression. Overexpression of the PNPLA3 Met148 variant was associated with a 1.75-fold increase in lactic acid, suggesting a shift to anaerobic metabolism and mitochondrial dysfunction. Together, these results suggest a critical role of PNPLA3 in the modulation of liver metabolism beyond its classical participation in triacylglycerol remodeling. Copyright © 2014 the American Physiological Society.

Evaluation of Interferon Resistance in Newly Established Genotype 1b Hepatitis C Virus Cell Culture System

PubMed Central

Taniguchi, Miki; Tasaka-Fujita, Megumi; Nakagawa, Mina; Watanabe, Takako; Kawai-Kitahata, Fukiko; Otani, Satoshi; Goto, Fumio; Nagata, Hiroko; Kaneko, Shun; Nitta, Sayuri; Murakawa, Miyako; Nishimura-Sakurai, Yuki; Azuma, Seishin; Itsui, Yasuhiro; Mori, Kenichi; Yagi, Shintaro; Kakinuma, Sei; Asahina, Yasuhiro; Watanabe, Mamoru

2016-01-01

Background and Aims: The hepatitis C virus (HCV) genotype 1b is known to exhibit treatment resistance with respect to interferon (IFN) therapy. Substitution of amino acids 70 and 91 in the core region of the 1b genotype is a significant predictor of liver carcinogenesis and poor response to pegylated-IFN-α and ribavirin therapy. However, the molecular mechanism has not yet been clearly elucidated because of limitations of the HCV genotype 1b infectious model. Recently, the TPF1-M170T HCV genotype 1b cell culture system was established, in which the clone successfully replicates and infects Huh-7-derived Huh7-ALS32.50 cells. Therefore, the purpose of this study was to compare IFN resistance in various HCV clones using this system. Methods: HCV core amino acid substitutions R70Q and L91M were introduced to the TPF1-M170T clone and then transfected into Huh7-ALS32.50 cells. To evaluate the production of each virus, intracellular HCV core antigens were measured. Results were confirmed with Western blot analysis using anti-NS5A antibodies, and IFN sensitivity was subsequently measured. Results: Each clone was transfected successfully compared with JFH-1, with a significant difference in intracellular HCV core antigen (p < 0.05), an indicator of continuous HCV replication. Among all clones, L91M showed the highest increase in the HCV core antigen and HCV protein. There was no significant resistance against IFN treatment in core substitutions; however, IFN sensitivity was significantly different between the wildtype core and JFH-1 (p < 0.05). Conclusions: A novel genotype 1b HCV cell culture was constructed with core amino acid substitutions, which demonstrated IFN resistance of genotype 1b. This system will be useful for future analyses into the mechanisms of HCV genotype 1b treatment. PMID:27047766

Acoustic Impedance Analysis with High-Frequency Ultrasound for Identification of Fatty Acid Species in the Liver.

PubMed

Ito, Kazuyo; Yoshida, Kenji; Maruyama, Hitoshi; Mamou, Jonathan; Yamaguchi, Tadashi

2017-03-01

Acoustic properties of free fatty acids present in the liver were studied as a possible basis for non-invasive ultrasonic diagnosis of non-alcoholic steatohepatitis. Acoustic impedance was measured for the following types of tissue samples: Four pathologic types of mouse liver, five kinds of FFAs in solvent and five kinds of FFAs in cultured Huh-7 cells. A transducer with an 80-MHz center frequency was incorporated into a scanning acoustic microscopy system. Acoustic impedance was calculated from the amplitude of the signal reflected from the specimen surface. The Kruskal-Wallis test revealed statistically significant differences (p < 0.01) in acoustic impedance not only among pathologic types, but also among the FFAs in solvent and in cultured Huh-7 cells. These results suggest that each of the FFAs, especially palmitate, oleate and palmitoleate acid, can be distinguished from each other, regardless of whether they were in solution or absorbed by cells. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Spherically-clustered porous Au-Ag alloy nanoparticle prepared by partial inhibition of galvanic replacement and its application for efficient multimodal therapy.

PubMed

Jang, Hongje; Min, Dal-Hee

2015-03-24

The polyvinylpyrrolidone (PVP)-coated spherically clustered porous gold-silver alloy nanoparticle (PVP-SPAN) was prepared by low temperature mediated, partially inhibited galvanic replacement reaction followed by silver etching process. The prepared porous nanostructures exhibited excellent photothermal conversion efficiency under irradiation of near-infrared light (NIR) and allowed a high payload of both doxorubicin (Dox) and thiolated dye-labeled oligonucleotide, DNAzyme (FDz). Especially, PVP-SPAN provided 10 times higher loading capacity for oligonucleotide than conventional hollow nanoshells due to increased pore diameter and surface-to-volume ratio. We demonstrated highly efficient chemo-thermo-gene multitherapy based on codelivery of Dox and FDz with NIR-mediated photothermal therapeutic effect using a model system of hepatitis C virus infected human liver cells (Huh7 human hepatocarcinoma cell line containing hepatitis C virus NS3 gene replicon) compared to conventional hollow nanoshells.

Characterization of Cladosporols from the Marine Algal-Derived Endophytic Fungus Cladosporium cladosporioides EN-399 and Configurational Revision of the Previously Reported Cladosporol Derivatives.

PubMed

Li, Hong-Lei; Li, Xiao-Ming; Mándi, Attila; Antus, Sándor; Li, Xin; Zhang, Peng; Liu, Yang; Kurtán, Tibor; Wang, Bin-Gui

2017-10-06

Four new cladosporol derivatives, cladosporols F-I (1-4), the known cladosporol C (5), and its new epimer, cladosporol J (6), were isolated and identified from the marine algal-derived endophytic fungus Cladosporium cladosporioides EN-399. Their structures were determined by detailed interpretation of NMR and MS data, and the absolute configurations were established on the basis of TDDFT-ECD and OR calculations. The configurational assignment of cladosporols F (1) and G (2) showed that the previously reported absolute configuration of cladosporol A and all the related cladosporols need to be revised from (4'R) to (4'S). Compounds 1-6 showed antibacterial activity against Escherichia coli, Micrococcus luteus, and Vibrio harveyi with MIC values ranging from 4 to 128 μg/mL. Compound 3 showed significant cytotoxicity against A549, Huh7, and LM3 cell lines with IC 50 values of 5.0, 1.0, and 4.1 μM, respectively, and compound 5 showed activity against H446 cell line with IC 50 value of 4.0 μM.

Recombinant Hepatitis E virus like particles can function as RNA nanocarriers.

PubMed

Panda, Subrat Kumar; Kapur, Neeraj; Paliwal, Daizy; Durgapal, Hemlata

2015-06-24

Assembled virus-like particles (VLPs) without genetic material, with structure similar to infectious virions, have been successfully used as vaccines. We earlier described in vitro assembly, characterisation and tissue specific receptor dependent Clathrin mediated entry of empty HEV VLPs, produced from Escherichia coli expressed HEV capsid protein (pORF2). Similar VLP's have been described as a potential candidate vaccine (Hecolin) against HEV. We have attempted to use such recombinant assembled Hepatitis E virus (HEV) VLPs as a carrier for heterologous RNA with protein coding sequence fused in-frame with HEV 5' region (containing cap and encapsidation signal) and investigated, if the relevant protein could be expressed and elicit an immune response in vivo. In vitro transcribed red fluorescent protein (RFP)/Hepatitis B virus surface antigen (HBsAg) RNA, fused to 5'-HEV sequence with cap and encapsidation signal (1-249 nt), was packaged into the recombinant HEV-VLPs and incubated with five different cell lines (Huh7, A549, Vero, HeLa and SiHa). The pORF2-VLPs could specifically transfer exogenous coding RNA into Huh7 and A549 cells. In vivo, Balb/c mice were immunized (intramuscular injections) with 100 µg pORF2-VLP encapsidated with 5'-methyl-G-HEV (1-249 nt)-HBsAg RNA, blood samples were collected and screened by ELISA for anti-pORF2 and anti-HBsAg antibodies. Humoral immune response could be elicited in Balb/c mice against both HEV capsid protein and cargo RNA encoded HBsAg protein. These findings suggest that other than being a possible vaccine, HEV pORF2-VLPs can be used as a promising non-replicative tissue specific gene delivery system.

Erythrocyte membrane based cationic polymer-mcDNA complexes as an efficient gene delivery system.

PubMed

Huang, Ping; Zhao, Jing; Wei, Chiju; Hou, Xiaohu; Chen, Pingzhang; Tan, Yan; He, Cheng-Yi; Wang, Zhiyong; Chen, Zhi-Ying

2016-12-20

Gene therapy has great promise for the treatment of obtained and inherited serious diseases. However, the lack of safe and efficient gene delivery systems remains a barrier for their clinical application. Here, we reported a potential gene delivery vehicle composed of the erythrocyte membrane and cationic polymers, for example the XtremeGENE from Roche and the ε-caprolactone modified polyethylenimine. In addition to high efficiency, this system showed negligible cytotoxicity compared to the two cationic polymers alone in various cell lines, including human embryonic kidney cells (293T), human liver cancer cells (Huh7 and HepG2), murine dendritic cells (DC2.4) and human umbilical cord mesenchymal stem cells (Hu-MSCs). Moreover, the results of confocal laser scanning microscopy and flow cytometry suggested that the cell uptake of this gene vector was improved and might be introduced by the fusion interaction between the erythrocyte membrane and targeted cells.Thus, all the results revealed that the erythrocyte membrane based gene delivery system might be able to serve as an excellent gene delivery system.

Hepatitis C Virus Strain-Dependent Usage of Apolipoprotein E Modulates Assembly Efficiency and Specific Infectivity of Secreted Virions.

PubMed

Weller, Romy; Hueging, Kathrin; Brown, Richard J P; Todt, Daniel; Joecks, Sebastian; Vondran, Florian W R; Pietschmann, Thomas

2017-09-15

Hepatitis C virus (HCV) is extraordinarily diverse and uses entry factors in a strain-specific manner. Virus particles associate with lipoproteins, and apolipoprotein E (ApoE) is critical for HCV assembly and infectivity. However, whether ApoE dependency is common to all HCV genotypes remains unknown. Therefore, we compared the roles of ApoE utilizing 10 virus strains from genotypes 1 through 7. ApoA and ApoC also support HCV assembly, so they may contribute to virus production in a strain-dependent fashion. Transcriptome sequencing (RNA-seq) revealed abundant coexpression of ApoE, ApoB, ApoA1, ApoA2, ApoC1, ApoC2, and ApoC3 in primary hepatocytes and in Huh-7.5 cells. Virus production was examined in Huh-7.5 cells with and without ApoE expression and in 293T cells where individual apolipoproteins (ApoE1, -E2, -E3, -A1, -A2, -C1, and -C3) were provided in trans All strains were strictly ApoE dependent. However, ApoE involvement in virus production was strain and cell type specific, because some HCV strains poorly produced infectious virus in ApoE-expressing 293T cells and because ApoE knockout differentially affected virus production of HCV strains in Huh-7.5 cells. ApoE allelic isoforms (ApoE2, -E3, and -E4) complemented virus production of HCV strains to comparable degrees. All tested strains assembled infectious progeny with ApoE in preference to other exchangeable apolipoproteins (ApoA1, -A2, -C1, and -C3). The specific infectivity of HCV particles was similar for 293T- and Huh-7.5-derived particles for most strains; however, it differed by more than 100-fold in some viruses. Collectively, this study reveals strain-dependent and host cell-dependent use of ApoE during HCV assembly. These differences relate to the efficacy of virus production and also to the properties of released virus particles and therefore govern viral fitness at the level of assembly and cell entry. IMPORTANCE Chronic HCV infections are a major cause of liver disease. HCV is highly variable, and strain-specific determinants modulate the response to antiviral therapy, the natural course of infection, and cell entry factor usage. Here we explored whether host factor dependency of HCV in particle assembly is modulated by strain-dependent viral properties. We showed that all examined HCV strains, which represent all seven known genotypes, rely on ApoE expression for assembly of infectious progeny. However, the degree of ApoE dependence is modulated in a strain-specific and cell type-dependent manner. This indicates that HCV strains differ in their assembly properties and host factor usage during assembly of infectious progeny. Importantly, these differences relate not only to the efficiency of virus production and release but also to the infectiousness of virus particles. Thus, strain-dependent features of HCV modulate ApoE usage, with implications for virus fitness at the level of assembly and cell entry. Copyright © 2017 Weller et al.

RPLP1 and RPLP2 Are Essential Flavivirus Host Factors That Promote Early Viral Protein Accumulation

PubMed Central

Campos, Rafael K.; Wong, Benjamin; Lu, Yi-Fan; Shi, Pei-Yong; Pompon, Julien

2016-01-01

ABSTRACT The Flavivirus genus contains several arthropod-borne viruses that pose global health threats, including dengue viruses (DENV), yellow fever virus (YFV), and Zika virus (ZIKV). In order to understand how these viruses replicate in human cells, we previously conducted genome-scale RNA interference screens to identify candidate host factors. In these screens, we identified ribosomal proteins RPLP1 and RPLP2 (RPLP1/2) to be among the most crucial putative host factors required for DENV and YFV infection. RPLP1/2 are phosphoproteins that bind the ribosome through interaction with another ribosomal protein, RPLP0, to form a structure termed the ribosomal stalk. RPLP1/2 were validated as essential host factors for DENV, YFV, and ZIKV infection in two human cell lines: A549 lung adenocarcinoma and HuH-7 hepatoma cells, and for productive DENV infection of Aedes aegypti mosquitoes. Depletion of RPLP1/2 caused moderate cell-line-specific effects on global protein synthesis, as determined by metabolic labeling. In A549 cells, global translation was increased, while in HuH-7 cells it was reduced, albeit both of these effects were modest. In contrast, RPLP1/2 knockdown strongly reduced early DENV protein accumulation, suggesting a requirement for RPLP1/2 in viral translation. Furthermore, knockdown of RPLP1/2 reduced levels of DENV structural proteins expressed from an exogenous transgene. We postulate that these ribosomal proteins are required for efficient translation elongation through the viral open reading frame. In summary, this work identifies RPLP1/2 as critical flaviviral host factors required for translation. IMPORTANCE Flaviviruses cause important diseases in humans. Examples of mosquito-transmitted flaviviruses include dengue, yellow fever and Zika viruses. Viruses require a plethora of cellular factors to infect cells, and the ribosome plays an essential role in all viral infections. The ribosome is a complex macromolecular machine composed of RNA and proteins and it is responsible for protein synthesis. We identified two specific ribosomal proteins that are strictly required for flavivirus infection of human cells and mosquitoes: RPLP1 and RPLP2 (RPLP1/2). These proteins are part of a structure known as the ribosomal stalk and help orchestrate the elongation phase of translation. We show that flaviviruses are particularly dependent on the function of RPLP1/2. Our findings suggest that ribosome composition is an important factor for virus translation and may represent a regulatory layer for translation of specific cellular mRNAs. PMID:27974556

Studies of Antiviral Activity and Cytotoxicity of Wrightia tinctoria and Morinda citrifolia

PubMed Central

Selvam, P.; Murugesh, N.; Witvrouw, M.; Keyaerts, E.; Neyts, J.

2009-01-01

Different extracts of leaf parts of Wrightia tinctoria and fruit powder of Morinda citrifolia have been studied against replication of HIV-1(IIIB) in MT-4 cells and HCV in Huh 5.2 cells. Chloroform extract of Wrightia tinctoria exhibited a maximum protection of 48% against the cytopathic effect of HIV-1(IIIB) in MT-4 cells. Fruit juice of Morinda citrifolia exhibited a displayed marked cytotoxic activity in lymphocyte (MT-4) cells (CC50: 0.19 mg/ml). The 50% effective concentration for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells by Morinda citrifolia was 0.98 μg/ml and by chloroform extract of Wrightia tinctoria was 10 μg/ml. The concentration that reduced the growth of exponentially proliferating Huh 5-2 cells by 50% was greater than 50 μg/ml. PMID:20376221

Exosomes from Hepatitis C Infected Patients Transmit HCV Infection and Contain Replication Competent Viral RNA in Complex with Ago2-miR122-HSP90

PubMed Central

Kodys, Karen; Bala, Shashi; Szabo, Gyongyi

2014-01-01

Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naïve individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H+-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naïve cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies. PMID:25275643

Streptococcal serum opacity factor promotes cholesterol ester metabolism and bile acid secretion in vitro and in vivo.

PubMed

Gillard, Baiba K; Rodriguez, Perla J; Fields, David W; Raya, Joe L; Lagor, William R; Rosales, Corina; Courtney, Harry S; Gotto, Antonio M; Pownall, Henry J

2016-03-01

Plasma high density lipoprotein-cholesterol (HDL-C) concentrations negatively correlate with atherosclerotic cardiovascular disease. HDL is thought to have several atheroprotective functions, which are likely distinct from the epidemiological inverse relationship between HDL-C levels and risk. Specifically, strategies that reduce HDL-C while promoting reverse cholesterol transport (RCT) may have therapeutic value. The major product of the serum opacity factor (SOF) reaction versus HDL is a cholesteryl ester (CE)-rich microemulsion (CERM), which contains apo E and the CE of ~400,000 HDL particles. Huh7 hepatocytes take up CE faster when delivered as CERM than as HDL, in part via the LDL-receptor (LDLR). Here we compared the final RCT step, hepatic uptake and subsequent intracellular processing to cholesterol and bile salts for radiolabeled HDL-, CERM- and LDL-CE by Huh7 cells and in vivo in C57BL/6J mice. In Huh7 cells, uptake from LDL was greater than from CERM (2-4X) and HDL (5-10X). Halftimes for [(14)C]CE hydrolysis were 3.0±0.2, 4.4±0.6 and 5.4±0.7h respectively for HDL, CERM and LDL-CE. The fraction of sterols secreted as bile acids was ~50% by 8h for all three particles. HDL, CERM and LDL-CE metabolism in mice showed efficient plasma clearance of CERM-CE, liver uptake and metabolism, and secretion as bile acids into the gall bladder. This work supports the therapeutic potential of the SOF reaction, which diverts HDL-CE to the LDLR, thereby increasing hepatic CE uptake, and sterol disposal as bile acids. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel Cell Culture-Adapted Genotype 2a Hepatitis C Virus Infectious Clone

PubMed Central

Date, Tomoko; Kato, Takanobu; Kato, Junko; Takahashi, Hitoshi; Morikawa, Kenichi; Akazawa, Daisuke; Murayama, Asako; Tanaka-Kaneko, Keiko; Sata, Tetsutaro; Tanaka, Yasuhito; Mizokami, Masashi

2012-01-01

Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones. PMID:22787209

Silver nanoparticles induce endoplasmatic reticulum stress response in zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Christen, Verena; Capelle, Martinus; Fent, Karl, E-mail: karl.fent@fhnw.ch

2013-10-15

Silver nanoparticles (AgNPs) find increasing applications, and therefore humans and the environment are increasingly exposed to them. However, potential toxicological implications are not sufficiently known. Here we investigate effects of AgNPs (average size 120 nm) on zebrafish in vitro and in vivo, and compare them to human hepatoma cells (Huh7). AgNPs are incorporated in zebrafish liver cells (ZFL) and Huh7, and in zebrafish embryos. In ZFL cells AgNPs lead to induction of reactive oxygen species (ROS), endoplasmatic reticulum (ER) stress response, and TNF-α. Transcriptional alterations also occur in pro-apoptotic genes p53 and Bax. The transcriptional profile differed in ZFL andmore » Huh7 cells. In ZFL cells, the ER stress marker BiP is induced, concomitant with the ER stress marker ATF-6 and spliced XBP-1 after 6 h and 24 h exposure to 0.5 g/L and 0.05 g/L AgNPs, respectively. This indicates the induction of different pathways of the ER stress response. Moreover, AgNPs induce TNF-α. In zebrafish embryos exposed to 0.01, 0.1, 1 and 5 mg/L AgNPs hatching was affected and morphological defects occurred at high concentrations. ER stress related gene transcripts BiP and Synv are significantly up-regulated after 24 h at 0.1 and 5 mg/L AgNPs. Furthermore, transcriptional alterations occurred in the pro-apoptotic genes Noxa and p21. The ER stress response was strong in ZFL cells and occurred in zebrafish embryos as well. Our data demonstrate for the first time that AgNPs lead to induction of ER stress in zebrafish. The induction of ER stress can have several consequences including the activation of apoptotic and inflammatory pathways. - Highlights: • Effects of silver nanoparticles (120 nm AgNPs) are investigated in zebrafish. • AgNPs induce all ER stress reponses in vitro in zebrafish liver cells. • AgNPs induce weak ER stress in zebrafish embryos. • AgNPs induce oxidative stress and transcripts of pro-apoptosis genes.« less

Influence of oxygen partial pressure on the characteristics of human hepatocarcinoma cells.

PubMed

Trepiana, Jenifer; Meijide, Susana; Navarro, Rosaura; Hernández, M Luisa; Ruiz-Sanz, José Ignacio; Ruiz-Larrea, M Begoña

2017-08-01

Most of the in vitro studies using liver cell lines have been performed under atmospheric oxygen partial pressure (21% O 2 ). However, the oxygen concentrations in the liver and cancer cells are far from this value. In the present study, we have evaluated the influence of oxygen on 1) the tumor cell lines features (growth, steady-state ROS levels, GSH content, activities of antioxidant enzymes, p66 Shc and SOD expressions, metalloproteinases secretion, migration, invasion, and adhesion) of human hepatocellular carcinoma cell lines, and b) the response of the cells to an oxidant stimulus (aqueous leaf extract of the V. baccifera plant species). For this purpose, three hepatocarcinoma cell lines with different p53 status, HepG2 (wild-type), Huh7 (mutated), and Hep3B (deleted), were cultured (6-30 days) under atmospheric (21%) and more physiological (8%) pO 2 . Results showed that after long-term culturing at 8% versus 21% O 2 , the cellular proliferation rate and the steady-state levels of mitochondrial O 2 - were unaffected. However, the intracellular basal ROS levels were higher independently of the characteristics of the cell line. Moreover, the lower pO 2 was associated with lower glutathione content, the induction of p66 Shc and Mn-SOD proteins, and increased SOD activity only in HepG2. This cell line also showed a higher migration rate, secretion of active metalloproteinases, and a faster invasion. HepG2 cells were more resistant to the oxidative stress induced by V. baccifera. Results suggest that the long-term culturing of human hepatoma cells at a low, more physiological pO 2 induces antioxidant adaptations that could be mediated by p53, and may alter the cellular response to a subsequent oxidant challenge. Data support the necessity of validating outcomes from studies performed with hepatoma cell cultures under ambient O 2 . Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Evaluation of the Selenotranscriptome Expression in Two Hepatocellular Carcinoma Cell Lines

PubMed Central

Guariniello, Stefano; Di Bernardo, Giovanni; Cammarota, Marcella; Castello, Giuseppe

2015-01-01

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and is still one of the most fatal cancers. Hence, it needs to identify always new putative markers to improve its diagnosis and prognosis. Since the selenium is able to fight the oxidative damage which is one of the major origins of cell damage as well as cancer, we have recently focused our attention on selenoprotein family and their involvement in HCC. In the present paper we have carried out a global analysis of the selenotranscriptome expression in HepG2 and Huh7 cells compared to the normal human hepatocytes by reverse transcription-qPCR (RT-qPCR). Our data showed that in both cells there are three downregulated (DIO1, DIO2, and SELO) and ten upregulated (GPX4, GPX7, SELK, SELM, SELN, SELT, SELV, SEP15, SEPW1, and TrxR1) genes. Additionally, interactomic studies were carried out to evaluate the ability of these down- and upregulated genes to interact between them as well as to identify putative HUB nodes representing the centers of correlation able to exercise a direct control over the coordinated genes. PMID:26199857

Third-generation oncolytic herpes simplex virus inhibits the growth of liver tumors in mice.

PubMed

Nakatake, Richi; Kaibori, Masaki; Nakamura, Yusuke; Tanaka, Yoshito; Matushima, Hideyuki; Okumura, Tadayoshi; Murakami, Takashi; Ino, Yasushi; Todo, Tomoki; Kon, Masanori

2018-03-01

Multimodality therapies are used to manage patients with hepatocellular carcinoma (HCC), although advanced HCC is incurable. Oncolytic virus therapy is probably the next major breakthrough in cancer treatment. The third-generation oncolytic herpes simplex virus type 1 (HSV-1) T-01 kills tumor cells without damaging the surrounding normal tissues. Here we investigated the antitumor effects of T-01 on HCC and the host's immune response to HCC cells. The cytopathic activities of T-01 were tested in 14 human and 1 murine hepatoma cell line in vitro. In various mouse xenograft models, HuH-7, KYN-2, PLC/PRF/5 and HepG2 human cells and Hepa1-6 murine cells were used to investigate the in vivo efficacy of T-01. T-01 was cytotoxic to 13 cell lines (in vitro). In mouse xenograft models of subcutaneous, orthotopic and peritoneal tumor metastasis in athymic mice (BALB/c nu/nu), the growth of tumors formed by the human HCC cell lines and hepatoblastoma cell line was inhibited by T-01 compared with that of mock-inoculated tumors. In a bilateral Hepa1-6 subcutaneous tumor model in C57BL/6 mice, the growth of tumors inoculated with T-01 was inhibited, as was the case for contralateral tumors. T-01 also significantly reduced tumor growth. T-01 infection significantly enhanced antitumor efficacy via T cell-mediated immune responses. Results demonstrate that a third-generation oncolytic HSV-1 may serve as a novel treatment for patients with HCC. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Three-dimensional single-particle tracking in live cells: news from the third dimension

NASA Astrophysics Data System (ADS)

Dupont, A.; Gorelashvili, M.; Schüller, V.; Wehnekamp, F.; Arcizet, D.; Katayama, Y.; Lamb, D. C.; Heinrich, D.

2013-07-01

Single-particle tracking (SPT) is of growing importance in the biophysical community. It is used to investigate processes such as drug and gene delivery, viral uptake, intracellular trafficking or membrane-bound protein mobility. Traditionally, SPT is performed in two dimensions (2D) because of its technical simplicity. However, life occurs in three dimensions (3D) and many methods have been recently developed to track particles in 3D. Now, is the third dimension worth the effort? Here we investigate the differences between the 2D and 3D analyses of intracellular transport with the 3D development of a time-resolved mean square displacement (MSD) analysis introduced previously. The 3D trajectories, and the 2D projections, of fluorescent nanoparticles were obtained with an orbital tracking microscope in two different cell types: in Dictyostelium discoideum ameba and in adherent, more flattened HuH-7 human cells. As expected from the different 3D organization of both cells’ cytoskeletons, a third of the active transport was lost upon projection in the ameba whereas the identification of the active phases was barely affected in the HuH-7 cells. In both cell types, we found intracellular diffusion to be anisotropic and the diffusion coefficient values derived from the 2D analysis were therefore biased.

Copper induces hepatocyte injury due to the endoplasmic reticulum stress in cultured cells and patients with Wilson disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oe, Shinji, E-mail: ooes@med.uoeh-u.ac.jp; Miyagawa, Koichiro, E-mail: koichiro@med.uoeh-u.ac.jp; Honma, Yuichi, E-mail: y-homma@med.uoeh-u.ac.jp

Copper is an essential trace element, however, excess copper is harmful to human health. Excess copper-derived oxidants contribute to the progression of Wilson disease, and oxidative stress induces accumulation of abnormal proteins. It is known that the endoplasmic reticulum (ER) plays an important role in proper protein folding, and that accumulation of misfolded proteins disturbs ER homeostasis resulting in ER stress. However, copper-induced ER homeostasis disturbance has not been fully clarified. We treated human hepatoma cell line (Huh7) and immortalized-human hepatocyte cell line (OUMS29) with copper and chemical chaperones, including 4-phenylbutyrate and ursodeoxycholic acid. We examined copper-induced oxidative stress, ERmore » stress and apoptosis by immunofluorescence microscopy and immunoblot analyses. Furthermore, we examined the effects of copper on carcinogenesis. Excess copper induced not only oxidative stress but also ER stress. Furthermore, excess copper induced DNA damage and reduced cell proliferation. Chemical chaperones reduced this copper-induced hepatotoxicity. Excess copper induced hepatotoxicity via ER stress. We also confirmed the abnormality of ultra-structure of the ER of hepatocytes in patients with Wilson disease. These findings show that ER stress plays a pivotal role in Wilson disease, and suggests that chemical chaperones may have beneficial effects in the treatment of Wilson disease.« less

Pre-S2 Start Codon Mutation of Hepatitis B Virus Subgenotype B3 Effects on NF-κB Expression and Activation in Huh7 Cell Lines.

PubMed

Siburian, Marlinang Diarta; Suriapranata, Ivet Marita; Wanandi, Septelia Inawati

2018-03-19

A cross-sectional study on hepatitis B patients in Indonesia showed association of pre-S2 start codon mutation (M120 V) with cirrhosis and hepatocellular carcinoma (HCC), which was dissimilar from studies from other populations where pre-S2 deletion mutation was more prevalent. Different mutation patterns were attributed to different hepatitis B virus (HBV) subgenotypes in each population study. HBV surface proteins are reported to induce the activation of NF-κB, a transcriptional factor known to play an important role in the development of liver disease. This study aimed to see the effects of HBs variants in HBV subgenotype B3 on the expression and activation of NF-κB as one of the mechanisms in inducing advanced liver disease. HBV subgenotypes B3, each carrying wild-type (wt) HBs, M120 V, and pre-S2 deletion mutation were isolated from three HCC patients. HBs genes were amplified and cloned into pcDNA3.1 and were transfected using Lipofectamine into a Huh7 cell line. NF-κB activation was measured through IκB-α expression, which is regulated by NF-κB. RNA expressions for HBs, IκB-α, and NF-κB subunit (p50) were evaluated using real-time PCR. M120 V mutant had a significantly higher mRNA level compared with wt and pre-S2 deletion mutant; however, there were no significant differences in HBs protein expressions. The transcription level of p50 was higher in M120 V mutation compared with HBs wild-type and pre-S2 deletion mutant. NF-κB activation was higher in HBs wild-type compared with the two mutant variants. Pre-S2 mutations had no effect on the increment of NF-κB activation. However, M120 V mutation may utilize a different pathway in liver disease progression that involves high expression of NF-κB subunit, p50.

Effect of Quercetin on Hepatitis C Virus Life Cycle: From Viral to Host Targets.

PubMed

Rojas, Ángela; Del Campo, Jose A; Clement, Sophie; Lemasson, Matthieu; García-Valdecasas, Marta; Gil-Gómez, Antonio; Ranchal, Isidora; Bartosch, Birke; Bautista, Juan D; Rosenberg, Arielle R; Negro, Francesco; Romero-Gómez, Manuel

2016-08-22

Quercetin is a natural flavonoid, which has been shown to have anti hepatitis C virus (HCV) properties. However, the exact mechanisms whereby quercetin impacts the HCV life cycle are not fully understood. We assessed the effect of quercetin on different steps of the HCV life cycle in Huh-7.5 cells and primary human hepatocytes (PHH) infected with HCVcc. In both cell types, quercetin significantly decreased i) the viral genome replication; ii) the production of infectious HCV particles and iii) the specific infectivity of the newly produced viral particles (by 85% and 92%, Huh7.5 and PHH respectively). In addition, when applied directly on HCV particles, quercetin reduced their infectivity by 65%, suggesting that it affects the virion integrity. Interestingly, the HCV-induced up-regulation of diacylglycerol acyltransferase (DGAT) and the typical localization of the HCV core protein to the surface of lipid droplets, known to be mediated by DGAT, were both prevented by quercetin. In conclusion, quercetin appears to have direct and host-mediated antiviral effects against HCV.

Effect of Quercetin on Hepatitis C Virus Life Cycle: From Viral to Host Targets

PubMed Central

Rojas, Ángela; Del Campo, Jose A.; Clement, Sophie; Lemasson, Matthieu; García-Valdecasas, Marta; Gil-Gómez, Antonio; Ranchal, Isidora; Bartosch, Birke; Bautista, Juan D.; Rosenberg, Arielle R.; Negro, Francesco; Romero-Gómez, Manuel

2016-01-01

Quercetin is a natural flavonoid, which has been shown to have anti hepatitis C virus (HCV) properties. However, the exact mechanisms whereby quercetin impacts the HCV life cycle are not fully understood. We assessed the effect of quercetin on different steps of the HCV life cycle in Huh-7.5 cells and primary human hepatocytes (PHH) infected with HCVcc. In both cell types, quercetin significantly decreased i) the viral genome replication; ii) the production of infectious HCV particles and iii) the specific infectivity of the newly produced viral particles (by 85% and 92%, Huh7.5 and PHH respectively). In addition, when applied directly on HCV particles, quercetin reduced their infectivity by 65%, suggesting that it affects the virion integrity. Interestingly, the HCV-induced up-regulation of diacylglycerol acyltransferase (DGAT) and the typical localization of the HCV core protein to the surface of lipid droplets, known to be mediated by DGAT, were both prevented by quercetin. In conclusion, quercetin appears to have direct and host-mediated antiviral effects against HCV. PMID:27546480

Hepatitis B virus PreS1 facilitates hepatocellular carcinoma development by promoting appearance and self-renewal of liver cancer stem cells.

PubMed

Liu, Zhixin; Dai, Xuechen; Wang, Tianci; Zhang, Chengcheng; Zhang, Wenjun; Zhang, Wei; Zhang, Qi; Wu, Kailang; Liu, Fang; Liu, Yingle; Wu, Jianguo

2017-08-01

Hepatitis B virus (HBV) is a major etiologic agent of hepatocellular carcinoma (HCC). However, the molecular mechanism by which HBV infection contributes to HCC development is not fully understood. Here, we initially showed that HBV stimulates the production of cancer stem cells (CSCs)-related markers (CD133, CD117 and CD90) and CSCs-related genes (Klf4, Sox2, Nanog, c-Myc and Oct4) and facilitates the self-renewal of CSCs in human hepatoma cells. Cellular and clinical studies revealed that HBV facilitates hepatoma cell growth and migration, enhances white blood cell (WBC) production in the sera of patients, stimulates CD133 and CD117 expression in HCC tissues, and promotes the CSCs generation of human hepatoma cells and clinical cancer tissues. Detailed studies revealed that PreS1 protein of HBV is required for HBV-mediated CSCs generation. PreS1 activates CD133, CD117 and CD90 expression in normal hepatocyte derived cell line (L02) and human hepatoma cell line (HepG2 and Huh-7); facilitates L02 cells migration, growth and sphere formation; and finally enhances the abilities of L02 cells and HepG2 cells to induce tumorigeneses in nude mice. Thus, PreS1 acts as a new oncoprotein to play a key role in the appearance and self-renewal of CSCs during HCC development. Copyright © 2017 Elsevier B.V. All rights reserved.

Repression of miR-17-5p with elevated expression of E2F-1 and c-MYC in non-metastatic hepatocellular carcinoma and enhancement of cell growth upon reversing this expression pattern

DOE Office of Scientific and Technical Information (OSTI.GOV)

El Tayebi, H.M.; Omar, K.; Hegy, S.

2013-05-10

Highlights: •The oncogenic miR-17-5p is downregulated in non-metastatic hepatocellular carcinoma patients. •E2F-1 and c-MYC transcripts are upregulated in non-metastatic HCC patients. •miR-17-5p forced overexpression inhibited E2F-1 and c-MYC expression in HuH-7 cells. •miR-17-5p mimicking increased HuH-7 cell growth, proliferation, migration and colony formation. •miR-17-5p is responsible for HCC progression among the c-MYC/E2F-1/miR-17-5p triad members. -- Abstract: E2F-1, c-MYC, and miR-17-5p is a triad of two regulatory loops: a negative and a positive loop, where c-MYC induces the expression of E2F-1 that induces the expression of miR-17-5p which in turn reverses the expression of E2F-1 to close the loop. In thismore » study, we investigated this triad for the first time in hepatocellular carcinoma (HCC), where miR-17-5p showed a significant down-regulation in 23 non-metastatic HCC biopsies compared to 10 healthy tissues; however, E2F-1 and c-MYC transcripts were markedly elevated. Forced over-expression of miR-17-5p in HuH-7 cells resulted in enhanced cell proliferation, growth, migration and clonogenicity with concomitant inhibition of E2F-1 and c-MYC transcripts expressions, while antagomirs of miR-17-5p reversed these events. In conclusion, this study revealed a unique pattern of expression for miR-17-5p in non-metastatic HCC patients in contrast to metastatic HCC patients. In addition we show that miR-17-5p is the key player among the triad that tumor growth and spread.« less

Proper design of silica nanoparticles combines high brightness, lack of cytotoxicity and efficient cell endocytosis

NASA Astrophysics Data System (ADS)

Rampazzo, Enrico; Voltan, Rebecca; Petrizza, Luca; Zaccheroni, Nelsi; Prodi, Luca; Casciano, Fabio; Zauli, Giorgio; Secchiero, Paola

2013-08-01

Silica-based luminescent nanoparticles (SiNPs) show promising prospects in nanomedicine in light of their chemical properties and versatility. In this study, we have characterized silica core-PEG shell SiNPs derivatized with PEG moieties (NP-PEG), with external amino- (NP-PEG-amino) or carboxy-groups (NP-PEG-carbo), both in cell cultures as well as in animal models. By using different techniques, we could demonstrate that these SiNPs were safe and did not exhibit appreciable cytotoxicity in different relevant cell models, of normal or cancer cell types, growing either in suspension (JVM-2 leukemic cell line and primary normal peripheral blood mononuclear cells) or in adherence (human hepatocarcinoma Huh7 and umbilical vein endothelial cells). Moreover, by multiparametric flow cytometry, we could demonstrate that the highest efficiency of cell uptake and entry was observed with NP-PEG-amino, with a stable persistence of the fluorescence signal associated with SiNPs in the loaded cell populations both in vitro and in vivo settings suggesting this as an innovative method for cell traceability and detection in whole organisms. Finally, experiments performed with the endocytosis inhibitor Genistein clearly suggested the involvement of a caveolae-mediated pathway in SiNP endocytosis. Overall, these data support the safe use of these SiNPs for diagnostic and therapeutic applications.Silica-based luminescent nanoparticles (SiNPs) show promising prospects in nanomedicine in light of their chemical properties and versatility. In this study, we have characterized silica core-PEG shell SiNPs derivatized with PEG moieties (NP-PEG), with external amino- (NP-PEG-amino) or carboxy-groups (NP-PEG-carbo), both in cell cultures as well as in animal models. By using different techniques, we could demonstrate that these SiNPs were safe and did not exhibit appreciable cytotoxicity in different relevant cell models, of normal or cancer cell types, growing either in suspension (JVM-2 leukemic cell line and primary normal peripheral blood mononuclear cells) or in adherence (human hepatocarcinoma Huh7 and umbilical vein endothelial cells). Moreover, by multiparametric flow cytometry, we could demonstrate that the highest efficiency of cell uptake and entry was observed with NP-PEG-amino, with a stable persistence of the fluorescence signal associated with SiNPs in the loaded cell populations both in vitro and in vivo settings suggesting this as an innovative method for cell traceability and detection in whole organisms. Finally, experiments performed with the endocytosis inhibitor Genistein clearly suggested the involvement of a caveolae-mediated pathway in SiNP endocytosis. Overall, these data support the safe use of these SiNPs for diagnostic and therapeutic applications. Electronic supplementary information (ESI) available: Synthetic procedures, 1H and 13C NMR spectra, TEM and DLS measurements, and absorption and emission spectra. See DOI: 10.1039/c3nr02563b

Design, Construction and Evaluation of 1a/JFH1 HCV Chimera by Replacing the Intergenotypic Variable Region

PubMed Central

Ghasemi, Faezeh; Ghayour-Mobarhan, Majid; Pasdar, Alireza; Pourianfar, Hamid; Reza Aghasadeghi, Mohammad; Gouklani, Hamed; Meshkat, Zahra

2016-01-01

Background The E2 glycoprotein is an important encoded hepatitis C virus (HCV) protein that contains three different variable regions. Objectives The aim of the present study was to construct an HCV 1a/JFH1 chimeric virus by replacing the intergenotypic variable region (igVR) fragment of the highly variable region of the E2 gene of the Japanese Fulminant hepatitis genotype 2a JFH1 virus with a similar region of HCV genotype 1a. This chimera was produced as a model virus with the ability to be cultured. We analyzed the adapted virus and the variations of nucleic acids within it. Methods Specific primers were designed for the igVR of HCV genotype 1a followed by the overlap-PCR method for the synthesis of the desired DNA fragment. The amplified igVR-1a chimera gene and pFL-J6/JFH were digested by KpnI and BsiWI restriction enzymes, and the fragment was ligated into pFL-J6/JFH. The recombinant vector was transformed into Escherichia coli JM109 strain competent cells. All clones were confirmed by colony PCR using specific primers, and the confirmed recombinant vector was sequenced. The recombinant vector was targeted for RNA synthesis by T7 RNA polymerase enzyme. RNA transfection was performed in the Huh7.5 cell line. Virus production in several passages and the evaluated viral load were studied using quantitative real-time PCR and ELISA methods. After 30 passages, the RNA virus was extracted and cloned in PCDNA3.1 vector, and was then sequenced Results Quantitative real-time PCR results showed 11,292,514 copies/mL of chimeric virus production in cell culture. The virus production was confirmed using ELISA, which showed a virus core production of 808.2 pg/mL. The results of cloning and sequencing showed that some of the nucleic acids in the chimera virus were changed, affecting the viral behavior in the cell culture. Conclusions Real-time PCR and ELISA showed high levels of production of 1a/JFH1 chimeric HCV in the Huh7.5 cell culture. The constructed virus can be used for future studies, including the development of new HCV drugs and vaccines. PMID:27882063

Downregulation of transcription factor GATA4 sensitizes human hepatoblastoma cells to doxorubicin-induced apoptosis.

PubMed

Soini, Tea; Pihlajoki, Marjut; Kyrönlahti, Antti; Andersson, Leif C; Wilson, David B; Heikinheimo, Markku

2017-03-01

Hepatoblastoma, the most common type of pediatric liver cancer, is treated with a combination of surgery and chemotherapy. An essential drug in the treatment of hepatoblastoma is doxorubicin, which in high doses is cardiotoxic. This adverse effect is due to downregulation of cardiac expression of transcription factor GATA4, leading in turn to diminished levels of anti-apoptotic BCL2 (B-cell lymphoma 2) protein family members. GATA4 is also expressed in early fetal liver, but absent from normal postnatal hepatocytes. However, GATA4 is highly expressed in hepatoblastoma tissue. In this study, we assessed the role of GATA4 in doxorubicin-induced apoptosis of hepatoblastoma cells. Herein, we demonstrate that doxorubicin decreases GATA4 expression and alters the expression pattern of BCL2 family members, most profoundly that of BCL2 and BAK, in the HUH6 hepatoblastoma cell line. Silencing of GATA4 by siRNA prior to doxorubicin treatment sensitizes HUH6 cells to the apoptotic effect of this drug by further shifting the balance of BCL2 family members to the pro-apoptotic direction. Specifically, expression levels of anti-apoptotic BCL2 were decreased and pro-apoptotic BID were increased after GATA4 silencing. On the whole, our results indicate that since high endogenous levels of transcription factor GATA4 likely protect hepatoblastoma cells from doxorubicin-induced apoptosis, these cells can be rendered more sensitive to the drug by downregulation of GATA4.

Synthetic lipophilic antioxidant BO-653 suppresses HCV replication.

PubMed

Yasui, Fumihiko; Sudoh, Masayuki; Arai, Masaaki; Kohara, Michinori

2013-02-01

The influence of the intracellular redox state on the hepatitis C virus (HCV) life cycle is poorly understood. This study demonstrated the anti-HCV activity of 2,3-dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butylbenzofuran (BO-653), a synthetic lipophilic antioxidant, and examined whether BO-653's antioxidant activity is integral to its anti-HCV activity. The anti-HCV activity of BO-653 was investigated in HuH-7 cells bearing an HCV subgenomic replicon (FLR3-1 cells) and in HuH-7 cells infected persistently with HCV (RMT-tri cells). BO-653 inhibition of HCV replication was also compared with that of several hydrophilic and lipophilic antioxidants. BO-653 suppressed HCV replication in FLR3-1 and RMT-tri cells in a concentration-dependent manner. The lipophilic antioxidants had stronger anti-HCV activities than the hydrophilic antioxidants, and BO-653 displayed the strongest anti-HCV activity of all the antioxidants examined. Therefore, the anti-HCV activity of BO-653 was examined in chimeric mice harboring human hepatocytes infected with HCV. The combination treatment of BO-653 and polyethylene glycol-conjugated interferon-α (PEG-IFN) decreased serum HCV RNA titer more than that seen with PEG-IFN alone. These findings suggest that both the lipophilic property and the antioxidant activity of BO-653 play an important role in the inhibition of HCV replication. Copyright © 2012 Wiley Periodicals, Inc.

Chalcone-Induced Apoptosis through Caspase-Dependent Intrinsic Pathways in Human Hepatocellular Carcinoma Cells.

PubMed

Ramirez-Tagle, Rodrigo; Escobar, Carlos A; Romero, Valentina; Montorfano, Ignacio; Armisén, Ricardo; Borgna, Vincenzo; Jeldes, Emanuel; Pizarro, Luis; Simon, Felipe; Echeverria, Cesar

2016-02-22

Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers worldwide. Chemoprevention of HCC can be achieved through the use of natural or synthetic compounds that reverse, suppress or prevent the development of cancer progression. In this study, we investigated the antiproliferative effects and the mechanism of action of two compounds, 2,3,4'-trimethoxy-2'-hydroxy-chalcone (CH1) and 3'-bromo-3,4-dimethoxy-chalcone (CH2), over human hepatoma cells (HepG2 and Huh-7) and cultured mouse hepatocytes (HepM). Cytotoxic effects were observed over the HepG2 and Huh-7, and no effects were observed over the HepM. For HepG2 cells, treated separately with each chalcone, typical apoptotic laddering and nuclear condensation were observed. Additionally, the caspases and Bcl-2 family proteins activation by using Western blotting and immunocytochemistry were studied. Caspase-8 was not activated, but caspase-3 and -9 were both activated by chalcones in HepG2 cells. Chalcones also induced reactive oxygen species (ROS) accumulation after 4, 8 and 24 h of treatment in HepG2 cells. These results suggest that apoptosis in HepG2 was induced through: (i) a caspase-dependent intrinsic pathway; and (ii) by alterations in the cellular levels of Bcl-2 family proteins, and also, that the chalcone moiety could be a potent candidate as novel anticancer agents acting on human hepatomas.

The Role of the Sonic Hedgehog Pathway for Prostate Cancer Progression

DTIC Science & Technology

2007-02-01

hepatocellular carcinomas : through transcriptional activation of the ligand Shh (Carcinogenesis 27: 1334-40, 2006). Second, our studies of Su(Fu...in hepatocellular carcinomas . Fig. 3 shows that the sonic hedgehog promoter activity is high in Huh7 cells but low in HepG2 cells. In the presence of... hepatocellular carcinomas and prostate cancer. LNCaP cells 0 50 100 150 200 250 300 Vector (-1800 to -200) (-680 to -200) R el at iv e lu ci fe ra

Effective elimination of liver cancer stem-like cells by CD90 antibody targeted thermosensitive magnetoliposomes

PubMed Central

Yang, Rui; An, Li Y.; Miao, Qin F.; Li, Feng M.; Han, Yong; Wang, Hui X.; Liu, Dang P.; Chen, Rong; Tang, Sha Q.

2016-01-01

Aim To investigate the use of thermosensitive magnetoliposomes (TMs) loaded with magnetic iron oxide (Fe3O4) and the anti-cancer stem cell marker CD90 (CD90@TMs) to target and kill CD90+ liver cancer stem cells (LCSCs). Methods The hepatocellular carcinoma cell line Huh7 was used to separate CD90+ LCSCs by magnetic-activated cell sorting. CD90@TMs was characterized and their ability to target CD90+ LCSCs was determined. Experiments were used to investigate whether CD90@TMs combined with magnetic hyperthermia could effectively eliminate CD90+ LCSCs. Results The present study demonstrated that CD90+ LCSCs with stem cells properties were successfully isolated. We also successfully prepared CD90@TMs that was almost spherical and uniform with an average diameter of 130±4.6 nm and determined that magnetic iron oxide could be incorporated and retained a superparamagnetic response. CD90@TMs showed good targeting and increased inhibition of CD90+ LCSCs in vitro and in vivo compared to TMs. Conclusion CD90@TMs can be used for controlled and targeted delivery of anticancer drugs, which may offer a promising alternative for HCC therapy. PMID:27145285

Therapeutic potential of Taraxacum officinale against HCV NS5B polymerase: In-vitro and In silico study.

PubMed

Rehman, Sidra; Ijaz, Bushra; Fatima, Nighat; Muhammad, Syed Aun; Riazuddin, Sheikh

2016-10-01

Discovery of alternative and complementary regimens for HCV infection treatment is a need of time from clinical as well as economical point of views. Low cost of bioactive natural compounds production, high biochemical diversity and inexistent/milder side effects contribute to new therapies. Aim of this study is to clarify anti-HCV role of Taraxacum officinale, a natural habitat plant rich of flavonoids. In this study, methanol extract of T. officinale leaves was initially analyzed for its cytotoxic activity in human hepatoma (Huh-7) and CHO cell lines. Hepatoma cells were transfected with pCR3.1/Flagtag/HCV NS5B gene cloned vector (genotype 1a) along with T. officinale extract. Considering NS5B polymerase as potential therapeutic drug target, twelve phytochemicals of T. officinale were selected as ligands for molecular interaction with NS5B protein using Molecular Operating Environment (MOE) software. Sofosbuvir (Sovaldi: brand name) currently approved as new anti-HCV drug, was used as standard in current study for comparative analysis in computational docking screening. HCV NS5B polymerase as name indicates plays key role in viral genome replication. On the basis of which NS5B gene is targeted for determining antiviral role of T. officinale extract and 65% inhibition of NS5B expression was documented at nontoxic dose concentration (200μg/ml) using Real-time PCR. In addition, 57% inhibition of HCV replication was recorded when incubating Huh-7 cells with high titer serum of HCV infected patients along with leaves extract. Phytochemicals for instance d-glucopyranoside (-31.212 Kcal/mol), Quercetin (-29.222 Kcal/mol), Luteolin (-26.941 Kcal/mol) and some others displayed least binding energies as compared to standard drug Sofosbuvir (-21.0746 Kcal/mol). Results of our study strongly revealed that T. officinale leaves extract potentially blocked the viral replication and NS5B gene expression without posing any toxic effect on normal fibroblast cells of body. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

On the applicability of [18F]FBPA to predict L-BPA concentration after amino acid preloading in HuH-7 liver tumor model and the implication for liver boron neutron capture therapy.

PubMed

Grunewald, Catrin; Sauberer, Michael; Filip, Thomas; Wanek, Thomas; Stanek, Johann; Mairinger, Severin; Rollet, Sofia; Kudejova, Petra; Langer, Oliver; Schütz, Christian; Blaickner, Matthias; Kuntner, Claudia

2017-01-01

In recent years extra-corporal application of boron neutron capture therapy (BNCT) was evaluated for liver primary tumors or liver metastases. A prerequisite for such a high-risk procedure is proof of preferential delivery and high uptake of a 10 B-pharmaceutical in liver malignancies. In this work we evaluated in a preclinical tumor model if [ 18 F]FBPA tissue distribution measured with PET is able to predict the tissue distribution of [ 10 B]L-BPA. Tumor bearing mice (hepatocellular carcinoma cell line, HuH-7) were either subject of a [ 18 F]FBPA-PET scan with subsequent measurement of radioactivity content in extracted organs using a gamma counter or injected with [ 10 B]L-BPA with tissue samples analyzed by prompt gamma activation analysis (PGAA) or quantitative neutron capture radiography (QNCR). The impact of L-tyrosine, L-DOPA and L-BPA preloading on the tissue distribution of [ 18 F]FBPA and [ 10 B]L-BPA was evaluated and the pharmacokinetics of [ 18 F]FBPA investigated by compartment modeling. We found a significant correlation between [ 18 F]FBPA and [ 10 B]L-BPA uptake in tumors and various organs as well as high accumulation levels in pancreas and kidneys as reported in previous studies. Tumor-to-liver ratios of [ 18 F]FBPA ranged from 1.2 to 1.5. Preloading did not increase the uptake of [ 18 F]FBPA or [ 10 B]L-BPA in any organ and compartment modeling showed no statistically significant differences in [ 18 F]FBPA tumor kinetics. [ 18 F]FBPA-PET predicts [ 10 B]L-BPA concentration after amino acid preloading in HuH-7 hepatocellular carcinoma models. Preloading had no effect on tumor uptake of [ 18 F]FBPA. Despite differences in chemical structure and administered dose [ 18 F]FBPA and [ 10 B]L-BPA demonstrate an equivalent biodistribution in a preclinical tumor model. IMPLICATIONS FOR PATIENT CARE: [ 18 F]FBPA-PET is suitable for treatment planning and dose calculations in BNCT applications for liver malignancies. However, alternative tracers with more favorable tumor-to-liver ratios should be investigated. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanism of gemcitabine-induced suppression of human cholangiocellular carcinoma cell growth.

PubMed

Toyota, Yuka; Iwama, Hisakazu; Kato, Kiyohito; Tani, Joji; Katsura, Akiko; Miyata, Miwa; Fujiwara, Shintaro; Fujita, Koji; Sakamoto, Teppei; Fujimori, Takayuki; Okura, Ryoichi; Kobayashi, Kiyoyuki; Tadokoro, Tomoko; Mimura, Shima; Nomura, Takako; Miyoshi, Hisaaki; Morishita, Asahiro; Kamada, Hideki; Yoneyama, Hirohito; Okano, Keiichi; Suzuki, Yasuyuki; Masaki, Tsutomu

2015-10-01

Although gemcitabine (2',2'-difluorocytidine monohydrochloride) is a common anticancer agent of cholangiocellular carcinoma (CCC), its growth inhibitory effects and gemcitabine resistance in CCC cells are poorly understood. Our aims were to uncover the mechanism underlying the antitumor effect of gemcitabine and to analyze the mechanism regulating in vitro CCC cell gemcitabine resistance. In addition, we sought to identify miRNAs associated with the antitumor effects of gemcitabine in CCCs. Using a cell proliferation assay and flow cytometry, we examined the ability of gemcitabine to inhibit cell proliferation in three types of human CCC cell lines (HuCCT-1, Huh28, TKKK). We also employed western blotting to investigate the effects of gemcitabine on cell cycle-related molecules in CCC cells. In addition, we used array chips to assess gemcitabine-mediated changes in angiogenic molecules and activated tyrosine kinase receptors in CCC cells. We used miRNA array chips to comprehensively analyze gemcitabine-induced miRNAs and examined clusters of differentially expressed miRNAs in cells with and without gemcitabine treatment. Gemcitabine inhibited cell proliferation in a dose- and time-dependent manner in HuCCT-1 cells, whereas cell proliferation was unchanged in Huh28 and TKKK cells. Gemcitabine inhibited cell cycle progression in HuCCT-1 cells from G0/G1 to S phase, resulting in G1 cell cycle arrest due to the reduction of cyclin D1 expression. In addition, gemcitabine upregulated the angiogenic molecules IL-6, IL-8, ENA-78 and MCP-1. In TKKK cells, by contrast, gemcitabine did not arrest the cell cycle or modify angiogenic molecules. Furthermore, in gemcitabine-sensitive HuCCT-1 cells, gemcitabine markedly altered miRNA expression. The miRNAs and angiogenic molecules altered by gemcitabine contribute to the inhibition of tumor growth in vitro.

Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nemoto, Kiyomitsu, E-mail: nemoto@u-shizuoka-ken.ac.jp; Ikeda, Ayaka; Yoshida, Chiaki

Highlights: ► Nobiletin-mediated alterations of gene expression were examined with DNA microarrays. ► Three organ-derived cell lines were treated with 100 μM nobiletin for 24 h. ► In all cell lines, 3 endoplasmic reticulum stress-responsive genes were up-regulated. ► Some cell cycle-regulating and oxidative stress-promoting genes were down-regulated. ► These alterations may contribute to nobiletin-mediated biological effects. -- Abstract: Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitromore » and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines – 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells – were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 μM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and suppressions of oxidative stress and cell proliferation.« less

Expression of Clonorchis sinensis GIIIsPLA2 protein in baculovirus-infected insect cells and its overexpression facilitating epithelial-mesenchymal transition in Huh7 cells via AKT pathway.

PubMed

Shang, Mei; Xie, Zhizhi; Tang, Zeli; He, Lei; Wang, Xiaoyun; Wang, Caiqin; Wu, Yinjuan; Li, Ye; Zhao, Lu; Lv, Zhiyue; Wu, Zhongdao; Huang, Yan; Yu, Xinbing; Li, Xuerong

2017-04-01

Although prior studies confirmed that group III secretory phospholipase A 2 of Clonorchis sinensis (CsGIIIsPLA 2 ) had stimulating effect on liver fibrosis by binding to LX-2 cells, large-scale expression of recombinant protein and its function in the progression of hepatoma are worth exploring. Because of high productivity and low lipopolysaccharides (LPS) in the Sf9-baculovirus expression system, we firstly used this system to express the coding region of CsGIIIsPLA 2 . The molecular weight of recombinant CsGIIIsPLA 2 protein was about 34 kDa. Further investigation showed that most of the recombinant protein presented intracellular expression in Sf9 insect cell nucleus and could be detected only into cell debris, which made the protein purification and further functional study difficult. Therefore, to study the role of CsGIIIsPLA 2 in hepatocellular carcinoma (HCC) progression, CsGIIIsPLA 2 overexpression Huh7 cell model was applied. Cell proliferation, migration, and the expression level of epithelial-mesenchymal transition (EMT)-related molecules (E-cadherin, N-cadherin, α-catenin, Vimentin, p300, Snail, and Slug) along with possible mechanism were measured. The results indicated that CsGIIIsPLA 2 overexpression not only inhibited cell proliferation and promoted migration and EMT but also enhanced the phosphorylation of AKT in HCC cells. In conclusion, this study supported that CsGIIIsPLA 2 overexpression suppressed cell proliferation and induced EMT through the AKT pathway.

RNA interference mediated in human primary cells via recombinant baculoviral vectors.

PubMed

Nicholson, Linda J; Philippe, Marie; Paine, Alan J; Mann, Derek A; Dolphin, Colin T

2005-04-01

The success of RNA interference (RNAi) in mammalian cells, mediated by siRNAs or shRNA-generating plasmids, is dependent, to an extent, upon transfection efficiency. This is a particular problem with primary cells, which are often difficult to transfect using cationic lipid vehicles. Effective RNAi in primary cells is thus best achieved with viral vectors, and retro-, adeno-, and lentivirus RNAi systems have been described. However, the use of such human viral vectors is inherently problematic, e.g., Class 2 status and requirement of secondary helper functions. Although insect cells are their natural host, baculoviruses also transduce a range of vertebrate cell lines and primary cells with high efficiency. The inability of baculoviral vectors to replicate in mammalian cells, their Class 1 status, and the simplicity of their construction make baculovirus an attractive alternative gene delivery vector. We have developed a baculoviral-based RNAi system designed to express shRNAs and GFP from U6 and CMV promoters, respectively. Transduction of Saos2, HepG2, Huh7, and primary human hepatic stellate cells with a baculoviral construct expressing shRNAs targeting lamin A/C resulted in effective knockdown of the corresponding mRNA and protein. Development of this baculoviral-based system provides an additional shRNA delivery option for RNAi-based investigations in mammalian cells.

Intracellular localization of pregnane X receptor in HepG2 cells cultured by the hanging drop method.

PubMed

Yokobori, Kosuke; Kobayashi, Kaoru; Azuma, Ikuko; Akita, Hidetaka; Chiba, Kan

2017-10-01

Pregnane X receptor (PXR) is localized in the cytoplasm of liver cells, whereas it is localized in the nucleus of monolayer-cultured HepG2 cells. Since cultured cells are affected by the microenvironment in which they are grown, we studied the effect of three-dimensional (3D) culture on the localization of PXR in HepG2 cells using the hanging drop method. The results showed that PXR was retained in the cytoplasm of HepG2 cells and other human hepatocarcinoma cell lines (FLC5, FLC7 and Huh7) when they were cultured by the hanging drop method. Treatment with rifampicin, a ligand of PXR, translocated PXR from the cytoplasm to nucleus and increased expression levels of CYP3A4 mRNA in HepG2 cells cultured by the hanging drop method. These findings suggest that 3D culture is a key factor determining the intracellular localization of PXR in human hepatocarcinoma cells and that PXR that becomes retained in the cytoplasm of HepG2 cells with 3D culture has functions of nuclear translocation and regulation of target genes in response to human PXR ligands. Three-dimensionally cultured hepatocarcinoma cells would be a useful tool to evaluate induction potency of drug candidates and also to study mechanisms of nuclear translocation of PXR by human PXR ligands. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

EGF receptor targeted lipo-oligocation polyplexes for antitumoral siRNA and miRNA delivery

NASA Astrophysics Data System (ADS)

Müller, Katharina; Klein, Philipp M.; Heissig, Philipp; Roidl, Andreas; Wagner, Ernst

2016-11-01

Antitumoral siRNA and miRNA delivery was demonstrated by epidermal growth factor receptor (EGFR) targeted oligoaminoamide polyplexes. For this purpose, the T-shaped lipo-oligomer 454 was used to complex RNA into a core polyplex, which was subsequently functionalized with the targeting peptide ligand GE11 via a polyethylene glycol (PEG) linker. To this end, free cysteines on the surface of 454 polyplex were coupled with a maleimide-PEG-GE11 reagent (Mal-GE11). Resulting particles with sizes of 120-150 nm showed receptor-mediated uptake into EGFR-positive T24 bladder cancer cells, MDA-MB 231 breast cancer cells and Huh7 liver cancer cells. Furthermore, these formulations led to ligand-dependent gene silencing. RNA interference (RNAi) triggered antitumoral effects were observed for two different therapeutic RNAs, a miRNA-200c mimic or EG5 siRNA. Using polyplexes modified with a ratio of 0.8 molar equivalents of Mal-GE11, treatment of T24 or MDA-MB 231 cancer cells with miR-200c led to the expected decreased proliferation and migration, changes in cell cycle and enhanced sensitivity towards doxorubicin. Delivery of EG5 siRNA into Huh7 cells resulted in antitumoral activity with G2/M arrest, triggered by loss of mitotic spindle separation and formation of mono-astral spindles. These findings demonstrate the potential of GE11 ligand-containing RNAi polyplexes for cancer treatment.

Hepcidin suppression in β-thalassemia is associated with the down-regulation of atonal homolog 8.

PubMed

Upanan, Supranee; McKie, Andrew T; Latunde-Dada, Gladys O; Roytrakul, Sittiruk; Uthaipibull, Chairat; Pothacharoen, Peraphan; Kongtawelert, Prachya; Fucharoen, Suthat; Srichairatanakool, Somdet

2017-08-01

Atonal homolog 8 (ATOH8) is defined as a positive regulator of hepcidin transcription, which links erythropoietic activity with iron-sensing molecules. In the present study, we investigated the association between hepcidin and ATOH8 expression in β-thalassemia. We found that inhibition of hepcidin expression in β-thalassemia is correlated with reduced ATOH8 expression. Hepatic hepcidin 1 (Hamp1) and Atoh8 mRNA expression were down-regulated in β-thalassemic mice. Hepcidin (HAMP) and ATOH8 mRNA expression were consistently suppressed in Huh7 cells cultured in medium supplemented with β-thalassemia patient serum. The Huh7 cells, which were transfected with ATOH8-FLAG expression plasmid and cultured in the supplemented medium, exhibited increased levels of ATOH8 mRNA, ATOH8-FLAG protein, pSMAD1,5,8, and HAMP mRNA. Interestingly, over-expression of ATOH8 reversed the effects of hepcidin suppression induced by the β-thalassemia patient sera. In conclusion, hepcidin suppression in β-thalassemia is associated with the down-regulation of ATOH8 in response to anemia. We, therefore, suggest that ATOH8 is an important transcriptional regulator of hepcidin in β-thalassemia.

CXCL7 promotes proliferation and invasion of cholangiocarcinoma cells.

PubMed

Guo, Qian; Jian, Zhixiang; Jia, Baoqing; Chang, Liang

2017-02-01

CXCL7 is an important chemoattractant cytokine, which signals through binding to its receptor CXCR2. Recent studies have demonstrated that the CXCL7/CXCR2 signaling plays a promoting role in several common malignancies, including lung, renal, colon, and breast cancer. However, the regulatory role of CXCL7, in cholangiocarcinoma, as well as the underlying mechanism, has not been previously reported. Herein, we found more positive expression of CXCL7 in cholangiocarcinoma tissues compared to adjacent non-tumor tissues. High CXCL7 expression was significantly correlated with poor differentiation, lymph node metastasis, vascular invasion and advanced clinical stage, but was not associated with age, gender, or tumor size. Besides, the expression of CXCL7 was significantly associated with the Ki67 expression, but not associated with CA199, AFP, or P53 expression in cholangiocarcinoma. Moreover, the overall survival of cholangiocarcinoma patients with high CXCL7 expression was significantly shorter than those with low CXCL7 expression. In vitro study indicated that CXCL7 and CXCR2 were also positively expressed in several common cholangiocarcinoma cell lines, including HuCCT1, HuH28, QBC939, EGI-1, OZ and WITT. SiRNA-induced inhibition of CXCL7 significantly reduced the proliferation and invasion of QBC939 cells. On the contrary, overexpression of CXCL7 markedly promoted these malignant phenotypes of QBC939 cells. Of note, the conditioned medium of CXCL7-overexpresing human hepatic stellate cells could also promote the proliferation and invasion of QBC939 cells, suggesting that CXCL7 may also play an oncogenic role in cholangiocarcinoma in a paracrine-dependent manner, not only in an autocrine-dependent manner. Molecular assay data suggested that the AKT signaling pathway was involved in the CXCL7-mediated malignant phenotypes of QBC939 cells. In summary, our study suggests that CXCL7 plays a promoting role in regulating the growth and metastasis of cholangiocarcinoma.

6-Shogaol induces cell cycle arrest and apoptosis in human hepatoma cells through pleiotropic mechanisms.

PubMed

Wu, Jung-Ju; Omar, Hany A; Lee, Ying-Ray; Teng, Yen-Ni; Chen, Pin-Shern; Chen, Yu-Chung; Huang, Hsiao-Shan; Lee, Kuan-Han; Hung, Jui-Hsiang

2015-09-05

Shogaols are a group of the active constituents of ginger that have been identified to have various biological activities. The aim of the current study was to investigate the antitumor activity of 6-shogaol in hepatocellular carcinoma (HCC) and the possible involvement of reactive oxygen species as a putative mechanism of action. HCC cell lines, HepG2 and Huh-7, were used to study the in vitro anti-cancer activity of 6-shogaol via the application of various molecular biology techniques. Results showed that 6-shogaol effectively inhibited the cell viability, caused cell cycle arrest at G2/M phase and induced apoptosis in HCC cells as indicated by MTT assay, DAPI nuclear staining, annexin V assay, cell cycle analysis, and activation of caspase-3. Western blot analysis revealed the ability of 6-shogaol to target cancer survival signaling pathways mediated by mitogen-activated protein kinase (MAPK), 5' AMP-activated protein kinase (AMPK) and Akt. In addition, 6-Shogaol induced alteration of cyclin proteins expression and caused cleavage of protein kinase C delta. Furthermore, 6-Shogaol was able to induce the production of reactive oxygen species and endoplasmic reticulum (ER) stress-associated proteins and the consequent activation of autophagy in HepG2 cells. Taken together, the current study highlights evidences that 6-shogaol induces apoptosis, modulates cyclins expression and targets cancer survival signaling pathways in HCC cell lines, at least in part, via the production of reactive oxygen species. These findings support 6-shogaol's clinical promise as a potential candidate for HCC therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Chalcone-Induced Apoptosis through Caspase-Dependent Intrinsic Pathways in Human Hepatocellular Carcinoma Cells

PubMed Central

Ramirez-Tagle, Rodrigo; Escobar, Carlos A.; Romero, Valentina; Montorfano, Ignacio; Armisén, Ricardo; Borgna, Vincenzo; Jeldes, Emanuel; Pizarro, Luis; Simon, Felipe; Echeverria, Cesar

2016-01-01

Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers worldwide. Chemoprevention of HCC can be achieved through the use of natural or synthetic compounds that reverse, suppress or prevent the development of cancer progression. In this study, we investigated the antiproliferative effects and the mechanism of action of two compounds, 2,3,4′-trimethoxy-2′-hydroxy-chalcone (CH1) and 3′-bromo-3,4-dimethoxy-chalcone (CH2), over human hepatoma cells (HepG2 and Huh-7) and cultured mouse hepatocytes (HepM). Cytotoxic effects were observed over the HepG2 and Huh-7, and no effects were observed over the HepM. For HepG2 cells, treated separately with each chalcone, typical apoptotic laddering and nuclear condensation were observed. Additionally, the caspases and Bcl-2 family proteins activation by using Western blotting and immunocytochemistry were studied. Caspase-8 was not activated, but caspase-3 and -9 were both activated by chalcones in HepG2 cells. Chalcones also induced reactive oxygen species (ROS) accumulation after 4, 8 and 24 h of treatment in HepG2 cells. These results suggest that apoptosis in HepG2 was induced through: (i) a caspase-dependent intrinsic pathway; and (ii) by alterations in the cellular levels of Bcl-2 family proteins, and also, that the chalcone moiety could be a potent candidate as novel anticancer agents acting on human hepatomas. PMID:26907262

Diphenyl difluoroketone: a potent chemotherapy candidate for human hepatocellular carcinoma.

PubMed

Liang, Yingjian; Yin, Dalong; Hou, Limin; Zheng, Tongsen; Wang, Jiabei; Meng, Xianzhi; Lu, Zhaoyang; Song, Xuan; Pan, Shangha; Jiang, Hongchi; Liu, Lianxin

2011-01-01

Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, was recently reported to inhibit proliferation of various cancer cells significantly. Here we try to determine the effect and mechanism of EF24 on hepatocellular carcinoma. 2 µM EF24 was found to inhibit the proliferation of PLC/PRF/5, Hep3B, HepG2, SK-HEP-1 and Huh 7 cell lines. However, even 8 µM EF24 treatment did not affect the proliferation of normal liver LO2 cells. Accordingly, 20 mg/kg/d EF24 inhibited the growth of the tumor xenografts conspicuously while causing no apparent change in liver, spleen or body weight. In addition, significant apoptosis and G(2)/M phase cell cycle arrest were found using flow cytometry. Besides, caspases and PARP activation and features typical of apoptosis including fragmented nuclei with condensed chromatin were also observed. Furthermore, the mechanism was targeted at the reduction of nuclear factor kappa b (NF-κB) pathway and the NF-κB-regulated gene products Bcl-2, COX-2, Cyclin B1. Our study has offered a strategy that EF24 being a therapeutic agent for hepatocellular carcinoma.

Diphenyl Difluoroketone: A Potent Chemotherapy Candidate for Human Hepatocellular Carcinoma

PubMed Central

Hou, Limin; Zheng, Tongsen; Wang, Jiabei; Meng, Xianzhi; Lu, Zhaoyang; Song, Xuan; Pan, Shangha; Jiang, Hongchi; Liu, Lianxin

2011-01-01

Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, was recently reported to inhibit proliferation of various cancer cells significantly. Here we try to determine the effect and mechanism of EF24 on hepatocellular carcinoma. 2 µM EF24 was found to inhibit the proliferation of PLC/PRF/5, Hep3B, HepG2, SK-HEP-1 and Huh 7 cell lines. However, even 8 µM EF24 treatment did not affect the proliferation of normal liver LO2 cells. Accordingly, 20 mg/kg/d EF24 inhibited the growth of the tumor xenografts conspicuously while causing no apparent change in liver, spleen or body weight. In addition, significant apoptosis and G2/M phase cell cycle arrest were found using flow cytometry. Besides, caspases and PARP activation and features typical of apoptosis including fragmented nuclei with condensed chromatin were also observed. Furthermore, the mechanism was targeted at the reduction of nuclear factor kappa b (NF-κB) pathway and the NF-κB–regulated gene products Bcl-2, COX-2, Cyclin B1. Our study has offered a strategy that EF24 being a therapeutic agent for hepatocellular carcinoma. PMID:21901145

Phosphorylated Heat Shock Protein 20 (HSPB6) Regulates Transforming Growth Factor-α-Induced Migration and Invasion of Hepatocellular Carcinoma Cells.

PubMed

Matsushima-Nishiwaki, Rie; Toyoda, Hidenori; Nagasawa, Tomoaki; Yasuda, Eisuke; Chiba, Naokazu; Okuda, Seiji; Maeda, Atsuyuki; Kaneoka, Yuji; Kumada, Takashi; Kozawa, Osamu

2016-01-01

Human hepatocellular carcinoma (HCC) is one of the major malignancies in the world. Small heat shock proteins (HSPs) are reported to play an important role in the regulation of a variety of cancer cell functions, and the functions of small HSPs are regulated by post-translational modifications such as phosphorylation. We previously reported that protein levels of a small HSP, HSP20 (HSPB6), decrease in vascular invasion positive HCC compared with those in the negative vascular invasion. Therefore, in the present study, we investigated whether HSP20 is implicated in HCC cell migration and the invasion using human HCC-derived HuH7 cells. The transforming growth factor (TGF)-α-induced migration and invasion were suppressed in the wild-type-HSP20 overexpressed cells in which phosphorylated HSP20 was detected. Phospho-mimic-HSP20 overexpression reduced the migration and invasion compared with unphosphorylated HSP20 overexpression. Dibutyryl cAMP, which enhanced the phosphorylation of wild-type-HSP20, significantly reduced the TGF-α-induced cell migration of wild-type HSP20 overexpressed cells. The TGF-α-induced cell migration was inhibited by SP600125, a c-Jun N-terminal kinases (JNK) inhibitor. In phospho-mimic-HSP20 overexpressed HuH7 cells, TGF-α-stimulated JNK phosphorylation was suppressed compared with the unphosphorylated HSP20 overexpressed cells. Moreover, the level of phospho-HSP20 protein in human HCC tissues was significantly correlated with tumor invasion. Taken together, our findings strongly suggest that phosphorylated HSP20 inhibits TGF-α-induced HCC cell migration and invasion via suppression of the JNK signaling pathway.

In vitro evaluation of the Aurora kinase inhibitor VX-680 for Hepatoblastoma.

PubMed

Dewerth, Alexander; Wonner, Timo; Lieber, Justus; Ellerkamp, Verena; Warmann, Steven W; Fuchs, Jörg; Armeanu-Ebinger, Sorin

2012-06-01

Hepatoblastoma (HB) has a poor prognosis in advanced stages. The aim of this study was to enhance effectiveness of chemotherapy with antineoplastic kinase inhibitors. Viability was monitored in HB cells (HUH6, HepT1) in monolayer and spheroid cultures treated with kinase inhibitors VX-680, Wee1-InhibitorII, and SU11274 alone or in combination with cisplatin (CDDP) using MTT assays. Apoptosis was revealed by Caspase-3 assay. Western blot and immunohistochemical analyses were performed to determine histone H3 phosphorylation. Among the kinase inhibitors strongest anti-proliferative effect on HB cells was documented for VX-680. HUH6 cells responded more sensitively to the Aurora kinase inhibitor as HepT1 cells (IC(50) 8 and 16.6 μM, respectively). While VX-680 and CDDP showed no additive effects, the combination of VX-680 and histone deacetylase inhibitor SAHA had a synergistic effect on the proliferation of HUH6 cells. The inhibition with VX-680 led to reduced histone H3 phosphorylation, to an increase of apoptotic cells, and to morphological changes such as vacuolization and swelling of the cells and nuclei. The data provide evidence that VX-680 might improve treatment results in HB with increased Aurora kinase activity by inhibiting cell proliferation and induction of apoptosis.

HCV Genotype 6a Escape From and Resistance to Velpatasvir, Pibrentasvir, and Sofosbuvir in Robust Infectious Cell Culture Models.

PubMed

Pham, Long V; Ramirez, Santseharay; Gottwein, Judith M; Fahnøe, Ulrik; Li, Yi-Ping; Pedersen, Jannie; Bukh, Jens

2018-06-01

Chronic liver diseases caused by hepatitis C virus (HCV) genotype 6 are prevalent in Asia, and millions of people require treatment with direct-acting antiviral regimens, such as NS5A inhibitor velpatasvir combined with the NS5B polymerase inhibitor sofosbuvir. We developed infectious cell culture models of HCV genotype 6a infection to study the effects of these inhibitors and the development of resistance. The consensus sequences of strains HK2 (MG717925) and HK6a (MG717928), originating from serum of patients with chronic HCV infection, were determined by Sanger sequencing of genomes amplified by reverse-transcription polymerase chain reaction. In vitro noninfectious full-length clones of these 6a strains were subsequently adapted in Huh7.5 cells, primarily by using substitutions identified in JFH1-based Core-NS5A and Core-NS5B genotype 6a recombinants. We studied the efficacy of NS5A and NS5B inhibitors in concentration-response assays. We examined the effects of long-term culture of Huh7.5 cells incubated with velpatasvir and sofosbuvir singly or combined following infection with passaged full-length HK2 or HK6a recombinant viruses. Resistance-associated substitutions (RAS) were identified by Sanger and next-generation sequencing, and their effects on viral fitness and in drug susceptibility were determined in reverse-genetic experiments. Adapted full-length HCV genotype 6a recombinants HK2cc and HK6acc had fast propagation kinetics and high infectivity titers. Compared with an HCV genotype 1a recombinant, HCV genotype 6a recombinants of strains HK2 and HK6a were equally sensitive to daclatasvir, elbasvir, velpatasvir, pibrentasvir, and sofosbuvir, but less sensitive to ledipasvir, ombitasvir, and dasabuvir. Long-term exposure of HCV genotype 6a-infected Huh7.5 cells with a combination of velpatasvir and sofosbuvir resulted in clearance of the virus, but the virus escaped the effects of single inhibitors via emergence of the RAS L31V in NS5A (conferring resistance to velpatasvir) and S282T in NS5B (conferring resistance to sofosbuvir). Engineered recombinant genotype 6a viruses with single RAS mediated resistance to velpatasvir or sofosbuvir. HCV genotype 6a viruses with RAS NS5A-L31V or NS5B-S282T were however, able to propagate and escape in Huh7.5 cells exposed to the combination of velpatasvir and sofosbuvir. Further, HCV genotype 6a with NS5A-L31V was able to propagate and escape in the presence of pibrentasvir with emergence of NS5A-L28S, conferring a high level of resistance to this inhibitor. Strains of HCV genotype 6a isolated from patients can be adapted to propagate in cultured cells, permitting studies of the complete life cycle for this important genotype. The combination of velpatasvir and sofosbuvir is required to block propagation of original HCV genotype 6a, which quickly becomes resistant to single inhibitors via the rapid emergence and persistence of RAS. These features of HCV genotype 6a could compromise treatment. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Anti-hepatocarcinoma effects of berberine nanosuspension against human HepG2 and Huh7 cells as well as H22 tumor bearing mice

NASA Astrophysics Data System (ADS)

Wang, Zhi-ping; Wu, Jun-biao; Zhou, Qun; Wang, Yi-fei; Chen, Tongsheng

2014-09-01

Hepatocarcinoma, a malignant cancer, threaten human life badly. It is a current issue to seek the effective natural remedy from plant to treat cancer due to the resistance of the advanced hepatocarcinoma to chemotherapy. Berberine (Ber), an isoquinoline derivative alkaloid, has a wide range of pharmacological properties and is considered to have anti-hepatocarcinoma effects. However its low oral bioavailability restricts its wide application. In this report, Ber nanosuspension (Ber-NS) composed of Ber and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) was prepared by high pressure homogenization technique. Both in vitro and in vivo anti-hepatocarcinoma effects of Ber-NS relative to effcacy of bulk Ber were evaluated. The particle size and zeta potential of Ber-NS were 73.1 +/- 3.7 nm and 6.99 +/- 0.17 mV, respectively. Ber-NS exhibited significant inhibitory effects against human HepG2 and Huh7 cells, and the corresponding IC50 values were 8.1 and 4.7 μg/ml (18.3 and 6.5 μg/ml of Ber solution). In vivo studies also showed higher antitumor efficacy, and inhibition rates was 63.7% (41.4 % of Ber solution) at 100 mg/kg intragastric administration in the H22 solid tumor bearing mice. These results suggest that the delivery of Ber as a nanosuspension is a promising approach for treating hepatocarcinoma.

Azathioprine desensitizes liver cancer cells to insulin-like growth factor 1 and causes apoptosis when it is combined with bafilomycin A1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hernández-Breijo, Borja; Monserrat, Jorge; Román, Irene D.

Hepatoblastoma is a primary liver cancer that affects children, due to the sensitivity of this tumor to insulin-like growth factor 1 (IGF-1). In this paper we show that azathioprine (AZA) is capable of inhibiting IGF1-mediated signaling cascade in HepG2 cells. The efficiency of AZA on inhibition of proliferation differs in the evaluated cell lines as follows: HepG2 (an experimental model of hepatoblastoma) > Hep3B (derived from a hepatocellular carcinoma) > HuH6 (derived from a hepatoblastoma) ≫ HuH7 (derived from a hepatocellular carcinoma) = Chang Liver cells (a non-malignant cellular model). The effect of AZA in HepG2 cells has been provenmore » to derive from activation of Ras/ERK/TSC2, leading to activation of mTOR/p70S6K in a sustained manner. p70S6K phosphorylates IRS-1 in serine 307 which leads to the uncoupling between IRS-1 and p85 (the regulatory subunit of PI3K) and therefore causing the lack of response of HepG2 to IGF-1. As a consequence, proliferation induced by IGF-1 is inhibited by AZA and autophagy increases leading to senescence of HepG2 cells. Our results suggest that AZA induces the autophagic process in HepG2 activating senescence, and driving to deceleration of cell cycle but not to apoptosis. However, when simultaneous to AZA treatment the autophagy was inhibited by bafilomycin A1 and the degradation of regulatory proteins of cell cycle (e.g. Rb, E2F, and cyclin D1) provoked apoptosis. In conclusion, AZA induces resistance in hepatoblastoma cells to IGF-1, which leads to autophagy activation, and causes apoptosis when it is combined with bafilomycin A1. We are presenting here a novel mechanism of action of azathioprine, which could be useful in treatment of IGF-1 dependent tumors, especially in its combination with other drugs. - Highlights: • Azathioprine activated Ras/ERK/TSC-2/mTOR/p70S6K signaling pathway in HepG2 cells. • Azathioprine inhibited IGF-1-mediated signaling cascade. • Azathioprine induced autophagy leading to cell cycle arrest. • Cells died by apoptosis when azathioprine was combined with bafilomycin A1.« less

Rab1a regulates sorting of early endocytic vesicles

PubMed Central

Mukhopadhyay, Aparna; Quiroz, Jose A.

2014-01-01

We previously reported that Rab1a is associated with asialoorosomucoid (ASOR)-containing early endocytic vesicles, where it is required for their microtubule-based motility. In Rab1a knockdown (KD) cell lines, ASOR failed to segregate from its receptor and, consequently, did not reach lysosomes for degradation, indicating a defect in early endosome sorting. Although Rab1 is required for Golgi/endoplasmic reticulum trafficking, this process was unaffected, likely due to retained expression of Rab1b in these cells. The present study shows that Rab1a has a more general role in endocytic vesicle processing that extends to EGF and transferrin (Tfn) trafficking. Compared with results in control Huh7 cells, EGF accumulated in aggregates within Rab1a KD cells, failing to reach lysosomal compartments. Tfn, a prototypical example of recycling cargo, accumulated in a Rab11-mediated slow-recycling compartment in Rab1a KD cells, in contrast to control cells, which sort Tfn into a fast-recycling Rab4 compartment. These data indicate that Rab1a is an important regulator of early endosome sorting for multiple cargo species. The effectors and accessory proteins recruited by Rab1a to early endocytic vesicles include the minus-end-directed kinesin motor KifC1, while others remain to be discovered. PMID:24407591

[Effect of Hepatitis C virus proteins on the production of proinflammatory and profibrotic cytokines in Huh7.5 human hepatoma cells].

PubMed

Masalova, O V; Lesnova, E I; Permyakova, K Yu; Samokhvalov, E I; Ivanov, A V; Kochetkov, S N; Kushch, A A

2016-01-01

Hepatitis C virus (HCV) is a widespread dangerous human pathogen. Up to 80% of HCV-infected individuals develop chronic infection, which is often accompanied by liver inflammation and fibrosis and, at terminal stages, liver cirrhosis and cancer. Treatment of patients with end-stage liver disease is often ineffective, and even patients with suppressed HCV replication have higher risk of death as compared with noninfected subjects. Therefore, investigating the mechanisms that underlie HCV pathogenesis and developing treatments for virus-associated liver dysfunction remain an important goal. The effect of individual HCV proteins on the production of proinflammatory and profibrotic cytokines in hepatocellular carcinoma Huh7.5 cells was analyzed in a systematic manner. Cells were transfected with plasmids encoding HCV proteins. Cytokine production and secretion was accessed by immunocytochemistry and ELISA of the culture medium, and transcription of the cytokine genes was assessed using reverse transcription and PCR. HCV proteins proved to differ in effect on cytokine production. Downregulation of interleukin 6 (IL-6) production was observed in cells expressing the HCV core, NS3, and NS5A proteins. Production of transforming growth factor β1 (TGF-β1) was lower in cells expressing the core proteins, NS3, or E1/E2 glycoproteins. A pronounced increase in production and secretion of tumor necrosis factor α (TNF-α) was observed in response to expression of the HCV E1/E2 glycoproteins. A higher biosynthesis, but a lower level in the cell culture medium, was detected for interleukin 1β (IL-1β) in cells harboring NS4 and IL-6 in cells expressing NS5В. The finding was possibly explained by protein-specific retention and consequent accumulation of the respective cytokines in the cell.

LncRNA-uc002mbe.2 Interacting with hnRNPA2B1 Mediates AKT Deactivation and p21 Up-Regulation Induced by Trichostatin in Liver Cancer Cells.

PubMed

Chen, Ting; Gu, Chengxin; Xue, Cailin; Yang, Tao; Zhong, Yun; Liu, Shiming; Nie, Yuqiang; Yang, Hui

2017-01-01

Long non-coding RNAs (lncRNAs) have been implicated in liver carcinogenesis. We previously showed that the induction of lncRNA-uc002mbe.2 is positively associated with the apoptotic effect of trichostatin A (TSA) in hepatocellular carcinoma (HCC) cells. The current study further analyzed the role of uc002mbe.2 in TSA-induced liver cancer cell death. The level of uc002mbe.2 was markedly increased by TSA in the cytoplasm of HCC cells. Knockdown of uc002mbe.2 prohibited TSA-induced G2/M cell cycle arrest, p21 induction, and apoptosis of Huh7 cells and reversed the TSA-mediated decrease in p-AKT. RNA pull-down and RNA-binding protein immunoprecipitation (RIP) assays revealed that TSA induced an interaction between uc002mbe.2 and heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) in Huh7 cells. This interaction mediated AKT deactivation and p21 induction in liver cancer cells. In an athymic xenograft mouse model, knockdown of uc002mbe.2 significantly prohibited the TSA-mediated reduction in tumor size and weight. In addition, the ability of TSA to reduce hnRNPA2B1 and p-AKT levels and induce p21 in the xenograft tumors was prevented by uc002mbe.2 knockdown. Therefore, the interaction of uc002mbe.2 and hnRNPA2B1 in mediating AKT deactivation and p21 induction is involved in the cytostatic effect of trichostatin in liver cancer cells.

Sirtuin 3 enhanced drug sensitivity of human hepatoma cells through glutathione S-transferase pi 1/JNK signaling pathway

PubMed Central

Cai, Xue-Fei; Zhang, Wen-Lu; Ren, Ji-Hua; Zhou, Li; Chen, Xiang; Chen, Ke; Li, Wan-Yu; Liu, Bo; Yang, Qiu-Xia; Cheng, Sheng-Tao; Huang, Li-Xia; Huang, Ai-Long; Chen, Juan

2016-01-01

SIRT3, a class III histone deacetylase, has been implicated in various cancers as a novel therapeutic target. In hepatocellular carcinoma (HCC), we previously reported that SIRT3 induced cell apoptosis by regulating GSK-3β/Bax signaling pathway. Downregulation of SIRT3 in HCC cells facilitates tumor cell survival. In this study, we found that chemotherapeutic agents (doxorubicin, cisplatin and epirubicin) and sorafenib treatment downregulated SIRT3 mRNA and protein levels in three HCC cell lines. MTS assay found that SIRT3 overexpression sensitized liver cancer cells to chemotherapeutic agents and sorafenib in SMMC-7721, Huh-7 and PLC/PRF/5 cell lines. Moreover, SIRT3 overexpression promoted chemotherapeutic agents-induced or sorafenib-induced apoptosis as evidenced by flow cytometry, enhanced PARP cleavage and enhanced Caspase-9 cleavage in three HCC cells. In contrast, SIRT3 silencing increased drug resistance of HCC cells to chemotherapeutic agents. Mechanistic study found that SIRT3 downregulated the mRNA and protein levels of glutathione S-transferase pi 1 (GSTP1), which is a member of phase II detoxification enzymes families involved in metabolizing for chemotherapeutic agents. Moreover, SIRT3 decreased the amount of GSTP1 that was associated with JNK, which finally contributed the activation of JNK activity and activation of downstream target c-Jun and Bim. Importantly, GSTP1 overexpression or JNK inhibitor abolished SIRT3-induced apoptosis in HCC cells exposed to chemotherapeutic agents. Finally, there was a negative correlation between SIRT3 expression and GSTP1 expression in human HCC tissues. Together, our findings revealed SIRT3 could enhance the drug sensitivity of HCC cells to an array of chemotherapeutic agents. SIRT3 may serve as a potential target for improving the chemosensitivity of HCC patients. PMID:27367026

Sirtuin 3 enhanced drug sensitivity of human hepatoma cells through glutathione S-transferase pi 1/JNK signaling pathway.

PubMed

Tao, Na-Na; Zhou, Hong-Zhong; Tang, Hua; Cai, Xue-Fei; Zhang, Wen-Lu; Ren, Ji-Hua; Zhou, Li; Chen, Xiang; Chen, Ke; Li, Wan-Yu; Liu, Bo; Yang, Qiu-Xia; Cheng, Sheng-Tao; Huang, Li-Xia; Huang, Ai-Long; Chen, Juan

2016-08-02

SIRT3, a class III histone deacetylase, has been implicated in various cancers as a novel therapeutic target. In hepatocellular carcinoma (HCC), we previously reported that SIRT3 induced cell apoptosis by regulating GSK-3β/Bax signaling pathway. Downregulation of SIRT3 in HCC cells facilitates tumor cell survival. In this study, we found that chemotherapeutic agents (doxorubicin, cisplatin and epirubicin) and sorafenib treatment downregulated SIRT3 mRNA and protein levels in three HCC cell lines. MTS assay found that SIRT3 overexpression sensitized liver cancer cells to chemotherapeutic agents and sorafenib in SMMC-7721, Huh-7 and PLC/PRF/5 cell lines. Moreover, SIRT3 overexpression promoted chemotherapeutic agents-induced or sorafenib-induced apoptosis as evidenced by flow cytometry, enhanced PARP cleavage and enhanced Caspase-9 cleavage in three HCC cells. In contrast, SIRT3 silencing increased drug resistance of HCC cells to chemotherapeutic agents. Mechanistic study found that SIRT3 downregulated the mRNA and protein levels of glutathione S-transferase pi 1 (GSTP1), which is a member of phase II detoxification enzymes families involved in metabolizing for chemotherapeutic agents. Moreover, SIRT3 decreased the amount of GSTP1 that was associated with JNK, which finally contributed the activation of JNK activity and activation of downstream target c-Jun and Bim. Importantly, GSTP1 overexpression or JNK inhibitor abolished SIRT3-induced apoptosis in HCC cells exposed to chemotherapeutic agents. Finally, there was a negative correlation between SIRT3 expression and GSTP1 expression in human HCC tissues. Together, our findings revealed SIRT3 could enhance the drug sensitivity of HCC cells to an array of chemotherapeutic agents. SIRT3 may serve as a potential target for improving the chemosensitivity of HCC patients.

The interplay between hepatic stellate cells and hepatocytes in an in vitro model of NASH.

PubMed

Barbero-Becerra, Varenka J; Giraudi, Pablo J; Chávez-Tapia, Norberto C; Uribe, Misael; Tiribelli, Claudio; Rosso, Natalia

2015-10-01

A complex interplay exists between hepatocytes and hepatic stellate cells (HSC) in hepatic fibrogenesis. The activation of HSCs after liver injury leads to production of extracellular matrix (ECM). Co-culture models could be useful to mimic the liver microenvironment. This study evaluates the effect of free fatty acids (FFA) on HSC cells and the interplay with hepatocytes via both soluble-mediator and cell-cell contact. The human hepatocyte cell line (HuH7) and HSC cells (LX2) were exposed to FFA for 24 h in 3 different experimental set-ups: (A) monoculture of HSC; (B) Transwell® system (effect of soluble mediators); and (C) Simultaneous Co-Culture (SCC) (cell-to-cell connections). Intracellular FFA accumulation was assessed qualitatively (microscopy) and quantitatively (flow cytometry); the activation of HSC (alpha smooth muscle actin, α-SMA) expression of ECM components were quantified by RT-PCR. FFA exposure induces intracellular fat accumulation in all the experimental set-up but the expression of α-SMA was significantly increased only in SCC. On the contrary, the expression of ECM was substantially decreased in the transwell® system. The HSC activation is independent of FFA accumulation but requires cell-to-cell interaction with hepatocyte. On the contrary, the gene regulation of ECM components seems to occur through paracrine mediators. Copyright © 2015 Elsevier Ltd. All rights reserved.

Combined treatment with silibinin and either sorafenib or gefitinib enhances their growth-inhibiting effects in hepatocellular carcinoma cells

PubMed Central

Gu, Ha Ra; Choi, Su Jin; Lee, Jae Cheol; Kim, You Cheoul; Han, Chul Ju; Kim, Jin; Yang, Ki Young; Kim, Yeon Joo; Noh, Geum Youb; No, So Hyeon; Jeong, Jae-Hoon

2015-01-01

Background/Aims Silibinin, the main component of silymarin, is used as a hepatoprotectant and exhibits anticancer effects against various cancer cells. This study evaluated the effects of a combination of silibinin with either gefitinib or sorafenib on hepatocellular carcinoma (HCC) cells. Methods Several different human HCC cell lines were used to test the growth-inhibiting effects and cell toxicity of silibinin both alone and in combination with either gefitinib or sorafenib. The cell viability and growth inhibition were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue staining, and a colony-forming assay. Furthermore, changes in epidermal growth factor receptor (EGFR)-related signals were evaluated by Western blot analysis. Results Gefitinib, sorafenib, and silibinin individually exhibited dose-dependent antiproliferative effects on HCC cells. Combined treatment with silibinin enhanced the gefitinib-induced growth-inhibiting effects in some HCC cell lines. The combination effect of gefitinib and silibinin was synergistic in the SNU761 cell line, but was only additive in the Huh-BAT cell line. The combination effect may be attributable to inhibition of EGFR-dependent Akt signaling. Enhanced growth-inhibiting effects were also observed in HCC cells treated with a combination of sorafenib and silibinin. Conclusions Combined treatment with silibinin enhanced the growth-inhibiting effects of both gefitinib and sorafenib. Therefore, the combination of silibinin with either sorafenib or gefitinib could be a useful treatment approach for HCC in the future. PMID:25834802

Combined treatment with silibinin and either sorafenib or gefitinib enhances their growth-inhibiting effects in hepatocellular carcinoma cells.

PubMed

Gu, Ha Ra; Park, Su Cheol; Choi, Su Jin; Lee, Jae Cheol; Kim, You Cheoul; Han, Chul Ju; Kim, Jin; Yang, Ki Young; Kim, Yeon Joo; Noh, Geum Youb; No, So Hyeon; Jeong, Jae-Hoon

2015-03-01

Silibinin, the main component of silymarin, is used as a hepatoprotectant and exhibits anticancer effects against various cancer cells. This study evaluated the effects of a combination of silibinin with either gefitinib or sorafenib on hepatocellular carcinoma (HCC) cells. Several different human HCC cell lines were used to test the growth-inhibiting effects and cell toxicity of silibinin both alone and in combination with either gefitinib or sorafenib. The cell viability and growth inhibition were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue staining, and a colony-forming assay. Furthermore, changes in epidermal growth factor receptor (EGFR)-related signals were evaluated by Western blot analysis. Gefitinib, sorafenib, and silibinin individually exhibited dose-dependent antiproliferative effects on HCC cells. Combined treatment with silibinin enhanced the gefitinib-induced growth-inhibiting effects in some HCC cell lines. The combination effect of gefitinib and silibinin was synergistic in the SNU761 cell line, but was only additive in the Huh-BAT cell line. The combination effect may be attributable to inhibition of EGFR-dependent Akt signaling. Enhanced growth-inhibiting effects were also observed in HCC cells treated with a combination of sorafenib and silibinin. Combined treatment with silibinin enhanced the growth-inhibiting effects of both gefitinib and sorafenib. Therefore, the combination of silibinin with either sorafenib or gefitinib could be a useful treatment approach for HCC in the future.

Synthesis and biological evaluation of some novel pyrido[1,2-a]pyrimidin-4-ones as antimalarial agents.

PubMed

Mane, Uttam R; Mohanakrishnan, D; Sahal, Dinkar; Murumkar, Prashant R; Giridhar, Rajani; Yadav, Mange Ram

2014-05-22

Novel pyrido[1,2-a]pyrimidin-4-ones have been synthesized and evaluated for their antimalarial activity by SYBR Green I assay against erythrocytic stages of chloroquine (CQ) sensitive Pf 3D7 strain. The antimalarial screening of 42 different compounds revealed that 3-Fluorobenzyl(4-oxo-4H-pyrido [1,2-a]pyrimidin-3-yl)carbamate (21, IC50 value 33 μM) and 4-Oxo-N-[4-(trifluoromethyl)benzyl]-4H-pyrido[1,2-a]pyrimidine-3-carboxamide (37, IC50 value 37 μM) showed moderate antimalarial activity. Cytotoxicity study was performed against mammalian cell line (Huh-7) by using the MTT assay for the moderately active compounds. Structural activity relationship (SAR) studies displayed that B-ring unsubstituted pyrido[1,2-a]pyrimidine scaffold is responsible for the antimalarial activities of the evaluated derivatives. This SAR based antimalarial screening supported that pyrido[1,2-a]pyrimidin-4-one can be considered as a lead heterocyclic structure for further development of more potent derivatives for antimalarial activity. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

A locked nucleic acid antisense oligonucleotide (LNA) silences PCSK9 and enhances LDLR expression in vitro and in vivo.

PubMed

Gupta, Nidhi; Fisker, Niels; Asselin, Marie-Claude; Lindholm, Marie; Rosenbohm, Christoph; Ørum, Henrik; Elmén, Joacim; Seidah, Nabil G; Straarup, Ellen Marie

2010-05-17

The proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in the etiology of familial hypercholesterolemia (FH) and is also an attractive therapeutic target to reduce low density lipoprotein (LDL) cholesterol. PCSK9 accelerates the degradation of hepatic low density lipoprotein receptor (LDLR) and low levels of hepatic PCSK9 activity are associated with reduced levels of circulating LDL-cholesterol. The present study presents the first evidence for the efficacy of a locked nucleic acid (LNA) antisense oligonucleotide (LNA ASO) that targets both human and mouse PCSK9. We employed human hepatocytes derived cell lines HepG2 and HuH7 and a pancreatic mouse beta-TC3 cell line known to express high endogenous levels of PCSK9. LNA ASO efficiently reduced the mRNA and protein levels of PCSK9 with a concomitant increase in LDLR protein levels after transfection in these cells. In vivo efficacy of LNA ASO was further investigated in mice by tail vein intravenous administration of LNA ASO in saline solution. The level of PCSK9 mRNA was reduced by approximately 60%, an effect lasting more than 16 days. Hepatic LDLR protein levels were significantly up-regulated by 2.5-3 folds for at least 8 days and approximately 2 fold for 16 days. Finally, measurement of liver alanine aminotransferase (ALT) levels revealed that long term LNA ASO treatment (7 weeks) does not cause hepatotoxicity. LNA-mediated PCSK9 mRNA inhibition displayed potent reduction of PCSK9 in cell lines and mouse liver. Our data clearly revealed the efficacy and safety of LNA ASO in reducing PCSK9 levels, an approach that is now ready for testing in primates. The major significance and take home message of this work is the development of a novel and promising approach for human therapeutic intervention of the PCSK9 pathway and hence for reducing some of the cardiovascular risk factors associated with the metabolic syndrome.

Nardilysin promotes hepatocellular carcinoma through activation of signal transducer and activator of transcription 3.

PubMed

Kasai, Yosuke; Toriguchi, Kan; Hatano, Etsuro; Nishi, Kiyoto; Ohno, Mikiko; Yoh, Tomoaki; Fukuyama, Keita; Nishio, Takahiro; Okuno, Masayuki; Iwaisako, Keiko; Seo, Satoru; Taura, Kojiro; Kurokawa, Masato; Kunichika, Makoto; Uemoto, Shinji; Nishi, Eiichiro

2017-05-01

Nardilysin (NRDC) is a metalloendopeptidase of the M16 family. We previously showed that NRDC activates inflammatory cytokine signaling, including interleukin-6-signal transducer and activator of transcription 3 (STAT3) signaling. NRDC has been implicated in the promotion of breast, gastric and esophageal cancer, as well as the development of liver fibrosis. In this study, we investigated the role of NRDC in the promotion of hepatocellular carcinoma (HCC), both clinically and experimentally. We found that NRDC expression was upregulated threefold in HCC tissue compared to the adjacent non-tumor liver tissue, which was confirmed by immunohistochemistry and western blotting. We also found that high serum NRDC was associated with large tumor size (>3 cm, P = 0.016) and poor prognosis after hepatectomy (median survival time 32.0 vs 73.9 months, P = 0.003) in patients with hepatitis C (n = 120). Diethylnitrosamine-induced hepatocarcinogenesis was suppressed in heterozygous NRDC-deficient mice compared to their wild-type littermates. Gene silencing of NRDC with miRNA diminished the growth of Huh-7 and Hep3B spheroids in vitro. Notably, phosphorylation of STAT3 was decreased in NRDC-depleted Huh-7 spheroids compared to control spheroids. The effect of a STAT3 inhibitor (S3I-201) on the growth of Huh-7 spheroids was reduced in NRDC-depleted cells relative to controls. Our results show that NRDC is a promising prognostic marker for HCC in patients with hepatitis C, and that NRDC promotes tumor growth through activation of STAT3. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Tannic Acid Inhibits Hepatitis C Virus Entry into Huh7.5 Cells

PubMed Central

Hagedorn, Curt H.

2015-01-01

Chronic infection with the hepatitis C virus (HCV) is a cause of cirrhosis and hepatocellular carcinoma worldwide. Although antiviral therapy has dramatically improved recently, a number of patients remain untreated and some do not clear infection with treatment. Viral entry is an essential step in initiating and maintaining chronic HCV infections. One dramatic example of this is the nearly 100% infection of newly transplanted livers in patients with chronic hepatitis C. HCV entry inhibitors could play a critical role in preventing HCV infection of newly transplanted livers. Tannic acid, a polymer of gallic acid and glucose molecules, is a plant-derived polyphenol that defends some plants from insects and microbial infections. It has been shown to have a variety of biological effects, including antiviral activity, and is used as a flavoring agent in foods and beverages. In this study, we demonstrate that tannic acid is a potent inhibitor of HCV entry into Huh7.5 cells at low concentrations (IC50 5.8 μM). It also blocks cell-to-cell spread in infectious HCV cell cultures, but does not inhibit HCV replication following infection. Moreover, experimental results indicate that tannic acid inhibits an early step of viral entry, such as the docking of HCV at the cell surface. Gallic acid, tannic acid’s structural component, did not show any anti-HCV activity including inhibition of HCV entry or replication at concentrations up to 25 μM. It is possible the tannin structure is related on the effect on HCV inhibition. Tannic acid, which is widely distributed in plants and foods, has HCV antiviral activity in cell culture at low micromolar concentrations, may provide a relative inexpensive adjuvant to direct-acting HCV antivirals and warrants future investigation. PMID:26186636

Release of Infectious Hepatitis C Virus from Huh7 Cells Occurs via a trans-Golgi Network-to-Endosome Pathway Independent of Very-Low-Density Lipoprotein Secretion

PubMed Central

Mankouri, Jamel; Walter, Cheryl; Stewart, Hazel; Bentham, Matthew; Park, Wei Sun; Heo, Won Do; Fukuda, Mitsunori

2016-01-01

ABSTRACT The release of infectious hepatitis C virus (HCV) particles from infected cells remains poorly characterized. We previously demonstrated that virus release is dependent on the endosomal sorting complex required for transport (ESCRT). Here, we show a critical role of trans-Golgi network (TGN)-endosome trafficking during the assembly, but principally the secretion, of infectious virus. This was demonstrated by both small interfering RNA (siRNA)-mediated silencing of TGN-associated adaptor proteins and a panel of dominant negative (DN) Rab GTPases involved in TGN-endosome trafficking steps. Importantly, interfering with factors critical for HCV release did not have a concomitant effect on secretion of triglycerides, ApoB, or ApoE, indicating that particles are likely released from Huh7 cells via pathways distinct from that of very-low-density lipoprotein (VLDL). Finally, we show that HCV NS2 perturbs TGN architecture, redistributing TGN membranes to closely associate with HCV core protein residing on lipid droplets. These findings support the notion that HCV hijacks TGN-endosome trafficking to facilitate particle assembly and release. Moreover, although essential for assembly and infectivity, the trafficking of mature virions is seemingly independent of host lipoproteins. IMPORTANCE The mechanisms by which infectious hepatitis C virus particles are assembled and released from the cell are poorly understood. We show that the virus subverts host cell trafficking pathways to effect the release of virus particles and disrupts the structure of the Golgi apparatus, a key cellular organelle involved in secretion. In addition, we demonstrate that the mechanisms used by the virus to exit the cell are distinct from those used by the cell to release lipoproteins, suggesting that the virus effects a unique modification to cellular trafficking pathways. PMID:27226379

Arylacetamide deacetylase: a novel host factor with important roles in the lipolysis of cellular triacylglycerol stores, VLDL assembly and HCV production.

PubMed

Nourbakhsh, Mahra; Douglas, Donna N; Pu, Christopher Hao; Lewis, Jamie T; Kawahara, Toshiyasu; Lisboa, Luiz F; Wei, Enhui; Asthana, Sonal; Quiroga, Ariel D; Law, Lok Man John; Chen, Chao; Addison, William R; Nelson, Randy; Houghton, Michael; Lehner, Richard; Kneteman, Norman M

2013-08-01

Very low density lipoproteins (VLDLs) are triacylglycerol (TG)-rich lipoproteins produced by the human liver. VLDLs derive the majority of their TG cargo from the lipolysis of TG stored in hepatocellular lipid droplets (LDs). Important roles for LDs and the VLDL secretory pathway in the cell culture production of infectious hepatitis C virus (HCV) have been established. We hypothesized that TG lipolysis and VLDL production are impaired during HCV infection so that these cellular processes can be diverted towards HCV production. We used an HCV permissive cell culture system (JFH-1/HuH7.5 cells) to examine the relationship between TG lipolysis, VLDL assembly, and the HCV lifecycle using standard biochemical approaches. Lipolysis of cellular TG and VLDL production were impaired in HCV infected cells during the early peak of viral infection. This was partially explained by an apparent deficiency of a putative TG lipase, arylacetamide deacetylase (AADAC). The re-introduction of AADAC to infected cells restored cellular TG lipolysis, indicating a role for HCV-mediated downregulation of AADAC in this process. Defective lipolysis of cellular TG stores and VLDL production were also observed in HuH7.5 cells stably expressing a short hairpin RNA targeting AADAC expression, proving AADAC deficiency contributes to these defective pathways. Finally, impaired production of HCV was observed with AADAC knockdown cells, demonstrating a role for AADAC in the HCV lifecycle. This insight into the biology of HCV infection and possibly pathogenesis identifies AADAC as a novel and translationally relevant therapeutic target. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

MicroRNA-9 enhances sensitivity to cetuximab in epithelial phenotype hepatocellular carcinoma cells through regulation of the eukaryotic translation initiation factor 5A-2.

PubMed

Xue, Fei; Liang, Yuntian; Li, Zhenrong; Liu, Yanhui; Zhang, Hongwei; Wen, Yu; Yan, Lei; Tang, Qiang; Xiao, Erhui; Zhang, Dongyi

2018-01-01

Hepatocellular carcinoma (HCC) is one of the most widespread malignant human tumors worldwide. Treatment options include radiotherapy, surgical intervention and chemotherapy; however, drug resistance is an ongoing treatment concern. In the present study, the effects of a microRNA (miR/miRNA), miR-9, on the sensitivity of HCC cell lines to the epidermal growth factor receptor inhibitor, cetuximab, were examined. miR-9 has been proposed to serve a role in tumorigenesis and tumor progression. In the present study, bioinformatics analyses identified the eukaryotic translation initiation factor 5A2 (eIF-5A-2) as a target of miR-9. The expression levels of miR-9 and eIF-5A-2 were examined by reverse transcription-quantitative polymerase chain reaction and HCC cell lines were transfected with miR-9 mimics and inhibitors to determine the effects of the miRNA on cell proliferation and viability. The miR-9 mimic was revealed to significantly increase the sensitivity of epithelial phenotype HCC cells (Hep3B and Huh7) to cetuximab, while the miR-9 inhibitor triggered the opposite effect. There were no significant differences in sensitivity to cetuximab observed in mesenchymal phenotype HCC cells (SNU387 and SNU449). Cells lines displaying high expression levels of eIF-5A-2 were more resistant to cetuximab. Transfection of cells with a miR-9 mimic resulted in downregulation of the expression of eIF-5A-2 mRNA, while an miR-9 inhibitor increased expression. When expression of eIF-5A-2 was knocked down with siRNA, the effects of miR-9 on cetuximab sensitivity were no longer observed. Taken together, these data support a role for miR-9 in enhancing the sensitivity of epithelial phenotype HCC cells to cetuximab through regulation of eIF-5A-2.

Targeting Phosphatidylinositide3-Kinase/Akt pathway by BKM120 for radiosensitization in hepatocellular carcinoma

PubMed Central

Liu, Wei-Lin; Gao, Ming; Tzen, Kai-Yuan; Tsai, Chiao-Ling; Hsu, Feng-Ming; Cheng, Ann-Lii; Cheng, Jason Chia-Hsien

2014-01-01

Tumor control of hepatocellular carcinoma by radiotherapy remains unsatisfactory. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a critical role in inhibiting cancer cell death. Elevated PI3K/Akt activity is associated with increased cellular resistance to irradiation. Our aim was to determine whether the inhibition of PI3K/Akt activity by a PI3K inhibitor, BKM120, contributes to the increased sensitivity of liver cancer cells to irradiation. The hepatocellular carcinoma cell lines (Huh7 and BNL) were used to evaluate the in vitro synergism between BKM120 and irradiation. Balb/c mice bearing ectopic BNL xenografts were treated with BKM120 and/or radiotherapy to assess the in vivo response. BKM120 increased cell killing by radiation, increased the expression of apoptotic markers, and suppressed the repair of radiation-induced DNA double-strand breaks. BKM120 pretreatment inhibited radiation-induced Akt phosphorylation and enhanced the tumor-suppressive effect and radiation-induced tumor cell apoptosis in ectopic xenografts. Inhibition of mTOR phosphorylation by rapamycin enhanced the radiosensitivity of BKM120-treated hepatocellular carcinoma cells. The synergism between BKM120 and irradiation likely inhibits the activation of Akt by radiation, leading to increased cell apoptosis and suppression of DNA-double-strand breaks repair in hepatocellular carcinoma cells. These data suggest that the BKM120/radiation combination may be a strategy worthy of clinical trials. PMID:25004403

Potential of Equine Herpesvirus 1 as a Vector for Immunization

PubMed Central

Trapp, Sascha; von Einem, Jens; Hofmann, Helga; Köstler, Josef; Wild, Jens; Wagner, Ralf; Beer, Martin; Osterrieder, Nikolaus

2005-01-01

Key problems using viral vectors for vaccination and gene therapy are antivector immunity, low transduction efficiencies, acute toxicity, and limited capacity to package foreign genetic information. It could be demonstrated that animal and human cells were efficiently transduced with equine herpesvirus 1 (EHV-1) reconstituted from viral DNA maintained and manipulated in Escherichia coli. Between 13 and 23% of primary human CD3+, CD4+, CD8+, CD11b+, and CD19+ cells and more than 70% of CD4+ MT4 cells or various human tumor cell lines (MeWo, Huh7, HeLa, 293T, or H1299) could be transduced with one infectious unit of EHV-1 per cell. After intranasal instillation of EHV-1 into mice, efficient transgene expression in lungs was detectable. Successful immunization using EHV-1 was shown after delivery of the human immunodeficiency virus type 1 Pr55gag precursor by the induction of a Gag-specific CD8+ immune response in mice. Because EHV-1 was not neutralized by human sera containing high titers of antibodies directed against human herpesviruses 1 to 5, it is concluded that this animal herpesvirus has enormous potential as a vaccine vector, because it is able to efficiently transduce a variety of animal and human cells, has high DNA packaging capacity, and can conveniently be maintained and manipulated in prokaryotic cells. PMID:15827159

Antituberculosis compounds from a deep-sea-derived fungus Aspergillus sp. SCSIO Ind09F01.

PubMed

Luo, Xiaowei; Zhou, Xuefeng; Lin, Xiuping; Qin, Xiaochu; Zhang, Tianyu; Wang, Junfeng; Tu, Zhengchao; Yang, Bin; Liao, Shengrong; Tian, Yongqi; Pang, Xiaoyan; Kaliyaperumal, Kumaravel; Li, Jian Lin; Tao, Huaming; Liu, Yonghong

2017-08-01

Eleven diketopiperazine and fumiquinazoline alkaloids (1-11) together with a tetracyclic triterpenoid helvolic acid (12) were obtained from the cultures of a deep-sea derived fungus Aspergillus sp. SCSIO Ind09F01. The structures of these compounds (1-12) were determined mainly by the extensive NMR, ESIMS spectra data and by comparison with previously described compounds. Besides, anti-tuberculosis, cytotoxic, antibacterial, COX-2 inhibitory and antiviral activities of these compounds were evaluated. Gliotoxin (3), 12,13-dihydroxy-fumitremorgin C (11) and helvolic acid (12) exhibited very strong anti-tuberculosis activity towards Mycobacterium tuberculosis with the prominent MIC 50 values of <0.03, 2.41 and 0.894 μM, respectively, which was here reported for the first time. Meanwhile gliotoxin also displayed significant selective cytotoxicities against K562, A549 and Huh-7 cell lines with the IC 50 values of 0.191, 0.015 and 95.4 μM, respectively.

Low-dose chemotherapy of hepatocellular carcinoma through triggered-release from bilayer-decorated magnetoliposomes.

PubMed

Chen, Yanjing; Chen, Yuan; Xiao, Da; Bose, Arijit; Deng, Ruitang; Bothun, Geoffrey D

2014-04-01

Low-dose (LD) chemotherapy is a promising treatment strategy that may be improved by controlled delivery. Polyethylene glycol-stabilized bilayer-decorated magnetoliposomes (dMLs) have been designed as a stimuli-responsive LD chemotherapy drug delivery system and tested in vitro using Huh-7 hepatocellular carcinoma cell line. The dMLs contained hydrophobic superparamagnetic iron oxide nanoparticles within the lipid bilayer and doxorubicin hydrochloride (DOX, 2 μM) within the aqueous core. Structural analysis by cryogenic transmission electron microscopy and dynamic light scattering showed that the assemblies were approximately 120 nm in diameter. Furthermore, the samples consisted of a mixture of dMLs and bare liposomes (no nanoparticles), which provided dual burst and spontaneous DOX release profiles, respectively. Cell viability results show that the cytotoxicity of DOX-loaded dMLs was similar to that of bare dMLs (∼10%), which indicates that spontaneous DOX leakage had little cytotoxic effect. However, when subjected to a physiologically acceptable radiofrequency (RF) electromagnetic field, cell viability was reduced up to 40% after 8h and significant cell death (>90%) was observed after 24h. The therapeutic mechanism was intracellular RF-triggered DOX release from the dMLs and not intracellular hyperthermia due to nanoparticle heating via magnetic losses. Copyright © 2014 Elsevier B.V. All rights reserved.

Low-Dose Chemotherapy of Hepatocellular Carcinoma through Triggered-Release from Bilayer-Decorated Magnetoliposomes

PubMed Central

Chen, Yanjing; Chen, Yuan; Xiao, Da; Bose, Arijit; Deng, Ruitang; Bothun, Geoffrey D.

2014-01-01

Low-dose (LD) chemotherapy is a promising treatment strategy that may be improved by controlled delivery. Polyethylene glycol-stabilized bilayer-decorated magnetoliposomes (dMLs) have been designed as a stimuli-responsive LD chemotherapy drug delivery system and tested in vitro using Huh-7 hepatocellular carcinoma cell line. The dMLs contained hydrophobic superparamagnetic iron oxide nanoparticles within the lipid bilayer and doxorubicin hydrochloride (DOX, 2 µM) within the aqueous core. Structural analysis by cryogenic transmission electron microscopy and dynamic light scattering showed that the assemblies were approximately 120 nm in diameter. Furthermore, the samples consisted of a mixture of dMLs and bare liposomes (no nanoparticles), which provided dual burst and spontaneous DOX release profiles, respectively. Cell viability results show that the cytotoxicity of DOX-loaded dMLs was similar to that of bare dMLs (~10%), which indicates that spontaneous DOX leakage had little cytotoxic effect. However, when subjected to a physiologically acceptable radiofrequency (RF) electromagnetic field, cell viability was reduced up to 40% after 8 h and complete cell death was observed after 24 h. The therapeutic mechanism was intracellular RF-triggered DOX release from the dMLs and not intracellular hyperthermia due to nanoparticle heating via magnetic losses. PMID:24549047

Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

PubMed Central

Gomes, Rafael G. B.; da Silva, Camila T.; Taniguchi, Juliana B.; No, Joo Hwan; Lombardot, Benoit; Schwartz, Olivier; Hansen, Michael A. E.; Freitas-Junior, Lucio H.

2013-01-01

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity - inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis. PMID:24205414

Down-regulation of Transducin-Like Enhancer of Split protein 4 in hepatocellular carcinoma promotes cell proliferation and epithelial-Mesenchymal-Transition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Xiao-cai; Xiao, Cui-cui; Li, Hua

Background: Transducin-Like Enhancer of Split protein 4 (TLE4) has been reported to be involved in some subsets of acute myeloid leukemia and colorectal cancer. In the present study, we aimed to explore the role of TLE4 in tumorigenesis and cancer progression in hepatocellular carcinoma (HCC). Methods: The expression pattern of TLE4 in HCC was determined by Western-blot and qRT-PCR, gain-of-function and loss-of-function was used to explore the biological role of TLE4 in HCC cells. A xenograft model was established to confirm its effects on proliferation. Results: The protein expression levels of TLE4 were significantly down-regulated in HCC tissues compared tomore » matched adjacent normal liver tissues. In vitro, down-regulation of TLE4 in Huh7 or SMMC-7721 promoted cell proliferation and ectopical expression of TLE4 in Hep3B or Bel-7404 suppressed cell proliferation. In addition, the cell colony formation ability was enhanced after down-regulation of TLE4 expression in Huh-7 but suppressed after over-expression in Hep3B. Furthermore, down-regulation of TLE4 increased the cell invasion ability, as well as increased the expression level of Vimentin and decreased that of E-cadherin, indicating a phenotype of epithelial-mesenchymal transition (EMT) in HCC cells. On the contrary, ectopical expression of TLE4 in HCC cells decreased the cell invasion ability and inhibited EMT. In vivo, compared to control group, xenograft tumor volumes were significantly decreased in TLE4 overexpression group. Conclusions: These results demonstrated that TLE4 might play important regulatory roles in cellular proliferation and EMT process in HCC. - Highlights: • TLE4 is significantly down-regulated in HCC samples. • Down regulated of TLE4 in HCC cells promotes cell proliferation. • Down regulated of TLE4 in HCC cells promotes epithelial-to-mesenchymal transition.« less

The Combination of Arginine Deprivation and 5-Fluorouracil Improves Therapeutic Efficacy in Argininosuccinate Synthetase Negative Hepatocellular Carcinoma

PubMed Central

Thongkum, Angkana; Wu, Chunjing; Li, Ying-Ying; Wangpaichitr, Medhi; Navasumrit, Panida; Parnlob, Varabhorn; Sricharunrat, Thaniya; Bhudhisawasdi, Vajarabhongsa; Ruchirawat, Mathuros; Savaraj, Niramol

2017-01-01

Argininosuccinate synthetase (ASS), a key enzyme to synthesize arginine is down regulated in many tumors including hepatocellular carcinoma (HCC). Similar to previous reports, we have found the decrease in ASS expression in poorly differentiated HCC. These ASS(-) tumors are auxotrophic for arginine. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine, has shown activity in these tumors, but the antitumor effect is not robust and hence combination treatment is needed. Herein, we have elucidated the effectiveness of ADI-PEG20 combined with 5-Fluorouracil (5-FU) in ASS(-)HCC by targeting urea cycle and pyrimidine metabolism using four HCC cell lines as model. SNU398 and SNU387 express very low levels of ASS or ASS(-) while Huh-1, and HepG2 express high ASS similar to normal cells. Our results showed that the augmented cytotoxic effect of combination treatment only occurs in SNU398 and SNU387, and not in HepG2 and Huh-1 (ASS(+)) cells, and is partly due to reduced anti-apoptotic proteins X-linked inhibitor of apoptosis protein (XIAP), myeloid leukemia cell differentiation protein (Mcl-1) and B-cell lymphoma-2 (Bcl-2). Importantly, lack of ASS also influences essential enzymes in pyrimidine synthesis (carbamoyl-phosphate synthetase2, aspartate transcarbamylase and dihydrooratase (CAD) and thymidylate synthase (TS)) and malate dehydrogenase-1 (MDH-1) in TCA cycle. ADI-PEG20 treatment decreased these enzymes and made them more vulnerable to 5-FU. Transfection of ASS restored these enzymes and abolished the sensitivity to ADI-PEG20 and combination treatment. Overall, our data suggest that ASS influences multiple enzymes involved in 5-FU sensitivity. Combining ADI-PEG20 and 5-FU may be effective to treat ASS(-)hepatoma and warrants further clinical investigation. PMID:28587170

The Combination of Arginine Deprivation and 5-Fluorouracil Improves Therapeutic Efficacy in Argininosuccinate Synthetase Negative Hepatocellular Carcinoma.

PubMed

Thongkum, Angkana; Wu, Chunjing; Li, Ying-Ying; Wangpaichitr, Medhi; Navasumrit, Panida; Parnlob, Varabhorn; Sricharunrat, Thaniya; Bhudhisawasdi, Vajarabhongsa; Ruchirawat, Mathuros; Savaraj, Niramol

2017-06-01

Argininosuccinate synthetase (ASS), a key enzyme to synthesize arginine is down regulated in many tumors including hepatocellular carcinoma (HCC). Similar to previous reports, we have found the decrease in ASS expression in poorly differentiated HCC. These ASS(-) tumors are auxotrophic for arginine. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine, has shown activity in these tumors, but the antitumor effect is not robust and hence combination treatment is needed. Herein, we have elucidated the effectiveness of ADI-PEG20 combined with 5-Fluorouracil (5-FU) in ASS(-)HCC by targeting urea cycle and pyrimidine metabolism using four HCC cell lines as model. SNU398 and SNU387 express very low levels of ASS or ASS(-) while Huh-1, and HepG2 express high ASS similar to normal cells. Our results showed that the augmented cytotoxic effect of combination treatment only occurs in SNU398 and SNU387, and not in HepG2 and Huh-1 (ASS(+)) cells, and is partly due to reduced anti-apoptotic proteins X-linked inhibitor of apoptosis protein (XIAP), myeloid leukemia cell differentiation protein (Mcl-1) and B-cell lymphoma-2 (Bcl-2). Importantly, lack of ASS also influences essential enzymes in pyrimidine synthesis (carbamoyl-phosphate synthetase2, aspartate transcarbamylase and dihydrooratase (CAD) and thymidylate synthase (TS)) and malate dehydrogenase-1 (MDH-1) in TCA cycle. ADI-PEG20 treatment decreased these enzymes and made them more vulnerable to 5-FU. Transfection of ASS restored these enzymes and abolished the sensitivity to ADI-PEG20 and combination treatment. Overall, our data suggest that ASS influences multiple enzymes involved in 5-FU sensitivity. Combining ADI-PEG20 and 5-FU may be effective to treat ASS(-)hepatoma and warrants further clinical investigation.

A microfluidic co-culture system to monitor tumor-stromal interactions on a chip

PubMed Central

Menon, Nishanth V.; Cao, Bin; Lim, Mayasari; Kang, Yuejun

2014-01-01

The living cells are arranged in a complex natural environment wherein they interact with extracellular matrix and other neighboring cells. Cell-cell interactions, especially those between distinct phenotypes, have attracted particular interest due to the significant physiological relevance they can reveal for both fundamental and applied biomedical research. To study cell-cell interactions, it is necessary to develop co-culture systems, where different cell types can be cultured within the same confined space. Although the current advancement in lab-on-a-chip technology has allowed the creation of in vitro models to mimic the complexity of in vivo environment, it is still rather challenging to create such co-culture systems for easy control of different colonies of cells. In this paper, we have demonstrated a straightforward method for the development of an on-chip co-culture system. It involves a series of steps to selectively change the surface property for discriminative cell seeding and to induce cellular interaction in a co-culture region. Bone marrow stromal cells (HS5) and a liver tumor cell line (HuH7) have been used to demonstrate this co-culture model. The cell migration and cellular interaction have been analyzed using microscopy and biochemical assays. This co-culture system could be used as a disease model to obtain biological insight of pathological progression, as well as a tool to evaluate the efficacy of different drugs for pharmaceutical studies. PMID:25553194

Silencing the Girdin gene enhances radio-sensitivity of hepatocellular carcinoma via suppression of glycolytic metabolism.

PubMed

Yu, Li; Sun, Yifan; Li, Jingjing; Wang, Yan; Zhu, Yuxing; Shi, Yong; Fan, Xiaojun; Zhou, Jianda; Bao, Ying; Xiao, Jie; Cao, Ke; Cao, Peiguo

2017-08-15

Radiotherapy has been used increasingly to treat primary hepatocellular carcinoma. Clinically, the main cause of radiotherapy failure is cellular radioresistance, conferred via glycolytic metabolism. Our previous study demonstrated that Girdin is upregulated in primary hepatocellular carcinoma and promotes the invasion and metastasis of tumor cells. However, whether Girdin underlies the radio-sensitivity of hepatocellular carcinoma remains unclear. A short hairpin RNA (shRNA) was used to silence CCDC88A (encoding Girdin), and real-time PCR was performed to determine CCDC88A mRNA expression. Then, cell proliferation, colony formation, flow cytometric, scratch, and transwell assays were to examine the influence of Girdin silencing on cellular radiosensitivity. Glycolysis assays were conducted to exam cell glycolysis process. Western blotting was performed to explore the signaling pathway downstream of Girdin. Finally, animal experiments were performed to demonstrate the effect of CCDC88A silencing on the radiosensitivity of hepatoma in vivo. shRNA-induced Girdin silencing suppressed glycolysis and enhanced the radio-sensitivity of hepatic cell lines, HepG2 and Huh-7. Furthermore, silencing of Girdin inhibited the PI3K/AKT/HIF-1α signaling pathway, which is a central regulator of glycolysis. Girdin can regulate glycolysis in hepatocellular carcinoma cells through the PI3K/AKT/HIF-1α signaling pathway, which decreases the sensitivity of tumor cells to radiotherapy.

Intensive hypermethylation of the CpG island of Ras association domain family 1A in hepatitis B virus-associated hepatocellular carcinomas.

PubMed

Zhong, Sheng; Yeo, Winnie; Tang, Mandy W; Wong, Nathalie; Lai, Paul B S; Johnson, Phillip J

2003-08-15

The human Ras association domain family 1A gene (RASSF1A) is a newly isolated tumor suppressor gene. In this study, we analyzed the methylation status of the promoter region of RASSF1A using bisulfite sequencing and PCR-RFLP in four liver cancer cell lines (Hep3B, HepG(2), SK-HEP-1, and Huh-7) and a cohort of 43 hepatitis B virus-associated hepatocellular carcinoma (HCC) tissues and their corresponding nontumor tissue specimens. The methylation of the CpG islands in the RASSF1A promoter was not detected in 4 samples of normal liver tissue or 10 samples of peripheral blood mononuclear cells from normal subjects. However, the CpG islands were completely methylated, and transcription of the RASSF1A was silenced in the four cell lines. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine reactivated the expression of RASSF1A in the Hep3B and HepG2 cells. In 41 of 43 (95%) HCC specimens studied, the promoter region of RASSF1A was intensively methylated at its CpG sites. Although heterogeneous methylation was also detected in 16 of the 23 (70%) corresponding nontumorous tissues analyzed, the level of methylation was significantly lower than in the corresponding tumor tissues. HCC has the highest incidence of promoter methylation of RASSF1A among all malignancies yet reported suggesting that hypermethylation of the CpG island promoter of RASSF1A may play an important pathological role in this tumor.

Prognostic significance of catalase expression and its regulatory effects on hepatitis B virus X protein (HBx) in HBV-related advanced hepatocellular carcinomas

PubMed Central

Cho, Mi-Young; Cheong, Jae Youn; Lim, Wonchung; Jo, Sujin; Lee, Youngsoo; Wang, Hee-Jung; Han, Kyou-Hoon; Cho, Hyeseong

2014-01-01

Hepatitis B virus X protein (HBx) plays a role in liver cancer development. We previously showed that ROS increased HBx levels and here, we investigated the role of antioxidants in the regulation of HBx expression and their clinical relevance. We found that overexpression of catalase induced a significant loss in HBx levels. The cysteine null mutant of HBx (Cys−) showed a dramatic reduction in its protein stability. In clonogenic proliferation assays, Huh7-X cells produced a significant number of colonies whereas Huh7-Cys− cells failed to generate them. The Cys at position 69 of HBx was crucial to maintain its protein stability and transactivation function in response to ROS. Among 50 HBV-related hepatocellular carcinoma (HCC) specimens, 72% of HCCs showed lower catalase levels than those of surrounding non-tumor tissues. In advanced stage IV, catalase levels in non-tumor tissues were increased whereas those in tumors were further reduced. Accordingly, patients with a high T/N ratio for catalase showed significantly longer survival than those with a low T/N ratio. Together, catalase expression in HCC patients can be clinically useful for prediction of patient survival, and restoration of catalase expression in HCCs could be an important strategy for intervention in HBV-induced liver diseases. PMID:25361011

Allosteric MEK1/2 Inhibitor Refametinib (BAY 86-9766) in Combination with Sorafenib Exhibits Antitumor Activity in Preclinical Murine and Rat Models of Hepatocellular Carcinoma12

PubMed Central

Schmieder, Roberta; Puehler, Florian; Neuhaus, Roland; Kissel, Maria; Adjei, Alex A; Miner, Jeffrey N; Mumberg, Dominik; Ziegelbauer, Karl; Scholz, Arne

2013-01-01

OBJECTIVE: The objectives of the study were to evaluate the allosteric mitogen-activated protein kinase kinase (MEK) inhibitor BAY 86-9766 in monotherapy and in combination with sorafenib in orthotopic and subcutaneous hepatocellular carcinoma (HCC) models with different underlying etiologies in two species. DESIGN: Antiproliferative potential of BAY 86-9766 and synergistic effects with sorafenib were studied in several HCC cell lines. Relevant pathway signaling was studied in MH3924a cells. For in vivo testing, the HCC cells were implanted subcutaneously or orthotopically. Survival and mode of action (MoA) were analyzed. RESULTS: BAY 86-9766 exhibited potent antiproliferative activity in HCC cell lines with half-maximal inhibitory concentration values ranging from 33 to 762 nM. BAY 86-9766 was strongly synergistic with sorafenib in suppressing tumor cell proliferation and inhibiting phosphorylation of the extracellular signal-regulated kinase (ERK). BAY 86-9766 prolonged survival in Hep3B xenografts, murine Hepa129 allografts, and MH3924A rat allografts. Additionally, tumor growth, ascites formation, and serum alpha-fetoprotein levels were reduced. Synergistic effects in combination with sorafenib were shown in Huh-7, Hep3B xenografts, and MH3924A allografts. On the signaling pathway level, the combination of BAY 86-9766 and sorafenib led to inhibition of the upregulatory feedback loop toward MEK phosphorylation observed after BAY 86-9766 monotreatment. With regard to the underlying MoA, inhibition of ERK phosphorylation, tumor cell proliferation, and microvessel density was observed in vivo. CONCLUSION: BAY 86-9766 shows potent single-agent antitumor activity and acts synergistically in combination with sorafenib in preclinical HCC models. These results support the ongoing clinical development of BAY 86-9766 and sorafenib in advanced HCC. PMID:24204195

Hepatitis C virus inhibitor synergism suggests multistep interactions between heat-shock protein 90 and hepatitis C virus replication

PubMed Central

Kubota, Naoko; Nomoto, Masataka; Hwang, Gi-Wook; Watanabe, Toshihiko; Kohara, Michinori; Wakita, Takaji; Naganuma, Akira; Kuge, Shusuke

2016-01-01

AIM: To address the effect of heat-shock protein 90 (HSP90) inhibitors on the release of the hepatitis C virus (HCV), a cell culture-derived HCV (JFH1/HCVcc) from Huh-7 cells was examined. METHODS: We quantified both the intracellular and extracellular (culture medium) levels of the components (RNA and core) of JFH-1/HCVcc. The intracellular HCV RNA and core levels were determined after the JFH1/HCVcc-infected Huh-7 cells were treated with radicicol for 36 h. The extracellular HCV RNA and core protein levels were determined from the medium of the last 24 h of radicicol treatment. To determine the possible role of the HSP90 inhibitor in HCV release, we examined the effect of a combined application of low doses of the HSP90 inhibitor radicicol and the RNA replication inhibitors cyclosporin A (CsA) or interferon. Finally, we statistically examined the combined effect of radicicol and CsA using the combination index (CI) and graphical representation proposed by Chou and Talalay. RESULTS: We found that the HSP90 inhibitors had greater inhibitory effects on the HCV RNA and core protein levels measured in the medium than inside the cells. This inhibitory effect was observed in the presence of a low level of a known RNA replication inhibitor (CsA or interferon-α). Treating the cells with a combination of radicicol and cyclosporin A for 24 h resulted in significant synergy (CI < 1) that affected the release of both the viral RNA and the core protein. CONCLUSION: In addition to having an inhibitory effect on RNA replication, HSP90 inhibitors may interfere with an HCV replication step that occurs after the synthesis of viral RNA, such as assembly and release. PMID:26925202

Ribonucleoside Analogue That Blocks Replication of Bovine Viral Diarrhea and Hepatitis C Viruses in Culture

PubMed Central

Stuyver, Lieven J.; Whitaker, Tony; McBrayer, Tamara R.; Hernandez-Santiago, Brenda I.; Lostia, Stefania; Tharnish, Phillip M.; Ramesh, Mangala; Chu, Chung K.; Jordan, Robert; Shi, Junxing; Rachakonda, Suguna; Watanabe, Kyoichi A.; Otto, Michael J.; Schinazi, Raymond F.

2003-01-01

A base-modified nucleoside analogue, β-d-N4-hydroxycytidine (NHC), was found to have antipestivirus and antihepacivirus activities. This compound inhibited the production of cytopathic bovine viral diarrhea virus (BVDV) RNA in a dose-dependant manner with a 90% effective concentration (EC90) of 5.4 μM, an observation that was confirmed by virus yield assays (EC90 = 2 μM). When tested for hepatitis C virus (HCV) replicon RNA reduction in Huh7 cells, NHC had an EC90 of 5 μM on day 4. The HCV RNA reduction was incubation time and nucleoside concentration dependent. The in vitro antiviral effect of NHC was additive with recombinant alpha interferon-2a and could be prevented by the addition of exogenous cytidine and uridine but not of other natural ribo- or 2′-deoxynucleosides. When HCV RNA replicon cells were cultured in the presence of increasing concentrations of NHC (up to 40 μM) for up to 45 cell passages, no resistant replicon was selected. Similarly, resistant BVDV could not be selected after 20 passages. NHC was phosphorylated to the triphosphate form in Huh7 cells, but in cell-free HCV NS5B assays, synthetic NHC-triphosphate (NHC-TP) did not inhibit the polymerization reaction. Instead, NHC-TP appeared to serve as a weak alternative substrate for the viral polymerase, thereby changing the mobility of the product in polyacrylamide electrophoresis gels. We speculate that incorporated nucleoside analogues with the capacity of changing the thermodynamics of regulatory secondary structures (with or without introducing mutations) may represent an important class of new antiviral agents for the treatment of RNA virus infections, especially HCV. PMID:12499198

Sorafenib and triptolide as combination therapy for hepatocellular carcinoma.

PubMed

Alsaied, Osama A; Sangwan, Veena; Banerjee, Sulagna; Krosch, Tara C; Chugh, Rohit; Saluja, Ashok; Vickers, Selwyn M; Jensen, Eric H

2014-08-01

Sorafenib is the only drug approved by the Food and Drug Administration for metastatic hepatocellular carcinoma (HCC). Triptolide, a diterpene triepoxide, exhibits antineoplastic properties in multiple tumor cell types. In this study, we examined the effects of these agents and their combination on HCC in vitro and in vivo models. HuH-7 and PLC/PRF/5 cells were treated with triptolide (50 nM), sorafenib (1.25 or 2.5 μM), or a combination of both. Cell viability assay (CCK-8), caspase 3&7 activation, and nuclear factor κB assays were performed. For in vivo studies, 40 mice were implanted with subcutaneous HuH7 tumors and divided into four treatment groups (n = 10); saline control, sorafenib 10 mg/kg PO daily (S), Minnelide (a prodrug of triptolide) 0.21 mg/kg intraperitoneally7 daily (M), and combination of both (C). Tumor volumes were assessed weekly. The combination of triptolide and sorafenib was superior to either drug alone in inducing apoptosis and decreasing viability, whereas triptolide alone was sufficient to decrease nuclear factor κB activity. After 2 weeks of treatment, tumor growth inhibition rates were S = 59%, M = 84%, and C = 93%, whereas tumor volumes in control animals increased by 9-fold. When crossed over to combination treatment, control mice tumor growth volumes plateaued over the following 4 weeks. The combination of sorafenib and triptolide is superior to single drug treatment in increasing cell death and apoptosis in vitro. Combining sorafenib with Minnelide inhibited tumor growth with greater efficacy than single-agent treatments. Importantly, in vivo combination treatment allowed for using a lesser dose of sorafenib (10 mg/kg), which is less than 10% of currently prescribed dose for HCC patients. Therefore, combination treatment could have translational potential in the management of HCC. Copyright © 2014 Mosby, Inc. All rights reserved.

The Relative Biological Effectiveness for Carbon and Oxygen Ion Beams Using the Raster-Scanning Technique in Hepatocellular Carcinoma Cell Lines

PubMed Central

Habermehl, Daniel; Ilicic, Katarina; Dehne, Sarah; Rieken, Stefan; Orschiedt, Lena; Brons, Stephan; Haberer, Thomas; Weber, Klaus-Josef; Debus, Jürgen; Combs, Stephanie E.

2014-01-01

Background Aim of this study was to evaluate the relative biological effectiveness (RBE) of carbon (12C) and oxygen ion (16O)-irradiation applied in the raster-scanning technique at the Heidelberg Ion beam Therapy center (HIT) based on clonogenic survival in hepatocellular carcinoma cell lines compared to photon irradiation. Methods Four human HCC lines Hep3B, PLC, HepG2 and HUH7 were irradiated with photons, 12C and 16O using a customized experimental setting at HIT for in-vitro trials. Cells were irradiated with increasing physical photon single doses of 0, 2, 4 and 6 Gy and heavy ionsingle doses of 0, 0.125, 0.5, 1, 2, 3 Gy (12C and 16O). SOBP-penetration depth and extension was 35 mm +/−4 mm and 36 mm +/−5 mm for carbon ions and oxygen ions respectively. Mean energy level and mean linear energy transfer (LET) were 130 MeV/u and 112 keV/um for 12C, and 154 MeV/u and 146 keV/um for 16O. Clonogenic survival was computated and realtive biological effectiveness (RBE) values were defined. Results For all cell lines and both particle modalities α- and β-values were determined. As expected, α-values were significantly higher for 12C and 16O than for photons, reflecting a steeper decline of the initial slope of the survival curves for high-LET beams. RBE-values were in the range of 2.1–3.3 and 1.9–3.1 for 12C and 16O, respectively. Conclusion Both irradiation with 12C and 16O using the rasterscanning technique leads to an enhanced RBE in HCC cell lines. No relevant differences between achieved RBE-values for 12C and 16O were found. Results of this work will further influence biological-adapted treatment planning for HCC patients that will undergo particle therapy with 12C or 16O. PMID:25460352

Plasmodium berghei Δp52&p36 parasites develop independent of a parasitophorous vacuole membrane in Huh-7 liver cells.

PubMed

Ploemen, Ivo H J; Croes, Huib J; van Gemert, Geert-Jan J; Wijers-Rouw, Mietske; Hermsen, Cornelus C; Sauerwein, Robert W

2012-01-01

The proteins P52 and P36 are expressed in the sporozoite stage of the murine malaria parasite Plasmodium berghei. Δp52&p36 sporozoites lacking expression of both proteins are severely compromised in their capability to develop into liver stage parasites and abort development soon after invasion; presumably due to the absence of a parasitophorous vacuole membrane (PVM). However, a small proportion of P. berghei Δp52&p36 parasites is capable to fully mature in hepatocytes causing breakthrough blood stage infections. We have studied the maturation of replicating Δp52&p36 parasites in cultured Huh-7 hepatocytes. Approximately 50% of Δp52&p36 parasites developed inside the nucleus of the hepatocyte but did not complete maturation and failed to produce merosomes. In contrast cytosolic Δp52&p36 parasites were able to fully mature and produced infectious merozoites. These Δp52&p36 parasites developed into mature schizonts in the absence of an apparent parasitophorous vacuole membrane as shown by immunofluorescence and electron microscopy. Merozoites derived from these maturing Δp52&p36 liver stages were infectious for C57BL/6 mice.

p55PIK regulates alpha-fetoprotein expression through the NF-κB signaling pathway.

PubMed

Ye, Guoguo; Sun, Ge; Cheng, Zhikui; Zhang, Lei; Hu, Kanghong; Xia, Xianmin; Zhou, Yin

2017-12-15

Alpha-fetoprotein (AFP) is regarded as a diagnostic and prognostic biomarker and a potential therapeutic target for hepatocellular carcinoma (HCC). However, the regulation of AFP expression in HCC remains poorly understood. This study aimed to investigate the mechanism by which AFP expression is regulated by p55PIK, an isoform of PI3K. Human HCC cell lines (HepG2 and Huh-7) were treated with p55PIK specific competitive inhibitor or shRNA, or p55PIK overexpression vector, in the absence or presence of NF-κB inhibitor PDTC. AFP expression was detected by quantitative real-time PCR and Western blotting. NF-κB responsive elements in AFP enhancer region were characterized by luciferase reporter assay. p55PIK significantly stimulated the expression of AFP by activating NF-κB signaling pathway in HCC cells. Furthermore, two NF-κB binding sites in AFP enhancer region were identified to be primarily responsible for p55PIK mediated upregulation of AFP expression. p55PIK/NF-κB signaling plays an important role in the upregulation of AFP expression in HCC. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of β-catenin protein in hepatocellular carcinoma and its relationship with alpha-fetoprotein.

PubMed

Ren, Ya-Jun; Huang, Tao; Yu, Hong-Lu; Zhang, Li; He, Qian-Jin; Xiong, Zhi-Fan; Peng, Hua

2016-12-01

This study aimed to investigate the expression of β-catenin in hepatocellular carcinoma (HCC) tissues and its relationship with α-fetoprotein (AFP) in HCC. Immunohistochemistry was used to determine the expression of β-catenin in normal liver tissues (n=10), liver cirrhosis tissues (n=20), and primary HCC tissues (n=60). The relationship between β-catenin expression and clinical parameters of HCC was investigated. Real-time PCR and Western blotting were used to detect the mRNA and protein expression levels of β-catenin in the liver cancer cell line SMMC-7721 transfected with a plasmid encoding AFP, and also the mRNA and protein expression levels of β-catenin were measured in the liver cancer cell line Huh7 before and after the transfection with AFP shRNA plasmids. The results showed that β-catenin was only expressed on the cell membrane in normal liver tissues. Its localization to the cytoplasm and nucleus of cells was observed in a small proportion of cirrhotic tissues or adjacent HCC tissues, and such ectopic expression of β-catenin was predominant in HCC tissues. The abnormal expression of β-catenin was correlated with serum AFP levels, cancer cell differentiation and vascular invasion (P<0.05). Additionally, the increased expression of AFP resulted in the upregulation of β-catenin mRNA and protein levels, while knockdown of AFP with AFP shRNA led to significantly decreased β-catenin mRNA and protein levels (P<0.05). It was suggested that the abnormal expression of β-catenin is implicated in hepatic carcinogenesis and development. AFP can lead to increased expression of β-catenin, which may account for the poor prognosis of AFP-associated HCC patients.

DLK1 as a potential target against cancer stem/progenitor cells of hepatocellular carcinoma.

PubMed

Xu, Xiao; Liu, Rui-Fang; Zhang, Xin; Huang, Li-Yu; Chen, Fei; Fei, Qian-Lan; Han, Ze-Guang

2012-03-01

Delta-like 1 homolog (DLK1; Drosophila) is a hepatic stem/progenitor cell marker in fetal livers that plays a vital role in oncogenesis of hepatocellular carcinoma (HCC). The aim of this study is to investigate whether DLK1 could serve as a potential therapeutic target against cancer stem/progenitor cells of HCC. DLK1(+) and DLK1(-) cells were sorted by fluorescence-activated cell sorting and magnetic-activated cell sorting, respectively, and then were evaluated by flow cytometry. The biological behaviors of these isolated cells and those with DLK1 knockdown were assessed by growth curve, colony formation assay, spheroid colony formation, chemoresistance, and in vivo tumorigenicity. Adenovirus-mediated RNA interference was used to knockdown the endogenous DLK1. We found that DLK1(+) population was less than 10% in almost all 17 HCC cell lines examined. DLK1(+) HCC cells showed stronger ability of chemoresistance, colony formation, spheroid colony formation, and in vivo tumorigenicity compared with DLK1(-) cells. The DLK1(+) HCC cells could generate the progeny without DLK1 expression. Furthermore, DLK1 knockdown could suppress the ability of proliferation, colony formation, spheroid colony formation, and in vivo tumorigenicity of Hep3B and Huh-7 HCC cells. Our data suggested that DLK1(+) HCC cells have characteristics similar to those of cancer stem/progenitor cells. RNA interference against DLK1 can suppress the malignant behaviors of HCC cells, possibly through directly disrupting cancer stem/progenitor cells, which suggested that DLK1 could be a potential therapeutic target against the HCC stem/progenitor cells.

Sorafenib and FH535 in combination act synergistically on hepatocellular carcinoma by targeting cell bioenergetics and mitochondrial function.

PubMed

Turcios, Lilia; Vilchez, Valery; Acosta, Luis F; Poyil, Pratheeshkumar; Butterfield, David Allan; Mitov, Mihail; Marti, Francesc; Gedaly, Roberto

2017-06-01

Treatment of advanced hepatocellular carcinoma (HCC) remains a challenge due to the high tumor heterogeneity. In the present study, we aim to evaluate the impact of the β-catenin inhibitor, FH535, alone or in combination with the Ras/Raf/MAPK inhibitor Sorafenib, on the bioenergetics profiles of the HCC cell lines Huh7 and PLC/PRF/5. Single low-dose treatments with FH535 or Sorafenib promoted different effects on mitochondrial respiration and glycolysis in a cell type specific manner. However, the combination of these drugs significantly reduced both mitochondrial respiration and glycolytic rates regardless of the HCC cells. The significant changes in mitochondrial respiration observed in cells treated with the Sorafenib-FH535 combination may correspond to differential targeting of ETC complexes and changes in substrate utilization mediated by each drug. Moreover, the bioenergetics changes and the loss of mitochondrial membrane potential that were evidenced by treatment of HCC cells with the combination of FH535 and Sorafenib, preceded the induction of cell apoptosis. Overall, our results demonstrated that Sorafenib-FH535 drug combination induce the disruption of the bioenergetics of HCC by the simultaneous targeting of mitochondrial respiration and glycolytic flux that leads the synergistic effect on inhibition of cell proliferation. These findings support the therapeutic potential of combinatory FH535-Sorafenib treatment of the HCC heterogeneity by the simultaneous targeting of different molecular pathways. Copyright © 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Resveratrol as a Pan-HDAC Inhibitor Alters the Acetylation Status of Jistone Proteins in Human-Derived Hepatoblastoma Cells

PubMed Central

Böcker, Alexander; Busch, Christian; Weiland, Timo; Noor, Seema; Leischner, Christian; Schleicher, Sabine; Mayer, Mascha; Weiss, Thomas S.; Bischoff, Stephan C.; Lauer, Ulrich M.; Bitzer, Michael

2013-01-01

The polyphenolic alcohol resveratrol has demonstrated promising activities for the prevention and treatment of cancer. Different modes of action have been described for resveratrol including the activation of sirtuins, which represent the class III histone deacetylases (HDACs). However, little is known about the activity of resveratrol on the classical HDACs of class I, II and IV, although these classes are involved in cancer development or progression and inhibitors of HDACs (HDACi) are currently under investigation as promising novel anticancer drugs. We could show by in silico docking studies that resveratrol has the chemical structure to inhibit the activity of different human HDAC enzymes. In vitro analyses of overall HDAC inhibition and a detailed HDAC profiling showed that resveratrol inhibited all eleven human HDACs of class I, II and IV in a dose-dependent manner. Transferring this molecular mechanism into cancer therapy strategies, resveratrol treatment was analyzed on solid tumor cell lines. Despite the fact that hepatocellular carcinoma (HCC) is known to be particularly resistant against conventional chemotherapeutics, treatment of HCC with established HDACi already has shown promising results. Testing of resveratrol on hepatoma cell lines HepG2, Hep3B and HuH7 revealed a dose-dependent antiproliferative effect on all cell lines. Interestingly, only for HepG2 cells a specific inhibition of HDACs and in turn a histone hyperacetylation caused by resveratrol was detected. Additional testing of human blood samples demonstrated a HDACi activity by resveratrol ex vivo. Concluding toxicity studies showed that primary human hepatocytes tolerated resveratrol, whereas in vivo chicken embryotoxicity assays demonstrated severe toxicity at high concentrations. Taken together, this novel pan-HDACi activity opens up a new perspective of resveratrol for cancer therapy alone or in combination with other chemotherapeutics. Moreover, resveratrol may serve as a lead structure for chemical optimization of bioavailability, pharmacology or HDAC inhibition. PMID:24023672

Vitamin K2 downregulates the expression of fibroblast growth factor receptor 3 in human hepatocellular carcinoma cells.

PubMed

Cao, Ke; Liu, Weidong; Nakamura, Hideji; Enomoto, Hirayuki; Yamamoto, Teruhisa; Saito, Masaki; Imanishi, Hiroyasu; Shimomura, Soji; Cao, Peiguo; Nishiguchi, Shuhei

2009-11-01

Vitamin K2 exerts an antitumor activity on human hepatocellular carcinoma (HCC), however, its inhibitory mechanism has not yet been clarified. This study was designed to identify the attractive target molecule of vitamin K2 and shed some light on its effects on fibroblast growth factor receptor (FGFR)3 in HCC cells. The changes in the gene expression of HuH-7 after vitamin K2 treatment were evaluated by a DNA chip analysis. The mRNA and protein levels of FGFR were evaluated by semiquantitative reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and western blot analysis. The promoter activity of the FGFR3 gene was measured by a dual-luciferase assay. The DNA chip analysis revealed different inhibitory rates of gene expression of FGFR3 (60.6%) and FGFR1 (19.4%) after vitamin K2 treatment. Vitamin K2 suppresses the proliferation of HuH-7 in a dose-dependent manner and its inhibitory rate reached approximately 61.8% at the dose of 30 microM. FGFR3 mRNA was significantly reduced based on semiquantitative RT-PCR and decreased 61.5% by a real-time PCR method after vitamin K2 treatment, but FGFR1 mRNA was not. The level of FGFR3 protein was also reduced by vitamin K2 treatment. The luciferase assay demonstrated that vitamin K2 significantly suppressed the promoter activity of FGFR3. Furthermore, the FGFR3-ERK1/2 signaling pathway was suppressed by vitamin K2 treatment. These findings suggest that vitamin K2 may suppress the proliferation of HCC cells through the downregulation of the FGFR3 expression. The transcriptional suppression of FGFR3 may be a novel mechanism of the vitamin K2 action for HCC cells.

Clinical Predictors of Liver Fibrosis in Patients With Chronic Hepatitis B Virus Infection From Children to Adults.

PubMed

Wu, Jia-Feng; Song, Shih-Hsi; Lee, Chee-Seng; Chen, Huey-Ling; Ni, Yen-Hsuan; Hsu, Hong-Yuan; Wu, Tzee-Chung; Chang, Mei-Hwei

2018-04-11

This study aimed to elucidate predictors of liver fibrosis in patients with chronic hepatitis B virus (HBV) infection. Transient elastography was performed to define liver stiffness in 533 patients with chronic HBV infection (mean age ± standard deviation, 30.72 ± 0.57 years). Protein array was performed on serum samples and lysates of Huh7 cells transfected with HBV mutants; the results were confirmed by enzyme-linked immunosorbent assay. Single-nucleotide polymorphisms in the gene encoding interleukin 1β (IL-1β) were examined in patients with chronic HBV infection with and without liver fibrosis. Male sex, age ≥18 years, and serum α-fetoprotein level >3.6 ng/mL were independent predictors of a liver stiffness measurement of ≥7 kPa (P = .005, .019, and <.001, respectively). HBV e antigen (HBeAg)-negative hepatitis is associated with increased liver stiffness (P < .001). Elevation of the serum IL-1β level was demonstrated in subjects with liver fibrosis. IL-1β was upregulated in Huh7 cells transfected with HBV mutants associated with HBeAg-negative hepatitis. The AA genotype at rs16944 and the CC genotype at rs1143627 in the gene encoding IL-1β were associated with higher serum IL-1β levels and liver fibrosis. Male sex, age ≥18 years, elevated α-fetoprotein level, and HBeAg-negative hepatitis are risk factors for liver fibrosis. IL-1β is involved in the progression of liver fibrosis in subjects with HBeAg-negative hepatitis.

Cytokine-like Activity of Liver Type Fatty Acid Binding Protein (L-FABP) Inducing Inflammatory Cytokine Interleukin-6

PubMed Central

Kim, Hyunwoo; Gil, Gaae; Lee, Siyoung; Kwak, Areum; Jo, Seunghyun; Kim, Ensom; Nguyen, Tam T.; Kim, Sinae; Jhun, Hyunjhung; Kim, Somi; Kim, Miyeon; Lee, Youngmin

2016-01-01

It has been reported that fatty acid binding proteins (FABPs) do not act only as intracellular mediators of lipid responses but also have extracellular functions. This study aimed to investigate whether extracellular liver type (L)-FABP has a biological activity and to determined serum L-FABP levels in patients with end-stage renal disease (ESRD). We isolated L-FABP complementary deoxyribonucleic acid (cDNA) from the Huh7 human hepatocarcinoma cell line and expressed the recombinant L-FABP protein in Escherichia coli. A549 lung carcinoma and THP-1 monocytic cells were stimulated with the human recombinant L-FABP. Human whole blood cells were also treated with the human recombinant L-FABP or interleukin (IL)-1α. IL-6 levels were measured in cell culture supernatants using IL-6 enzyme-linked immunosorbent assay (ELISA). Human recombinant L-FABP induced IL-6 in a dose-dependent manner in A549, THP-1 cells, and whole blood cells. The blood samples of healthy volunteers and patients with ESRD were taken after an overnight fast. The serum levels of L-FABP in healthy volunteers and ESRD patients were quantified with L-FABP ELISA. The values of L-FABP in patients with ESRD were significantly lower than those in the control group. Our results demonstrated the biological activity of L-FABP in human cells suggesting L-FABP can be a mediator of inflammation. PMID:27799875

Discovery of 1-((2R,4aR,6R,7R,7aR)-2-Isopropoxy-2-oxidodihydro-4H,6H-spiro[furo[3,2-d][1,3,2]dioxaphosphinine-7,2'-oxetan]-6-yl)pyrimidine-2,4(1H,3H)-dione (JNJ-54257099), a 3'-5'-Cyclic Phosphate Ester Prodrug of 2'-Deoxy-2'-Spirooxetane Uridine Triphosphate Useful for HCV Inhibition.

PubMed

Jonckers, Tim H M; Tahri, Abdellah; Vijgen, Leen; Berke, Jan Martin; Lachau-Durand, Sophie; Stoops, Bart; Snoeys, Jan; Leclercq, Laurent; Tambuyzer, Lotke; Lin, Tse-I; Simmen, Kenny; Raboisson, Pierre

2016-06-23

JNJ-54257099 (9) is a novel cyclic phosphate ester derivative that belongs to the class of 2'-deoxy-2'-spirooxetane uridine nucleotide prodrugs which are known as inhibitors of the HCV NS5B RNA-dependent RNA polymerase (RdRp). In the Huh-7 HCV genotype (GT) 1b replicon-containing cell line 9 is devoid of any anti-HCV activity, an observation attributable to inefficient prodrug metabolism which was found to be CYP3A4-dependent. In contrast, in vitro incubation of 9 in primary human hepatocytes as well as pharmacokinetic evaluation thereof in different preclinical species reveals the formation of substantial levels of 2'-deoxy-2'-spirooxetane uridine triphosphate (8), a potent inhibitor of the HCV NS5B polymerase. Overall, it was found that 9 displays a superior profile compared to its phosphoramidate prodrug analogues (e.g., 4) described previously. Of particular interest is the in vivo dose dependent reduction of HCV RNA observed in HCV infected (GT1a and GT3a) human hepatocyte chimeric mice after 7 days of oral administration of 9.

Purification and characterization of a novel type i ribosome inactivating protein, pachyerosin, from Pachyrhizus erosus seeds, and preparation of its immunotoxin against human hepatoma cells.

PubMed

Guo, Jin-Lin; Cheng, Yuan-Liu; Qiu, Yi; Shen, Cai-Hong; Yi, Bin; Peng, Cheng

2014-07-01

Pachyrhizus erosus seeds have a high protein content and are used in China due to their cytotoxic effect. Here we report the biological and pharmacological activity of the protein extracts from P. erosus seeds. A novel ribosome-inactivating protein, pachyerosin, from P. erosus seeds was successively purified to homogeneity using ammonium sulfate precipitation, DEAE-sepharose FF, and Sephacryl S-200. Pachyerosin showed to be a type I ribosome-inactivating protein with a molecular mass of 29 kDa and an isoelectric point of 9.19. It strongly inhibited protein synthesis of rabbit reticulocyte lysate with an IC50 of 0.37 ng/mL and showed N-glycosidase activity on rat liver ribosomes with an EC50 of 85.9 pM. The N-terminal 27 amino acids of pachyerosin revealed a 60.71% sequence identity with abrin A from the seeds of Abrus precatorius. With the aim of targeting the delivery of pachyerosin, immunotoxin was prepared by conjugating pachyerosin with anti-human AFP monoclonal antibodies SM0736. The immunotoxin pachyerosin-SM0736 efficiently inhibited the growth of the human hepatoma cell line HuH-7 with an IC50 of 0.050 ± 0.004 nM, 2360 times lower than that of pachyerosin and 430 times lower than that of the immunotoxin against human gastric cancer cell line SGC7901. These results imply that pachyerosin may be used as a new promising anticancer agent. Georg Thieme Verlag KG Stuttgart · New York.

Functional Characterization of Glycine N-Methyltransferase and Its Interactive Protein DEPDC6/DEPTOR in Hepatocellular Carcinoma

PubMed Central

Yen, Chia-Hung; Lu, Yao-Cheng; Li, Chung-Hsien; Lee, Cheng-Ming; Chen, Chia-Yen; Cheng, Ming-Yuan; Huang, Shiu-Feng; Chen, Kuen-Feng; Cheng, Ann-Lii; Liao, Li-Ying; Lee, Yan-Hwa Wu; Chen, Yi-Ming Arthur

2012-01-01

Glycine N-methyltransferase (GNMT) is a tumor suppressor for hepatocellular carcinoma (HCC). High rates of Gnmt knockout mice developed HCC. Epigenetic alteration and dysregulation of several pathways including wingless-type MMTV integration site (Wnt), mitogen-activated protein kinase (MAPK) and Janus kinase and signal transducer and activator of transcription (JAK-STAT) are associated with HCC development in Gnmt knockout mice. We hypothesized that GNMT may regulate signal transduction through interacting with other proteins directly. In this report, we identified a mammalian target of rapamycin (mTOR) inhibitor (DEP domain containing MTOR-interacting protein [DEPDC6/DEPTOR]) as a GNMT-binding protein by using yeast two-hybrid screening. Fluorescence resonance energy transfer assay demonstrated that the C-terminal half of GNMT interact with the PSD-95/Dlg1/ZO-1 (PDZ) domain of DEPDC6/DEPTOR. Immunohistochemical staining showed that 27.5% (14/51) of HCC patients had higher expression levels of DEPDC6/DEPTOR in the tumorous tissues than in tumor-adjacent tissues, especially among HCC patients with hepatitis B viral infection (odds ratio 10.3, 95% confidence interval [CI] 1.05–11.3) or patients with poor prognosis (death hazard ratio 4.51, 95% CI 1.60–12.7). In terms of molecular mechanism, knockdown of DEPDC6/DEPTOR expression in HuH-7 cells caused S6K and 4E-BP activation, but suppressed Akt. Overexpression of DEPDC6/DEPTOR activated Akt and increased survival of HCC cells. Overexpression of GNMT caused activation of mTOR/raptor downstream signaling and delayed G2/M cell cycle progression, which altogether resulted in cellular senescence. Furthermore, GNMT reduced proliferation of HuH-7 cells and sensitized them to rapamycin treatment both in vitro and in vivo. In conclusion, GNMT regulates HCC growth in part through interacting with DEPDC6/DEPTOR and modulating mTOR/raptor signaling pathway. Both GNMT and DEPDC6/DEPTOR are potential targets for developing therapeutics for HCC. PMID:22160218

The organic solute transporters alpha and beta are induced by hypoxia in human hepatocytes

PubMed Central

Schaffner, Carlos A; Mwinyi, Jessica; Gai, Zhibo; Thasler, Wolfgang E; Eloranta, Jyrki J; Kullak-Ublick, Gerd A

2015-01-01

Background & Aims The organic solute transporters alpha and beta (OSTα-OSTβ) form a heterodimeric transporter located at the basolateral membrane of intestinal epithelial cells and hepatocytes. Liver injury caused by ischaemia-reperfusion, cancer, inflammation or cholestasis can induce a state of hypoxia in hepatocytes. Here, we studied the effect of hypoxia on the expression of OSTα-OSTβ. Methods OSTα-OSTβ expression was measured in Huh7 cells and primary human hepatocytes (PHH) exposed to chenodeoxycholic acid (CDCA), hypoxia or both. OSTα-OSTβ promoter activity was analysed in luciferase reporter gene assays. Binding of hypoxia-inducible factor-1 alpha (HIF-1α) to the OSTα-OSTβ gene promoters was studied in electrophoretic mobility shift assays (EMSA). Results Expression of OSTα and OSTβ increased in PHH under conditions of hypoxia. Exposure of Huh7 cells or PHH to CDCA (50 μM) enhanced the effect of hypoxia on OSTα mRNA levels. In luciferase assays and EMSA, the inducing effect of low oxygen could be assigned to HIF-1α, which binds to hypoxia responsive elements (HRE) in the OSTα and OSTβ gene promoters. Site-directed mutagenesis of either the predicted HRE or the bile acid responsive FXR binding site abolished inducibility of the OSTα promoter, indicating that both elements need to be intact for induction by hypoxia and CDCA. In a rat model of chronic renal failure, the known increase in hepatic OSTα expression was associated with an increase in HIF-1α protein levels. Conclusion OSTα-OSTβ expression is induced by hypoxia. FXR and HIF-1α bind in close proximity to the OSTα gene promoter and produce synergistic effects on OSTα expression. PMID:24703425

FASTmiR: an RNA-based sensor for in vitro quantification and live-cell localization of small RNAs

PubMed Central

Huang, Kun; Doyle, Francis; Wurz, Zachary E.; Tenenbaum, Scott A.; Hammond, Reza K.

2017-01-01

Abstract Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a variety of important regulatory roles in many eukaryotes. Their small size has made it challenging to study them directly in live cells. Here we describe an RNA-based fluorescent sensor for small RNA detection both in vitro and in vivo, adaptable for any small RNA. It utilizes an sxRNA switch for detection of miRNA–mRNA interactions combined with a fluorophore-binding sequence ‘Spinach’, a GFP-like RNA aptamer for which the RNA–fluorophore complex exhibits strong and consistent fluorescence under an excitation wavelength. Two example sensors, FASTmiR171 and FASTmiR122, can rapidly detect and quantify the levels of miR171 and miR122 in vitro. The sensors can determine relative levels of miRNAs in total RNA extracts with sensitivity similar to small RNA sequencing and northern blots. FASTmiR sensors were also used to estimate the copy number range of miRNAs in total RNA extracts. To localize and analyze the spatial distribution of small RNAs in live, single cells, tandem copies of FASTmiR122 were expressed in different cell lines. FASTmiR122 was able to quantitatively detect the differences in miR122 levels in Huh7 and HEK293T cells demonstrating its potential for tracking miRNA expression and localization in vivo. PMID:28586459

Evaluating Exosome Protein Content Changes Induced by Virus Activity Using SILAC Labeling and LC-MS/MS.

PubMed

Zhao, X; Xie, Y; Liu, J

2017-01-01

Exosomes are small membrane vesicles that are produced by cells and excreted into extracellular space. Contents of exosomes generally include lipid, membrane, and soluble proteins, and various types of coding and noncoding RNAs. Over the past decades, it has become clear that exosomes constitute an important vector for intercellular transport and communication with significant functional relevance. Evaluating exosome contents and their changes are vital for understanding its role in different physiological and pathological processes. Infection by certain pathogens, including viruses as well as intracellular bacteria, fungi, and parasites, has been shown to induce specific content changes in exosomes produced by infected cells. Evidences also indicate that exosomes produced by infected cells may actively participate in host-virus interactions, including immune responses. Studies of exosome content changes involve highly complex experimental and computational procedures, which can become even more complicated in the context of viral infections, due to the production and secretion of multiple virus-derived proteins and particles by infected cells. In this chapter, general and specific considerations relating to studies of exosome content changes induced by virus activities are discussed and illustrated with the detailed protocols previously used to identify protein content changes in Huh-7 cell exosomes induced by transfection with hepatitis B virus replicon plasmids, using SILAC labeling and LS-MS/MS. Hopefully, this would help enable more and further studies along similar lines and enhance the understanding of this new aspect of host-pathogen interactions. © 2017 Elsevier Inc. All rights reserved.

Establishment of an AAV Reverse Infection-Based Array

PubMed Central

Wang, Gang; Dong, Zheyue; Shen, Wei; Zheng, Gang; Wu, Xiaobing; Xue, Jinglun; Wang, Yue; Chen, Jinzhong

2010-01-01

Background The development of a convenient high-throughput gene transduction approach is critical for biological screening. Adeno-associated virus (AAV) vectors are broadly used in gene therapy studies, yet their applications in in vitro high-throughput gene transduction are limited. Principal Findings We established an AAV reverse infection (RI)-based method in which cells were transduced by quantified recombinant AAVs (rAAVs) pre-coated onto 96-well plates. The number of pre-coated rAAV particles and number of cells loaded per well, as well as the temperature stability of the rAAVs on the plates, were evaluated. As the first application of this method, six serotypes or hybrid serotypes of rAAVs (AAV1, AAV2, AAV5/5, AAV8, AAV25 m, AAV28 m) were compared for their transduction efficiencies using various cell lines, including BHK21, HEK293, BEAS-2BS, HeLaS3, Huh7, Hepa1-6, and A549. AAV2 and AAV1 displayed high transduction efficiency; thus, they were deemed to be suitable candidate vectors for the RI-based array. We next evaluated the impact of sodium butyrate (NaB) treatment on rAAV vector-mediated reporter gene expression and found it was significantly enhanced, suggesting that our system reflected the biological response of target cells to specific treatments. Conclusions/Significance Our study provides a novel method for establishing a highly efficient gene transduction array that may be developed into a platform for cell biological assays. PMID:20976058

Adenovirus E1A and E1B-19K Proteins Protect Human Hepatoma Cells from Transforming Growth Factor β1-induced Apoptosis

PubMed Central

Tarakanova, Vera L.; Wold, William S. M.

2009-01-01

Primary and some transformed hepatocytes undergo apoptosis in response to transforming growth factor β1 (TGFβ). We report that infection with species C human adenovirus conferred resistance to TGFβ-induced apoptosis in human hepatocellular carcinoma cells (Huh-7). Protection against TGFβ-mediated cell death in adenovirus-infected cells correlated with the maintenance of normal nuclear morphology, lack of pro-caspases 8 and 3 processing, maintenance of the mitochondrial membrane potential, and lack of cellular DNA degradation. The TGFβ pro-apoptotic signaling pathway was blocked upstream of mitochondria in adenovirus-infected cells. Both the N-terminal sequences of the E1A proteins and the E1B-19K protein were necessary to protect infected cells against TGFβ-induced apoptosis. PMID:19854227

High Content Screening of a Kinase-Focused Library Reveals Compounds Broadly-Active against Dengue Viruses

PubMed Central

Li, Xiaolan; Milan Bonotto, Rafaela; No, Joo Hwan; Kim, Keum Hyun; Baek, Sungmin; Kim, Hee Young; Windisch, Marc Peter; Pamplona Mosimann, Ana Luiza; de Borba, Luana; Liuzzi, Michel; Hansen, Michael Adsetts Edberg; Nunes Duarte dos Santos, Claudia; Freitas-Junior, Lucio Holanda

2013-01-01

Dengue virus is a mosquito-borne flavivirus that has a large impact in global health. It is considered as one of the medically important arboviruses, and developing a preventive or therapeutic solution remains a top priority in the medical and scientific community. Drug discovery programs for potential dengue antivirals have increased dramatically over the last decade, largely in part to the introduction of high-throughput assays. In this study, we have developed an image-based dengue high-throughput/high-content assay (HT/HCA) using an innovative computer vision approach to screen a kinase-focused library for anti-dengue compounds. Using this dengue HT/HCA, we identified a group of compounds with a 4-(1-aminoethyl)-N-methylthiazol-2-amine as a common core structure that inhibits dengue viral infection in a human liver-derived cell line (Huh-7.5 cells). Compounds CND1201, CND1203 and CND1243 exhibited strong antiviral activities against all four dengue serotypes. Plaque reduction and time-of-addition assays suggests that these compounds interfere with the late stage of viral infection cycle. These findings demonstrate that our image-based dengue HT/HCA is a reliable tool that can be used to screen various chemical libraries for potential dengue antiviral candidates. PMID:23437413

Luteolin restricts dengue virus replication through inhibition of the proprotein convertase furin.

PubMed

Peng, Minhua; Watanabe, Satoru; Chan, Kitti Wing Ki; He, Qiuyan; Zhao, Ya; Zhang, Zhongde; Lai, Xiaoping; Luo, Dahai; Vasudevan, Subhash G; Li, Geng

2017-07-01

In many countries afflicted with dengue fever, traditional medicines are widely used as panaceas for illness, and here we describe the systematic evaluation of a widely known natural product, luteolin, originating from the "heat clearing" class of herbs. We show that luteolin inhibits the replication of all four serotypes of dengue virus, but the selectivity of the inhibition was weak. In addition, ADE-mediated dengue virus infection of human cell lines and primary PBMCs was inhibited. In a time-of-drug-addition study, luteolin was found to reduce infectious virus particle formation, but not viral RNA synthesis, in Huh-7 cells. During the virus life cycle, the host protease furin cleaves the pr moiety from prM protein of immature virus particles in the trans-Golgi network to produce mature virions. Analysis of virus particles from luteolin-treated cells revealed that prM was not cleaved efficiently. Biochemical interrogation of human furin showed that luteolin inhibited the enzyme activity in an uncompetitive manner, with Ki value of 58.6 μM, suggesting that treatment may restrict the virion maturation process. Luteolin also exhibited in vivo antiviral activity in mice infected with DENV, causing reduced viremia. Given the mode of action of luteolin and its widespread source, it is possible that it can be tested in combination with other dengue virus inhibitors. Copyright © 2017 Elsevier B.V. All rights reserved.

Synergistic effect of interleukin-17 and tumour necrosis factor-α on inflammatory response in hepatocytes through interleukin-6-dependent and independent pathways.

PubMed

Beringer, A; Thiam, N; Molle, J; Bartosch, B; Miossec, P

2018-04-20

The proinflammatory cytokines interleukin (IL)-17 and tumour necrosis factor (TNF)-α are targets for treatment in many chronic inflammatory diseases. Here, we examined their role in liver inflammatory response compared to that of IL-6. Human hepatoma cells (HepaRG, Huh7.5 and HepG2 cells) and primary human hepatocytes (PHH) were cultured with IL-6, IL-17 and/or TNF-α. To determine the contribution of the IL-6 pathway in the IL-17/TNF-α-mediated effect, an anti-IL-6 receptor antibody was used. IL-17 and TNF-α increased in synergy IL-6 secretion by HepaRG cells and PHH but not by Huh7.5 and HepG2 cells. This IL-17/TNF-α synergistic cooperation enhanced the levels of C-reactive protein (CRP) and aspartate aminotransferase (ASAT) in HepaRG cell and PHH cultures through the induction of IL-6. IL-17/TNF-α also up-regulated IL-8, monocyte chemoattractant protein (MCP)-1 and chemokine (C-C motif) ligand 20 (CCL20) chemokines in synergy through an IL-6-independent pathway. Interestingly, first exposure to IL-17, but not to TNF-α, was crucial for the initiation of the IL-17/TNF-α synergistic effect on IL-6 and IL-8 production. In HepaRG cells, IL-17 enhanced IL-6 mRNA stability resulting in increased IL-6 protein levels. The IL-17A/TNF-α synergistic effect on IL-6 and IL-8 induction was mediated through the activation of extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase, nuclear factor-κB and/or protein kinase B (Akt)-phosphatidylinositol 3-kinase signalling pathways. Therefore, the IL-17/TNF-α synergistic interaction mediates systemic inflammation and cell damage in hepatocytes mainly through IL-6 for CRP and ASAT induction. Independently of IL-6, the IL-17A/TNF-α combination may also induce immune cell recruitment by chemokine up-regulation. IL-17 and/or TNF-α neutralization can be a promising therapeutic strategy to control both systemic inflammation and liver cell attraction. © 2018 British Society for Immunology.

Hepatitis D virus replication is sensed by MDA5 and induces IFN-β/λ responses in hepatocytes.

PubMed

Zhang, Zhenfeng; Filzmayer, Christina; Ni, Yi; Sültmann, Holger; Mutz, Pascal; Hiet, Marie-Sophie; Vondran, Florian W R; Bartenschlager, Ralf; Urban, Stephan

2018-07-01

Hepatitis B virus (HBV) and D virus (HDV) co-infections cause the most severe form of viral hepatitis. HDV induces an innate immune response, but it is unknown how the host cell senses HDV and if this defense affects HDV replication. We aim to characterize interferon (IFN) activation by HDV, identify the responsible sensor and evaluate the effect of IFN on HDV replication. HDV and HBV susceptible hepatoma cell lines and primary human hepatocytes (PHH) were used for infection studies. Viral markers and cellular gene expression were analyzed at different time points after infection. Pattern recognition receptors (PRRs) required for HDV-mediated IFN activation and the impact on HDV replication were studied using stable knock-down or overexpression of the PRRs. Microarray analysis revealed that HDV but not HBV infection activated a broad range of interferon stimulated genes (ISGs) in HepG2 NTCP cells. HDV strongly activated IFN-β and IFN-λ in cell lines and PHH. HDV induced IFN levels remained unaltered upon RIG-I (DDX58) or TLR3 knock-down, but were almost completely abolished upon MDA5 (IFIH1) depletion. Conversely, overexpression of MDA5 but not RIG-I and TLR3 in HuH7.5 NTCP cells partially restored ISG induction. During long-term infection, IFN levels gradually diminished in both HepG2 NTCP and HepaRG NTCP cell lines. MDA5 depletion had little effect on HDV replication despite dampening HDV-induced IFN response. Moreover, treatment with type I or type III IFNs did not abolish HDV replication. Active replication of HDV induces an IFN-β/λ response, which is predominantly mediated by MDA5. This IFN response and exogenous IFN treatment have only a moderate effect on HDV replication in vitro indicating the adaption of HDV replication to an IFN-activated state. In contrast to hepatitis B virus, infection with hepatitis D virus induces a strong IFN-β/λ response in innate immune competent cell lines. MDA5 is the key sensor for the recognition of hepatitis D virus replicative intermediates. An IFN-activated state did not prevent hepatitis D virus replication in vitro, indicating that hepatitis D virus is resistant to self-induced innate immune responses and therapeutic IFN treatment. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Fatty acid binding protein 5 promotes tumor angiogenesis and activates the IL6/STAT3/VEGFA pathway in hepatocellular carcinoma.

PubMed

Pan, Long; Xiao, Heng; Liao, Rui; Chen, Qingsong; Peng, Chong; Zhang, Yuchi; Mu, Tong; Wu, Zhongjun

2018-06-25

Tumor angiogenesis is an essential process for facilitating tumor growth and metastasis. Fatty acid binding protein 5(FABP5)is highly expressed in hepatocellular carcinoma (HCC). Thus, we investigated the role of FABP5 in tumor angiogenesis during HCC development. In this study, the protein and mRNA levels of FABP5 in matched HCC and adjacent noncancerous liver tissues from 43 patients were determined using immunohistochemistry and real-time quantitative PCR, respectively. Two HCC cell lines (Huh7 and SMMC-7721) and human umbilical vein endothelial cells (HUVECS) were used to investigate the pro-angiogenic effect of FABP5 by tube formation, CCK8 and Transwell migration assays. The expression levels of interleukin 6 (IL6) and vascular endothelial growth factor A (VEGFA) secreted from HCC cells were detected by enzyme-linked immunosorbent assay (ELISA). In 43 HCC patients, the expression of FABP5 mRNA was positively correlated with intratumoral VEGFA mRNA expression. FABP5 mRNA expression was also associated with adverse HCC characteristics. In vitro, cell viability, cell migration and tube formation in HUVECs were enhanced with increasing expression of FABP5 in HCC cells. Downregulation of FABP5 expression inhibited the IL6/STAT3/VEGFA pathway in HCC cells and inhibited tumor angiogenesis. FABP5 was shown to promote angiogenesis and activate the IL6/STAT3/VEGFA pathway in HCC. FABP5 may be a potential antiangiogenic target in the treatment of HCC. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

JA, a new type of polyunsaturated fatty acid isolated from Juglans mandshurica Maxim, limits the survival and induces apoptosis of heptocarcinoma cells.

PubMed

Gao, Xiu-Li; Lin, Hua; Zhao, Wei; Hou, Ya-Qin; Bao, Yong-Li; Song, Zhen-Bo; Sun, Lu-Guo; Tian, Shang-Yi; Liu, Biao; Li, Yu-Xin

2016-03-01

Juglans mandshurica Maxim (Juglandaceae) is a famous folk medicine for cancer treatment and some natural compounds isolated from it have been studied extensively. Previously we isolated a type of ω-9 polyunsaturated fatty acid (JA) from the bark of J. mandshurica, however little is known about its activity and the underlying mechanisms. In this study, we studied anti-tumor activity of JA on several human cancer cell lines. Results showed that JA is cytotoxic to HepG2, MDA-MB-231, SGC-7901, A549 and Huh7 cells at a concentration exerting minimal toxic effects on L02 cells. The selective toxicity of JA was better than other classical anti-cancer drugs. Further investigation indicated that JA could induce cell apoptosis, characterized by chromatin condensation, DNA fragmentation and activation of the apoptosis-associated proteins such as Caspase-3 and PARP-1. Moreover, we investigated the cellular apoptosis pathway involved in the apoptosis process in HepG2 cells. We found that proteins involved in mitochondrion (cleaved-Caspase-9, Apaf-1, HtrA2/Omi, Bax, and Mitochondrial Bax) and endocytoplasmic reticulum (XBP-1s, GRP78, cleaved-Caspase-7 and cleaved-Caspase-12) apoptotic pathways were up-regulated when cells were treated by JA. In addition, a morphological change in the mitochondrion was detected. Furthermore, we found that JA could inhibit DNA synthesis and induce G2/M cell cycle arrest. The expression of G2-to-M transition related proteins, such as CyclinB1 and phosphorylated-CDK1, were reduced. In contrast, the G2-to-M inhibitor p21 was increased in JA-treated cells. Overall, our results suggest that JA can induce mitochondrion- and endocytoplasmic reticulum-mediated apoptosis, and G2/M phase arrest in HepG2 cells, making it a promising therapeutic agent against hepatoma.

Enhancing the antiviral potency of ER α-glucosidase inhibitor IHVR-19029 against hemorrhagic fever viruses in vitro and in vivo.

PubMed

Ma, Julia; Zhang, Xuexiang; Soloveva, Veronica; Warren, Travis; Guo, Fang; Wu, Shuo; Lu, Huagang; Guo, Jia; Su, Qing; Shen, Helen; Solon, Eric; Comunale, Mary Ann; Mehta, Anand; Guo, Ju-Tao; Bavari, Sina; Du, Yanming; Block, Timothy M; Chang, Jinhong

2018-02-01

Targeting host functions essential for viral replication has been considered as a broad spectrum and resistance-refractory antiviral approach. However, only a few host functions have, thus far, been validated as broad-spectrum antiviral targets in vivo. ER α-glucosidases I and II have been demonstrated to be essential for the morphogenesis of many enveloped viruses, including members from four families of viruses causing hemorrhagic fever. In vivo antiviral efficacy of various iminosugar-based ER α-glucosidase inhibitors has been reported in animals infected with Dengue, Japanese encephalitis, Ebola, Marburg and influenza viruses. Herein, we established Huh7.5-derived cell lines with ER α-glucosidase I or II knockout using CRISPR/Cas9 and demonstrated that the replication of Dengue, Yellow fever and Zika viruses was reduced by only 1-2 logs in the knockout cell lines. The results clearly indicate that only a partial suppression of viral replication can possibly be achieved with a complete inhibition of ER-α-glucosidases I or II by their inhibitors. We therefore explore to improve the antiviral efficacy of a lead iminosugar IHVR-19029 through combination with another broad-spectrum antiviral agent, favipiravir (T-705). Indeed, combination of IHVR-19029 and T-705 synergistically inhibited the replication of Yellow fever and Ebola viruses in cultured cells. Moreover, in a mouse model of Ebola virus infection, combination of sub-optimal doses of IHVR-19029 and T-705 significantly increased the survival rate of infected animals. We have thus proved the concept of combinational therapeutic strategy for the treatment of viral hemorrhagic fevers with broad spectrum host- and viral- targeting antiviral agents. Copyright © 2017 Elsevier B.V. All rights reserved.

Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Wei, E-mail: detachedy@yahoo.com.cn; Sun, Ting; Cao, Jianping

2012-05-01

Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1{alpha} (HIF-1{alpha}) and miR-210 expression and cell arrest in the G{sub 0}/G{sub 1} phase inmore » all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G{sub 0}/G{sub 1} phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. -- Highlights: Black-Right-Pointing-Pointer miR-210 downregulation radiosensitized hypoxic hepatoma. Black-Right-Pointing-Pointer AIFM3 was identified as a direct target gene of miR-210. Black-Right-Pointing-Pointer miR-210 might be a therapeutic target to hypoxic hepatoma.« less

MEK-ERK inhibition potentiates WAY-600-induced anti-cancer efficiency in preclinical hepatocellular carcinoma (HCC) models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Kaifeng, E-mail: kaifeng_wangdr@sina.com; Fan, Yaohua; Chen, Gongying

The search for novel anti-hepatocellular carcinoma (HCC) agents is important. Mammalian target of rapamycin (mTOR) hyper-activation plays a pivotal role in promoting HCC tumorigenesis and chemoresistance. The current preclinical study evaluated the potential anti-HCC activity by a potent mTOR kinase inhibitor, WAY-600. We showed that WAY-600 inhibited survival and proliferation of HCC cell lines (HepG2 and Huh7) and primary human HCC cells. Caspase-dependent apoptosis was activated by WAY-600 in above HCC cells. Reversely, caspase inhibitors largely attenuated WAY-600's lethality against HCC cells. At the signaling level, WAY-600 blocked mTOR complex 1/2 (mTORC1/2) assemble and activation, yet activated MEK-ERK pathway inmore » HCC cells. MEK-ERK inhibitors, PD-98059 and MEK-162, or MEK1/2 shRNA significantly potentiated WAY-600's cytotoxicity in HCC cells. Further studies showed that WAY-600 intraperitoneal (i.p.) administration in nude mice inhibited p-AKT Ser-473 and displayed significant anti-cancer activity against HepG2 xenografts. Remarkably, co-administration of MEK-162 further potentiated WAY-600's anti-HCC activity in vivo. These preclinical results demonstrate the potent anti-HCC activity by WAY-600, either alone or with MEK-ERK inhibitors. -- Highlights: •WAY-600 inhibits HCC cell survival and proliferation in vitro. •WAY-600 activates caspase-dependent apoptosis in HCC cells. •WAY-600 blocks mTORC1/2 activation, but activates MEK-ERK in HCC cells. •MEK-ERK inhibitors or MEK1/2 shRNA enhances WAY-600's cytotoxicity against HCC cells. •MEK-162 co-administration potentiates WAY-600-induced the anti-HepG2 tumor efficacy.« less

Taurolithocholate-induced MRP2 retrieval involves MARCKS phosphorylation by protein kinase Cϵ in HUH-NTCP Cells.

PubMed

Schonhoff, Christopher M; Webster, Cynthia R L; Anwer, M Sawkat

2013-07-01

Taurolithocholate (TLC) acutely inhibits the biliary excretion of multidrug-resistant associated protein 2 (Mrp2) substrates by inducing Mrp2 retrieval from the canalicular membrane, whereas cyclic adenosine monophosphate (cAMP) increases plasma membrane (PM)-MRP2. The effect of TLC may be mediated via protein kinase Cϵ (PKCϵ). Myristoylated alanine-rich C kinase substrate (MARCKS) is a membrane-bound F-actin crosslinking protein and is phosphorylated by PKCs. MARCKS phosphorylation has been implicated in endocytosis, and the underlying mechanism appears to be the detachment of phosphorylated myristoylated alanine-rich C kinase substrate (pMARCKS) from the membrane. The aim of the present study was to test the hypothesis that TLC-induced MRP2 retrieval involves PKCϵ-mediated MARCKS phosphorylation. Studies were conducted in HuH7 cells stably transfected with sodium taurocholate cotransporting polypeptide (HuH-NTCP cells) and in rat hepatocytes. TLC increased PM-PKCϵ and decreased PM-MRP2 in both HuH-NTCP cells and hepatocytes. cAMP did not affect PM-PKCϵ and increased PM-MRP2 in these cells. In HuH-NTCP cells, dominant-negative (DN) PKCϵ reversed TLC-induced decreases in PM-MRP2 without affecting cAMP-induced increases in PM-MRP2. TLC, but not cAMP, increased MARCKS phosphorylation in HuH-NTCP cells and hepatocytes. TLC and phorbol myristate acetate increased cytosolic pMARCKS and decreased PM-MARCKS in HuH-NTCP cells. TLC failed to increase MARCKS phosphorylation in HuH-NTCP cells transfected with DN-PKCϵ, and this suggested PKCϵ-mediated phosphorylation of MARCKS by TLC. In HuH-NTCP cells transfected with phosphorylation-deficient MARCKS, TLC failed to increase MARCKS phosphorylation or decrease PM-MRP2. Taken together, these results support the hypothesis that TLC-induced MRP2 retrieval involves TLC-mediated activation of PKCϵ followed by MARCKS phosphorylation and consequent detachment of MARCKS from the membrane. Copyright © 2013 American Association for the Study of Liver Diseases.

MicroRNA-122 Down-Regulation Is Involved in Phenobarbital-Mediated Activation of the Constitutive Androstane Receptor

PubMed Central

Shizu, Ryota; Shindo, Sawako; Yoshida, Takemi; Numazawa, Satoshi

2012-01-01

Constitutive androstane receptor (CAR) is a nuclear receptor that regulates the transcription of target genes, including CYP2B and 3A. Phenobarbital activates CAR, at least in part, in an AMP-activated protein kinase (AMPK)-dependent manner. However, the precise mechanisms underlying phenobarbital activation of AMPK are still unclear. In the present study, it was demonstrated that phenobarbital administration to mice decreases hepatic miR-122, a liver-enriched microRNA involved in both hepatic differentiation and function. The time-course change in the phenobarbital-mediated down-regulation of miR-122 was inversely correlated with AMPK activation. Phenobarbital decreased primary miR-122 to approximately 25% of the basal level as early as 1 h and suppressed transactivity of mir-122 promoter in HuH-7 cells, suggesting that the down-regulation occurred at the transcriptional level. AMPK activation by metformin or 5-aminoimidazole-4-carboxamide 1-β-D-ribonucleoside had no evident effect on miR-122 levels. An inhibitory RNA specific for miR-122 increased activated AMPK and CAR-mediated trancactivation of the phenobarbital-responsive enhancer module in HepG2 cells. Conversely, the reporter activity induced by the ectopic CAR was almost completely suppressed by co-transfection with the miR-122 mimic RNA. GFP-tagged CAR was expressed in the cytoplasm in addition to the nucleus in the majority of HuH-7 cells in which miR-122 was highly expressed. Co-transfection of the mimic or the inhibitor RNA for miR-122 further increased or decreased, respectively, the number of cells that expressed GFP-CAR in the cytoplasm. Taken together, these results suggest that phenobarbital-mediated down-regulation of miR-122 is an early and important event in the AMPK-dependent CAR activation and transactivation of its target genes. PMID:22815988

Long non-coding RNA linc-cdh4-2 inhibits the migration and invasion of HCC cells by targeting R-cadherin pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Yunzhen; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou 350025; Wang, Gaoxiong

Long non-coding RNAs (LncRNAs) have played very important roles in the malignancy behaviors of hepatocellular carcinoma (HCC). Linc-cdh4-2 (TCONS-00027978) is a novel LncRNA that has been identified in HCC tissues from our previous study. Overexpression of linc-cdh4-2 in HCC cell lines (SK-Hep-1 and Huh7) significantly decreases the migration and invasion abilities of these cells, while knockdown the expression of linc-cdh4-2 significantly increases the migration and invasion abilities. Interestingly, neither the over expression nor the knock down of linc-cdh4-2 could affect the viability and proliferation of HCC cells. Mechanistically, the linc-cdh4-2 could up-regulate the protein level of R-cadherin through direct bindingmore » that might improve the protein stability. Over expression of linc-cdh4-2 could significantly increase the protein levels of R-cadherin and decrease the protein levels of small GTPase RAC1, and vice-versa. Further knockdown R-cadherin in linc-cdh4-2 stably overexpressed cells, could significantly upregulate the protein levels of RAC1 and improve the cell migration and invasion abilities. Taken together, the novel linc-cdh4-2 may negatively regulate the motility of the HCC cells through targeting R-cadherin-RAC1 signaling pathway. - Highlights: • Linc-cdh4-2 negatively related with the invasion and metastasis ability of HCC cells. • Linc-cdh4-2 could up-regulate the protein level of R-cadherin through direct binding. • Knockdown of R-cadherin increases the migration and invasion abilities of HCC cell. • Knockdown of R-cadherin could significantly upregulate the protein levels of RAC1.« less

A Scalable Perfusion Culture System with Miniature Peristaltic Pumps for Live-Cell Imaging Assays with Provision for Microfabricated Scaffolds

PubMed Central

Balakrishnan, Sreenath; Suma, M.S.; Raju, Shilpa R.; Bhargav, Santosh D.B.; Arunima, S.; Das, Saumitra

2015-01-01

Abstract We present a perfusion culture system with miniature bioreactors and peristaltic pumps. The bioreactors are designed for perfusion, live-cell imaging studies, easy incorporation of microfabricated scaffolds, and convenience of operation in standard cell culture techniques. By combining with miniature peristaltic pumps—one for each bioreactor to avoid cross-contamination and to maintain desired flow rate in each—we have made a culture system that facilitates perfusion culture inside standard incubators. This scalable system can support multiple parallel perfusion experiments. The major components are fabricated by three-dimensional printing using VeroWhite, which we show to be amenable to ex vivo cell culture. Furthermore, the components of the system can be reused, thus making it economical. We validate the system and illustrate its versatility by culturing primary rat hepatocytes, live imaging the growth of mouse fibroblasts (NIH 3T3) on microfabricated ring-scaffolds inserted into the bioreactor, performing perfusion culture of breast cancer cells (MCF7), and high-magnification imaging of hepatocarcinoma cells (HuH7). PMID:26309810

Synergistic anti-tumour effects of tetrandrine and chloroquine combination therapy in human cancer: a potential antagonistic role for p21

PubMed Central

Mei, Liufeng; Chen, Yicheng; Wang, Zhimeng; Wang, Jian; Wan, Jiali; Yu, Chunrong; Liu, Xin; Li, Wenhua

2015-01-01

Background and Purpose Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the Chinese medicinal herb Stephaniae tetrandrae, has a long history in Chinese clinical applications to treat diverse diseases. Tetrandrine induced apoptosis or, at low concentrations, autophagy of human hepatocellular carcinoma cells. Here we have tested the effects of inhibitors of autophagy such as chloroquine, on the response to low concentrations of tetrandrine in cancer cells. Experimental Approach Cultures of several cancer cell lines, including Huh7, U251, HCT116 and A549 cells, were exposed to tetrandrine, chloroquine or a combination of these compounds. Cell viability and content of reactive oxygen species (ROS) were measured and synergy assessed by calculation of the combination index. Western blot and RT-PCR assays were also used along with fluorescence microscopy and histochemical techniques. Key Results Combinations of tetrandrine and chloroquine were more cytotoxic than the same concentrations used separately and these effects showed synergy. Such effects involved increased ROS generation and were dependent on caspase-3 but independent of Akt activity. Blockade of tetrandrine-induced autophagy with 3-methyladenine or bafilomycin-A1 induced apoptosis in cancer cells. Lack of p21 protein (p21−/− HCT116 cells) increased sensitivity to the apoptotic effects of the combination of tetrandrine and chloroquine. In a tumour xenograft model in mice, combined treatment with tetrandrine and chloroquine induced ROS accumulation and cell apoptosis, and decreased tumour growth. Conclusions and Implications The combinations of tetrandrine and chloroquine exhibited synergistic anti-tumour activity, in vitro and in vivo. Our results suggest a novel therapeutic strategy for tumour treatment. PMID:25521075

Hepatitis C virus depends on E-cadherin as an entry factor and regulates its expression in epithelial-to-mesenchymal transition.

PubMed

Li, Qisheng; Sodroski, Catherine; Lowey, Brianna; Schweitzer, Cameron J; Cha, Helen; Zhang, Fang; Liang, T Jake

2016-07-05

Hepatitis C virus (HCV) enters the host cell through interactions with a cascade of cellular factors. Although significant progress has been made in understanding HCV entry, the precise mechanisms by which HCV exploits the receptor complex and host machinery to enter the cell remain unclear. This intricate process of viral entry likely depends on additional yet-to-be-defined cellular molecules. Recently, by applying integrative functional genomics approaches, we identified and interrogated distinct sets of host dependencies in the complete HCV life cycle. Viral entry assays using HCV pseudoparticles (HCVpps) of various genotypes uncovered multiple previously unappreciated host factors, including E-cadherin, that mediate HCV entry. E-cadherin silencing significantly inhibited HCV infection in Huh7.5.1 cells, HepG2/miR122/CD81 cells, and primary human hepatocytes at a postbinding entry step. Knockdown of E-cadherin, however, had no effect on HCV RNA replication or internal ribosomal entry site (IRES)-mediated translation. In addition, an E-cadherin monoclonal antibody effectively blocked HCV entry and infection in hepatocytes. Mechanistic studies demonstrated that E-cadherin is closely associated with claudin-1 (CLDN1) and occludin (OCLN) on the cell membrane. Depletion of E-cadherin drastically diminished the cell-surface distribution of these two tight junction proteins in various hepatic cell lines, indicating that E-cadherin plays an important regulatory role in CLDN1/OCLN localization on the cell surface. Furthermore, loss of E-cadherin expression in hepatocytes is associated with HCV-induced epithelial-to-mesenchymal transition (EMT), providing an important link between HCV infection and liver cancer. Our data indicate that a dynamic interplay among E-cadherin, tight junctions, and EMT exists and mediates an important function in HCV entry.

3β-Hydroxysterol Δ24-Reductase on the Surface of Hepatitis C Virus-Related Hepatocellular Carcinoma Cells Can Be a Target for Molecular Targeting Therapy

PubMed Central

Saito, Makoto; Takano, Takashi; Nishimura, Tomohiro; Kohara, Michinori; Tsukiyama-Kohara, Kyoko

2015-01-01

In our previous study, we demonstrated that 3β-hydroxysterol Δ24-reductase (DHCR24) was overexpressed in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), and that its expression was induced by HCV. Using a monoclonal antibody against DHCR24 (2-152a MAb), we found that DHCR24 was specifically expressed on the surface of HCC cell lines. Based on these findings, we aimed to establish a novel targeting strategy using 2-152a MAb to treat HCV-related HCC. In the present study, we examined the antitumor activity of 2-152a MAb. In the presence of complement, HCC-derived HuH-7 cells were killed by treatment with 2-152a MAb, which was mediated by complement-dependent cytotoxicity (CDC). In addition, the antigen recognition domain of 2-152a MAb was responsible for the unique anti-HCV activity. These findings demonstrate the feasibility of using 2-152a MAb for antibody therapy against HCV-related HCC. In addition, surface DHCR24 on HCC cells exhibited a functional property, agonist-induced internalization. We showed that 2-152a MAb-mediated binding of a cytotoxic agent (a saponin-conjugated secondary antibody) to surface DHCR24 led to significant cytotoxicity. This suggests that surface DHCR24 on HCC cells can function as a carrier for internalization. Therefore, surface DHCR24 could be a valuable target for HCV-related HCC therapy, and 2-152a MAb appears to be useful for this targeted therapy. PMID:25875901

Hepatitis virus infection affects DNA methylation in mice with humanized livers.

PubMed

Okamoto, Yasuyuki; Shinjo, Keiko; Shimizu, Yasuhiro; Sano, Tsuyoshi; Yamao, Kenji; Gao, Wentao; Fujii, Makiko; Osada, Hirotaka; Sekido, Yoshitaka; Murakami, Shuko; Tanaka, Yasuhito; Joh, Takashi; Sato, Shinya; Takahashi, Satoru; Wakita, Takaji; Zhu, Jingde; Issa, Jean-Pierre J; Kondo, Yutaka

2014-02-01

Cells of tumors associated with chronic inflammation frequently have altered patterns of DNA methylation, including hepatocellular carcinomas. Chronic hepatitis has also been associated with aberrant DNA methylation, but little is known about their relationship. Pyrosequencing was used to determine the methylation status of cultured Huh7.5.1 hepatoma cells after hepatitis C virus (HCV) infection. We also studied mice with severe combined immunodeficiency carrying the urokinase-type plasminogen activator transgene controlled by an albumin promoter (urokinase-type plasminogen activator/severe combined immunodeficient mice), in which up to 85% of hepatocytes were replaced by human hepatocytes (chimeric mice). Mice were given intravenous injections of hepatitis B virus (HBV) or HCV, liver tissues were collected, and DNA methylation profiles were determined at different time points after infection. We also compared methylation patterns between paired samples of hepatocellular carcinomas and adjacent nontumor liver tissues from patients. No reproducible changes in DNA methylation were observed after infection of Huh7.5.1 cells with HCV. Livers from HBV- and HCV-infected mice had genome-wide, time-dependent changes in DNA methylation, compared with uninfected urokinase-type plasminogen activator/severe combined immunodeficient mice. There were changes in 160 ± 63 genes in HBV-infected and 237 ± 110 genes in HCV-infected mice. Methylation of 149 common genes increased in HBV- and HCV-infected mice; methylation of some of these genes also increased in hepatocellular carcinoma samples from patients compared with nontumor tissues. Expression of Ifng, which is expressed by natural killer cells, increased significantly in chimeric livers, in concordance with induction of DNA methylation, after infection with HBV or HCV. Induction of Ifng was reduced after administration of an inhibitor of natural killer cell function (anti-asialo GM1). In chimeric mice with humanized livers, infection with HBV and HCV appears to activate a natural kill cell-dependent innate immune response. This contributes to the induction and accumulation of aberrant DNA methylation in human hepatocytes. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Antitumor effect of the novel sphingosine kinase 2 inhibitor ABC294640 is enhanced by inhibition of autophagy and by sorafenib in human cholangiocarcinoma cells.

PubMed

Ding, Xiwei; Chaiteerakij, Roongruedee; Moser, Catherine D; Shaleh, Hassan; Boakye, Jeffrey; Chen, Gang; Ndzengue, Albert; Li, Ying; Zhou, Yanling; Huang, Shengbing; Sinicrope, Frank A; Zou, Xiaoping; Thomas, Melanie B; Smith, Charles D; Roberts, Lewis R

2016-04-12

Sphingosine kinase 2 (Sphk2) has an oncogenic role in cancer. A recently developed first-in-class Sphk2 specific inhibitor ABC294640 displays antitumor activity in many cancer models. However, the role of Sphk2 and the antitumor activity of its inhibitor ABC294640 are not known in cholangiocarcinoma. We investigated the potential of targeting Sphk2 for the treatment of cholangiocarcinoma. We found that Sphk2 is overexpressed in five established human cholangiocarcinoma cell lines (WITT, HuCCT1, EGI-1, OZ and HuH28) and a new patient-derived cholangiocarcinoma cell line (LIV27) compared to H69 normal cholangiocytes. Inhibition of Sphk2 by ABC294640 inhibited proliferation and induced caspase-dependent apoptosis. Furthermore, we found that ABC294640 inhibited STAT3 phosphorylation, one of the key signaling pathways regulating cholangiocarcinoma cell proliferation and survival. ABC294640 also induced autophagy. Inhibition of autophagy by bafilomycin A1 or chloroquine potentiated ABC294640-induced cytotoxicity and apoptosis. In addition, ABC294640 in combination with sorafenib synergistically inhibited cell proliferation of cholangiocarcinoma cells. Strong decreases in STAT3 phosphorylation were observed in WITT and HuCCT1 cells exposed to the ABC294640 and sorafenib combination. These findings provide novel evidence that Sphk2 may be a rational therapeutic target in cholangiocarcinoma. Combinations of ABC294640 with sorafenib and/or autophagy inhibitors may provide novel strategies for the treatment of cholangiocarcinoma.

A mPEG-PLGA-b-PLL copolymer carrier for adriamycin and siRNA delivery.

PubMed

Liu, Peifeng; Yu, Hui; Sun, Ying; Zhu, Mingjie; Duan, Yourong

2012-06-01

A amphiphilic block copolymer composed of conventional monomethoxy (polyethylene glycol)-poly (d,l-lactide-co-glycolide)-poly (l-lysine) (mPEG-PLGA-b-PLL) was synthesized. The chemical structure of this copolymer and its precursors was confirmed by Fourier Transform Infrared Spectroscopy (FTIR), (1)H Nuclear Magnetic Resonance ((1)H NMR) and Gel Permeation Chromatography (GPC). The copolymer was used to prepare nanoparticles (NPs) that were then loaded with either the anti-cancer drug adriamycin or small interfering RNA-negative (siRNA) using a double emulsion method. MTT assays used to study the in vitro cytotoxicity of mPEG-PLGA-b-PLL NPs showed that these particles were not toxic in huh-7 hepatic carcinoma cells. Confocal laser scanning microscopy (CLSM) and flow cytometer analysis results demonstrated efficient mPEG-PLGA-b-PLL NPs-mediated delivery of both adriamycin and siRNA into the cells. In vivo the targeting delivery of adriamycin or siRNA mediated by mPEG-PLGA-b-PLL NPs in the huh-7 hepatic carcinoma-bearing mice was evaluated using a fluorescence imaging system. The targeting delivery results and froze section analysis confirmed that drug or siRNA is deliver to tumor more efficiently by mPEG-PLGA-b-PLL NPs than free drug or Lipofectamine™2000. The high efficiency delivery of mPEG-PLGA-b-PLL NPs mainly due to the enhancement of cellular uptake. These results imply that mPEG-PLGA-b-PLL NPs have a great potential to be used as an effective carriers for adriamycin or siRNA. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Biochemical Characterization of the Active Anti-Hepatitis C Virus Metabolites of 2,6-Diaminopurine Ribonucleoside Prodrug Compared to Sofosbuvir and BMS-986094

PubMed Central

Ehteshami, Maryam; Tao, Sijia; Ozturk, Tugba; Zhou, Longhu; Cho, Jong Hyun; Zhang, Hongwang; Amiralaei, Sheida; Shelton, Jadd R.; Lu, Xiao; Khalil, Ahmed; Domaoal, Robert A.; Stanton, Richard A.; Suesserman, Justin E.; Lin, Biing; Lee, Sam S.; Amblard, Franck; Whitaker, Tony; Coats, Steven J.

2016-01-01

Ribonucleoside analog inhibitors (rNAI) target the hepatitis C virus (HCV) RNA-dependent RNA polymerase nonstructural protein 5B (NS5B) and cause RNA chain termination. Here, we expand our studies on β-d-2′-C-methyl-2,6-diaminopurine-ribonucleotide (DAPN) phosphoramidate prodrug 1 (PD1) as a novel investigational inhibitor of HCV. DAPN-PD1 is metabolized intracellularly into two distinct bioactive nucleoside triphosphate (TP) analogs. The first metabolite, 2′-C-methyl-GTP, is a well-characterized inhibitor of NS5B polymerase, whereas the second metabolite, 2′-C-methyl-DAPN-TP, behaves as an adenosine base analog. In vitro assays suggest that both metabolites are inhibitors of NS5B-mediated RNA polymerization. Additional factors, such as rNAI-TP incorporation efficiencies, intracellular rNAI-TP levels, and competition with natural ribonucleotides, were examined in order to further characterize the potential role of each nucleotide metabolite in vivo. Finally, we found that although both 2′-C-methyl-GTP and 2′-C-methyl-DAPN-TP were weak substrates for human mitochondrial RNA (mtRNA) polymerase (POLRMT) in vitro, DAPN-PD1 did not cause off-target inhibition of mtRNA transcription in Huh-7 cells. In contrast, administration of BMS-986094, which also generates 2′-C-methyl-GTP and previously has been associated with toxicity in humans, caused detectable inhibition of mtRNA transcription. Metabolism of BMS-986094 in Huh-7 cells leads to 87-fold higher levels of intracellular 2′-C-methyl-GTP than DAPN-PD1. Collectively, our data characterize DAPN-PD1 as a novel and potent antiviral agent that combines the delivery of two active metabolites. PMID:27216050

Impedimetric quantification of the formation process and the chemosensitivity of cancer cell colonies suspended in 3D environment.

PubMed

Lei, Kin Fong; Wu, Zong-Ming; Huang, Chia-Hao

2015-12-15

In cancer research, colony formation assay is a gold standard for the investigation of the development of early tumors and the effects of cytotoxic agents on tumors in vitro. Quantification of cancer cell colonies suspended in hydrogel is currently achieved by manual counting under microscope. It is challenging to microscopically quantify the colony number and size without subjective bias. In this work, impedimetric quantification of cancer cell colonies suspended in hydrogel was successfully developed and provides a quantitative and objective method to describe the colony formation process and the development of colony size during the culture course. A biosensor embedded with a pair of parallel plate electrodes was fabricated for the impedimetric quantification. Cancer cell (cell line: Huh-7) were encapsulated in methyl cellulose hydrogel and cultured to gradually form cancer cell colonies suspended in 3D environment. At pre-set schedule during the culture course, small volume (50 μL) of colonies/MC hydrogel was collected, mixed with measurement hydrogel, and loaded to the biosensor for measurement. Hence, the colony formation process could be quantitatively represented by a colony index and a colony size index calculated from electrical impedance. Based on these developments, chemosensitivity of cancer cell colonies under different concentrations of anti-cancer drug, i.e., doxorubicin, was quantitatively investigated to study the efficacy of anti-cancer drug. Also, dose-response curve was constructed to calculate the IC50 value, which is an important indicator for chemosensitivity assay. These results showed the impedimetric quantification is a promising technique for the colony formation assay. Copyright © 2015 Elsevier B.V. All rights reserved.

The miR-199-dynamin regulatory axis controls receptor-mediated endocytosis.

PubMed

Aranda, Juan F; Canfrán-Duque, Alberto; Goedeke, Leigh; Suárez, Yajaira; Fernández-Hernando, Carlos

2015-09-01

Small non-coding RNAs (microRNAs) are important regulators of gene expression that modulate many physiological processes; however, their role in regulating intracellular transport remains largely unknown. Intriguingly, we found that the dynamin (DNM) genes, a GTPase family of proteins responsible for endocytosis in eukaryotic cells, encode the conserved miR-199a and miR-199b family of miRNAs within their intronic sequences. Here, we demonstrate that miR-199a and miR-199b regulate endocytic transport by controlling the expression of important mediators of endocytosis such as clathrin heavy chain (CLTC), Rab5A, low-density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1). Importantly, miR-199a-5p and miR-199b-5p overexpression markedly inhibits CLTC, Rab5A, LDLR and Cav-1 expression, thus preventing receptor-mediated endocytosis in human cell lines (Huh7 and HeLa). Of note, miR-199a-5p inhibition increases target gene expression and receptor-mediated endocytosis. Taken together, our work identifies a new mechanism by which microRNAs regulate intracellular trafficking. In particular, we demonstrate that the DNM, miR-199a-5p and miR-199b-5p genes act as a bifunctional locus that regulates endocytosis, thus adding an unexpected layer of complexity in the regulation of intracellular trafficking. © 2015. Published by The Company of Biologists Ltd.

PNPLA3 is regulated by glucose in human hepatocytes, and its I148M mutant slows down triglyceride hydrolysis.

PubMed

Perttilä, Julia; Huaman-Samanez, Carolina; Caron, Sandrine; Tanhuanpää, Kimmo; Staels, Bart; Yki-Järvinen, Hannele; Olkkonen, Vesa M

2012-05-15

Liver fat is increased in carriers of the minor G allele in rs738409 (I148M amino acid substitution) in patatin-like phospholipase domain-containing 3 (PNPLA3)/adiponutrin. We studied transcriptional regulation of PNPLA3 in immortalized human hepatocytes (IHH) and human hepatoma cells (HuH7) and the impact of PNPLA3 I148M mutant on hepatocyte triglyceride metabolism. Studies in IHH showed that silencing of the carbohydrate response element-binding protein (ChREBP) abolished induction of PNPLA3 mRNA by glucose. Glucose-dependent binding of ChREBP to a newly identified carbohydrate response element in the PNPLA3 promoter was demonstrated by chromatin immunoprecipitation. Adenoviral overexpression of mouse ChREBP in IHH failed to induce PNPLA3 mRNA. [(3)H]acetate or [(3)H]oleate incorporation with 1-h pulse labeling or 18-h [(3)H]oleate labeling in HuH7 cells showed no effect of PNPLA3 I148M on triglyceride (TG) synthesis in the absence of free fatty acid (FFA) loading. Increased [(3)H]oleate accumulation into triglycerides in I148M-expressing cells was observed after 18 h of labeling in the presence of 200 μM FFA-albumin complexes. This was accompanied by increased PNPLA3 protein levels. The rate of hydrolysis of [(3)H]TG during lipid depletion was decreased significantly by PNPLA3 I148M. Our results suggest that PNPLA3 is regulated in human hepatocytes by glucose via ChREBP. PNPLA3 I148M enhances cellular accumulation of [(3)H]TG in the presence of excess FFA, which is known to stabilize PNPLA3 protein. These data do not exclude an effect of PNPLA3 I148M on hepatocyte lipogenesis but show that the mutant increases the stability of triglycerides.

Up-Regulation of MicroRNA-190b Plays a Role for Decreased IGF-1 That Induces Insulin Resistance in Human Hepatocellular Carcinoma

PubMed Central

Hung, Tzu-Min; Ho, Cheng-Maw; Liu, Yen-Chun; Lee, Jia-Ling; Liao, Yow-Rong; Wu, Yao-Ming; Ho, Ming-Chih; Chen, Chien-Hung; Lai, Hong-Shiee; Lee, Po-Huang

2014-01-01

Background & Aims Insulin-like growth factor, (IGF)-1, is produced mainly by the liver and plays important roles in promoting growth and regulating metabolism. Previous study reported that development of hepatocellular carcinoma (HCC) was accompanied by a significant reduction in serum IGF-1 levels. Here, we hypothesized that dysregulation of microRNAs (miRNA) in HCC can modulate IGF-1 expression post-transcriptionally. Methods The miRNAs expression profiles in a dataset of 29 HCC patients were examined using illumina BeadArray. Specific miRNA (miR)-190b, which was significantly up-regulated in HCC tumor tissues when compared with paired non-tumor tissues, was among those predicted to interact with 3′-untranslated region (UTR) of IGF-1. In order to explore the regulatory effects of miR-190b on IGF-1 expression, luciferase reporter assay, quantitative real-time PCR, western blotting and immunofluorecence analysis were performed in HCC cells. Results Overexpression of miR-190b in Huh7 cells attenuated the expression of IGF-1, whereas inhibition of miR-190b resulted in up-regulation of IGF-1. Restoration of IGF-1 expression reversed miR-190b-mediated impaired insulin signaling in Huh7 cells, supporting that IGF-1 was a direct and functional target of miR-190b. Additionally, low serum IGF-1 level was associated with insulin resistance and poor overall survival in HCC patients. Conclusions Increased expression of miR-190 may cause decreased IGF-1 in HCC development. Insulin resistance appears to be a part of the physiopathologic significance of decreased IGF-1 levels in HCC progression. This study provides a novel miRNA-mediated regulatory mechanism for controlling IGF-1 expression in HCC and elucidates the biological relevance of this interaction in HCC. PMID:24586785

Lipid-induced Signaling Causes Release of Inflammatory Extracellular Vesicles from Hepatocytes

PubMed Central

Hirsova, Petra; Ibrahim, Samar H.; Krishnan, Anuradha; Verma, Vikas K.; Bronk, Steven F.; Werneburg, Nathan W.; Charlton, Michael R.; Shah, Vijay H.; Malhi, Harmeet; Gores, Gregory J.

2016-01-01

BACKGROUND & AIMS Hepatocyte cellular dysfunction and death induced by lipids, and macrophage-associated inflammation are characteristics of nonalcoholic steatohepatitis (NASH). The fatty acid palmitate can activate death receptor 5 (DR5) on hepatocytes, leading to their death, but little is known about how this process contributes to macrophage-associated inflammation. We investigated whether lipid-induced DR5 signaling results in release of extracellular vesicles (EV) from hepatocytes, and whether these can induce an inflammatory macrophage phenotype. METHODS Primary mouse and human hepatocytes and Huh7 cells were incubated with palmitate, its metabolite lysophosphatidylcholine, or diluent (control). The released EV were isolated, characterized, quantified, and applied to macrophages. C57BL/6 mice were placed on chow or a diet high in fat, fructose, and cholesterol to induce NASH. Some mice were also given the ROCK1 inhibitor fasudil; 2 weeks later, serum EVs were isolated and characterized by immunoblot and nanoparticle-tracking analyses. Livers were collected and analyzed by histology, immunohistochemistry, and quantitative PCR. RESULTS Incubation of primary hepatocytes and Huh7 cells with palmitate or lysophosphatidylcholine increased their release of EV, compared with control cells. This release was reduced by inactivating mediators of the DR5 signaling pathway or ROCK1 inhibition. Hepatocyte-derived EV contained TRAIL and induced expression of interleukin-1, beta (Il1b) and Il6 mRNAs in mouse bone marrow-derived macrophages. Activation of macrophages required DR5 and RIP1. Administration of the ROCK1 inhibitor fasudil to mice with NASH reduced serum levels of EV; this reduction was associated with decreased liver injury, inflammation, and fibrosis. CONCLUSIONS Lipids, which stimulate DR5, induce release of hepatocyte EV, which activate an inflammatory phenotype in macrophages. Strategies to inhibit ROCK1-dependent release of EV by hepatocytes might be developed for treatment of patients with NASH. PMID:26764184

Lack of evidence for a liver or intestinal miRNA regulation involved in the hypertriglyceridemic effect of APOC3 3'UTR variant SstI.

PubMed

Dancer, Marine; Caussy, Cyrielle; Di Filippo, Mathilde; Moulin, Philippe; Marçais, Christophe; Charrière, Sybil

2016-12-01

APOC3 is a major regulator of triglycerides metabolism. Several APOC3 variants are associated with hypertriglyceridemia (HTG). Our aim was to establish the potential regulation of APOC3 3'UTR variants associated with HTG by liver or intestinal miRNAs. We sequenced APOC3 3'UTR in 100 type 2 diabetic (TD2) patients with severe HTG (TG > 15 mmol/L) (HTG group) compared to 100 normotriglyceridemic patients (NTG group). We performed in silico studies to identify potential loss of miRNA binding induced by APOC3 3'UTR variants. We also performed in vitro studies to test the functionality of miRNA/APOC3 variants interactions: APOC3 3'UTR plasmids coupled with a firefly luciferase reporter were transfected in HepG2, HuH-7 and Caco-2 cells. We identified only two variants: SstI (rs5128) and BbvI (rs5225) in APOC3 3'UTR in the 2 groups of patients. Only the SstI-S2 rare allele was significantly associated with HTG (allele frequency 19,5% in HTG group vs. 9,5% in NTG group, p = 0.0045). In silico studies predicted a potential loss in the binding of 5 miRNAs induced by the S2 variant. These 5 miRNAs are all endogenously expressed in human liver and intestine, as well as in the cell models studied. However, in vitro, the S2 variant did not modulate APOC3 3'UTR reporter gene expression in HepG2, HuH-7 and Caco-2 cells. Our results do not confirm the hypothesis of a direct regulation of the APOC3 SstI variant by hepatic or intestinal miRNAs. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Development of hepatitis C virus genotype 3a cell culture system.

PubMed

Kim, Sulyi; Date, Tomoko; Yokokawa, Hiroshi; Kono, Tamaki; Aizaki, Hideki; Maurel, Patrick; Gondeau, Claire; Wakita, Takaji

2014-12-01

Hepatitis C virus (HCV) genotype 3a infection poses a serious health problem worldwide. A significant association has been reported between HCV genotype 3a infections and hepatic steatosis. Nevertheless, virological characterization of genotype 3a HCV is delayed due to the lack of appropriate virus cell culture systems. In the present study, we established the first infectious genotype 3a HCV system by introducing adaptive mutations into the S310 strain. HCV core proteins had different locations in JFH-1 and S310 virus-infected cells. Furthermore, the lipid content in S310 virus-infected cells was higher than Huh7.5.1 cells and JFH-1 virus-infected cells as determined by the lipid droplet staining area. This genotype 3a infectious cell culture system may be a useful experimental model for studying genotype 3a viral life cycles, molecular mechanisms of pathogenesis, and genotype 3a-specific antiviral drug development. © 2014 by the American Association for the Study of Liver Diseases.

Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase.

PubMed

Kaushik, Nidhi; Subramani, Chandru; Anang, Saumya; Muthumohan, Rajagopalan; Shalimar; Nayak, Baibaswata; Ranjith-Kumar, C T; Surjit, Milan

2017-11-01

Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection. IMPORTANCE Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used to treat HEV cases, there are known side effects and limitations of such therapy. Our discovery of the ability of zinc salts to block HEV replication by virtue of their ability to inhibit the activity of viral RdRp is important because these findings pave the way to test the efficacy of zinc supplementation therapy in HEV-infected patients. Since zinc supplementation therapy is known to be safe in healthy individuals and since high-dose zinc is used in the treatment of Wilson's disease, it may be possible to control HEV-associated health problems following a similar treatment regimen. Copyright © 2017 American Society for Microbiology.

Dietary Polyphenols Increase Paraoxonase 1 Gene Expression by an Aryl Hydrocarbon Receptor-Dependent Mechanism

PubMed Central

Gouédard, Cédric; Barouki, Robert; Morel, Yannick

2004-01-01

Human paraoxonase 1 (PON-1) is a serum high-density lipoprotein-associated enzyme mainly secreted by the liver. It has endogenous and exogenous substrates and displays protective properties with respect to cardiovascular disease and organophosphate intoxication. In the HuH7 human hepatoma cell line, PON-1 activity and mRNA levels were increased by dietary polyphenolic compounds such as quercetin but also by toxic ligands of the aryl hydrocarbon receptor (AhR) such as 3-methylcholanthrene (3-MC). However, the 2,3,7,8-tetrachlorobenzo(p)dioxin (TCDD) was a poor inducer. Transient and stable transfection assays indicated that these compounds increased the PON-1 gene promoter activity in an AhR-dependent manner, since their effect was inhibited by 7-keto-cholesterol and AhR-directed short interfering RNA. Deletions and mutations studies showed that a xenobiotic responsive element (XRE)-like sequence within the PON-1 promoter mediated the effect of 3-MC and quercetin. In contrast with consensus XREs from the cytochrome P450 1A1 gene, the PON-1 XRE-like element mediated preferentially the effect of quercetin compared to the results seen with TCDD. Furthermore, AhR binding to this element was preferentially activated by quercetin. These observations provide a molecular mechanism for the regulation of the cardioprotective enzyme PON-1 by polyphenols. They suggest also that AhR ligands may differentially regulate gene expression depending on the DNA target sequence. PMID:15169886

Identification of biomarkers associated with partial epithelial to mesenchymal transition in the secretome of slug over-expressing hepatocellular carcinoma cells.

PubMed

Karaosmanoğlu, Oğuzhan; Banerjee, Sreeparna; Sivas, Hülya

2018-06-01

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Complete epithelial to mesenchymal transition (EMT) has long been considered as a crucial step for metastasis initiation. It has, however, become apparent that many carcinoma cells can metastasize without complete loss of epithelial traits or with incomplete gain of mesenchymal traits, i.e., partial EMT. Here, we aimed to determine the similarities and differences between complete and partial EMT through over-expression of the EMT-associated transcription factor Slug in different HCC-derived cell lines. Slug over-expressing HCC-derived HepG2 and Huh7 cells were assessed for their EMT, chemo-resistance and stemness features using Western blotting, qRT-PCR, neutral red uptake, doxorubicin accumulation and scratch wound healing assays. We also collected conditioned media from Slug over-expressing HCC cells and analyzed its exosomal protein content for the presence of chemo-resistance and partial EMT markers using MALDI-TOF/TOF and ELISA assays, respectively. We found that Slug over-expression resulted in the induction of both complete and partial EMT in the different HCC-derived cell lines tested. Complete EMT was characterized by downregulation of E-cadherin and upregulation of ZEB2. Partial EMT was characterized by upregulation of E-cadherin and downregulation of vimentin and ZEB2. Interestingly, we found that Slug induced chemo-resistance through downregulation of the ATP binding cassette (ABC) transporter ABCB1 and upregulation of the ABC transporter ABCG2, as well as through expression of CD133, a stemness marker that exhibited a similar expression pattern in cells with either a complete or a partial EMT phenotype. In addition, we found that Slug-mediated partial EMT was associated with enhanced exosomal secretion of post-translationally modified fibronectin 1 (FN1), collagen type II alpha 1 (COL2A1) and native fibrinogen gamma chain (FGG). From our data we conclude that the exosomal proteins identified may be considered as potential non-invasive biomarkers for chemo-resistance and partial EMT in HCC.

3-Decylcatechol induces autophagy-mediated cell death through the IRE1α/JNK/p62 in hepatocellular carcinoma cells

PubMed Central

Kim, Jin-A; Jo, In-Hwa; Han, Yeon Soo; Jo, Yong Hun; Kim, Kwang-Youn; Seo, Young-Kyo; Moon, Jae-Hak; Jung, Chang Hwa; Jeon, Tae-Il

2017-01-01

The natural, phenolic lipid urushiol exhibits both antioxidant and anticancer activities; however, its biological activity on hepatocellular carcinoma (HCC) has not been previously investigated. Here, we demonstrate that an urushiol derivative, 3-decylcatechol (DC), induces human HCC Huh7 cell death by induction of autophagy. DC initiates the autophagic process by activation of the mammalian target of rapamycin signaling pathway via Unc-51-like autophagy activating kinase 1, leading to autophagosome formation. The autophagy inhibitor, chloroquine, suppressed autolysosome formation and cell death induction by DC, indicating an autophagic cell death. Interestingly, DC also activated the endoplasmic reticulum (ER) stress response that promotes autophagy via p62 transcriptional activation involving the inositol-requiring enzyme 1α/c-Jun N-terminal kinase/c-jun pathway. We also show that cytosolic calcium mobilization is necessary for the ER stress response and autophagy induction by DC. These findings reveal a novel mechanism by which this urushiol derivative induces autophagic cell death in HCC. PMID:28938597

JTC801 Induces pH-dependent Death Specifically in Cancer Cells and Slows Growth of Tumors in Mice.

PubMed

Song, Xinxin; Zhu, Shan; Xie, Yangchun; Liu, Jiao; Sun, Lingyi; Zeng, Dexing; Wang, Pengcheng; Ma, Xiaochao; Kroemer, Guido; Bartlett, David L; Billiar, Timothy R; Lotze, Michael T; Zeh, Herbert J; Kang, Rui; Tang, Daolin

2018-04-01

Maintenance of acid-base homeostasis is required for normal physiology, metabolism, and development. It is not clear how cell death is activated in response to changes in pH. We performed a screen to identify agents that induce cell death in a pH-dependent manner (we call this alkaliptosis) in pancreatic ductal adenocarcinoma cancer (PDAC) cells and tested their effects in mice. We screened a library of 254 compounds that interact with G-protein-coupled receptors (GPCRs) to identify those with cytotoxic activity against a human PDAC cell line (PANC1). We evaluated the ability of JTC801, which binds the opiod receptor and has analgesic effects, to stimulate cell death in human PDAC cell lines (PANC1, MiaPaCa2, CFPAC1, PANC2.03, BxPc3, and CAPAN2), mouse pancreatic cancer-associated stellate cell lines, primary human pancreatic ductal epithelial cells, and 60 cancer cell lines (the NCI-60 panel). Genes encoding proteins in cell death and GPCR signaling pathways, as well as those that regulate nuclear factor-κB (NF-κB) activity, were knocked out, knocked down, or expressed from transgenes in cancer cell lines. JTC801 was administered by gavage to mice with xenograft tumors, C57BL/6 mice with orthographic pancreatic tumors grown from Pdx1-Cre;KRas G12D/+ ;Tp53 R172H/+ (KPC) cells, mice with metastases following tail-vein injection of KPC cells, and Pdx-1-Cre;Kras G12D/+ mice crossed with Hmgb1 flox/flox mice (KCH mice). Pancreata were collected from mice and analyzed for tumor growth and by histology and immunohistochemistry. We compared gene and protein expression levels between human pancreatic cancer tissues and patient survival times using online R2 genomic or immunohistochemistry analyses. Exposure of human PDAC cell lines (PANC1 and MiaPaCa2) to JTC801 did not induce molecular markers of apoptosis (cleavage of caspase 3 or poly [ADP ribose] polymerase [PARP]), necroptosis (interaction between receptor-interacting serine-threonine kinase 3 [RIPK3] and mixed lineage kinase domain like pseudokinase [MLKL]), or ferroptosis (degradation of glutathione peroxidase 4 [GPX4]). Inhibitors of apoptosis (Z-VAD-FMK), necroptosis (necrosulfonamide), ferroptosis (ferrostatin-1), or autophagy (hydroxychloroquine) did not prevent JTC801-induced death of PANC1 or MiaPaCa2 cells. The cytotoxic effects of JTC801 in immortalized fibroblast cell lines was not affected by disruption of genes that promote apoptosis (Bax -/- /Bak -/- cells), necroptosis (Ripk1 -/- , Ripk3 -/- , or Mlkl -/- cells), ferroptosis (Gpx4 -/- cells), or autophagy (Atg3 -/- , Atg5 -/- , Atg7 -/- , or Sqstm1 -/- cells). We found JTC801 to induce a pH-dependent form cell death (alkaliptosis) in cancer cells but not normal cells (hepatocytes, bone marrow CD34 + progenitor cells, peripheral blood mononuclear cells, or dermal fibroblasts) or healthy tissues of C57BL/6 mice. JTC801 induced alkaliptosis in cancer cells by activating NF-κB, which repressed expression of the carbonic anhydrase 9 gene (CA9), whose product regulates pH balance in cells. In analyses of Cancer Genome Atlas data and tissue microarrays, we associated increased tumor level of CA9 mRNA or protein with shorter survival times of patients with pancreatic, kidney, or lung cancers. Knockdown of CA9 reduced the protective effects of NF-κB inhibition on JTC801-induced cell death and intracellular alkalinization in PANC1 and MiaPaCa2 cell lines. Oral administration of JTC801 inhibited growth of xenograft tumors (from PANC1, MiaPaCa2, SK-MEL-28, PC-3, 786-0, SF-295, HCT116, OV-CAR3, and HuH7 cells), orthotropic tumors (from KPC cells), lung metastases (from KPC cells) of mice, and slowed growth of tumors in KCH mice. In a screen of agents that interact with GPCR pathways, we found JTC801 to induce pH-dependent cell death (alkaliptosis) specifically in cancer cells such as PDAC cells, by reducing expression of CA9. Levels of CA9 are increased in human cancer tissues. JTC801 might be developed for treatment of pancreatic cancer. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.

PubMed

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-04-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis.

Printing 2-Dimentional Droplet Array for Single-Cell Reverse Transcription Quantitative PCR Assay with a Microfluidic Robot

PubMed Central

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-01-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis. PMID:25828383

The water soluble axially disubstituted silicon phthalocyanines: photophysicochemical properties and in vitro studies.

PubMed

Göksel, Meltem; Biyiklioglu, Zekeriya; Durmuş, Mahmut

2017-08-01

Two novel silicon(IV) phthalocyanines bearing 1,3-bis[3(dimethylamino)phenoxy]propan-2-ol or 1,3-bis[3(diethylamino)phenoxy]propan-2-ol groups at their axial positions were synthesized. These phthalocyanines were converted into their water soluble derivatives by the quaternization reaction with methyl iodide. The quaternized phthalocyanines show excellent solubility aqueous solutions without any aggregation which makes them potential photosensitizers for use in photodynamic therapy (PDT). For this reason, the photophysical and photochemical properties such as fluorescence quantum yields, lifetimes, singlet oxygen generation and photodegradation of both non-ionic (3 and 5) and quaternized cationic silicon(IV) phthalocyanines were investigated. Furthermore, the cytotoxicity of PDT was determined by colorimetric proliferation assay against to hepatocellular carcinoma (HuH-7) cancer cells. In this study, the cells were incubated with a novel water soluble silicon(IV) phthalocyanine derivatives and thereafter the cells were illuminated using broad-band incoherent light source.

Evaluation of the role of three candidate human kinases in the conversion of the hepatitis C virus inhibitor 2'-C-methyl-cytidine to its 5'-monophosphate metabolite.

PubMed

Golitsina, Nina L; Danehy, Francis T; Fellows, Ross; Cretton-Scott, Erika; Standring, David N

2010-03-01

Nucleoside analogs are effective inhibitors of the hepatitis C virus (HCV) in the clinical setting. One such molecule, 2'-C-methyl-cytidine (2'-MeC), entered clinical development as NM283, a valine ester prodrug form of 2'-MeC possessing improved oral bioavailability. To be active against HCV, 2'-MeC must be converted to 2'-MeC triphosphate which inhibits NS5B, the HCV RNA-dependent RNA polymerase. Conversion of 2'-MeC to 2'-MeC monophosphate is the first step in 2'-MeC triphosphate production and is thought to be the rate-limiting step. Here we investigate which of three possible enzymes, deoxycytidine kinase (dCK), uridine-cytidine kinase 1 (UCK1), or uridine-cytidine kinase 2 (UCK2), mediate this first phosphorylation step. Purified recombinant enzymes UCK2 and dCK, but not UCK1, could phosphorylate 2'-MeC in vitro. However, siRNA knockdown experiments in three human cell lines (HeLa, Huh7 and HepG2) defined UCK2 and not dCK as the key kinase for the formation of 2'-MeC monophosphate in cultured human cells. These results underscore the importance of confirming enzymatic kinase data with appropriate cell-based assays. Finally, we present data suggesting that inefficient phosphorylation by UCK2 likely limits the antiviral activity of 2'-MeC against HCV. This paves the way for the use of a nucleotide prodrug approach to overcome this limitation.

[Preclinical study of AV0012 early stage inhibitor of hepatitis C virus infection: I. In vitro ADME and pharmacokinetics].

PubMed

Ivashchenko, A V; Iamanushkin, P M; Mit'kin, O D; Ezhova, E V; Korzinov, O M; Shevkun, N A; Koriakova, A G; Karapetian, R N; Bychko, V V; Ivashchenko, A A; Agrba, V Z; Lapin, B A; Orlov, S V

2014-01-01

In vitro immunohistochemical investigations on the human hepatoma cell line (Huh7) infected with hepatitis C virus (HCV) strain JFH-1 showed that AV0012 compound blocks the early stages of viral infection. AV0012 also blocked viral infection spread in tissue culture through the secreted virus and through tight cell-to-cell contact. AV0012 is a specific inhibitor of HCV but not of related pestivirus, flaviviruses and other RNA-containing viruses such as bovine diarrhea (BVDV), Venezuelan equine encephalitis (strain TC-83), dengue type 2 (New Guinea), yellow fever (strain 17D), west Nile fever, parainfluenza (type 3) virus, RSV (strain A2), and Rhinovirus (type 2 strain HGP). It is established that human serum does not significantly affect the antiviral activity of AV0012 in vitro. The drug combination studies with AV0012 and interferon alpha 2a in vitro showed that the two inhibitors act additively, which makes possible the use of this combination in clinical tests. AV0012 is highly soluble and stable in aqueous solutions and murine blood plasma, has limited metabolic stability, low binding to human plasma proteins, high permeability through biological membranes, and only interacts with isoenzymes 2D6 and 3A4 of human cytochrome P450. In animal pharmacokinetic studies, AV0012 was rapidly absorbed into the blood stream upon oral administration, showed sufficiently long half-elimination times, and had high oral bioavailability that reached 92% in monkeys. Further preclinical development of AV0012 is in progress.

Identification of small molecule inhibitors of the Chikungunya virus nsP1 RNA capping enzyme.

PubMed

Feibelman, Kristen M; Fuller, Benjamin P; Li, Linfeng; LaBarbera, Daniel V; Geiss, Brian J

2018-06-01

Chikungunya virus (CHIKV) is an arthropod-borne alphavirus. Alphaviruses are positive strand RNA viruses that require a 5' cap structure to direct translation of the viral polyprotein and prevent degradation of the viral RNA genome by host cell nucleases. Formation of the 5' RNA cap is orchestrated by the viral protein nsP1, which binds GTP and provides the N-7 methyltransferase and guanylyltransferase activities that are necessary for cap formation. Viruses with aberrant nsP1 activity are unable to replicate effectively suggesting that nsP1 is a promising target for antiviral drug discovery. Given the absence of commercially available antiviral therapies for CHIKV, it is imperative to identify compounds that could be developed as potential therapeutics. This study details a high-throughput screen of 3051 compounds from libraries containing FDA-approved drugs, natural products, and known bioactives against CHIKV nsP1 using a fluorescence polarization-based GTP competition assay. Several small molecule hits from this screen were able to compete with GTP for the CHIKV nsP1 GTP binding site at low molar concentrations. Compounds were also evaluated with an orthogonal assay that measured the ability of nsP1 to perform the guanylation step of the capping reaction in the presence of inhibitor. In addition, live virus assays with CHIKV and closely related alphavirus, Sindbis virus, were used in conjunction with cell toxicity assays to determine the antiviral activity of compounds in cell culture. The naturally derived compound lobaric acid was found to inhibit CHIKV nsP1 GTP binding and guanylation as well as attenuate viral growth in vitro at both 24 hpi and 48 hpi in hamster BHK21 and human Huh 7 cell lines. These data indicate that development of lobaric acid and further exploration of CHIKV nsP1 as a drug target may aid in the progress of anti-alphaviral drug development strategies. Copyright © 2018. Published by Elsevier B.V.

A liver enhancer in the fibrinogen gene cluster.

PubMed

Fort, Alexandre; Fish, Richard J; Attanasio, Catia; Dosch, Roland; Visel, Axel; Neerman-Arbez, Marguerite

2011-01-06

The plasma concentration of fibrinogen varies in the healthy human population between 1.5 and 3.5 g/L. Understanding the basis of this variability has clinical importance because elevated fibrinogen levels are associated with increased cardiovascular disease risk. To identify novel regulatory elements involved in the control of fibrinogen expression, we used sequence conservation and in silico-predicted regulatory potential to select 14 conserved noncoding sequences (CNCs) within the conserved block of synteny containing the fibrinogen locus. The regulatory potential of each CNC was tested in vitro using a luciferase reporter gene assay in fibrinogen-expressing hepatoma cell lines (HuH7 and HepG2). 4 potential enhancers were tested for their ability to direct enhanced green fluorescent protein expression in zebrafish embryos. CNC12, a sequence equidistant from the human fibrinogen alpha and beta chain genes, activates strong liver enhanced green fluorescent protein expression in injected embryos and their transgenic progeny. A transgenic assay in embryonic day 14.5 mouse embryos confirmed the ability of CNC12 to activate transcription in the liver. While additional experiments are necessary to prove the role of CNC12 in the regulation of fibrinogen, our study reveals a novel regulatory element in the fibrinogen locus that is active in the liver and may contribute to variable fibrinogen expression in humans.

Water-dispersible magnetic carbon nanotubes as T2-weighted MRI contrast agents.

PubMed

Liu, Yue; Hughes, Timothy C; Muir, Benjamin W; Waddington, Lynne J; Gengenbach, Thomas R; Easton, Christopher D; Hinton, Tracey M; Moffat, Bradford A; Hao, Xiaojuan; Qiu, Jieshan

2014-01-01

An efficient MRI T2-weighted contrast agent incorporating a potential liver targeting functionality was synthesized via the combination of superparamagnetic iron oxide (SPIO) nanoparticles with multiwalled carbon nanotubes (MWCNTs). Poly(diallyldimethylammonium chloride) (PDDA) was coated on the surface of acid treated MWCNTs via electrostatic interactions and SPIO nanoparticles modified with a potential targeting agent, lactose-glycine adduct (Lac-Gly), were subsequently immobilized on the surface of the PDDA-MWCNTs. A narrow magnetic hysteresis loop indicated that the product displayed superparamagnetism at room temperature which was further confirmed by ZFC (zero field cooling)/FC (field cooling) curves measured by SQUID. The multifunctional MWCNT-based magnetic nanocomposites showed low cytotoxicity in vitro to HEK293 and Huh7 cell lines. Enhanced T2 relaxivities were observed for the hybrid material (186 mM(-1) s(-1)) in comparison with the pure magnetic nanoparticles (92 mM(-1) s(-1)) due to the capacity of the MWCNTs to "carry" more nanoparticles as clusters. More importantly, after administration of the composite material to an in vivo liver cancer model in mice, a significant increase in tumor to liver contrast ratio (277%) was observed in T2 weighted magnetic resonance images. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Interference of hepatitis C virus RNA replication by short interfering RNAs

NASA Astrophysics Data System (ADS)

Kapadia, Sharookh B.; Brideau-Andersen, Amy; Chisari, Francis V.

2003-02-01

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Current therapy of patients with chronic HCV infection includes treatment with IFN in combination with ribavirin. Because most treated patients do not resolve the infection, alternative treatment is essential. RNA interference (RNAi) is a recently discovered antiviral mechanism present in plants and animals that induces double-stranded RNA degradation. Using a selectable subgenomic HCV replicon cell culture system, we have shown that RNAi can specifically inhibit HCV RNA replication and protein expression in Huh-7 cells that stably replicate the HCV genome, and that this antiviral effect is independent of IFN. These results suggest that RNAi may represent a new approach for the treatment of persistent HCV infection.

The anti-hepatocellular carcinoma cell activity by a novel mTOR kinase inhibitor CZ415

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Wei; Research Center of Blood Transfusion Medicine, Education Ministry Key Laboratory of Laboratory Medicine, Zhejiang Provincial People's Hospital, People’s Hospital of Hangzhou medical College, Hangzhou; Chen, Bingyu

Dysregulation of mammalian target of rapamycin (mTOR) in hepatocellular carcinoma (HCC) represents a valuable treatment target. Recent studies have developed a highly-selective and potent mTOR kinase inhibitor, CZ415. Here, we showed that nM concentrations of CZ415 efficiently inhibited survival and induced apoptosis in HCC cell lines (HepG2 and Huh-7) and primary-cultured human HCC cells. Meanwhile, CZ415 inhibited proliferation of HCC cells, more potently than mTORC1 inhibitors (rapamycin and RAD001). CZ415 was yet non-cytotoxic to the L02 human hepatocytes. Mechanistic studies showed that CZ415 disrupted assembly of mTOR complex 1 (mTORC1) and mTORC2 in HepG2 cells. Meanwhile, activation of mTORC1 (p-S6K1)more » and mTORC2 (p-AKT, Ser-473) was almost blocked by CZ415. In vivo studies revealed that oral administration of CZ415 significantly suppressed HepG2 xenograft tumor growth in severe combined immuno-deficient (SCID) mice. Activation of mTORC1/2 was also largely inhibited in CZ415-treated HepG2 tumor tissue. Together, these results show that CZ415 blocks mTORC1/2 activation and efficiently inhibits HCC cell growth in vitro and in vivo. - Highlights: • CZ415 is anti-survival and pro-apoptotic to hepatocellular carcinoma (HCC) cells. • CZ415 inhibits HCC cell proliferation, more efficiently than mTORC1 inhibitors. • CZ415 blocks assembly and activation of both mTORC1 and mTORC2 in HCC cells. • CZ415 oral administration inhibits HepG2 tumor growth in SCID mice. • mTORC1/2 activation in HepG2 tumor is inhibited with CZ415 administration.« less

Niemann-Pick type C2 protein regulates liver cancer progression via modulating ERK1/2 pathway: Clinicopathological correlations and therapeutical implications.

PubMed

Liao, Yi-Jen; Fang, Cheng-Chieh; Yen, Chia-Hung; Hsu, Shih-Ming; Wang, Chung-Kwe; Huang, Shiu-Feng; Liang, Yu-Chih; Lin, Ying-Yu; Chu, Yu-Tseng; Arthur Chen, Yi-Ming

2015-09-15

Primary hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the third leading cause of cancer-related death. It is important to identify new targets for early diagnosis and treatment of HCC. Niemann-Pick type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in HCC tumorigenesis. In this study, we showed that NPC2 is abundantly expressed in normal liver, but is downregulated in human HCC tissues. The patients with NPC2 downregulation expressed much higher α-fetoprotein, multiple tumor type, vascular invasion, later pathological stage and shorter survival rate. Knockdown NPC2 in liver cancer cell lines promote cell proliferation, migration and xenograft tumorigenesis. In contrast, NPC2 overexpression inhibits HuH7 promoted tumor growth. Furthermore, administration of hepatotropic adeno-associated virus 8 (AAV8) delivered NPC2 decreased the inflammatory infiltration, the expression of two early HCC markers-glypican 3 and survivin and suppressed the spontaneous HCC development in mice. To identify the NPC2-dependent mechanism, we emphasized on the status of MAPK/ERK signaling. MEK1/2 inhibitor treatment demonstrated that the expression of NPC2 affected the activation of ERK1/2 but not MEK1/2. In addition, cholesterol trafficking inhibitor treatment did not alter the cell proliferation and the activation of MEK/ERK. In conclusion, our study demonstrates that NPC2 may play an important role in negatively regulate cell proliferation and ERK1/2 activation that were independent of cholesterol accumulation. AAV-NPC2 may thus represent a new treatment strategy for liver cancer. © 2015 UICC.

Screening of Dengue Virus Antiviral Activity of Marine Seaweeds by an In Situ Enzyme-Linked Immunosorbent Assay

PubMed Central

Koishi, Andrea Cristine; Zanello, Paula Rodrigues; Bianco, Éverson Miguel; Bordignon, Juliano; Nunes Duarte dos Santos, Claudia

2012-01-01

Dengue is a significant public health problem worldwide. Despite the important social and clinical impact, there is no vaccine or specific antiviral therapy for prevention and treatment of dengue virus (DENV) infection. Considering the above, drug discovery research for dengue is of utmost importance; in addition natural marine products provide diverse and novel chemical structures with potent biological activities that must be evaluated. In this study we propose a target-free approach for dengue drug discovery based on a novel, rapid, and economic in situ enzyme-linked immunosorbent assay and the screening of a panel of marine seaweed extracts. The in situ ELISA was standardized and validated for Huh7.5 cell line infected with all four serotypes of DENV, among them clinical isolates and a laboratory strain. Statistical analysis showed an average S/B of 7.2 and Z-factor of 0.62, demonstrating assay consistency and reliability. A panel of fifteen seaweed extracts was then screened at the maximum non-toxic dose previously determined by the MTT and Neutral Red cytotoxic assays. Eight seaweed extracts were able to reduce DENV infection of at least one serotype tested. Four extracts (Phaeophyta: Canistrocarpus cervicornis, Padina gymnospora; Rhodophyta: Palisada perforate; Chlorophyta: Caulerpa racemosa) were chosen for further evaluation, and time of addition studies point that they might act at an early stage of the viral infection cycle, such as binding or internalization. PMID:23227238

Genome-Wide Transcriptional Reorganization Associated with Senescence-to-Immortality Switch during Human Hepatocellular Carcinogenesis

PubMed Central

Konu, Ozlen; Yuzugullu, Haluk; Gursoy-Yuzugullu, Ozge; Ozturk, Nuri; Ozen, Cigdem; Ozdag, Hilal; Erdal, Esra; Karademir, Sedat; Sagol, Ozgul; Mizrak, Dilsa; Bozkaya, Hakan; Ilk, Hakki Gokhan; Ilk, Ozlem; Bilen, Biter; Cetin-Atalay, Rengul; Akar, Nejat; Ozturk, Mehmet

2013-01-01

Senescence is a permanent proliferation arrest in response to cell stress such as DNA damage. It contributes strongly to tissue aging and serves as a major barrier against tumor development. Most tumor cells are believed to bypass the senescence barrier (become “immortal”) by inactivating growth control genes such as TP53 and CDKN2A. They also reactivate telomerase reverse transcriptase. Senescence-to-immortality transition is accompanied by major phenotypic and biochemical changes mediated by genome-wide transcriptional modifications. This appears to happen during hepatocellular carcinoma (HCC) development in patients with liver cirrhosis, however, the accompanying transcriptional changes are virtually unknown. We investigated genome-wide transcriptional changes related to the senescence-to-immortality switch during hepatocellular carcinogenesis. Initially, we performed transcriptome analysis of senescent and immortal clones of Huh7 HCC cell line, and identified genes with significant differential expression to establish a senescence-related gene list. Through the analysis of senescence-related gene expression in different liver tissues we showed that cirrhosis and HCC display expression patterns compatible with senescent and immortal phenotypes, respectively; dysplasia being a transitional state. Gene set enrichment analysis revealed that cirrhosis/senescence-associated genes were preferentially expressed in non-tumor tissues, less malignant tumors, and differentiated or senescent cells. In contrast, HCC/immortality genes were up-regulated in tumor tissues, or more malignant tumors and progenitor cells. In HCC tumors and immortal cells genes involved in DNA repair, cell cycle, telomere extension and branched chain amino acid metabolism were up-regulated, whereas genes involved in cell signaling, as well as in drug, lipid, retinoid and glycolytic metabolism were down-regulated. Based on these distinctive gene expression features we developed a 15-gene hepatocellular immortality signature test that discriminated HCC from cirrhosis with high accuracy. Our findings demonstrate that senescence bypass plays a central role in hepatocellular carcinogenesis engendering systematic changes in the transcription of genes regulating DNA repair, proliferation, differentiation and metabolism. PMID:23691139

Functional evaluation of synthetic flavonoids and chalcones for potential antiviral and anticancer properties.

PubMed

Mateeva, Nelly; Eyunni, Suresh V K; Redda, Kinfe K; Ononuju, Ucheze; Hansberry, Tony D; Aikens, Cecilia; Nag, Anita

2017-06-01

Flavonoids, stilbenes, and chalcones are plant secondary metabolites that often possess diverse biological activities including anti-inflammatory, anti-cancer, and anti-viral activities. The wide range of bioactivities poses a challenge to identify their targets. Here, we studied a set of synthetically generated flavonoids and chalcones to evaluate for their biological activity, and compared similarly substituted flavonoids and chalcones. Substituted chalcones, but not flavonoids, showed inhibition of viral translation without significantly affecting viral replication in cells infected with hepatitis C virus (HCV). We suggest that the chalcones used in this study inhibit mammalian target of rapamycin (mTOR) pathway by ablating phosphorylation of ribosomal protein 6 (rps6), and also the kinase necessary for phosphorylating rps6 in Huh7.5 cells (pS6K1). In addition, selected chalcones showed inhibition of growth in Ishikawa, MCF7, and MDA-MB-231 cells resulting an IC 50 of 1-6µg/mL. When similarly substituted flavonoids were used against the same set of cancer cells, we did not observe any inhibitory effect. Together, we report that chalcones show potential for anti-viral and anti-cancer activities compared to similarly substituted flavonoids. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antiviral effects of Curcuma longa L. against dengue virus in vitro and in vivo

NASA Astrophysics Data System (ADS)

Ichsyani, M.; Ridhanya, A.; Risanti, M.; Desti, H.; Ceria, R.; Putri, D. H.; Sudiro, T. M.; Dewi, B. E.

2017-12-01

Dengue is the most common infective disease caused by dengue virus (DENV) and endemic diseases in tropical and subtropical areas. Until now, there is no specific antiviral for dengue infection. It is known that viral load is related to disease severity. Curcuma longa L. (turmeric) with curcumin as major active compound has been identified for its antiviral effect. This study to determine antiviral effect of C. longa extract on DENV-2 in vitro and in vivo along with its toxicity in liver and kidney of ddY mice. Antiviral activity (IC50) and toxicity (CC50) in vitro was examined on Huh7it-1 cells by focus assay and a MTT assay, respectively. To determine the selectivity index (SI), we used CC50 and IC50 value. The safe doses obtained were used for toxicity tests of liver and kidney with histopathological and biochemical observations. The C. longa extracts was given orally with dose of 0.147 mg/mL for each mice at 2 hours after injected with DENV-2 infected Huh7it-1 cells. Serum was collected from intraorbital at 6 hours and 24 hours after infection and focus assay was used to determine viral load. In this study, the acquired value of IC50 was 17,91 μg/mL whereas the value of CC50 was 85,4 μg/mL. The value of SI of C. longa was 4.8. In vivo, we found that C. longa remarkable reduced of viral load after 24 hour. Histopathological examination showed no specific abnormalities in liver and kidney. There was no significant increase in levels of SGPT, SGOT, urea, and creatinine. From this study it can be concluded that C. longa could potentially be used as antiviral against DENV with low cytotoxicity and effective inhibition.

[Low-molecular-weight regulators of biogenic polyamine metabolism affect cytokine production and expression of hepatitis С virus proteins in Huh7.5 human hepatocarcinoma cells].

PubMed

Masalova, O V; Lesnova, E I; Samokhvalov, E I; Permyakova, K Yu; Ivanov, A V; Kochetkov, S N; Kushch, A A

2017-01-01

Hepatitis C virus (HCV) induces the expression of the genes of proinflammatory cytokines, the excessive production of which may cause cell death, and contribute to development of liver fibrosis and hepatocarcinoma. The relationship between cytokine production and metabolic disorders in HCV-infected cells remains obscure. The levels of biogenic polyamines, spermine, spermidine, and their precursor putrescine, may be a potential regulator of these processes. The purpose of the present work was to study the effects of the compounds which modulate biogenic polyamines metabolism on cytokine production and HCV proteins expression. Human hepatocarcinoma Huh7.5 cells have been transfected with the plasmids that encode HCV proteins and further incubated with the following low-molecular compounds that affect different stages of polyamine metabolism: (1) difluoromethylornithine (DFMO), the inhibitor of ornithine decarboxylase, the enzyme that catalyzes the biosynthesis of polyamines; (2) N,N'-bis(2,3-butane dienyl)-1,4-diaminobutane (MDL72.527), the inhibitor of proteins involved in polyamine degradation; and (3) synthetic polyamine analog N^(I),N^(II)-diethylnorspermine (DENSpm), an inducer of polyamine degradation enzyme. The intracellular accumulation and secretion of cytokines (IL-6, IL-1β, TNF-α, and TGF-β) was assessed by immunocytochemistry and in the immunoenzyme assay, while the cytokine gene expression was studied using reverse transcription and PCR. The effects of the compounds under analysis on the expression of HCV proteins were analyzed using the indirect immunofluorescence with anti-HCV monoclonal antibodies. It has been demonstrated that, in cells transfected with HCV genes, DFMO reduces the production of three out of four tested cytokines, namely, TNF-α and TGF-β in cells that express HCV core, Е1Е2, NS3, NS5A, and NS5B proteins, and IL-1β in the cells that express HCV core, Е1Е2, and NS3 proteins. MDL72527 and DENSpm decreased cytokine production to a lesser extent. Incubation with DFMO led to a 28-32% decrease in the number of cells expressing NS5B or NS5A, both of which are key components of the HCV replication complex. The results obtained in the work indicate that a further detailed study of the antiviral activity of DFMO is required in order to assess its potential as an anti-hepatitis C therapeutic agent.

Knockdown of AMPKα decreases ATM expression and increases radiosensitivity under hypoxia and nutrient starvation in an SV40-transformed human fibroblast cell line, LM217.

PubMed

Murata, Yasuhiko; Hashimoto, Takuma; Urushihara, Yusuke; Shiga, Soichiro; Takeda, Kazuya; Jingu, Keiichi; Hosoi, Yoshio

2018-01-22

Presence of unperfused regions containing cells under hypoxia and nutrient starvation contributes to radioresistance in solid human tumors. It is well known that hypoxia causes cellular radioresistance, but little is known about the effects of nutrient starvation on radiosensitivity. We have reported that nutrient starvation induced decrease of mTORC1 activity and decrease of radiosensitivity in an SV40-transformed human fibroblast cell line, LM217, and that nutrient starvation induced increase of mTORC1 activity and increase of radiosensitivity in human liver cancer cell lines, HepG2 and HuH6 (Murata et al., BBRC 2015). Knockdown of mTOR using small interfering RNA (siRNA) for mTOR suppressed radiosensitivity under nutrient starvation alone in HepG2 cells, which suggests that mTORC1 pathway regulates radiosensitivity under nutrient starvation alone. In the present study, effects of hypoxia and nutrient starvation on radiosensitivity were investigated using the same cell lines. LM217 and HepG2 cells were used to examine the effects of hypoxia and nutrient starvation on cellular radiosensitivity, mTORC1 pathway including AMPK, ATM, and HIF-1α, which are known as regulators of mTORC1 activity, and glycogen storage, which is induced by HIF-1 and HIF-2 under hypoxia and promotes cell survival. Under hypoxia and nutrient starvation, AMPK activity and ATM expression were increased in LM217 cells and decreased in HepG2 cells compared with AMPK activity under nutrient starvation alone or ATM expression under hypoxia alone. Under hypoxia and nutrient starvation, radiosensitivity was decreased in LM217 cells and increased in HepG2 cells compared with radiosensitivity under hypoxia alone. Under hypoxia and nutrient starvation, knockdown of AMPK decreased ATM activity and increased radiation sensitivity in LM217 cells. In both cell lines, mTORC1 activity was decreased under hypoxia and nutrient starvation. Under hypoxia alone, knockdown of mTOR slightly increased ATM expression but did not affect radiosensitivity in LM217. Under hypoxia and nutrient starvation, HIF-1α expression was suppressed and glycogen storage was reduced. Our data suggest that AMPK regulates ATM expression and partially regulates radiosensitivity under hypoxia and nutrient starvation. The molecular mechanism underlying the induction of ATM expression by AMPK remains to be elucidated. Copyright © 2017. Published by Elsevier Inc.

EF24 inhibits tumor growth and metastasis via suppressing NF-kappaB dependent pathways in human cholangiocarcinoma

PubMed Central

Yin, Da-long; Liang, Ying-jian; Zheng, Tong-sen; Song, Rui-peng; Wang, Jia-bei; Sun, Bo-shi; Pan, Shang-ha; Qu, Lian-dong; Liu, Jia-ren; Jiang, Hong-chi; Liu, Lian-xin

2016-01-01

A synthetic monoketone analog of curcumin, termed 3, 5-bis (2-flurobenzylidene) piperidin-4-one (EF24), has been reported to inhibit the growth of a variety of cancer cells both in vitro and in vivo. However, whether EF24 has anticancer effects on cholangiocarcinoma (CCA) cells and the mechanisms remain to be investigated. The aim of our study was to evaluate the molecular mechanisms underlying the anticancer effects of EF24 on CCA tumor growth and metastasis. Cell proliferation, apoptosis, migration, invasion, tumorigenesis and metastasis were examined. EF24 exhibited time- and dose-dependent inhibitory effects on HuCCT-1, TFK-1 and HuH28 human CCA cell lines. EF24 inhibited CCA cell proliferation, migration, and induced G2/M phase arrest. EF24 induced cell apoptosis along with negative regulation of NF-κB- X-linked inhibitor of apoptosis protein (XIAP) signaling pathway. XIAP inhibition by lentivirus mediated RNA interference enhanced EF24-induced apoptosis, while XIAP overexpression reduced it in CCA cells. In vivo, EF24 significantly suppressed the growth of CCA tumor xenografts and tumor metastasis while displaying low toxicity levels. Our findings indicate that EF24 is a potent antitumor agent that inhibits tumor growth and metastasis by inhibiting NF-κB dependent signaling pathways. EF24 may represent a novel approach for CCA treatment. PMID:27571770

EF24 inhibits tumor growth and metastasis via suppressing NF-kappaB dependent pathways in human cholangiocarcinoma.

PubMed

Yin, Da-Long; Liang, Ying-Jian; Zheng, Tong-Sen; Song, Rui-Peng; Wang, Jia-Bei; Sun, Bo-Shi; Pan, Shang-Ha; Qu, Lian-Dong; Liu, Jia-Ren; Jiang, Hong-Chi; Liu, Lian-Xin

2016-08-30

A synthetic monoketone analog of curcumin, termed 3, 5-bis (2-flurobenzylidene) piperidin-4-one (EF24), has been reported to inhibit the growth of a variety of cancer cells both in vitro and in vivo. However, whether EF24 has anticancer effects on cholangiocarcinoma (CCA) cells and the mechanisms remain to be investigated. The aim of our study was to evaluate the molecular mechanisms underlying the anticancer effects of EF24 on CCA tumor growth and metastasis. Cell proliferation, apoptosis, migration, invasion, tumorigenesis and metastasis were examined. EF24 exhibited time- and dose-dependent inhibitory effects on HuCCT-1, TFK-1 and HuH28 human CCA cell lines. EF24 inhibited CCA cell proliferation, migration, and induced G2/M phase arrest. EF24 induced cell apoptosis along with negative regulation of NF-κB- X-linked inhibitor of apoptosis protein (XIAP) signaling pathway. XIAP inhibition by lentivirus mediated RNA interference enhanced EF24-induced apoptosis, while XIAP overexpression reduced it in CCA cells. In vivo, EF24 significantly suppressed the growth of CCA tumor xenografts and tumor metastasis while displaying low toxicity levels. Our findings indicate that EF24 is a potent antitumor agent that inhibits tumor growth and metastasis by inhibiting NF-κB dependent signaling pathways. EF24 may represent a novel approach for CCA treatment.

Miniature fiber optic spectrometer-based quantitative fluorescence resonance energy transfer measurement in single living cells.

PubMed

Chai, Liuying; Zhang, Jianwei; Zhang, Lili; Chen, Tongsheng

2015-03-01

Spectral measurement of fluorescence resonance energy transfer (FRET), spFRET, is a widely used FRET quantification method in living cells today. We set up a spectrometer-microscope platform that consists of a miniature fiber optic spectrometer and a widefield fluorescence microscope for the spectral measurement of absolute FRET efficiency (E) and acceptor-to-donor concentration ratio (R(C)) in single living cells. The microscope was used for guiding cells and the spectra were simultaneously detected by the miniature fiber optic spectrometer. Moreover, our platform has independent excitation and emission controllers, so different excitations can share the same emission channel. In addition, we developed a modified spectral FRET quantification method (mlux-FRET) for the multiple donors and multiple acceptors FRET construct (mD∼nA) sample, and we also developed a spectra-based 2-channel acceptor-sensitized FRET quantification method (spE-FRET). We implemented these modified FRET quantification methods on our platform to measure the absolute E and R(C) values of tandem constructs with different acceptor/donor stoichiometries in single living Huh-7 cells.

Photodynamic effect of photosensitizer-loaded hollow silica nanoparticles for hepatobiliary malignancies: an in vitro and in vivo study

NASA Astrophysics Data System (ADS)

Deng, Xiaofeng; Xiong, Li; Wen, Yu; Liu, Zhongtao; Pei, Dongni; Huang, Yaxun; Miao, Xiongying

2014-03-01

Background and aims: Nanoparticles have been explored recently as an efficient delivery system for photosensitizers in photodynamic therapy. In this study, polyhematoporphyrin (C34H38N4NaO5,) was loaded into hollow silica nanoparticles (HSNP) by one-step wet chemical-based synthetic route. We evaluate the efficacy and safety of polyhematoporphyrin-loaded HSNP with hepatobiliary malignant cells and in vivo models. Methods: Human liver cancer, cholangiocarcinoma and gallbladder cancer cells were cultured with the HSNP and cellular viability was determined by MTT assay. Apoptotic and necrotic cells were measured by flow cytometry. Finally, we investigate its effect in vivo. Results: In MTT assay, the cell viability of QBC939, Huh-7, GBC-SD and HepG2 cells of the HSNP was 6.4+/-1.3%, 6.5+/-1.2%, 3.7+/-1.2% and 4.7+/-2.0%, respectively, which were significant different from that of free polyhematoporphyrin 62.4+/-4.7%, 62.5+/-6.0%, 33.4+/-6.5% and 44.3+/-1.9%. Flow cytometry demonstrated the laser-induced cell death with polyhematoporphyrin-loaded HSNP was much more severe. Similarly, in vivo results of each kind of cell revealed 14 days post-photoradiated, tumor sizes of the HSNP group were significantly smaller. Administration of the HSNP without illumination cannot cause killing effect both in vitro and in vivo experiments. Conclusions: HSNP is a desirable delivery system in photodynamic therapy for hepatobiliary malignacies, with improved aqueous solubility, stability and transport efficiency of photosensitizers.

Novel perspectives for hepatitis A virus therapy revealed by comparative analysis of hepatitis C virus and hepatitis A virus RNA replication.

PubMed

Esser-Nobis, Katharina; Harak, Christian; Schult, Philipp; Kusov, Yuri; Lohmann, Volker

2015-08-01

Hepatitis A virus (HAV) and hepatitis C virus (HCV) are two positive-strand RNA viruses sharing a similar biology, but causing opposing infection outcomes, with HAV always being cleared and HCV establishing persistence in the majority of infections. To gain deeper insight into determinants of replication, persistence, and treatment, we established a homogenous cell-culture model allowing a thorough comparison of RNA replication of both viruses. By screening different human liver-derived cell lines with subgenomic reporter replicons of HAV as well as of different HCV genotypes, we found that Huh7-Lunet cells supported HAV- and HCV-RNA replication with similar efficiency and limited interference between both replicases. HAV and HCV replicons were similarly sensitive to interferon (IFN), but differed in their ability to establish persistent replication in cell culture. In contrast to HCV, HAV replicated independently from microRNA-122 and phosphatidylinositol 4-kinase IIIα and β (PI4KIII). Both viruses were efficiently inhibited by cyclosporin A and NIM811, a nonimmunosuppressive analog thereof, suggesting an overlapping dependency on cyclophilins for replication. However, analysis of a broader set of inhibitors revealed that, in contrast to HCV, HAV does not depend on cyclophilin A, but rather on adenosine-triphosphate-binding cassette transporters and FK506-binding proteins. Finally, silibinin, but not its modified intravenous formulation, efficiently inhibited HAV genome replication in vitro, suggesting oral silibinin as a potential therapeutic option for HAV infections. We established a cell-culture model enabling comparative studies on RNA replication of HAV and HCV in a homogenous cellular background with comparable replication efficiency. We thereby identified new host cell targets and potential treatment options for HAV and set the ground for future studies to unravel determinants of clearance and persistence. © 2015 by the American Association for the Study of Liver Diseases.

Statins Suppress Ebola Virus Infectivity by Interfering with Glycoprotein Processing.

PubMed

Shrivastava-Ranjan, Punya; Flint, Mike; Bergeron, Éric; McElroy, Anita K; Chatterjee, Payel; Albariño, César G; Nichol, Stuart T; Spiropoulou, Christina F

2018-05-01

Ebola virus (EBOV) infection is a major public health concern due to high fatality rates and limited effective treatments. Statins, widely used cholesterol-lowering drugs, have pleiotropic mechanisms of action and were suggested as potential adjunct therapy for Ebola virus disease (EVD) during the 2013-2016 outbreak in West Africa. Here, we evaluated the antiviral effects of statin (lovastatin) on EBOV infection in vitro Statin treatment decreased infectious EBOV production in primary human monocyte-derived macrophages and in the hepatic cell line Huh7. Statin treatment did not interfere with viral entry, but the viral particles released from treated cells showed reduced infectivity due to inhibition of viral glycoprotein processing, as evidenced by decreased ratios of the mature glycoprotein form to precursor form. Statin-induced inhibition of infectious virus production and glycoprotein processing was reversed by exogenous mevalonate, the rate-limiting product of the cholesterol biosynthesis pathway, but not by low-density lipoprotein. Finally, statin-treated cells produced EBOV particles devoid of the surface glycoproteins required for virus infectivity. Our findings demonstrate that statin treatment inhibits EBOV infection and suggest that the efficacy of statin treatment should be evaluated in appropriate animal models of EVD. IMPORTANCE Treatments targeting Ebola virus disease (EVD) are experimental, expensive, and scarce. Statins are inexpensive generic drugs that have been used for many years for the treatment of hypercholesterolemia and have a favorable safety profile. Here, we show the antiviral effects of statins on infectious Ebola virus (EBOV) production. Our study reveals a novel molecular mechanism in which statin regulates EBOV particle infectivity by preventing glycoprotein processing and incorporation into virus particles. Additionally, statins have anti-inflammatory and immunomodulatory effects. Since inflammation and dysregulation of the immune system are characteristic features of EVD, statins could be explored as part of EVD therapeutics.

Assessment of α-fetoprotein targeted HSV1-tk expression in hepatocellular carcinoma with in vivo imaging.

PubMed

Park, Ju Hui; Kim, Kwang Il; Lee, Kyo Chul; Lee, Yong Jin; Lee, Tae Sup; Chung, Wee Sup; Lim, Sang Moo; Kang, Joo Hyun

2015-02-01

Tumor-specific enhancer/promoter is applicable for targeting gene expression in tumors and helpful for tumor-targeting imaging and therapy. We aimed to acquire α-fetoprotein (AFP)-producing hepatocellular carcinoma (HCC) specific images using adenovirus containing HSV1-tk gene controlled by AFP enhancer/promoter and evaluate in vivo ganciclovir (GCV)-medicated therapeutic effects on AFP-targeted HSV1-tk expression with (18)F-FDG positron emission tomography (PET). Recombinant adenovirus expressing HSV1-tk under AFP enhancer/promoter was produced (AdAFP-TK) and the expression levels were evaluated by RT-PCR and (125)I-IVDU uptake. GCV-mediated HSV1-tk cytotoxicity was determined by MTT assay. After the mixture of AdAFP-fLuc and AdAFP-TK was administrated, bioluminescent images (BLIs) and (18)F-FHBG PET images were obtained in tumor-bearing mice. In vivo therapeutic effects of AdAFP-TK and GCV in the HuH-7 xenograft model were monitored by (18)F-FDG PET. When infected with AdAFP-TK, cell viability in HuH-7 was reduced, but those in HT-29 and SK-Hep-1 were not significantly decreased at any GCV concentration less than 100 μM. AFP-targeted fLuc and HSV1-tk expression were clearly visualized by BLI and (18)F-FHBG PET images in AFP-producing HCC, respectively. In vivo GCV-mediated tumor growth inhibition by AFP-targeted HSV1-tk expression was monitored by (18)F-FDG PET. Recombinant AdAFP-TK could be applied for AFP-targeted HCC gene therapy and imaging in AFP-producing HCC.

Host apolipoprotein B messenger RNA-editing enzyme catalytic polypeptide-like 3G is an innate defensive factor and drug target against hepatitis C virus.

PubMed

Peng, Zong-Gen; Zhao, Zhi-Yun; Li, Yan-Ping; Wang, Yu-Ping; Hao, Lan-Hu; Fan, Bo; Li, Yu-Huan; Wang, Yue-Ming; Shan, Yong-Qiang; Han, Yan-Xing; Zhu, Yan-Ping; Li, Jian-Rui; You, Xue-Fu; Li, Zhuo-Rong; Jiang, Jian-Dong

2011-04-01

Host cellular factor apolipoprotein B messenger RNA (mRNA)-editing enzyme catalytic polypeptide-like 3G (hA3G) is a cytidine deaminase that inhibits a group of viruses including human immunodeficiency virus-1 (HIV-1). In the continuation of our research on hA3G, we found that hA3G stabilizing compounds significantly inhibited hepatitis C virus (HCV) replication. Therefore, this study investigated the role of hA3G in HCV replication. Introduction of external hA3G into HCV-infected Huh7.5 human hepatocytes inhibited HCV replication; knockdown of endogenous hA3G enhanced HCV replication. Exogenous HIV-1 virion infectivity factor (Vif) decreased intracellular hA3G and therefore enhanced HCV proliferation, suggesting that the presence of Vif might be an explanation for the HIV-1/HCV coinfection often observed in HIV-1(+) individuals. Treatment of the HCV-infected Huh7.5 cells with RN-5 or IMB-26, two known hA3G stabilizing compounds, increased intracellular hA3G and accordingly inhibited HCV replication. The compounds inhibit HCV through increasing the level of hA3G incorporated into HCV particles, but not through inhibiting HCV enzymes. However, G/A hypermutation in the HCV genome were not detected, suggesting a new antiviral mechanism of hA3G in HCV, different from that in HIV-1. Stabilization of hA3G by RN-5 was safe in vivo. hA3G appears to be a cellular restrict factor against HCV and could be a potential target for drug discovery. 2011 American Association for the Study of Liver Diseases.

Effects of an oral iron chelator, deferasirox, on advanced hepatocellular carcinoma.

PubMed

Saeki, Issei; Yamamoto, Naoki; Yamasaki, Takahiro; Takami, Taro; Maeda, Masaki; Fujisawa, Koichi; Iwamoto, Takuya; Matsumoto, Toshihiko; Hidaka, Isao; Ishikawa, Tsuyoshi; Uchida, Koichi; Tani, Kenji; Sakaida, Isao

2016-10-28

To evaluate the inhibitory effects of deferasirox (DFX) against hepatocellular carcinoma (HCC) through basic and clinical studies. In the basic study, the effect of DFX was investigated in three hepatoma cell lines (HepG2, Hep3B, and Huh7), as well as in an N-nitrosodiethylamine-induced murine HCC model. In the clinical study, six advanced HCC patients refractory to chemotherapy were enrolled. The initial dose of DFX was 10 mg/kg per day and was increased by 10 mg/kg per day every week, until the maximum dose of 30 mg/kg per day. The duration of a single course of DFX therapy was 28 consecutive days. In the event of dose-limiting toxicity (according to the Common Terminology Criteria for Adverse Events v.4.0), DFX dose was reduced. Administration of DFX inhibited the proliferation of hepatoma cell lines and induced the activation of caspase-3 in a dose-dependent manner in vitro . In the murine model, DFX treatment significantly suppressed the development of liver tumors ( P < 0.01), and significantly upregulated the mRNA expression levels of hepcidin ( P < 0.05), transferrin receptor 1 ( P < 0.05), and hypoxia inducible factor-1α ( P < 0.05) in both tumor and non-tumor tissues, compared with control mice. In the clinical study, anorexia and elevated serum creatinine were observed in four and all six patients, respectively. However, reduction in DFX dose led to decrease in serum creatinine levels in all patients. After the first course of DFX, one patient discontinued the therapy. We assessed the tumor response in the remaining five patients; one patient exhibited stable disease, while four patients exhibited progressive disease. The one-year survival rate of the six patients was 17%. We demonstrated that DFX inhibited HCC in the basic study, but not in the clinical study due to dose-limiting toxicities.

Self-assembled Monolayer Mediated Surface Environment Modification of Poly(vinylpyrrolidone)-Coated Hollow Au-Ag Nanoshells for Enhanced Loading of Hydrophobic Drug and Efficient Multimodal Therapy.

PubMed

Jang, Hongje; Kim, Dong-Eun; Min, Dal-Hee

2015-06-17

Hollow Au-Ag bimetallic nanoshell possessing hydrophobic interior space and hydrophilic exterior surface was prepared and its application as a chemo-thermo-gene therapeutic agent based on its high payload of multiple drugs having different water solubility was demonstrated. The multifunctional drug delivery system is based on the hydrophobic interior created by the self-assembled monolayer (SAM) of hexanethiol onto the inner surface of the hollow metallic nanoshells whereas the outer surface was mostly coated by hydrophilic biocompatible polymer. The nanoshells having surface environment modified by hexanethiol SAMs provided high capacity both for hydrophilic DNAzyme (Dz) to induce gene silencing and for hydrophobic SN38 (7-ethyl-10-hydroxycamptothecin), anticancer drug. The release of the loaded Dz and SN38 was independently triggered by an acidic environment and by photothermal temperature elevation upon irradiation, respectively. The chemo-thermo-gene multitherapy based on the present nanoshells having modified surface environment showed high efficacy in quantitative cell-based assays using Huh7 human liver cell containing hepatitis C viral NS3 gene replicon RNA.

Interferon-lambda (IFN-λ) induces signal transduction and gene expression in human hepatocytes, but not in lymphocytes or monocytes

PubMed Central

Dickensheets, Harold; Sheikh, Faruk; Park, Ogyi; Gao, Bin; Donnelly, Raymond P.

2013-01-01

This study compared the ability of IFN-α and IFN-λ to induce signal transduction and gene expression in primary human hepatocytes, PBLs, and monocytes. IFN-α drug products are widely used to treat chronic HCV infection; however, IFN-α therapy often induces hematologic toxicities as a result of the broad expression of IFNARs on many cell types, including most leukocytes. rIFN-λ1 is currently being tested as a potential alternative to IFN-α for treating chronic HCV. Although IFN-λ has been shown to be active on hepatoma cell lines, such as HepG2 and Huh-7, its ability to induce responses in primary human hepatocytes or leukocytes has not been examined. We found that IFN-λ induces activation of Jak/STAT signaling in mouse and human hepatocytes, and the ability of IFN-λ to induce STAT activation correlates with induction of numerous ISGs. Although the magnitude of ISG expression induced by IFN-λ in hepatocytes was generally lower than that induced by IFN-α, the repertoire of regulated genes was quite similar. Our findings demonstrate that although IFN-α and IFN-λ signal through distinct receptors, they induce expression of a common set of ISGs in hepatocytes. However, unlike IFN-α, IFN-λ did not induce STAT activation or ISG expression by purified lymphocytes or monocytes. This important functional difference may provide a clinical advantage for IFN-λ as a treatment for chronic HCV infection, as it is less likely to induce the leukopenias that are often associated with IFN-α therapy. PMID:23258595

The natural compound silvestrol is a potent inhibitor of Ebola virus replication.

PubMed

Biedenkopf, Nadine; Lange-Grünweller, Kerstin; Schulte, Falk W; Weißer, Aileen; Müller, Christin; Becker, Dirk; Becker, Stephan; Hartmann, Roland K; Grünweller, Arnold

2017-01-01

The DEAD-box RNA helicase eIF4A, which is part of the heterotrimeric translation initiation complex in eukaryotes, is an important novel drug target in cancer research because its helicase activity is required to unwind extended and highly structured 5'-UTRs of several proto-oncogenes. Silvestrol, a natural compound isolated from the plant Aglaia foveolata, is a highly efficient, non-toxic and specific inhibitor of eIF4A. Importantly, 5'-capped viral mRNAs often contain structured 5'-UTRs as well, which may suggest a dependence on eIF4A for their translation by the host protein synthesis machinery. In view of the recent Ebola virus (EBOV) outbreak in West Africa, the identification of potent antiviral compounds is urgently required. Since Ebola mRNAs are 5'-capped and harbor RNA secondary structures in their extended 5'-UTRs, we initiated a BSL4 study to analyze silvestrol in EBOV-infected Huh-7 cells and in primary human macrophages for its antiviral activity. We observed that silvestrol inhibits EBOV infection at low nanomolar concentrations, as inferred from large reductions of viral titers. This correlated with an almost complete disappearance of EBOV proteins, comparable in effect to the translational shutdown of expression of the proto-oncoprotein PIM1, a cellular kinase known to be affected by silvestrol. Effective silvestrol concentrations were non-toxic in the tested cell systems. Thus, silvestrol appears to be a promising first-line drug for the treatment of acute EBOV and possibly other viral infections. Copyright © 2016 Elsevier B.V. All rights reserved.

OSBPL10, a novel candidate gene for high triglyceride trait in dyslipidemic Finnish subjects, regulates cellular lipid metabolism.

PubMed

Perttilä, Julia; Merikanto, Krista; Naukkarinen, Jussi; Surakka, Ida; Martin, Nicolas W; Tanhuanpää, Kimmo; Grimard, Vinciane; Taskinen, Marja-Riitta; Thiele, Christoph; Salomaa, Veikko; Jula, Antti; Perola, Markus; Virtanen, Ismo; Peltonen, Leena; Olkkonen, Vesa M

2009-08-01

Analysis of variants in three genes encoding oxysterol-binding protein (OSBP) homologues (OSBPL2, OSBPL9, OSBPL10) in Finnish families with familial low high-density lipoprotein (HDL) levels (N = 426) or familial combined hyperlipidemia (N = 684) revealed suggestive linkage of OSBPL10 single-nucleotide polymorphisms (SNPs) with extreme end high triglyceride (TG; >90th percentile) trait. Prompted by this initial finding, we carried out association analysis in a metabolic syndrome subcohort (Genmets) of Health2000 examination survey (N = 2,138), revealing association of multiple OSBPL10 SNPs with high serum TG levels (>95th percentile). To investigate whether OSBPL10 could be the gene underlying the observed linkage and association, we carried out functional experiments in the human hepatoma cell line Huh7. Silencing of OSBPL10 increased the incorporation of [(3)H]acetate into cholesterol and both [(3)H]acetate and [(3)H]oleate into triglycerides and enhanced the accumulation of secreted apolipoprotein B100 in growth medium, suggesting that the encoded protein ORP10 suppresses hepatic lipogenesis and very-low-density lipoprotein production. ORP10 was shown to associate dynamically with microtubules, consistent with its involvement in intracellular transport or organelle positioning. The data introduces OSBPL10 as a gene whose variation may contribute to high triglyceride levels in dyslipidemic Finnish subjects and provides evidence for ORP10 as a regulator of cellular lipid metabolism.

Evaluation of Antitrypanosomal Dihydroquinolines for Hepatotoxicity, Mutagenicity, and Methemoglobin Formation In Vitro

PubMed Central

Werbovetz, Karl A.; Riccio, Edward S.; Furimsky, Anna; Richard, Julian V.; He, Shanshan; Iyer, Lalitha; Mirsalis, Jon

2014-01-01

N 1-benzylated dihydroquinolin-6-ols and their corresponding esters display exceptional activity against African trypanosomes in vitro, and administration of members of this class of compounds to trypanosome-infected mice results in cures in a first stage African trypanosomiasis model. Since a quinone imine intermediate has been implicated in the antiparasitic mechanism of action of these compounds, evaluation of the hepatotoxic, mutagenic, and methemoglobin-promoting effects of these agents was performed. 1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride (OSU-36.HCl) and 1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate (OSU-40) showed outstanding in vitro selectivity for T. brucei compared to the HepG2, Hep3B, Huh7 and PLC5 hepatocyte cell lines. OSU-36.HCl and 1-(2-methoxybenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate (OSU-75) were not mutagenic when screened in the Ames assay, with or without metabolic activation. The latter two compounds promoted time- and dose-dependent formation of methemoglobin when incubated in whole human blood, but such levels were below those typically required to produce symptoms of methemoglobinemia in humans. While compounds capable of quinone imine formation require careful evaluation, these in vitro studies indicate that antitrypanosomal dihydroquinolines merit further study as drug candidates against the neglected tropical disease human African trypanosomiasis. PMID:24819520

Evaluation of Antitrypanosomal Dihydroquinolines for Hepatotoxicity, Mutagenicity, and Methemoglobin Formation In Vitro.

PubMed

Werbovetz, Karl A; Riccio, Edward S; Furimsky, Anna; Richard, Julian V; He, Shanshan; Iyer, Lalitha; Mirsalis, Jon

2014-07-01

N1-Benzylated dihydroquinolin-6-ols and their corresponding esters display exceptional activity against African trypanosomes in vitro, and administration of members of this class of compounds to trypanosome-infected mice results in cures in a first-stage African trypanosomiasis model. Since a quinone imine intermediate has been implicated in the antiparasitic mechanism of action of these compounds, evaluation of the hepatotoxic, mutagenic, and methemoglobin-promoting effects of these agents was performed. 1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride and 1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate showed outstanding in vitro selectivity for Trypanosoma brucei compared to the HepG2, Hep3B, Huh7, and PLC5 hepatocyte cell lines. 1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride and 1-(2-methoxybenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate were not mutagenic when screened in the Ames assay, with or without metabolic activation. The latter 2 compounds promoted time- and dose-dependent formation of methemoglobin when incubated in whole human blood, but such levels were below those typically required to produce symptoms of methemoglobinemia in humans. Although compounds capable of quinone imine formation require careful evaluation, these in vitro studies indicate that antitrypanosomal dihydroquinolines merit further study as drug candidates against the neglected tropical disease human African trypanosomiasis. © The Author(s) 2014.

Inhibitory Effects of Caffeic Acid, a Coffee-Related Organic Acid, on the Propagation of Hepatitis C Virus.

PubMed

Tanida, Isei; Shirasago, Yoshitaka; Suzuki, Ryosuke; Abe, Ryo; Wakita, Takaji; Hanada, Kentaro; Fukasawa, Masayoshi

2015-01-01

Multipurpose cohort studies have demonstrated that coffee consumption reduces the risk of hepatocellular carcinoma (HCC). Given that one of the main causes of HCC is hepatitis C virus (HCV) infection, we examined the effect of caffeic acid, a major organic acid derived from coffee, on the propagation of HCV using an in vitro naïve HCV particle-infection and production system within human hepatoma-derived Huh-7.5.1-8 cells. When cells were treated with 1% coffee extract or 0.1% caffeic acid for 1-h post HCV infection, the amount of HCV particles released into the medium at 3 and 4 days post-infection considerably decreased. In addition, HCV-infected cells cultured with 0.001% caffeic acid for 4 days, also released less HCV particles into the medium. Caffeic acid treatment inhibited the initial stage of HCV infection (i.e., between virion entry and the translation of the RNA genome) in both HCV genotypes 1b and 2a. These results suggest that the treatment of cells with caffeic acid may inhibit HCV propagation.

Downregulated circular RNA hsa_circ_0001649 regulates proliferation, migration and invasion in cholangiocarcinoma cells.

PubMed

Xu, Yi; Yao, Yue; Zhong, Xiangyu; Leng, Kaiming; Qin, Wei; Qu, Lijun; Cui, Yunfu; Jiang, Xingming

2018-02-05

Cholangiocarcinoma (CCA) is one of the most aggressive malignancies with increasing worldwide incidence and is characterized by unfavorable prognosis due to its early invasive characteristics and poor response to chemotherapy or radiotherapy. Accumulating evidence has indicated that aberrantly expressed circular RNAs (circRNAs) are involved in cancer development and progression. However, their clinical values and biological roles in CCA remain unclear. Hsa_circ_0001649, a novel cancer-related circRNA, has been previously reported to be downregulated in hepatocellular carcinoma and gastric cancer. In the present study, qRT-PCR was carried out to measure the expression of hsa_circ_0001649 in CCA tissue samples and cell lines, and the correlation between hsa_circ_0001649 expression and clinicopathologic features was analyzed. The biological functions of hsa_circ_0001649 in CCA cells were evaluated both in vitro and in vivo. As a result, hsa_circ_0001649 was aberrantly downregulated in CCA tissues and cells, and this downregulation was associated with tumor size and differentiation grade in CCA. In addition, hsa_circ_0001649 overexpression caused tumor suppressive effects via inhibiting cell proliferation, migration and invasion; inducing cell apoptosis in KMBC and Huh-28 cells. On the contrary, silencing of hsa_circ_0001649 caused the opposite phenotypes. Furthermore, tumor xenograft study confirmed the in vitro results. Collectively, our findings suggest that hsa_circ_0001649 might be a rational CCA-related therapeutic target. Copyright © 2018 Elsevier Inc. All rights reserved.

Antitumor activity of YM155, a selective survivin suppressant, in combination with cisplatin in hepatoblastoma.

PubMed

Yu, Ying; Zhao, Xiaosu; Zhang, Yu; Kang, Yanling; Wang, Jiaqi; Liu, Yingchun

2015-07-01

Cisplatin (CDDP) is a chemotherapeutic drug that is often used for the treatment of hepatoblastoma. However, many patients acquire resistance to therapeutic agents leading to local and distant treatment failure. It has been shown that suppression survivin contributed to the inhibition of tumor growth and enhanced chemotherapeutic sensitivity in several types of cancer. The aim of the present study was to determine whether treatment with sepantronium bromide (YM155), a novel small molecule inhibitor of survivin, enhanced the sensitivity of CDDP to hepatoblastoma cells, leading to the therapeutic efficacy of cisplatin. In vitro and in vivo models were used to examine the anticancer efficacy of YM155, either as a monotherapy or in combination with CDDP to identify more effective therapeutics against hepatoblastoma. The results showed that survivin expression was upregulated in hepatoblastoma tissues and cell lines, and that YM155 inhibited survivin expression in hepatoblastoma cells in a dose-dependent manner. YM155 enhanced sensitivity of CDDP to human HepG2 and HuH-6 hepatoblastoma cells. The YM155 combination with CDDP in hepatoblastoma cells significantly decreased cell proliferation and formation, and induced cell apoptosis than either agent alone. In a mouse xenograft model, YM155 combined with CDDP significantly suppressed tumor growth compared to the monotherapy. Taken together, these findings suggested that the combination of YM155 and CDDP is a promising drug candidate for the treatment of hepatoblastoma.

B7-H4 as a Target for Breast Cancer Immunotherapy

DTIC Science & Technology

2012-06-01

lymphoma and leukemia cell lines. CEM, Karpas 299, and TLBR -1, cell lines derived from acute T-cell lymphoblastic leukemia, large cell anaplastic...Accomplishments  Generation of human B7-H4-Fc fusion protein (antigen).  Discovery of a B7-H4 receptor on CEM, Karpas 299, and TLBR -1 cell lines...CEM Karpas 299 TLBR -1 Jurkat B7-H4R Figure 3. B7-H4 binding to human T-cell lymphoma cell lines. Red

Indole-3- carbinol enhances sorafenib cytotoxicity in hepatocellular carcinoma cells: A mechanistic study.

PubMed

Abdelmageed, Mai M; El-Naga, Reem N; El-Demerdash, Ebtehal; Elmazar, Mohamed M

2016-09-09

Sorafenib is the only chemotherapeutic agent currently approved for unresectable hepatocellular carcinoma (HCC). However, poor response rates have been widely reported. Indole-3-carbinol (I3C) is a potential chemopreventive phytochemical. The present study aimed to explore the potential chemomodulatory effects of I3C on sorafenib in HCC cells as well as the possible underlying mechanisms. I3C exhibited a greater cytotoxicity in HepG2 cells compared to Huh-7 cells (p < 0.0001). Moreover, the co-treatment of HepG2 cells with I3C and sorafenib was more effective (p = 0.002). Accordingly, subsequent mechanistic studies were carried on HepG2 cells. The results show that the ability of I3C to enhance sorafenib cytotoxicity in HCC cells could be partially attributed to increasing the apoptotic activity and decreasing the angiogenic potentials. The combination had a negative effect on epithelial-mesenchymal transition (EMT). Increased NOX-1 expression was also observed which may indicate the involvement of NOX-1 in I3C chemomodulatory effects. Additionally, the combination induced cell cycle arrest at the G0/G1 phase. In conclusion, these findings provide evidence that I3C enhances sorafenib anti-cancer activity in HCC cells.

Indole-3- carbinol enhances sorafenib cytotoxicity in hepatocellular carcinoma cells: A mechanistic study

PubMed Central

Abdelmageed, Mai M.; El-Naga, Reem N.; El-Demerdash, Ebtehal; Elmazar, Mohamed M.

2016-01-01

Sorafenib is the only chemotherapeutic agent currently approved for unresectable hepatocellular carcinoma (HCC). However, poor response rates have been widely reported. Indole-3-carbinol (I3C) is a potential chemopreventive phytochemical. The present study aimed to explore the potential chemomodulatory effects of I3C on sorafenib in HCC cells as well as the possible underlying mechanisms. I3C exhibited a greater cytotoxicity in HepG2 cells compared to Huh-7 cells (p < 0.0001). Moreover, the co-treatment of HepG2 cells with I3C and sorafenib was more effective (p = 0.002). Accordingly, subsequent mechanistic studies were carried on HepG2 cells. The results show that the ability of I3C to enhance sorafenib cytotoxicity in HCC cells could be partially attributed to increasing the apoptotic activity and decreasing the angiogenic potentials. The combination had a negative effect on epithelial-mesenchymal transition (EMT). Increased NOX-1 expression was also observed which may indicate the involvement of NOX-1 in I3C chemomodulatory effects. Additionally, the combination induced cell cycle arrest at the G0/G1 phase. In conclusion, these findings provide evidence that I3C enhances sorafenib anti-cancer activity in HCC cells. PMID:27612096

Hepatitis C Virus Reveals a Novel Early Control in Acute Immune Response

PubMed Central

Arnaud, Noëlla; Dabo, Stéphanie; Akazawa, Daisuke; Fukasawa, Masayoshi; Shinkai-Ouchi, Fumiko; Hugon, Jacques; Wakita, Takaji; Meurs, Eliane F.

2011-01-01

Recognition of viral RNA structures by the intracytosolic RNA helicase RIG-I triggers induction of innate immunity. Efficient induction requires RIG-I ubiquitination by the E3 ligase TRIM25, its interaction with the mitochondria-bound MAVS protein, recruitment of TRAF3, IRF3- and NF-κB-kinases and transcription of Interferon (IFN). In addition, IRF3 alone induces some of the Interferon-Stimulated Genes (ISGs), referred to as early ISGs. Infection of hepatocytes with Hepatitis C virus (HCV) results in poor production of IFN despite recognition of the viral RNA by RIG-I but can lead to induction of early ISGs. HCV was shown to inhibit IFN production by cleaving MAVS through its NS3/4A protease and by controlling cellular translation through activation of PKR, an eIF2α-kinase containing dsRNA-binding domains (DRBD). Here, we have identified a third mode of control of IFN induction by HCV. Using HCVcc and the Huh7.25.CD81 cells, we found that HCV controls RIG-I ubiquitination through the di-ubiquitine-like protein ISG15, one of the early ISGs. A transcriptome analysis performed on Huh7.25.CD81 cells silenced or not for PKR and infected with JFH1 revealed that HCV infection leads to induction of 49 PKR-dependent genes, including ISG15 and several early ISGs. Silencing experiments revealed that this novel PKR-dependent pathway involves MAVS, TRAF3 and IRF3 but not RIG-I, and that it does not induce IFN. Use of PKR inhibitors showed that this pathway requires the DRBD but not the kinase activity of PKR. We then demonstrated that PKR interacts with HCV RNA and MAVS prior to RIG-I. In conclusion, HCV recruits PKR early in infection as a sensor to trigger induction of several IRF3-dependent genes. Among those, ISG15 acts to negatively control the RIG-I/MAVS pathway, at the level of RIG-I ubiquitination.These data give novel insights in the machinery involved in the early events of innate immune response. PMID:22022264

The role of the NLRP3 inflammasome and the activation of IL-1β in the pathogenesis of chronic viral hepatic inflammation.

PubMed

Molyvdas, Adam; Georgopoulou, Urania; Lazaridis, Nikolaos; Hytiroglou, Prodromos; Dimitriadis, Alexios; Foka, Pelagia; Vassiliadis, Themistoklis; Loli, Georgia; Phillipidis, Athanasios; Zebekakis, Pantelis; Germenis, Anastasios E; Speletas, Matthaios; Germanidis, Georgios

2018-05-23

Chronic viral hepatitis is a prevalent disease with major health implications. Its underlying pathophysiological mechanisms are not fully understood. IL-1β and the NLRP3 inflammasome involvement has been suggested in recent years, from in vitro data and data from peripheral blood samples. Therefore, we investigated IL-1β and the NLRP3 inflammasome in liver tissues in an effort to clarify their role in the pathophysiology of chronic viral hepatitis. We studied liver biopsies from patients with a new diagnosis of either chronic hepatitis B (CHB) and chronic hepatitis C (CHC) or patients with chronic hepatitis B in remission (CHB-rem). The biopsies were separated in two parts. The first part was sent to histology to determine the grade of inflammation and fibrosis. From the second part, RNA was extracted and converted to cDNA used in semi-quantitative Real-Time PCR to measure the levels of IL1B, CASP1, NLRP3, ASC and IL1RA. The cell lines used in the in vitro experiments were Huh7.5, LX2 and THP-1 in variety of combinations of monocultures, co-cultures and triple cultures with one of the cell lines infected with the JFH-1 HCV clone. From the cell cultures RNA was extracted and converted to cDNA. For cell lines, we focused in the expression of IL1B and NLRP3. The expression of IL1B, CASP1 and NLRP3 were found significantly different between our groups (p = 0.001, p = 0.001 and p = 0.038, respectively). CHB patients displayed significantly higher IL1B and CASP1 mRNA levels compared to both CHB-rem and CHC patients. IL1B expression significantly correlates with liver biochemical data in CHB patients (AST: p = 0.006, r = 0.457; ALT p = 0.002, r = 0.497). Finally, mRNA levels of IL1B in CHB patients significantly correlate with the degree of inflammation (p = 0.016) but not the stage of fibrosis (p = 0.362). Interestingly, the relative expression of IL1B in triple culture experiments in vitro was below of 1.5-fold, suggesting no activation of IL1B. Moreover, no activation of NLRP3 was demonstrated in all investigated in vitro conditions. IL-1β might play an important role in the pathogenesis of chronic hepatic inflammation from HBV, but not from HCV. Copyright © 2018 Elsevier Ltd. All rights reserved.

Gingerol Synergizes the Cytotoxic Effects of Doxorubicin against Liver Cancer Cells and Protects from Its Vascular Toxicity.

PubMed

Al-Abbasi, Fahad A; Alghamdi, Eman A; Baghdadi, Mohammed A; Alamoudi, Abdulmohsin J; El-Halawany, Ali M; El-Bassossy, Hany M; Aseeri, Ali H; Al-Abd, Ahmed M

2016-07-08

Hydroxyphenylalkanes and diarylheptanoids possess potential therapeutic value in different pathophysiological conditions, such as malignancy. In the current study, naturally isolated hydroxyphenylalkane and diarylheptanoid compounds were investigated for potential chemo-modulatory effects in addition to potential vascular protective roles with doxorubicin. Diarylheptanoids showed stronger antioxidant effects, in comparison to hydroxyphenylalkanes, as demonstrated by DPPH assay and amelioration of CCl₄-induced disturbed intracellular GSH/GSSG balance. Shogaol and 4'-methoxygingerol showed considerable cytotoxic effects against HCT116, HeLa, HepG2 and MCF7 cells, with IC50 values ranging from 3.1 to 19.4 µM. Gingerol significantly enhanced the cytotoxic profile of doxorubicin against HepG₂ and Huh7, cells decreasing its IC50s by 10- and 4-fold, respectively. Cell cycle distribution was studied using DNA cytometry. Doxorubicin alone induced cell accumulation at S-phase and G₂/M-phase, while in combination with gingerol it significantly induced cell cycle arrest at the G₂/M-phase. Additionally, the vascular protective effect of gingerol against doxorubicin (10 µM) was examined on isolated aortic rings. Co-incubation with 6-gingerol (30 µM) completely blocked the exaggerated vasoconstriction and impaired vascular relaxation induced by doxorubicin. In conclusion, despite its relatively weak antioxidant properties, gingerol protected from DOX-induced vascular damage, apparently not through a ROS scavenging mechanism. Besides, gingerol synergized the cytotoxic effects of DOX against liver cancer cells without influencing the cellular pharmacokinetics.

Differential In Vitro Effects of Intravenous versus Oral Formulations of Silibinin on the HCV Life Cycle and Inflammation

PubMed Central

Wagoner, Jessica; Morishima, Chihiro; Graf, Tyler N.; Oberlies, Nicholas H.; Teissier, Elodie; Pécheur, Eve-Isabelle; Tavis, John E.; Polyak, Stephen J.

2011-01-01

Silymarin prevents liver disease in many experimental rodent models, and is the most popular botanical medicine consumed by patients with hepatitis C. Silibinin is a major component of silymarin, consisting of the flavonolignans silybin A and silybin B, which are insoluble in aqueous solution. A chemically modified and soluble version of silibinin, SIL, has been shown to potently reduce hepatitis C virus (HCV) RNA levels in vivo when administered intravenously. Silymarin and silibinin inhibit HCV infection in cell culture by targeting multiple steps in the virus lifecycle. We tested the hepatoprotective profiles of SIL and silibinin in assays that measure antiviral and anti-inflammatory functions. Both mixtures inhibited fusion of HCV pseudoparticles (HCVpp) with fluorescent liposomes in a dose-dependent fashion. SIL inhibited 5 clinical genotype 1b isolates of NS5B RNA dependent RNA polymerase (RdRp) activity better than silibinin, with IC50 values of 40–85 µM. The enhanced activity of SIL may have been in part due to inhibition of NS5B binding to RNA templates. However, inhibition of the RdRps by both mixtures plateaued at 43–73%, suggesting that the products are poor overall inhibitors of RdRp. Silibinin did not inhibit HCV replication in subgenomic genotype 1b or 2a replicon cell lines, but it did inhibit JFH-1 infection. In contrast, SIL inhibited 1b but not 2a subgenomic replicons and also inhibited JFH-1 infection. Both mixtures inhibited production of progeny virus particles. Silibinin but not SIL inhibited NF-κB- and IFN-B-dependent transcription in Huh7 cells. However, both mixtures inhibited T cell proliferation to similar degrees. These data underscore the differences and similarities between the intravenous and oral formulations of silibinin, which could influence the clinical effects of this mixture on patients with chronic liver diseases. PMID:21297992

The VP40 protein of Marburg virus exhibits impaired budding and increased sensitivity to human tetherin following mouse adaptation.

PubMed

Feagins, Alicia R; Basler, Christopher F

2014-12-01

The Marburg virus VP40 protein is a viral matrix protein that spontaneously buds from cells. It also functions as an interferon (IFN) signaling antagonist by targeting Janus kinase 1 (JAK1). A previous study demonstrated that the VP40 protein of the Ravn strain of Marburg virus (Ravn virus [RAVV]) failed to block IFN signaling in mouse cells, whereas the mouse-adapted RAVV (maRAVV) VP40 acquired the ability to inhibit IFN responses in mouse cells. The increased IFN antagonist function of maRAVV VP40 mapped to residues 57 and 165, which were mutated during the mouse adaptation process. In the present study, we demonstrate that maRAVV VP40 lost the capacity to efficiently bud from human cell lines, despite the fact that both parental and maRAVV VP40s bud efficiently from mouse cell lines. The impaired budding in human cells corresponds with the appearance of protrusions on the surface of maRAVV VP40-expressing Huh7 cells and with an increased sensitivity of maRAVV VP40 to restriction by human tetherin but not mouse tetherin. However, transfer of the human tetherin cytoplasmic tail to mouse tetherin restored restriction of maRAVV VP40. Residues 57 and 165 were demonstrated to contribute to the failure of maRAVV VP40 to bud from human cells, and residue 57 was demonstrated to alter VP40 oligomerization, as assessed by coprecipitation assay, and to determine sensitivity to human tetherin. This suggests that RAVV VP40 acquired, during adaptation to mice, changes in its oligomerization potential that enhanced IFN antagonist function. However, this new capacity impaired RAVV VP40 budding from human cells. Filoviruses, which include Marburg viruses and Ebola viruses, are zoonotic pathogens that cause severe disease in humans and nonhuman primates but do not cause similar disease in wild-type laboratory strains of mice unless first adapted to these animals. Although mouse adaptation has been used as a method to develop small animal models of pathogenesis, the molecular determinants associated with filovirus mouse adaptation are poorly understood. Our study demonstrates how genetic changes that accrued during mouse adaptation of the Ravn strain of Marburg virus have impacted the budding function of the viral VP40 matrix protein. Strikingly, we find impairment of mouse-adapted VP40 budding function in human but not mouse cell lines, and we correlate the impairment with an increased sensitivity of VP40 to restriction by human but not mouse tetherin and with changes in VP40 oligomerization. These data suggest that there are functional costs associated with filovirus adaptation to new hosts and implicate tetherin as a filovirus host restriction factor. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Assembly of Hepatitis Delta Virus: Particle Characterization, Including the Ability To Infect Primary Human Hepatocytes▿

PubMed Central

Gudima, Severin; He, Yiping; Meier, Anja; Chang, Jinhong; Chen, Rongji; Jarnik, Michal; Nicolas, Emmanuelle; Bruss, Volker; Taylor, John

2007-01-01

Efficient assembly of hepatitis delta virus (HDV) was achieved by cotransfection of Huh7 cells with two plasmids: one to provide expression of the large, middle, and small envelope proteins of hepatitis B virus (HBV), the natural helper of HDV, and another to initiate replication of the HDV RNA genome. HDV released into the media was assayed for HDV RNA and HBV envelope proteins and characterized by rate-zonal sedimentation, immunoaffinity purification, electron microscopy, and the ability to infect primary human hepatocytes. Among the novel findings were that (i) immunostaining for delta antigen 6 days after infection with 300 genome equivalents (GE) per cell showed only 1% of cells as infected, but this was increased to 16% when 5% polyethylene glycol was present during infection; (ii) uninfected cells did not differ from infected cells in terms of albumin accumulation or the presence of E-cadherin at cell junctions; and (iii) sensitive quantitative real-time PCR assays detected HDV replication even when the multiplicity of infection was 0.2 GE/cell. In the future, this HDV assembly and infection system can be further developed to better understand the mechanisms shared by HBV and HDV for attachment and entry into host cells. PMID:17229685

miR-34a: Multiple Opposing Targets and One Destiny in Hepatocellular Carcinoma.

PubMed

Yacoub, Radwa Alaa; Fawzy, Injie Omar; Assal, Reem Amr; Hosny, Karim Adel; Zekri, Abdel-Rahman Nabawy; Esmat, Gamal; El Tayebi, Hend Mohamed; Abdelaziz, Ahmed Ihab

2016-12-28

Background and Aims: The role of miR-34a in hepatocellular carcinoma (HCC) is controversial and several unresolved issues remain, including its expression pattern and relevance to tumor etiology, tumor stage and prognosis, and finally, its impact on apoptosis. Methods: miR-34a expression was assessed in hepatitis C virus (HCV)-induced non-metastatic HCC tissues by RT-Q-PCR. Huh-7 cells were transfected with miR-34a mimics and the impact of miR-34a was examined on 84 pro-apoptotic/anti-apoptotic genes using PCR array; its net effect was tested on cell viability via MTT assay. Results: miR-34a expression was up-regulated in HCC tissues. Moreover, miR-34a induced a large set of pro-apoptotic/anti-apoptotic genes, with a net result of triggering apoptosis and repressing cell viability. Conclusions: HCC-related differential expression of miR-34a could be etiology-based or stage-specific, and low expression of miR-34a may predict poor prognosis. This study's findings also emphasize the role of miR-34a in apoptosis.

Corosolic Acid Induces Non-Apoptotic Cell Death through Generation of Lipid Reactive Oxygen Species Production in Human Renal Carcinoma Caki Cells.

PubMed

Woo, Seon Min; Seo, Seung Un; Min, Kyoung-Jin; Im, Seung-Soon; Nam, Ju-Ock; Chang, Jong-Soo; Kim, Shin; Park, Jong-Wook; Kwon, Taeg Kyu

2018-04-27

Corosolic acid is one of the pentacyclic triterpenoids isolated from Lagerstroemia speciose and has been reported to exhibit anti-cancer and anti-proliferative activities in various cancer cells. In the present study, we investigated the molecular mechanisms of corosolic acid in cancer cell death. Corosolic acid induces a decrease of cell viability and an increase of cell cytotoxicity in human renal carcinoma Caki cells. Corosolic acid-induced cell death is not inhibited by apoptosis inhibitor (z-VAD-fmk, a pan-caspase inhibitor), necroptosis inhibitor (necrostatin-1), or ferroptosis inhibitors (ferrostatin-1 and deferoxamine (DFO)). Furthermore, corosolic acid significantly induces reactive oxygen species (ROS) levels, but antioxidants ( N -acetyl-l-cysteine (NAC) and trolox) do not inhibit corosolic acid-induced cell death. Interestingly, corosolic acid induces lipid oxidation, and α-tocopherol markedly prevents corosolic acid-induced lipid peroxidation and cell death. Anti-chemotherapeutic effects of α-tocopherol are dependent on inhibition of lipid oxidation rather than inhibition of ROS production. In addition, corosolic acid induces non-apoptotic cell death in other renal cancer (ACHN and A498), breast cancer (MDA-MB231), and hepatocellular carcinoma (SK-Hep1 and Huh7) cells, and α-tocopherol markedly inhibits corosolic acid-induced cell death. Therefore, our results suggest that corosolic acid induces non-apoptotic cell death in cancer cells through the increase of lipid peroxidation.

B7-1 (CD80) as target for immunotoxin therapy for Hodgkin's disease.

PubMed Central

Vooijs, W. C.; Otten, H. G.; van Vliet, M.; van Dijk, A. J.; de Weger, R. A.; de Boer, M.; Bohlen, H.; Bolognesi, A.; Polito, L.; de Gast, G. C.

1997-01-01

In this preclinical study, the potential applicability of an anti-B7-1 immunotoxin (IT) for the treatment of Hodgkin's disease (HD) was investigated. Immunohistochemical analysis demonstrated strong expression of B7-1 on Hodgkin and Reed-Sternberg (R-S) cells and clear expression on dendritic cells, macrophages and some B-cells in tissues, but not on other tissue cells. Flow cytometric analysis demonstrated that B7-1 was expressed on a few monocytes, but not on CD34+ cells from bone marrow, resting T- or B-cells from peripheral blood or epithelial and endothelial cell lines. An anti-B7-1 immunotoxin containing the anti-B7-1 monoclonal antibody (MAb) B7-24 and saporin as toxin moiety was constructed and showed an affinity similar to that shown by the native MAb. It exhibited strong cytotoxicity against the B7-1+ B-cell line Raji (IC50 10(-11) M), R-S cell lines HDLM2, KM/H2 and L428 and also against a B7-1-transfected epithelial cell line, A431, whose parental line lacks expression of B7-1. In clonogenic assays with Raji cells or KM/H2 cells, a 3- or 4-log kill, respectively, was observed. No cytotoxicity was found against the B7-1- epithelial and endothelial cell lines or against haematopoietic progenitor cells. In conclusion, an anti-B7-1 immunotoxin was developed that had good cytotoxicity against R-S cell lines and that may be used in the elimination of R-S cells in vivo. A concomitant elimination of activated antigen-presenting cells may avoid development of antitoxin and anti-mouse Ig responses and allow repeated administration. Images Figure 1 PMID:9365164

Methylation of S100A8 is a promising diagnosis and prognostic marker in hepatocellular carcinoma.

PubMed

Liu, Kun; Zhang, Yuening; Zhang, Chengdong; Zhang, Qinle; Li, Jiatong; Xiao, Feifan; Li, Yingfang; Zhang, Ruoheng; Dou, Dongwei; Liang, Jiezhen; Qin, Jian; Lin, Zhidi; Zhao, Dong; Jiang, Min; Liang, Zhenxin; Su, Jie; Gupta, Vanaparthy Pranay; He, Min; Yang, Xiaoli

2016-08-30

The abnormality of DNA methylation is one of the major epigenetic alterations in the human hepatocellular carcinoma (HCC). We have assessed the global genomic DNA methylation profiles in human HCC patients by using the Infinium Human Methylation27 BeadChip. A CpG loci of S100A8 was found to be significantly hypomethylated in HCC.Pooled meta-analysis of five validation public datasets demonstrated its methylation level was significantly lower for HCC compared to paired adjacent normal tissues. Quantitative pyrosequencing analysis also showed that the S100A8 methylation level was decreased in cancer tissues (31.90%±13.31%) than that in the paired adjacent normal tissues (65.33%±3.64%, p<0.01). The area under the ROC curve (AUC) value was 0.950 (p<0.01). Kaplan-Meier survival curves revealed that hypomethylation of S100A8 was associated with shortened overall survival (OS) and progression-free survival (PFS) (log rank p<0.05). Multivariate Cox proportional hazards model also indicated significantly shorter OS (HR, 1.709; 95 % CI, 1.127-2.591) and PFS (HR, 1.767; 95 % CI, 1.168-2.974) were observed in the low-methylation-level group compared to the high-methylation-level group. Furthermore, S100A8 overexpression in Huh7 and MHCC-97H hepatoma cell lines led to increased cell proliferation, migration, invasion, and tumor growth. These findings suggested S100A8 methylation to be served as potential diagnosis and prognosis marker for HCC. S100A8 also may play as a tumor promoter in HCC.

Methylation of S100A8 is a promising diagnosis and prognostic marker in hepatocellular carcinoma

PubMed Central

Xiao, Feifan; Li, Yingfang; Zhang, Ruoheng; Dou, Dongwei; Liang, Jiezhen; Qin, Jian; Lin, Zhidi; Zhao, Dong; Jiang, Min; Liang, Zhenxin; Su, Jie; Gupta, Vanaparthy Pranay; He, Min; Yang, Xiaoli

2016-01-01

The abnormality of DNA methylation is one of the major epigenetic alterations in the human hepatocellular carcinoma (HCC). We have assessed the global genomic DNA methylation profiles in human HCC patients by using the Infinium Human Methylation27 BeadChip. A CpG loci of S100A8 was found to be significantly hypomethylated in HCC. Pooled meta-analysis of five validation public datasets demonstrated its methylation level was significantly lower for HCC compared to paired adjacent normal tissues. Quantitative pyrosequencing analysis also showed that the S100A8 methylation level was decreased in cancer tissues (31.90%±13.31%) than that in the paired adjacent normal tissues (65.33%±3.64%, p<0.01). The area under the ROC curve (AUC) value was 0.950 (p<0.01). Kaplan-Meier survival curves revealed that hypomethylation of S100A8 was associated with shortened overall survival (OS) and progression-free survival (PFS) (log rank p<0.05). Multivariate Cox proportional hazards model also indicated significantly shorter OS (HR, 1.709; 95 % CI, 1.127–2.591) and PFS (HR, 1.767; 95 % CI, 1.168–2.974) were observed in the low-methylation-level group compared to the high-methylation-level group. Furthermore, S100A8 overexpression in Huh7 and MHCC-97H hepatoma cell lines led to increased cell proliferation, migration, invasion, and tumor growth. These findings suggested S100A8 methylation to be served as potential diagnosis and prognosis marker for HCC. S100A8 also may play as a tumor promoter in HCC. PMID:27462864

Synthesis and optimization of cholesterol-based diquaternary ammonium Gemini Surfactant (Chol-GS) as a new gene delivery vector.

PubMed

Kim, Bieong-Kil; Doh, Kyung-Oh; Bae, Yun-Ui; Seu, Young-Bae

2011-01-01

Amongst a number of potential nonviral vectors, cationic liposomes have been actively researched, with both gemini surfactants and bola amphiphiles reported as being in possession of good structures in terms of cell viability and in vitro transfection. In this study, a cholesterol-based diquaternary ammonium gemini surfactant (Chol-GS) was synthesized and assessed as a novel nonviral gene vector. Chol-GS was synthesized from cholesterol by way of four reaction steps. The optimal efficiency was found to be at a weight ratio of 1:4 of lipid:DOPE (1,2-dioleoyl-L-alpha- glycero-3-phosphatidylethanolamine), and at a ratio of between 10:1~15:1 of liposome:DNA. The transfection efficiency was compared with commercial liposomes and with Lipofectamine, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE-C), and N-[1-(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTAP). The results indicate that the efficiency of Chol-GS is greater than that of all the tested commercial liposomes in COS7 and Huh7 cells, and higher than DOTAP and Lipofectamine in A549 cells. Confirmation of these findings was observed through the use of green fluorescent protein expression. Chol-GS exhibited a moderate level of cytotoxicity, at optimum concentrations for efficient transfection, indicating cell viability. Hence, the newly synthesized Chol-GS liposome has the potential of being an excellent nonviral vector for gene delivery.

A Concerted Action of Hepatitis C Virus P7 and Nonstructural Protein 2 Regulates Core Localization at the Endoplasmic Reticulum and Virus Assembly

PubMed Central

Boson, Bertrand; Granio, Ophélia; Bartenschlager, Ralf; Cosset, François-Loïc

2011-01-01

Hepatitis C virus (HCV) assembly remains a poorly understood process. Lipid droplets (LDs) are thought to act as platforms for the assembly of viral components. The JFH1 HCV strain replicates and assembles in association with LD-associated membranes, around which viral core protein is predominantly detected. In contrast, despite its intrinsic capacity to localize to LDs when expressed individually, we found that the core protein of the high-titer Jc1 recombinant virus was hardly detected on LDs of cell culture-grown HCV (HCVcc)-infected cells, but was mainly localized at endoplasmic reticulum (ER) membranes where it colocalized with the HCV envelope glycoproteins. Furthermore, high-titer cell culture-adapted JFH1 virus, obtained after long-term culture in Huh7.5 cells, exhibited an ER-localized core in contrast to non-adapted JFH1 virus, strengthening the hypothesis that ER localization of core is required for efficient HCV assembly. Our results further indicate that p7 and NS2 are HCV strain-specific factors that govern the recruitment of core protein from LDs to ER assembly sites. Indeed, using expression constructs and HCVcc recombinant genomes, we found that p7 is sufficient to induce core localization at the ER, independently of its ion-channel activity. Importantly, the combined expression of JFH1 or Jc1 p7 and NS2 induced the same differential core subcellular localization detected in JFH1- vs. Jc1-infected cells. Finally, results obtained by expressing p7-NS2 chimeras between either virus type indicated that compatibilities between the p7 and the first NS2 trans-membrane domains is required to induce core-ER localization and assembly of extra- and intra-cellular infectious viral particles. In conclusion, we identified p7 and NS2 as key determinants governing the subcellular localization of HCV core to LDs vs. ER and required for initiation of the early steps of virus assembly. PMID:21814513

Arginine deiminase expressed in vivo, driven by human telomerase reverse transcriptase promoter, displays high hepatoma targeting and oncolytic efficiency.

PubMed

Jiang, Hui; Guo, Song; Xiao, Dan; Bian, Xuzhao; Wang, Jie; Wang, Ying; Zhou, Huiting; Cai, Jun; Zheng, Zhongliang

2017-06-06

Arginine starvation has the potential to selectively treat both primary tumor and (micro) metastatic tissue with very low side effects. Arginine deiminase (ADI; EC 3.5.3.6), an arginine-degrading enzyme, has been studied as a potential anti-tumor drug for the treatment of arginine-auxotrophic tumors. Though ADI-PEG20 (pegylated ADI by PEG 20,000) already passed the phase I/II clinical trials [1], it is just used as adjuvant therapy because of its low efficiency and less targeting. Then, this paper discussed the efficiency of arginine starvation mediated by ADI expressed in cytoplasm for liver cancers. In order to guarantee the tumor targeting, human telomerase reverse transcriptase (hTERT) promoter was used to drive the expression of ADI in vivo. To access the anti-tumor efficiency of ADI, p53 gene was used as the positive control. Thus, ADI displayed obvious cytotoxicity to BEL7402 and HUH7 cell lines in cytoplasm. The apoptosis rates rose from 15% to nearly 60% after changing the expression vectors from pcDNA4 plasmid to adenovirus. Compared with p53-adenovirus, ADI-adenovirus showed the higher oncolytic activity in the intratumoral injection model of mice. Tumor disappeared after the treatment of ADI-adenovirus for two weeks, and the mice pulled through all. Therefore, ADI is an ideal anti-tumor gene for caner targeting therapy with the help of hTERT promoter.

The T47D cell line is an ideal experimental model to elucidate the progesterone-specific effects of a luminal A subtype of breast cancer.

PubMed

Yu, Sungryul; Kim, Taemook; Yoo, Kyung Hyun; Kang, Keunsoo

2017-05-06

Cell lines are often used as in vitro tools to mimic certain types of in vivo system; several cell lines, including MCF-7 and T47D, have been widely used in breast cancer studies without investigating the cell lines' characteristics. In this study, we compared the genome-wide binding profiles of ERα, PR, and P300, and the gene expression changes between MCF-7 and T47D cell lines that represent the luminal A subtype of breast cancer. Surprisingly, several thousand genes were differentially expressed under estrogenic condition. In addition, ERα, PR, and P300 binding to regulatory elements showed distinct genomic landscapes between MCF-7 and T47D cell lines in the same hormonal states. In particular, the T47D cell line was markedly susceptible to progesterone, whereas the MCF-7 cell line did not respond to progesterone in the presence of estrogen. Consistently, changes in the expression level of the PR-target gene, STAT5A, were only observed in the T47D cell line, not the MCF-7 cell line, when treated with progesterone. Overall, the results highlight the importance of selecting appropriate cell lines for breast cancer studies and suggest that T47D cell lines can be an ideal experimental model to elucidate the progesterone-specific effects of a luminal A subtype of breast cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Aeginetia indica Decoction Inhibits Hepatitis C Virus Life Cycle

PubMed Central

Lin, Cheng-Wei; Lo, Chieh-Wen; Tsai, Chia-Ni; Pan, Ting-Chun; Chen, Pin-Yin

2018-01-01

Chronic hepatitis C virus (HCV) infection is still a global epidemic despite the introduction of several highly effective direct-acting antivirals that are tagged with sky-high prices. The present study aimed to identify an herbal decoction that ameliorates HCV infection. Among six herbal decoctions tested, the Aeginetia indica decoction had the most profound effect on the HCV reporter activity in infected Huh7.5.1 liver cells in a dose- and time-dependent manner. The Aeginetia indica decoction exerted multiple inhibitory effects on the HCV life cycle. Pretreatment of the cells with the Aeginetia indica decoction prior to HCV infection reduced the HCV RNA and non-structural protein 3 (NS3) protein levels in the infected cells. The Aeginetia indica decoction reduced HCV internal ribosome entry site-mediated protein translation activity. It also reduced the HCV RNA level in the infected cells in association with reduced NS5A phosphorylation at serine 235, a predominant phosphorylation event indispensable to HCV replication. Thus, the Aeginetia indica decoction inhibits HCV infection, translation, and replication. Mechanistically, the Aeginetia indica decoction probably reduced HCV replication via reducing NS5A phosphorylation at serine 235. PMID:29315273

Variation of Keratin 7 Expression and Other Phenotypic Characteristics of Independent Isolates of Cadmium Transformed Human Urothelial Cells (UROtsa)

PubMed Central

Somji, Seema; Zhou, Xu Dong; Mehus, Aaron; Sens, Mary Ann; Garrett, Scott H.; Lutz, Krista L.; Dunlevy, Jane R.; Zheng, Yun; Sens, Donald. A.

2009-01-01

This laboratory has shown that a human urothelial cell line (UROtsa) transformed by cadmium (Cd+2) produced subcutaneous tumor heterotransplants that resemble human transitional cell carcinoma (TCC). In the present study, additional Cd+2 transformed cell lines were isolated to determine if independent exposures of the cell line to Cd+2 would result in malignantly transformed cell lines possessing similar phenotypic properties. Seven independent isolates were isolated and assessed for their doubling times, morphology, ability to heterotransplant subcutaneously and in the peritoneal cavity of nude mice and for the expression keratin 7. The 7 cell lines all displayed an epithelial morphology with no evidence of squamous differentiation. Doubling times were variable among the isolates, being significantly reduced or similar to the parental cells. All 7 isolates were able to form subcutaneous tumor heterotransplants with a TCC morphology and all heterotransplants displayed areas of squamous differentiation of the transitional cells. The degree of squamous differentiation varied among the isolates. In contrast to subcutaneous tumor formation, only 1 isolate of the Cd+2 transformed cells (UTCd#1) was able to effectively colonize multiple sites within the peritoneal cavity. An analysis of keratin 7 expression showed no correlation with squamous differentiation for the subcutaneous heterotransplants generated from the 7 cell lines. Keratin 7 was expressed in 6 of the 7 cell lines and their subcutaneous tumor heterotransplants. Keratin 7 was not expressed in the cell line that was able to form tumors within the peritoneal cavity. These results show that individual isolates of Cd+2 transformed cells have both similarities and differences in their phenotype. PMID:19921857

Interferon sensitivity-determining region of hepatitis C virus influences virus production and interferon signaling

PubMed Central

Sugiyama, Ryuichi; Murayama, Asako; Nitta, Sayuri; Yamada, Norie; Tasaka-Fujita, Megumi; Masaki, Takahiro; Aly, Hussein Hassan; Shiina, Masaaki; Ryo, Akihide; Ishii, Koji; Wakita, Takaji; Kato, Takanobu

2018-01-01

The number of amino acid substitutions in the interferon (IFN) sensitivity-determining region (ISDR) of hepatitis C virus (HCV) NS5A is a strong predictor for the outcome of IFN-based treatment. To assess the involvement of ISDR in the HCV life cycle and to clarify the molecular mechanisms influencing IFN susceptibility, we used recombinant JFH-1 viruses with NS5A of the genotype 1b Con1 strain (JFH1/5ACon1) and with NS5A ISDR containing 7 amino acid substitutions (JFH1/5ACon1/i-7mut), and compared the virus propagation and the induction of interferon-stimulated genes (ISGs). By transfecting RNAs of these strains into HuH-7-derived cells, we found that the efficiency of infectious virus production of JFH1/5ACon1/i-7mut was attenuated compared with JFH1/5ACon1. After transfecting full-length HCV RNA into HepaRG cells, the mRNA expression of ISGs was sufficiently induced by IFN treatment in JFH1/5ACon1/i-7mut-transfected but not in JFH1/5ACon1-transfected cells. These data suggested that the NS5A-mediated inhibition of ISG induction was deteriorated by amino acid substitutions in the ISDR. In conclusion, using recombinant JFH-1 viruses, we demonstrated that HCV NS5A is associated with infectious virus production and the inhibition of IFN signaling, and amino acid substitutions in the NS5A ISDR deteriorate these functions. These observations explain the strain-specific evasion of IFN signaling by HCV. PMID:29464023

Toll-like receptor-4 is a target for suppression of proliferation and chemoresistance in HepG2 hepatoblastoma cells.

PubMed

Hsiao, Chih-Cheng; Chen, Po-Han; Cheng, Cheng-I; Tsai, Ming-Shian; Chang, Chih-Yang; Lu, Shang-Chieh; Hsieh, Ming-Chu; Lin, Yu-Chun; Lee, Po-Huang; Kao, Ying-Hsien

2015-11-01

Toll-like receptor-4 (TLR4) is known to influence growth and migration of hepatocellular tumors; however, its role in hepatoblastoma remains poorly understood. This study investigated the regulatory role of TLR4 in proliferation and chemoresistance of HepG2 hepatoblastoma cells. Treatment with lipopolysaccharide (LPS), a TLR4 agonist, was found to significantly upregulate TLR4 expression in HepG2 cells, but not in malignant Huh-7 and Sk-Hep1 hepatocellular carcinoma cells. Additionally, IL-6 enhanced LPS-induced TLR4 upregulation. LPS-stimulated TLR4 activation increased proliferation, nitric oxide synthase (NOS) expression, and NO production in HepG2 cells. Chemotherapeutic agents, cisplatin and doxorubicin, effectively inhibited TLR4 expression in HepG2 cells. Characterization of LPS-induced signaling activation and blockade with kinase inhibitors revealed the involvement of Akt and MAPK pathways in LPS-enhanced NO release from, and proliferation of HepG2 cells. Mechanistically, gene modifications as a result of TLR4 transfection and siRNA-mediated knockdown further demonstrated a crucial role for TLR4 in the regulation of NOS expression, cell proliferation, and chemoresistance in HepG2 cells. These findings suggest that targeting TLR4 expression and its cognate signaling may modulate proliferation and chemosensitivity in hepatoblastoma cells and serve as a potential therapeutic target. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Is “Huh?” a Universal Word? Conversational Infrastructure and the Convergent Evolution of Linguistic Items

PubMed Central

Dingemanse, Mark; Torreira, Francisco; Enfield, N. J.

2013-01-01

A word like Huh?–used as a repair initiator when, for example, one has not clearly heard what someone just said– is found in roughly the same form and function in spoken languages across the globe. We investigate it in naturally occurring conversations in ten languages and present evidence and arguments for two distinct claims: that Huh? is universal, and that it is a word. In support of the first, we show that the similarities in form and function of this interjection across languages are much greater than expected by chance. In support of the second claim we show that it is a lexical, conventionalised form that has to be learnt, unlike grunts or emotional cries. We discuss possible reasons for the cross-linguistic similarity and propose an account in terms of convergent evolution. Huh? is a universal word not because it is innate but because it is shaped by selective pressures in an interactional environment that all languages share: that of other-initiated repair. Our proposal enhances evolutionary models of language change by suggesting that conversational infrastructure can drive the convergent cultural evolution of linguistic items. PMID:24260108

Differential Susceptibility of Bighorn Sheep (Ovis canadensis) and Domestic Sheep (Ovis aries) Neutrophils to Mannheimia haemolytica Leukotoxin is not due to Differential Expression of Cell Surface CD18.

PubMed

Dassanayake, Rohana P; Shanthalingam, Sudarvili; Liu, Weiguo; Casas, Eduardo; Srikumaran, Subramaniam

2017-07-01

Bighornsheep ( Ovis canadensis ) are more susceptible to pneumonia caused by Mannheimia haemolytica than are domestic sheep ( Ovis aries ). Leukotoxin produced by M. haemolytica is the principal virulence factor involved in pneumonia pathogenesis. Although leukotoxin is cytolytic to all subsets of ruminant leukocytes, neutrophils are the most susceptible subset. Bighorn sheep neutrophils are four- to eightfold more susceptible to leukotoxin-induced cytolysis than are domestic sheep neutrophils. We hypothesized that the higher susceptibility of bighorn sheep neutrophils, in comparison to domestic sheep neutrophils, is due to higher expression of CD18, the receptor for leukotoxin on leukocytes. Our objective was to quantify CD18 expression on neutrophils of bighorn sheep and domestic sheep. Cell-surface CD18 expression on bighorn sheep and domestic sheep neutrophils was measured as antibody binding capacity of cells by flow cytometric analysis with two fluorochrome-conjugated anti-CD18 monoclonal antibodies (BAQ30A and HUH82A) and microspheres. Contrary to our expectations, CD18 expression was higher (P<0.0001) with monoclonal antibody BAQ30A and was higher (P<0.0002) as well with monoclonal antibody HUH80A on domestic sheep neutrophils in comparison to bighorn sheep neutrophils. These findings suggest that the higher in vitro susceptibility to leukotoxin of bighorn sheep neutrophils compared to domestic sheep neutrophils is not due to higher expression of the leukotoxin receptor CD18 on bighorn sheep neutrophils.

Fusion of CCL21 non-migratory active breast epithelial and breast cancer cells give rise to CCL21 migratory active tumor hybrid cell lines.

PubMed

Berndt, Benjamin; Haverkampf, Sonja; Reith, Georg; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S; Dittmar, Thomas

2013-01-01

The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells.

Fusion of CCL21 Non-Migratory Active Breast Epithelial and Breast Cancer Cells Give Rise to CCL21 Migratory Active Tumor Hybrid Cell Lines

PubMed Central

Reith, Georg; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S.; Dittmar, Thomas

2013-01-01

The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells. PMID:23667660

Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via NTCP-dependent uptake of enveloped virus particles.

PubMed

Tu, Thomas; Budzinska, Magdalena A; Vondran, Florian W R; Shackel, Nicholas A; Urban, Stephan

2018-02-07

Chronic infection by the Hepatitis B Virus (HBV) is the major contributor to liver disease worldwide. Though HBV replicates via a nuclear episomal DNA (cccDNA), integration of HBV DNA into the host cell genome is regularly observed in the liver of infected patients. While reported as a pro-oncogenic alteration, the mechanism(s) and timing of HBV DNA integration are not well-understood, chiefly due to the lack of in vitro infection models that have detectable integration events. Here, we have established an in vitro system in which integration can be reliably detected following HBV infection. We measured HBV DNA integration using inverse nested PCR in primary human hepatocytes, HepaRG-NTCP, HepG2-NTCP, and Huh7-NTCP cells after HBV infection. Integration was detected in all cell types at a rate of >1 per 10000 cells, with the most consistent detection in Huh7-NTCP cells. Integration rate remained stable between 3 and 9 days post-infection. HBV DNA integration was efficiently blocked by treatment with 200nM of the HBV entry inhibitor Myrcludex B, but not with 10μM Tenofovir, 100U Interferon alpha, or 1μM of the capsid assembly inhibitor GLS4. This suggests integration of HBV DNA occurs immediately after infection of hepatocytes and is likely independent of de novo HBV replication in this model. Site analysis revealed that HBV DNA integrations were distributed over the entire human genome. Further, integrated HBV DNA sequences were consistent with double-stranded linear HBV DNA being the major precursor. Thus, we have established an in vitro system to interrogate the mechanisms of HBV DNA integration. Importance Hepatitis B Virus (HBV) is a common blood-borne pathogen and, following a chronic infection, can cause liver cancer and liver cirrhosis. Integration of HBV DNA into the host genome occurs in all known members of the hepadnaviridae family, despite this form not being necessary for viral replication. HBV DNA integration has been reported to drive liver cancer formation and persistence of virus infection. However, when and the mechanism(s) by which HBV DNA integration occurs is not clear. Here, we have developed and characterized an in vitro system to reliably detect HBV DNA integrations that result from a true HBV infection event and that closely resemble those found in patient tissues. Using this model, we show that integration already occurs when the infection is first established. Importantly, we provide here a system to analyze molecular factors involved in HBV integration, which can be used to develop strategies to halt its formation. Copyright © 2018 American Society for Microbiology.

Role of RNA Interference (RNAi) in Dengue Virus Replication and Identification of NS4B as an RNAi Suppressor

PubMed Central

Kakumani, Pavan Kumar; Ponia, Sanket Singh; S, Rajgokul K.; Sood, Vikas; Chinnappan, Mahendran; Banerjea, Akhil C.; Medigeshi, Guruprasad R.; Malhotra, Pawan

2013-01-01

RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates; however, evidences for its contribution to mammalian antiviral defense are few. In the present study, we demonstrate the anti-dengue virus role of RNAi in mammalian cells. Dengue virus infection of Huh 7 cells decreased the mRNA levels of host RNAi factors, namely, Dicer, Drosha, Ago1, and Ago2, and in corollary, silencing of these genes in virus-infected cells enhanced dengue virus replication. In addition, we observed downregulation of many known human microRNAs (miRNAs) in response to viral infection. Using reversion-of-silencing assays, we further showed that NS4B of all four dengue virus serotypes is a potent RNAi suppressor. We generated a series of deletion mutants and demonstrated that NS4B mediates RNAi suppression via its middle and C-terminal domains, namely, transmembrane domain 3 (TMD3) and TMD5. Importantly, the NS4B N-terminal region, including the signal sequence 2K, which has been implicated in interferon (IFN)-antagonistic properties, was not involved in mediating RNAi suppressor activity. Site-directed mutagenesis of conserved residues revealed that a Phe-to-Ala (F112A) mutation in the TMD3 region resulted in a significant reduction of the RNAi suppression activity. The green fluorescent protein (GFP)-small interfering RNA (siRNA) biogenesis of the GFP-silenced line was considerably reduced by wild-type NS4B, while the F112A mutant abrogated this reduction. These results were further confirmed by in vitro dicer assays. Together, our results suggest the involvement of miRNA/RNAi pathways in dengue virus establishment and that dengue virus NS4B protein plays an important role in the modulation of the host RNAi/miRNA pathway to favor dengue virus replication. PMID:23741001

Evaluation of the Activity of Lamivudine and Zidovudine against Ebola Virus.

PubMed

Cong, Yu; Dyall, Julie; Hart, Brit J; DeWald, Lisa Evans; Johnson, Joshua C; Postnikova, Elena; Zhou, Huanying; Gross, Robin; Rojas, Oscar; Alexander, Isis; Josleyn, Nicole; Zhang, Tengfei; Michelotti, Julia; Janosko, Krisztina; Glass, Pamela J; Flint, Mike; McMullan, Laura K; Spiropoulou, Christina F; Mierzwa, Tim; Guha, Rajarshi; Shinn, Paul; Michael, Sam; Klumpp-Thomas, Carleen; McKnight, Crystal; Thomas, Craig; Eakin, Ann E; O'Loughlin, Kathleen G; Green, Carol E; Catz, Paul; Mirsalis, Jon C; Honko, Anna N; Olinger, Gene G; Bennett, Richard S; Holbrook, Michael R; Hensley, Lisa E; Jahrling, Peter B

2016-01-01

In the fall of 2014, an international news agency reported that patients suffering from Ebola virus disease (EVD) in Liberia were treated successfully with lamivudine, an antiviral drug used to treat human immunodeficiency virus-1 and hepatitis B virus infections. According to the report, 13 out of 15 patients treated with lamivudine survived and were declared free from Ebola virus disease. In this study, the anti-Ebola virus (EBOV) activity of lamivudine and another antiretroviral, zidovudine, were evaluated in a diverse set of cell lines against two variants of wild-type EBOV. Variable assay parameters were assessed to include different multiplicities of infection, lengths of inoculation times, and durations of dosing. At a multiplicity of infection of 1, lamivudine and zidovudine had no effect on EBOV propagation in Vero E6, Hep G2, or HeLa cells, or in primary human monocyte-derived macrophages. At a multiplicity of infection of 0.1, zidovudine demonstrated limited anti-EBOV activity in Huh 7 cells. Under certain conditions, lamivudine had low anti-EBOV activity at the maximum concentration tested (320 μM). However, lamivudine never achieved greater than 30% viral inhibition, and the activity was not consistently reproducible. Combination of lamivudine and zidovudine showed no synergistic antiviral activity. Independently, a set of in vitro experiments testing lamivudine and zidovudine for antiviral activity against an Ebola-enhanced green fluorescent protein reporter virus was performed at the Centers for Disease Control and Prevention. No antiviral activity was observed for either compound. A study evaluating the efficacy of lamivudine in a guinea pig model of EVD found no survival benefit. This lack of benefit was observed despite plasma lamivudine concentrations in guinea pig of about 4 μg/ml obtained in a separately conducted pharmacokinetics study. These studies found no evidence to support the therapeutic use of lamivudine for the treatment of EVD.

The Na+-Taurocholate Cotransporting Polypeptide Traffics with the Epidermal Growth Factor Receptor

PubMed Central

Wang, Xintao; Wang, Pijun; Wang, Wenjun; Murray, John W.; Wolkoff, Allan W.

2015-01-01

Na+-taurocholate cotransporting polypeptide (ntcp) mediates uptake of bile acids as well as serving as the receptor for hepatitis B virus in human liver. Previous studies showed that ntcp traffics on microtubules between the cell surface and endocytic vesicles. Specific inhibition of protein kinase C (PKC)ζ resulted in loss of microtubule-based motility of these vesicles in vitro and in living cells. The aim of the present study was to characterize the PKCζ target. Incubation of ntcp-containing endocytic vesicles with γ-32P-ATP revealed a 180 kDa phosphoglycoprotein that was identified as the EGF receptor (EGFR). Surface biotinylation of HuH7 cells expressing GFP-ntcp revealed substantially reduced trafficking of ntcp to the cell surface with EGFR knockdown. Microtubule-based motility of ntcp-containing endocytic vesicles was also significantly reduced when they were not associated with EGFR. Ntcp was also found to undergo cellular redistribution upon stimulation of cells with EGF, consistent with a model in which ntcp and EGF-EGFR internalize into common endocytic vesicles from which they segregate, trafficking EGF-EGFR to lysosomes and recycling ntcp to the plasma membrane. EGF regulation of ntcp trafficking may play a heretofore unanticipated role in subcellular targeting of ntcp ligands such as hepatitis B. PMID:26650232

Involvement of PPARγ in the antitumoral action of cannabinoids on hepatocellular carcinoma

PubMed Central

Vara, D; Morell, C; Rodríguez-Henche, N; Diaz-Laviada, I

2013-01-01

Cannabinoids exert antiproliferative effects in a wide range of tumoral cells, including hepatocellular carcinoma (HCC) cells. In this study, we examined whether the PPARγ-activated pathway contributed to the antitumor effect of two cannabinoids, Δ9-tetrahydrocannabinol (THC) and JWH-015, against HepG2 and HUH-7 HCC cells. Both cannabinoids increased the activity and intracellular level of PPARγ mRNA and protein, which was abolished by the PPARγ inhibitor GW9662. Moreover, genetic ablation with small interfering RNA (siRNA), as well as pharmacological inhibition of PPARγ decreased the cannabinoid-induced cell death and apoptosis. Likewise, GW9662 totally blocked the antitumoral action of cannabinoids in xenograft-induced HCC tumors in mice. In addition, PPARγ knockdown with siRNA caused accumulation of the autophagy markers LC3-II and p62, suggesting that PPARγ is necessary for the autophagy flux promoted by cannabinoids. Interestingly, downregulation of the endoplasmic reticulum stress-related protein tribbles homolog 3 (TRIB3) markedly reduced PPARγ expression and induced p62 accumulation, which was counteracted by overexpression of PPARγ in TRIB3-knocked down cells. Taken together, we demonstrate for the first time that the antiproliferative action of the cannabinoids THC and JWH-015 on HCC, in vitro and in vivo, are modulated by upregulation of PPARγ-dependent pathways. PMID:23640460

Regulation of hepatitis B virus ENI enhancer activity by hepatocyte-enriched transcription factor HNF3.

PubMed

Chen, M; Hieng, S; Qian, X; Costa, R; Ou, J H

1994-11-15

Hepatitis B virus (HBV) ENI enhancer can activate the expression of HBV and non-HBV genes in a liver-specific manner. By performing the electrophoretic mobility-shift assays, we demonstrated that the three related, liver-enriched, transcription factors, HNF3 alpha, HNF3 beta, and HNF3 gamma could all bind to the 2c site of HBV ENI enhancer. Mutations introduced in the 2c site to abolish the binding by HNF3 reduced the enhancer activity approximately 15-fold. Moreover, expression of HNF3 antisense sequences to suppress the expression of HNF3 in Huh-7 hepatoma cells led to reduction of the ENI enhancer activity. These results indicate that HNF3 positively regulates the ENI enhancer activity and this regulation is most likely mediated through the 2c site. The requirement of HNF3 for the ENI enhancer activity could explain the liver specificity of this enhancer element.

Prognostic significance of aminopeptidase-N (CD13) in hepatoblastoma.

PubMed

Saida, Satoshi; Watanabe, Ken-ichiro; Kato, Itaru; Fujino, Hisanori; Umeda, Katsutsugu; Okamoto, Shinya; Uemoto, Shinji; Hishiki, Tomoro; Yoshida, Hideo; Tanaka, Shiro; Adachi, Souichi; Niwa, Akira; Nakahata, Tatsutoshi; Heike, Toshio

2015-08-01

Hepatoblastoma is a rare childhood malignant tumor that originates from immature hepatic cells. Aminopeptidase-N(CD13), an ectopeptidase that promotes tumor invasion and metastasis, is expressed in fetal stage hepatic progenitor cells, although its role in hepatoblastoma remains unclear. The expression pattern of CD13 was investigated on immunohistochemistry in 30 tissue samples from 27 hepatoblastoma patients (16 with predominantly embryonal [pE] histology and 14 with predominantly fetal [pF] histology). Immunoreactive score (IRS) was used to quantify staining data, and the relationship between CD13 expression, clinicopathological factors, and clinical outcome was investigated. The biological function of CD13 was also examined in the hepatoblastoma cell lines Huh6 and HepG2. All specimens stained positive for CD13, with higher CD13 expression in pE than in pF hepatoblastoma samples (median IRS, 4; range, 2-9 vs 2; range, 1-4). Strong CD13 expression was correlated with vascular invasion. Five year event-free survival and overall survival were better in patients with CD13(low) than in those with CD13(high) tumors (100% vs 51.0%, P = 0.026; and 100% vs 74.0%, P = 0.114, respectively). A CD13-neutralizing antibody and the potent CD13 inhibitor, Ubenimex, suppressed invasive activity in HepG2 cells in vitro. CD13 expression is associated with hepatoblastoma invasiveness and could be a novel prognostic marker for hepatoblastoma. © 2015 Japan Pediatric Society.

Targeting PIM kinase as a therapeutic strategy in human hepatoblastoma

PubMed Central

Stafman, Laura L.; Mruthyunjayappa, Smitha; Waters, Alicia M.; Garner, Evan F.; Aye, Jamie M.; Stewart, Jerry E.; Yoon, Karina J.; Whelan, Kimberly; Mroczek-Musulman, Elizabeth; Beierle, Elizabeth A.

2018-01-01

Increasing incidence coupled with poor prognosis and treatments that are virtually unchanged over the past 20 years have made the need for the development of novel therapeutics for hepatoblastoma imperative. PIM kinases have been implicated as drivers of tumorigenesis in multiple cancers, including hepatocellular carcinoma. We hypothesized that PIM kinases, specifically PIM3, would play a role in hepatoblastoma tumorigenesis and that PIM kinase inhibition would affect hepatoblastoma in vitro and in vivo. Parameters including cell survival, proliferation, motility, and apoptosis were assessed in human hepatoblastoma cells following PIM3 knockdown with siRNA or treatment with the PIM inhibitor AZD1208. An in vivo model of human hepatoblastoma was utilized to study the effects of PIM inhibition alone and in combination with cisplatin. PIM kinases were found to be present in the human hepatoblastoma cell line, HuH6, and in a human hepatoblastoma patient-derived xenograft, COA67. PIM3 knockdown or inhibition with AZD1208 decreased cell survival, attachment independent growth, and motility. Additionally, inhibition of tumor growth was observed in a hepatoblastoma xenograft model in mice treated with AZD1208. Combination therapy with AZD1208 and cisplatin resulted in a significant increase in animal survival when compared to either treatment alone. The current studies showed that PIM kinase inhibition decreased human hepatoblastoma tumorigenicity both in vitro and in vivo, implying that PIM inhibitors may be useful as a novel therapeutic for children with hepatoblastoma.

Let-7 miRNA Precursors Co-express with LIN28B in Cervical Cells.

PubMed

Zamora-Contreras, Aida Margarita; Alvarez-Salas, Luis Marat

2018-01-01

The let-7 microRNAs (miRNAs) are frequently dysregulated in carcinogenic processes, including cervical cancer. LIN28 proteins regulate let-7 biogenesis by binding to conserved sequences within the pre-miRNA structure. Nevertheless, recent research has shown that some let-7 miRNAs may escape LIN28 regulation. Correlate pre-let-7 miRNAs and LIN28B levels in cervical cell lines with different malignancy and HPV content. Pre-let-7 levels were determined by RTqPCR. LIN28B and other let-7 targets were analyzed by immunoblot. In silico tools were used to correlate let-7 and LIN28B expression and to analyze prelet- 7 sequences and structures. Lin28B protein was detected in all tested cell lines although it was more expressed in tumor cell lines. High levels of pre-let-7c/f-1 and pre-miR-98 were present in almost all cell lines regardless malignancy and LIN28B expression. Pre-let-7g/i were mainly expressed in tumor cell lines, pre-let-7e and pre-let-7-a3 were absent in all cell lines and pre-let-7a-2 showed indistinct expression. LIN28B showed positive correlation with pre-let-7i/g/f-1 and pre-miR-98 in tumor cell lines, suggesting escape from regulation. Sequence alignment and analysis of pre-let-7 miRNAs showed distinctive structural features within the preE region that may influence the ideal pre-let-7 structuring for LIN28B interaction. Short preE-stems were present in pre-let-7 that may escape LIN28B regulation, but long preEstems were mostly associated with high-level pre-let-7 miRNAs. The observed differences of pre-let-7 levels in cervical cell lines may be the result of alternative preE structuring affecting interaction with LIN28B thus resulting in differential let-7 regulation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Anti-hepatitis C virus activity and synergistic effect of Nymphaea alba extracts and bioactive constituents in liver infected cells.

PubMed

Rehman, Sidra; Ashfaq, Usman Ali; Ijaz, Bushra; Riazuddin, Sheikh

2018-05-28

Without an effective vaccine, hepatitis C virus (HCV) remains a global threat, inflicting 170-300 million carriers worldwide at risk of cirrhosis and hepatocellular carcinoma (HCC). Though various direct acting antivirals have been redeemed the hepatitis C treatment, a few restraints persist including possible side effects, viral resistance emergence, excessive cost which restricts its availability to a common person. There is no preventive HCV vaccine available today so the discovery of potent antiviral natural flora and their bioactive constituents may help to develop preventive cures against HCV infection. In current study, we aim to clarify anti-HCV activity of methanol and acetone extracts along with the purified fractions of Pakistani local plant, Nymphaea alba L (N. alba) using Huh-7 cell line as transfection model. Synergistic study of purified fractions with interferon was performed using MDBK cell line (expressing interferon receptors) as transfection model. Recent study by our research group has observed potent anti-HCV NS3 protease activity of methanol and acetone extracts of N. alba. Effect of N. alba extracts, its fractions precisely, the N1 and N8 fractions on HCV replication was demonstrated by analyzing viral gene expression using in vitro transfection model. Considering NS3 protease as a dynamic drug target, fourteen phytochemicals of N. alba were selected as ligands for interaction with NS3 protein using Molecular Operating Environment (MOE) software. Boceprevir, FDA approved NS3 protease inhibitor, was used as standard for comparative study in docking screening. Herein we report 84% and 94% reduction of 3a genotype of HCV NS3/4A gene expression at mRNA level at non-toxic concentration. Specifically, two fractions 'N1' & 'N8' isolated from acetone extract suppressed HCV NS3 gene expression in transfected target cells with an EC 50 value of 37 ± 0.03 μg/ml and 20 ± 0.02 μg/ml respectively. Similarly, viral genotype 1a replication is strongly suppressed in target cells by N. alba flower extracts and purified fractions. Moreover, combination of fractions with standard antiviral drug displayed synergistic effects for inhibition of HCV replication. Phytochemicals including Isoquercetin, Hyperoside, Quercetin, Reynoutrin, Apigenin and Isokaempferide displayed minimum binding energies as compared to standard protease inhibitor. N. alba and its purified phytochemicals with new scaffolds might significantly serve as valuable and alternative regimen against HCV either alone or in combination with other potential anti-HCV agents. Copyright © 2018 Elsevier Ltd. All rights reserved.

Spontaneous Mutation at Amino Acid 544 of the Ebola Virus Glycoprotein Potentiates Virus Entry and Selection in Tissue Culture

PubMed Central

Ladner, Jason T.; Ettinger, Chelsea R.; Palacios, Gustavo

2017-01-01

ABSTRACT Ebolaviruses have a surface glycoprotein (GP1,2) that is required for virus attachment and entry into cells. Mutations affecting GP1,2 functions can alter virus growth properties. We generated a recombinant vesicular stomatitis virus encoding Ebola virus Makona variant GP1,2 (rVSV-MAK-GP) and observed emergence of a T544I mutation in the Makona GP1,2 gene during tissue culture passage in certain cell lines. The T544I mutation emerged within two passages when VSV-MAK-GP was grown on Vero E6, Vero, and BS-C-1 cells but not when it was passaged on Huh7 and HepG2 cells. The mutation led to a marked increase in virus growth kinetics and conferred a robust growth advantage over wild-type rVSV-MAK-GP on Vero E6 cells. Analysis of complete viral genomes collected from patients in western Africa indicated that this mutation was not found in Ebola virus clinical samples. However, we observed the emergence of T544I during serial passage of various Ebola Makona isolates on Vero E6 cells. Three independent isolates showed emergence of T544I from undetectable levels in nonpassaged virus or virus passaged once to frequencies of greater than 60% within a single passage, consistent with it being a tissue culture adaptation. Intriguingly, T544I is not found in any Sudan, Bundibugyo, or Tai Forest ebolavirus sequences. Furthermore, T544I did not emerge when we serially passaged recombinant VSV encoding GP1,2 from these ebolaviruses. This report provides experimental evidence that the spontaneous mutation T544I is a tissue culture adaptation in certain cell lines and that it may be unique for the species Zaire ebolavirus. IMPORTANCE The Ebola virus (Zaire) species is the most lethal species of all ebolaviruses in terms of mortality rate and number of deaths. Understanding how the Ebola virus surface glycoprotein functions to facilitate entry in cells is an area of intense research. Recently, three groups independently identified a polymorphism in the Ebola glycoprotein (I544) that enhanced virus entry, but they did not agree in their conclusions regarding its impact on pathogenesis. Our findings here address the origins of this polymorphism and provide experimental evidence showing that it is the result of a spontaneous mutation (T544I) specific to tissue culture conditions, suggesting that it has no role in pathogenesis. We further show that this mutation may be unique to the species Zaire ebolavirus, as it does not occur in Sudan, Bundibugyo, and Tai Forest ebolaviruses. Understanding the mechanism behind this mutation can provide insight into functional differences that exist in culture conditions and among ebolavirus glycoproteins. PMID:28539437

Triterpenoids principle of Wedelia calendulacea attenuated diethynitrosamine-induced hepatocellular carcinoma via down-regulating oxidative stress, inflammation and pathology via NF-kB pathway.

PubMed

Verma, Amita; Singh, Deepika; Anwar, Firoz; Bhatt, Prakash Chandra; Al-Abbasi, Fahad; Kumar, Vikas

2018-02-01

The aerial part of Wedelia calendulacea have been used in Ayurveda, Unani, Tibetan, Siddha and other folk medicine systems to protect the liver and renal tissue. Liver is considered as primary metabolizing site of body, which is prone to damage by endogenous and exogenous toxicants. A reason for liver toxicity, and major causes of the hepatocellular carcinoma (HCC). 19-α-Hydroxyurs-12(13)-ene-28 oic acid-3-O-β-D-glucopyranoside (HEG), a triterpenoids found in the higher plants, has been known to possess protective effect against various toxicants. The aim of the current study was to scrutinize the hepatoprotective mechanism of HEG against DEN-induced oxidative stress, hyperproliferation, inflammation and apoptosis tissue injury in Wistar rats. Invitro cell lines study of HEG scrutinized against the Hep-G2 and HuH-7 cells. A single dose of DEN (200 mg/kg) and double dose of phenobarbitol were administered to induce the liver damage in rats; the dose treatment of HEG was terminated at the end of 22 weeks. Macroscopical study was performed for the confirmation of hepatic nodules. The serum and hepatic samples were collected for further biochemical and histopathological analysis. Hepatic; non-hepatic; Phase I and II antioxidant enzymes were also examined. Additionally, we also scrutinized the inflammatory cytokines viz., tumor necrosis factor-α, interlukin-6, interlukin-1β, and Nuclear factor kappa beta (NF-kB), respectively. Histopathological study was also performed for analyzing the changes during the HCC. HEG confirmed the reduction of growth and deoxyribonucleic acid synthesis of both cell lines. DEN successfully induced the HCC in all group, which was significantly (p < 0.001) altered by the HEG in a dose-dependent manner. The decreased level of pro-inflammatory cytokines and altered membrane-bound enzyme activity were also observed. HEG inhibits the phase I, II and antioxidant enzymes at the effective dose-dependent manner, which were considered as the precursor of the HCC. The alteration of phase I, II and antioxidant enzymes confirmed the inhibition of inflammatory reaction and oxidative stress, which directly or indirectly inhibited the NF-kB expression. Collectively, we can conclude that the HEG inhibited the growth of Hepatocellular carcinoma via attenuating the NF-kB pathway.

A mechanism of apigenin-induced apoptosis is potentially related to anti-angiogenesis and anti-migration in human hepatocellular carcinoma cells.

PubMed

Kim, Bo Ra; Jeon, Young Keul; Nam, Myeong Jin

2011-07-01

Apigenin (APG) has been shown to have a strong anti-cancer effect on various cancer models via a programmed cell death, apoptosis. However, the fundamental mechanisms of these effects are still unclear. In the present study, we examined the question of whether or not APG can inhibit proliferation of hepatocellular carcinoma (HCC), huh-7 cells, resulting in apoptosis. In APG-treated cells, we observed typical features of apoptosis. To identify the proteins related to APG-induced apoptosis, we performed two-dimensional electrophoresis analysis and identified differentially expressed proteins. Among these proteins, we focused on vimentin, which plays a physiological role, such as cell migration and adhesion. We validated expression of vimentin in both mRNA and protein levels, verifying its decrease. In addition, we observed that APG down-regulated the expression levels of type I collagen, which collaborated with vimentin in cell migration and decreased the releasing amounts of VEGF and MMP-8, which are closely relevant to angiogenic activity. Finally, we confirmed the decreased capacity of cell migration due to down-regulation of vimentin, type I collagen, VEGF, and MMP-8 induced by APG. Based on the overall results, we suggested that vimentin was potentially associated with APG-induced apoptosis, as a key regulator in angiogenesis and migration. Copyright © 2011 Elsevier Ltd. All rights reserved.

Kurarinol induces hepatocellular carcinoma cell apoptosis through suppressing cellular signal transducer and activator of transcription 3 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shu, Guangwen; Yang, Jing; Zhao, Wenhao

Kurarinol is a flavonoid isolated from roots of the medical plant Sophora flavescens. However, its cytotoxic activity against hepatocellular carcinoma (HCC) cells and toxic effects on mammalians remain largely unexplored. Here, the pro-apoptotic activities of kurarinol on HCC cells and its toxic impacts on tumor-bearing mice were evaluated. The molecular mechanisms underlying kurarinol-induced HCC cell apoptosis were also investigated. We found that kurarinol dose-dependently provoked HepG2, Huh-7 and H22 HCC cell apoptosis. In addition, kurarinol gave rise to a considerable decrease in the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) in HCC cells. Suppression of STAT3more » signaling is involved in kurarinol-induced HCC cell apoptosis. In vivo studies showed that kurarinol injection substantially induced transplanted H22 cell apoptosis with low toxic impacts on tumor-bearing mice. Similarly, the transcriptional activity of STAT3 in transplanted tumor tissues was significantly suppressed after kurarinol treatment. Collectively, our current research demonstrated that kurarinol has the capacity of inducing HCC cell apoptosis both in vitro and in vivo with undetectable toxic impacts on the host. Suppressing STAT3 signaling is implicated in kurarinol-mediated HCC cell apoptosis. - Highlights: • Kurarinol induces hepatocellular carcinoma (HCC) cell apoptosis. • Kurarinol induces HCC cell apoptosis via inhibiting STAT3. • Kurarinol exhibits low toxic effects on tumor-bearing animals.« less

Ontogeny of con A and PHA responses of chicken blood cells in MHC-compatible lines 6(3) and 7(2).

PubMed

Fredericksen, T L; Gilmour, D G

1983-06-01

The development of T cell responsiveness to Con A and PHA was examined in two MHC-compatible inbred chicken lines, RPRL 6(3) and 7(2), at ages 2 to 118 days posthatching. These lines are respectively resistant or susceptible to Marek's disease, a naturally occurring, virally induced T cell lymphoma. Between-line comparisons were made of optimal in vitro responses of diluted serum-free blood cells to each mitogen in two groups of chicks tested over ages 2 to 63 and 41 to 118 days. Over 2 to 63 days, Con A responses increased with age at the same rate in each line, but 7(2) responses averaged 2.3 times higher than 6(3). The increase with age was dependent on blood lymphocyte counts, which also increased with age in parallel in both lines. In contrast, the between-line difference in responsiveness was dependent on intrinsic reactivity of cells as well as lymphocyte counts. Covariance analysis was used to estimate that line 7(2) was 1.4 times higher than 6(3) in intrinsic cell reactivity, after accounting for the effect of the twofold higher blood lymphocyte counts in 7(2), and that this intrinsic difference contributed almost one-half the total difference. Over 41 to 118 days Con A responses no longer increased with age, although lymphocyte counts were still increasing, and the line difference (2.6 times) was now almost entirely contributed by a 2.3-fold superiority of 7(2) blood cells in intrinsic reactivity. The line difference in PHA responses was the reverse of the above in young chicks, with 6(3) responses greater than 7(2) in spite of lower lymphocyte counts. In additional chicks tested over 5 to 26 days, intrinsic reactivity of 6(3) cells to PHA averaged 4.5 times higher than 7(2). There was an abrupt decline in intrinsic reactivity of line 6(3) blood cells between 26 and 41 days to a level equal with 7(2). After this age, line 7(2) responses were 1.8 times greater than those of 6(3), and this difference was dependent solely on lymphocyte count differences. The results suggest that different gene systems mediate blood cell responses to PHA as compared with Con A. The pattern of developmental differences between inbred lines indicates the existence of distinct or partly overlapping T cell subsets with different reactivities to PHA or Con A, and of higher suppressor activity of adherent cells in line 6(3) blood. Both these differences may be related to line 6(3) inherited resistance to Marek's disease.

Swine Influenza Virus PA and Neuraminidase Gene Reassortment into Human H1N1 Influenza Virus Is Associated with an Altered Pathogenic Phenotype Linked to Increased MIP-2 Expression.

PubMed

Dlugolenski, Daniel; Jones, Les; Howerth, Elizabeth; Wentworth, David; Tompkins, S Mark; Tripp, Ralph A

2015-05-01

Swine are susceptible to infection by both avian and human influenza viruses, and this feature is thought to contribute to novel reassortant influenza viruses. In this study, the influenza virus reassortment rate in swine and human cells was determined. Coinfection of swine cells with 2009 pandemic H1N1 virus (huH1N1) and an endemic swine H1N2 (A/swine/Illinois/02860/09) virus (swH1N2) resulted in a 23% reassortment rate that was independent of α2,3- or α2,6-sialic acid distribution on the cells. The reassortants had altered pathogenic phenotypes linked to introduction of the swine virus PA and neuraminidase (NA) into huH1N1. In mice, the huH1N1 PA and NA mediated increased MIP-2 expression early postinfection, resulting in substantial pulmonary neutrophilia with enhanced lung pathology and disease. The findings support the notion that swine are a mixing vessel for influenza virus reassortants independent of sialic acid distribution. These results show the potential for continued reassortment of the 2009 pandemic H1N1 virus with endemic swine viruses and for reassortants to have increased pathogenicity linked to the swine virus NA and PA genes which are associated with increased pulmonary neutrophil trafficking that is related to MIP-2 expression. Influenza A viruses can change rapidly via reassortment to create a novel virus, and reassortment can result in possible pandemics. Reassortments among subtypes from avian and human viruses led to the 1957 (H2N2 subtype) and 1968 (H3N2 subtype) human influenza pandemics. Recent analyses of circulating isolates have shown that multiple genes can be recombined from human, avian, and swine influenza viruses, leading to triple reassortants. Understanding the factors that can affect influenza A virus reassortment is needed for the establishment of disease intervention strategies that may reduce or preclude pandemics. The findings from this study show that swine cells provide a mixing vessel for influenza virus reassortment independent of differential sialic acid distribution. The findings also establish that circulating neuraminidase (NA) and PA genes could alter the pathogenic phenotype of the pandemic H1N1 virus, resulting in enhanced disease. The identification of such factors provides a framework for pandemic modeling and surveillance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Swine Influenza Virus PA and Neuraminidase Gene Reassortment into Human H1N1 Influenza Virus Is Associated with an Altered Pathogenic Phenotype Linked to Increased MIP-2 Expression

PubMed Central

Dlugolenski, Daniel; Jones, Les; Howerth, Elizabeth; Wentworth, David; Tompkins, S. Mark

2015-01-01

ABSTRACT Swine are susceptible to infection by both avian and human influenza viruses, and this feature is thought to contribute to novel reassortant influenza viruses. In this study, the influenza virus reassortment rate in swine and human cells was determined. Coinfection of swine cells with 2009 pandemic H1N1 virus (huH1N1) and an endemic swine H1N2 (A/swine/Illinois/02860/09) virus (swH1N2) resulted in a 23% reassortment rate that was independent of α2,3- or α2,6-sialic acid distribution on the cells. The reassortants had altered pathogenic phenotypes linked to introduction of the swine virus PA and neuraminidase (NA) into huH1N1. In mice, the huH1N1 PA and NA mediated increased MIP-2 expression early postinfection, resulting in substantial pulmonary neutrophilia with enhanced lung pathology and disease. The findings support the notion that swine are a mixing vessel for influenza virus reassortants independent of sialic acid distribution. These results show the potential for continued reassortment of the 2009 pandemic H1N1 virus with endemic swine viruses and for reassortants to have increased pathogenicity linked to the swine virus NA and PA genes which are associated with increased pulmonary neutrophil trafficking that is related to MIP-2 expression. IMPORTANCE Influenza A viruses can change rapidly via reassortment to create a novel virus, and reassortment can result in possible pandemics. Reassortments among subtypes from avian and human viruses led to the 1957 (H2N2 subtype) and 1968 (H3N2 subtype) human influenza pandemics. Recent analyses of circulating isolates have shown that multiple genes can be recombined from human, avian, and swine influenza viruses, leading to triple reassortants. Understanding the factors that can affect influenza A virus reassortment is needed for the establishment of disease intervention strategies that may reduce or preclude pandemics. The findings from this study show that swine cells provide a mixing vessel for influenza virus reassortment independent of differential sialic acid distribution. The findings also establish that circulating neuraminidase (NA) and PA genes could alter the pathogenic phenotype of the pandemic H1N1 virus, resulting in enhanced disease. The identification of such factors provides a framework for pandemic modeling and surveillance. PMID:25762737

Anti-HCV effect of Lentinula edodes mycelia solid culture extracts and low-molecular-weight lignin.

PubMed

Matsuhisa, Koji; Yamane, Seiji; Okamoto, Toru; Watari, Akihiro; Kondoh, Masuo; Matsuura, Yoshiharu; Yagi, Kiyohito

2015-06-19

Lentinula edodes mycelia solid culture extract (MSCE) contains several bioactive molecules, including some polyphenolic compounds, which exert immunomodulatory, antitumor, and hepatoprotective effects. In this study, we examined the anti-hepatitis C virus (HCV) activity of MSCE and low-molecular-weight lignin (LM-lignin), which is the active component responsible for the hepatoprotective effect of MSCE. Both MSCE and LM-lignin inhibited the entry of two HCV pseudovirus (HCVpv) types into Huh7.5.1 cells. LM-lignin inhibited HCVpv entry at a lower concentration than MSCE and inhibited the entry of HCV particles in cell culture (HCVcc). MSCE also inhibited HCV subgenome replication. LM-lignin had no effect on HCV replication, suggesting that MSCE contains additional active substances. We demonstrate here for the first time the anti-HCV effects of plant-derived LM-lignin and MSCE. The hepatoprotective effect of LM-lignin suggests that lignin derivatives, which can be produced in abundance from existing plant resources, may be effective in the treatment of HCV-related diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Complexity of expression of the intermediate filaments of six new human ovarian carcinoma cell lines: new expression of cytokeratin 20.

PubMed

Yanagibashi, T; Gorai, I; Nakazawa, T; Miyagi, E; Hirahara, F; Kitamura, H; Minaguchi, H

1997-01-01

Six permanent human ovarian carcinoma cell lines (OVISE, OVTOKO, OVMANA and OVSAYO from clear cell adenocarcinoma, and OVSAHO and OVKATE from serous papillary adenocarcinoma) were established from solid tumours. The cell lines have been in culture for 5-8 years, the passage number varying from 62 to 246. Immunohistochemical analysis has shown that five of the six cell lines express at least six cytokeratin (CK) polypeptides. OVISE and OVSAYO expressed CKs 6, 7, 8, 18, 19 and 15 and/or 16. OVTOKO was positive for CKs 7, 8, 18, 19 and 15 and/or 16. OVSAHO expressed CKs 6, 7, 8, 14, 18, 19 and 15 and/or 16. OVMANA expressed CKs 6, 7, 8, 18, 19, 20 and 15 and/or 16. OVKATE expressed CKs 6, 7, 8, 13, 17, 18, 19, 20 and 15 and/or 16. The expression of CK7, additional expression of vimentin, and clinical and histopathological findings enabled us to confirm that six cell lines had been established from primary ovarian cancers. Two of the six cell lines were positive for CK20, although CK20 was not expressed in the original tumours. The heterotransplanted tumours produced by CK20-positive cells also expressed CK20. This is the first report of ovarian carcinoma cell lines that express CK20 irrespective of their histological type. CK20 has been found in all colon carcinoma cell lines, but only in the mucinous type of ovarian tumours. These new ovarian carcinoma cell lines will therefore provide a relevant experimental system for elucidating the regulatory control mechanisms of intermediate filament expression.

The Therapeutic Potential of AN-7, a Novel Histone Deacetylase Inhibitor, for Treatment of Mycosis Fungoides/Sezary Syndrome Alone or with Doxorubicin

PubMed Central

Goldfeiz, Neta; Rephaeli, Ada; Nudelman, Abraham; Weitman, Michal; Tarasenko, Nataly; Gorovitz, Batia; Maron, Leah; Yehezkel, Shiran; Amitay-Laish, Iris; Lubin, Ido; Hodak, Emmilia

2016-01-01

The 2 histone deacetylase inhibitors (HDACIs) approved for the treatment of cutaneous T-cell lymphoma (CTCL) including mycosis fungoides/sezary syndrome (MF/SS), suberoylanilide hydroxamic acid (SAHA) and romidepsin, are associated with low rates of overall response and high rates of adverse effects. Data regarding combination treatments with HDACIs is sparse. Butyroyloxymethyl diethylphosphate (AN-7) is a novel HDACI, which was found to have selective anticancer activity in several cell lines and animal models. The aim of this study was to compare the anticancer effects of AN-7 and SAHA, either alone or combined with doxorubicin, on MF/SS cell lines and peripheral blood lymphocytes (PBL) from patients with Sezary syndrome (SPBL). MyLa cells, Hut78 cells, SPBL, and PBL from healthy normal individuals (NPBL) were exposed to the test drugs, and the findings were analyzed by a viability assay, an apoptosis assay, and Western blot. AN-7 was more selectively toxic to MyLa cells, Hut78 cells, and SPBL (relative to NPBL) than SAHA and also acted more rapidly. Both drugs induced apoptosis in MF/SS cell lines, SAHA had a greater effect on MyLa cell line, while AN-7 induced greater apoptosis in SPBL; both caused an accumulation of acetylated histone H3, but AN-7 was associated with earlier kinetics; and both caused a downregulation of the HDAC1 protein in MF/SS cell lines. AN-7 acted synergistically with doxorubicin in both MF/SS cell lines and SPBL, and antagonistically with doxorubicin in NPBL. By contrast, SAHA acted antagonistically with doxorubicin on MF/SS cell lines, SPBL, and NPBL, leaving <50% viable cells. In conclusion, AN-7 holds promise as a therapeutic agent in MF/SS and has several advantages over SAHA. Our data provide a rationale for combining AN-7, but not SAHA, with doxorubicin to induce the cell death in MF/SS. PMID:26752418

The Therapeutic Potential of AN-7, a Novel Histone Deacetylase Inhibitor, for Treatment of Mycosis Fungoides/Sezary Syndrome Alone or with Doxorubicin.

PubMed

Moyal, Lilach; Feldbaum, Nataly; Goldfeiz, Neta; Rephaeli, Ada; Nudelman, Abraham; Weitman, Michal; Tarasenko, Nataly; Gorovitz, Batia; Maron, Leah; Yehezkel, Shiran; Amitay-Laish, Iris; Lubin, Ido; Hodak, Emmilia

2016-01-01

The 2 histone deacetylase inhibitors (HDACIs) approved for the treatment of cutaneous T-cell lymphoma (CTCL) including mycosis fungoides/sezary syndrome (MF/SS), suberoylanilide hydroxamic acid (SAHA) and romidepsin, are associated with low rates of overall response and high rates of adverse effects. Data regarding combination treatments with HDACIs is sparse. Butyroyloxymethyl diethylphosphate (AN-7) is a novel HDACI, which was found to have selective anticancer activity in several cell lines and animal models. The aim of this study was to compare the anticancer effects of AN-7 and SAHA, either alone or combined with doxorubicin, on MF/SS cell lines and peripheral blood lymphocytes (PBL) from patients with Sezary syndrome (SPBL). MyLa cells, Hut78 cells, SPBL, and PBL from healthy normal individuals (NPBL) were exposed to the test drugs, and the findings were analyzed by a viability assay, an apoptosis assay, and Western blot. AN-7 was more selectively toxic to MyLa cells, Hut78 cells, and SPBL (relative to NPBL) than SAHA and also acted more rapidly. Both drugs induced apoptosis in MF/SS cell lines, SAHA had a greater effect on MyLa cell line, while AN-7 induced greater apoptosis in SPBL; both caused an accumulation of acetylated histone H3, but AN-7 was associated with earlier kinetics; and both caused a downregulation of the HDAC1 protein in MF/SS cell lines. AN-7 acted synergistically with doxorubicin in both MF/SS cell lines and SPBL, and antagonistically with doxorubicin in NPBL. By contrast, SAHA acted antagonistically with doxorubicin on MF/SS cell lines, SPBL, and NPBL, leaving <50% viable cells. In conclusion, AN-7 holds promise as a therapeutic agent in MF/SS and has several advantages over SAHA. Our data provide a rationale for combining AN-7, but not SAHA, with doxorubicin to induce the cell death in MF/SS.

Expression of Zinc Finger and BTB Domain-containing 7A in Colorectal Carcinoma.

PubMed

Joo, Jin Woo; Kim, Hyun-Soo; Do, Sung-Im; Sung, Ji-Youn

2018-05-01

Previous studies have revealed that zinc finger and BTB domain-containing 7A (ZBTB7A), an important proto-oncogene, plays multiple roles in carcinogenesis and is up-regulated in several human malignancies. However, the expression of ZBTB7A in colorectal carcinoma (CRC) has seldom been documented. In this study, we investigated the differential expression of ZBTB7A in CRC cell lines and tissues. Expression levels of ZBTB7A mRNA and protein were examined in CRC cell lines. ZBTB7A protein expression was also evaluated in tissue samples of normal colonic mucosa, high-grade dysplasia, and CRC using immunohistochemical staining. All CRC cell lines exhibited significantly higher ZBTB7A mRNA expression levels than did normal colonic epithelial cells. The ZBTB7A protein expression levels were clearly higher in the CRC cell lines than in the normal colonic epithelial cells. Consistent with the cell line data, immunostaining revealed that there were significant differences in ZBTB7A protein expression between tissue samples of CRC and normal colonic mucosa (p=0.048) and high-grade dysplasia (p=0.015). In addition, metastatic CRC exhibited significantly higher ZBTB7A protein expression levels than primary CRC (p=0.027). We demonstrated that ZBTB7A expression is up-regulated in CRC cell lines and tissues. Our data suggest that ZBTB7A is involved in the development and progression of CRC. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Milatuzumab-SN-38 conjugates for the treatment of CD74+ cancers.

PubMed

Govindan, Serengulam V; Cardillo, Thomas M; Sharkey, Robert M; Tat, Fatma; Gold, David V; Goldenberg, David M

2013-06-01

CD74 is an attractive target for antibody-drug conjugates (ADC), because it internalizes and recycles after antibody binding. CD74 mostly is associated with hematologic tumors but is expressed also in solid cancers. Therefore, ADCs of the humanized anti-CD74 antibody, milatuzumab, were examined for the therapy of CD74-expressing solid tumors. Milatuzumab-doxorubicin and two milatuzumab-SN-38 conjugates with cleavable linkers, differing in their stability in serum and how they release SN-38 in the lysosome, were prepared. CD74 expression was determined by flow cytometry and immunohistology. In vitro cytotoxicity and in vivo therapeutic studies were conducted in the human cancer cell lines A-375 (melanoma), HuH-7 and Hep-G2 (hepatoma), Capan-1 (pancreatic), NCI-N87 (gastric), and Raji Burkitt lymphoma. The milatuzumab-SN-38 ADC was compared with SN-38 ADCs prepared with anti-Trop-2 and anti-CEACAM6 antibodies in xenografts expressing their target antigens. Milatuzumab-doxorubicin was most effective in the lymphoma model, whereas in A-375 and Capan-1 solid tumors, only milatuzumab-SN-38 showed a therapeutic benefit. Despite much lower surface expression of CD74 than Trop-2 or CEACAM6, milatuzumab-SN-38 had similar efficacy in Capan-1 as anti-Trop-2-SN-38, but in NCI-N87, anti-CEACAM6 and anti-Trop-2 conjugates were superior. Studies in two hepatoma lines at a single dose level showed significant benefit over saline controls but not against an irrelevant immunoglobulin G conjugate. CD74 is a suitable target for ADCs in some solid tumor xenografts, with efficacy largely influenced by uniformity of CD74 expression and with SN-38 conjugates providing the best therapeutic responses; SN-38 conjugates were preferable in solid cancers, whereas doxorubicin ADC was better in lymphoma tested. ©2013 AACR

Expression of MUC1, MUC2, MUC3 and MUC4 mucin mRNAs in human pancreatic and intestinal tumor cell lines.

PubMed

Hollingsworth, M A; Strawhecker, J M; Caffrey, T C; Mack, D R

1994-04-15

We examined the steady-state expression levels of mRNA for the MUC1, MUC2, MUC3 and MUC4 gene products in 12 pancreatic tumor cell lines, 6 colon tumor cell lines, and one ileocecal tumor cell line. The results showed that 10 of 12 pancreatic tumor cell lines expressed MUC1 mRNA and that 7 of these 12 lines also expressed relatively high levels of MUC4 mRNA. In contrast, MUC2 mRNA was expressed at only low levels and MUC3 was not detected in the pancreatic tumor cell lines. All 7 intestinal tumor cell lines examined expressed MUC2, and 5 of 7 expressed MUC3; however only one expressed significant levels of MUC1 and 2 expressed low levels of MUC4 mRNA. This report of high levels of MUC4 mRNA expression by pancreatic tumor cells raises the possibility that mucin carbohydrate epitopes defined by antibodies such as DuPan 2 may be expressed on a second mucin core protein produced by pancreatic tumor cells.

Hepatitis C Virus core+1/ARF Protein Modulates the Cyclin D1/pRb Pathway and Promotes Carcinogenesis.

PubMed

Moustafa, Savvina; Karakasiliotis, Ioannis; Mavromara, Penelope

2018-05-01

Viruses often encompass overlapping reading frames and unconventional translation mechanisms in order to maximize the output from a minimum genome and to orchestrate their timely gene expression. Hepatitis C virus (HCV) possesses such an unconventional open reading frame (ORF) within the core-coding region, encoding an additional protein, initially designated ARFP, F, or core+1. Two predominant isoforms of core+1/ARFP have been reported, core+1/L, initiating from codon 26, and core+1/S, initiating from codons 85/87 of the polyprotein coding region. The biological significance of core+1/ARFP expression remains elusive. The aim of the present study was to gain insight into the functional and pathological properties of core+1/ARFP through its interaction with the host cell, combining in vitro and in vivo approaches. Our data provide strong evidence that the core+1/ARFP of HCV-1a stimulates cell proliferation in Huh7-based cell lines expressing either core+1/S or core+1/L isoforms and in transgenic liver disease mouse models expressing core+1/S protein in a liver-specific manner. Both isoforms of core+1/ARFP increase the levels of cyclin D1 and phosphorylated Rb, thus promoting the cell cycle. In addition, core+1/S was found to enhance liver regeneration and oncogenesis in transgenic mice. The induction of the cell cycle together with increased mRNA levels of cell proliferation-related oncogenes in cells expressing the core+1/ARFP proteins argue for an oncogenic potential of these proteins and an important role in HCV-associated pathogenesis. IMPORTANCE This study sheds light on the biological importance of a unique HCV protein. We show here that core+1/ARFP of HCV-1a interacts with the host machinery, leading to acceleration of the cell cycle and enhancement of liver carcinogenesis. This pathological mechanism(s) may complement the action of other viral proteins with oncogenic properties, leading to the development of hepatocellular carcinoma. In addition, given that immunological responses to core+1/ARFP have been correlated with liver disease severity in chronic HCV patients, we expect that the present work will assist in clarifying the pathophysiological relevance of this protein as a biomarker of disease progression. Copyright © 2018 American Society for Microbiology.

Transforming Growth Factor-β Promotes Liver Tumorigenesis in Mice via Up-regulation of Snail.

PubMed

Moon, Hyuk; Ju, Hye-Lim; Chung, Sook In; Cho, Kyung Joo; Eun, Jung Woo; Nam, Suk Woo; Han, Kwang-Hyub; Calvisi, Diego F; Ro, Simon Weonsang

2017-11-01

Transforming growth factor beta (TGF-β) suppresses early stages of tumorigenesis, but also contributes to migration and metastasis of cancer cells. A large number of human tumors contain mutations that inactivate its receptors, or downstream proteins such as Smad transcription factors, indicating that the TGF-β signaling pathway prevents tumor growth. We investigated the effects of TGF-β inhibition on liver tumorigenesis in mice. C57BL/6 mice received hydrodynamic tail-vein injections of transposons encoding HRAS G12V and a short hairpin RNA (shRNA) to down-regulate p53, or those encoding HRAS G12V and MYC, or those encoding HRAS G12V and TAZ S89A , to induce liver tumor formation; mice were also given injections of transposons encoding SMAD7 or shRNA against SMAD2, SMAD3, SMAD4, or SNAI1 (Snail), with or without ectopic expression of Snail. Survival times were compared, and livers were weighted and examined for tumors. Liver tumor tissues were analyzed by quantitative reverse-transcription PCR, RNA sequencing, immunoblots, and immunohistochemistry. We analyzed gene expression levels in human hepatocellular carcinoma samples deposited in The Cancer Genome Atlas. A cell proliferation assay was performed using human liver cancer cell lines (HepG2 and Huh7) stably expressing Snail or shRNA against Snail. TGF-β inhibition via overexpression of SMAD7 (or knockdown of SMAD2, SMAD3, or SMAD4) consistently reduced formation and growth of liver tumors in mice that expressed activated RAS plus shRNA against p53, or in mice that expressed activated RAS and TAZ. TGF-β signaling activated transcription of the Snail gene in liver tumors induced by HRAS G12V and shRNA against p53, and by activated RAS and TAZ. Knockdown of Snail reduced liver tumor formation in both tumor models. Ectopic expression of Snail restored liver tumorigenesis suppressed by disruption of TGF-β signaling. In human hepatocellular carcinoma, Snail expression correlated with TGF-β activation. Ectopic expression of Snail increased cellular proliferation, whereas Snail knockdown led to reduced proliferation in human hepatocellular carcinoma cells. In analyses of transgenic mice, we found TGF-β signaling to be required for formation of liver tumors upon expression of activated RAS and shRNA down-regulating p53, and upon expression of activated RAS and TAZ. Snail is the TGF-β target that is required for hepatic tumorigenesis in these models. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

FOXO/TXNIP pathway is involved in the suppression of hepatocellular carcinoma growth by glutamate antagonist MK-801

PubMed Central

2013-01-01

Background Accumulating evidence has suggested the importance of glutamate signaling in cancer growth, yet the signaling pathway has not been fully elucidated. N-methyl-D-aspartic acid (NMDA) receptor activates intracellular signaling pathways such as the extracellular-signal-regulated kinase (ERK) and forkhead box, class O (FOXO). Suppression of lung carcinoma growth by NMDA receptor antagonists via the ERK pathway has been reported. However, series of evidences suggested the importance of FOXO pathways for the regulation of normal and cancer cell growth. In the liver, FOXO1 play important roles for the cell proliferation such as hepatic stellate cells as well as liver metabolism. Our aim was to investigate the involvement of the FOXO pathway and the target genes in the growth inhibitory effects of NMDA receptor antagonist MK-801 in human hepatocellular carcinoma. Methods Expression of NMDAR1 in cancer cell lines from different tissues was examined by Western blot. NMDA receptor subunits in HepG2, HuH-7, and HLF were examined by reverse transcriptase polymerase chain reaction (RT-PCR), and growth inhibition by MK-801 and NBQX was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of MK-801 on the cell cycle were examined by flow cytometry and Western blot analysis. Expression of thioredoxin-interacting protein (TXNIP) and p27 was determined by real-time PCR and Western blotting. Activation of the FOXO pathway and TXNIP induction were examined by Western blotting, fluorescence microscopy, Chromatin immunoprecipitation (ChIP) assay, and reporter gene assay. The effects of TXNIP on growth inhibition were examined using the gene silencing technique. Results NMDA receptor subunits were expressed in all cell lines examined, and MK-801, but not NBQX, inhibited cell growth of hepatocellular carcinomas. Cell cycle analysis showed that MK-801 induced G1 cell cycle arrest by down-regulating cyclin D1 and up-regulating p27. MK-801 dephosphorylated Thr24 in FOXO1 and induced its nuclear translocation, thus increasing transcription of TXNIP, a tumor suppressor gene. Knock-down of TXNIP ameliorated the growth inhibitory effects of MK-801. Conclusions Our results indicate that functional NMDA receptors are expressed in hepatocellular carcinomas and that the FOXO pathway is involved in the growth inhibitory effects of MK-801. This mechanism could be common in hepatocellular carcinomas examined, but other mechanisms such as ERK pathway could exist in other cancer cells as reported in lung carcinoma cells. Altered expression levels of FOXO target genes including cyclin D1 and p27 may contribute to the inhibition of G1/S cell cycle transition. Induction of the tumor suppressor gene TXNIP plays an important role in the growth inhibition by MK-801. Our report provides new evidence that FOXO-TXNIP pathway play a role in the inhibition of the hepatocellular carcinoma growth by MK-801. PMID:24112473

An HPLC method associated with a thermodynamic analysis to compare the binding of TRAIL and its nanovectorized form to death receptors DR4 and DR5 and their relationship to cytotoxicity.

PubMed

Guillaume, Yves Claude; Lethier, Lydie; André, Claire

2016-11-15

TRAIL is a member of the TNF family of cytokines which induces apoptosis of cancer cells via its binding to its cognate receptors, DR5 a high affinity site and DR4 a site of low affinity. Our working group has recently demonstrated that nanovectorization of TRAIL with single wall carbon nanotubes (abbreviated NPT) enhanced TRAIL affinity to the high affinity site DR5 and increased pro apoptotic potential in different human tumor cell lines. In this paper, the DR4 low affinity site was immobilized on a chromatographic support and the effect of temperature on a wide temperature range 1°C-50°C was studied to calculate the thermodynamic parameters of the binding of TRAIL and NPT to DR4 and DR5 receptors. For the first time the heat capacity changes for the different binding processes were determined. At a physiological pH (7.4) the heat capacity changes for the binding of NPT to DR4 and DR5 were respectively equal to -0.91kJ/molK and -0.28kJ/molK and those obtained for the binding of TRAIL to DR4 and DR5 were respectively equal to -1.54kJ/molK and -1.05kJ/molK. By the use of differential scanning calorimetry (DSC), a phase transition (∼12°C for DR5, ∼4°C for DR4) between a disordered (low temperature) and an ordered (high temperature) solid like state visualized in the receptor structure confirmed the temperature dependence of binding affinity enthalpy ΔH for soluble TRAIL and its nanovectorized form to its cognate receptors. In the low temperature domain, the positive ΔH values contribute non-favourably to the free energy of binding, TRAIL and NPT described similar affinities for DR4 and DR5. For the high temperature domain, negative ΔH values indicated that van der Waals interactions and hydrogen bonding are engaged favourably at the ligand - receptor interface. Above 30°C, their rank-ordered affinities were thus strongly different in the sequence: TRAIL DR4

Cytoprotective effect of polysaccharide isolated from different mushrooms against 7-ketocholesterol induced damage in mouse liver cell line (BNL CL. 2).

PubMed

Kim, Joo-Shin; Chung, Hau Yin; Na, Keun

2007-01-01

Cytoprotective ability of polysaccharides isolated from different edible mushrooms was investigated on the 7-ketocholesterol-induced damaged cell line. Polysaccharide extracts from six different edible mushrooms-Flammulina velutipes, Peurotus ostreatus, Lentinus edodes, Agrocybe aegerita, Agaricus blazei, and Cordyceps militaris- were prepared by hot water extraction and alcohol precipitation. Cytoprotective ability was evaluated by measuring the viable cells of the normal embryonic liver cell line (BNL CL. 2) in the presence of 7-ketocholesterol. At 80 microg/mL of 7-ketocholesterol, cytotoxicity was very high with a loss of 98% of viable cells after 20 h of incubation. With the addition of 200 microg/mL of each polysaccharide isolate to the cell line containing 80 microg/mL of 7-ketocholesterol, polysaccharide isolates from both Flammulina velutipes and Peurotus ostreatus could significantly inhibit the 7-ketochoelsterol-induced cytotoxicity in the cells. But other polysaccharide isolates were not effective in inhibiting cell damage caused by the oxLDL-induced cytotoxicity.

Cytoprotective effect of polysaccharide isolated from different mushrooms against 7-ketocholesterol induced damage in mouse liver cell line (BNL CL. 2)

PubMed Central

Chung, Hau Yin; Na, Keun

2007-01-01

Cytoprotective ability of polysaccharides isolated from different edible mushrooms was investigated on the 7-ketocholesterol-induced damaged cell line. Polysaccharide extracts from six different edible mushrooms-Flammulina velutipes, Peurotus ostreatus, Lentinus edodes, Agrocybe aegerita, Agaricus blazei, and Cordyceps militaris- were prepared by hot water extraction and alcohol precipitation. Cytoprotective ability was evaluated by measuring the viable cells of the normal embryonic liver cell line (BNL CL. 2) in the presence of 7-ketocholesterol. At 80 µg/mL of 7-ketocholesterol, cytotoxicity was very high with a loss of 98% of viable cells after 20 h of incubation. With the addition of 200 µg/mL of each polysaccharide isolate to the cell line containing 80 µg/mL of 7-ketocholesterol, polysaccharide isolates from both Flammulina velutipes and Peurotus ostreatus could significantly inhibit the 7-ketochoelsterol-induced cytotoxicity in the cells. But other polysaccharide isolates were not effective in inhibiting cell damage caused by the oxLDL-induced cytotoxicity. PMID:20368935

Seven diverse human embryonic stem cell-derived chondrogenic clonal embryonic progenitor cell lines display site-specific cell fates.

PubMed

Sternberg, Hal; Kidd, Jennifer; Murai, James T; Jiang, Jianjie; Rinon, Ariel; Erickson, Isaac E; Funk, Walter D; Wang, Qian; Chapman, Karen B; Vangsness, C Thomas; West, Michael D

2013-03-01

The transcriptomes of seven diverse clonal human embryonic progenitor cell lines with chondrogenic potential were compared with that of bone marrow-derived mesenchymal stem cells (MSCs). The cell lines 4D20.8, 7PEND24, 7SMOO32, E15, MEL2, SK11 and SM30 were compared with MSCs using immunohistochemical methods, gene expression microarrays and quantitative real-time PCR. In the undifferentiated progenitor state, each line displayed unique combinations of site-specific markers, including AJAP1, ALDH1A2, BMP5, BARX1, HAND2, HOXB2, LHX1, LHX8, PITX1, TBX15 and ZIC2, but none of the lines expressed the MSC marker CD74. The lines showed diverse responses when differentiated in the presence of combinations of TGF-β3, BMP2, 4, 6 and 7 and GDF5, with the lines 4D20.8, SK11, SM30 and MEL2 showing osteogenic markers in some differentiation conditions. The line 7PEND24 showed evidence of regenerating articular cartilage and, in some conditions, markers of tendon differentiation. The scalability of site-specific clonal human embryonic stem cell-derived embryonic progenitor cell lines may provide novel models for the study of differentiation and methods for preparing purified and identified cells types for use in therapy.

The effect of syndecan-4 and glypican-1 knockdown on the proliferation and differentiation of turkey satellite cells differing in age and growth rates.

PubMed

Velleman, Sandra G; Clark, Daniel L; Tonniges, Jeffrey R

2018-09-01

Posthatch skeletal muscle growth requires myogenic satellite cells and the dynamic expression of cell membrane-associated proteins. The membrane associated heparan sulfate proteoglycans, syndecan-4 and glypican-1, link the satellite cell niche to the intracellular environment. Sydnecan-4 and glypican-1 are differentially expressed with age in turkey satellite cells and their over-expression impacts both satellite cell proliferation and differentiation, but their effect on satellite cells from lines with different growth potentials is not known. The objective of the current study was to determine if syndecan-4 and glypican-1 regulation of satellite cell proliferation and differentiation is affected by age and growth selection. Pectoralis major satellite cells isolated at 1 d, 7 and 16-wk of age from a Randombred Control 2 (RBC2) line and a 16-wk body weight (F) line selected from the RBC2 line turkeys were studied. Syndecan-4 and glypican-1 expression was knocked down in both lines. The F-line cells proliferated faster than RBC2 line cells regardless of age, while differentiation tended to be greater in RBC2 line cells than F-line cells at each age. Syndecan-4 knockdown decreased proliferation at 7- and 16-wk but not 1 d cells, and increased differentiation at 1 d and 7 wk but not 16 wk cells. Glypican-1 knockdown differentially affected proliferation depending on cell age, whereas differentiation was decreased for 7- and 16-wk but not 1 d cells. These data suggest syndecan-4 and glypican-1 differentially affected satellite cell function in an age-dependent manner, but had little impact on differences in proliferation and differentiation due to growth selection. Copyright © 2018. Published by Elsevier Inc.

Evasion of superinfection exclusion and elimination of primary viral RNA by an adapted strain of hepatitis C virus.

PubMed

Webster, Brian; Ott, Melanie; Greene, Warner C

2013-12-01

Cells that are productively infected by hepatitis C virus (HCV) are refractory to a second infection by HCV via a block in viral replication known as superinfection exclusion. The block occurs at a postentry step and likely involves translation or replication of the secondary viral RNA, but the mechanism is largely unknown. To characterize HCV superinfection exclusion, we selected for an HCV variant that could overcome the block. We produced a high-titer HC-J6/JFH1 (Jc1) viral genome with a fluorescent reporter inserted between NS5A and NS5B and used it to infect Huh7.5 cells containing a Jc1 replicon. With multiple passages of these infected cells, we isolated an HCV variant that can superinfect cells at high levels. Notably, the superinfectious virus rapidly cleared the primary replicon from superinfected cells. Viral competition experiments, using a novel strategy of sequence-barcoding viral strains, as well as superinfection of replicon cells demonstrated that mutations in E1, p7, NS5A, and the poly(U/UC) tract of the 3' untranslated region were important for superinfection. Furthermore, these mutations dramatically increased the infectivity of the virus in naive cells. Interestingly, viruses with a shorter poly(U/UC) and an NS5A domain II mutation were most effective in overcoming the postentry block. Neither of these changes affected viral RNA translation, indicating that the major barrier to postentry exclusion occurs at viral RNA replication. The evolution of the ability to superinfect after less than a month in culture and the concomitant exclusion of the primary replicon suggest that superinfection exclusion dramatically affects viral fitness and dynamics in vivo.

Replicative stress and alterations in cell cycle checkpoint controls following acetaminophen hepatotoxicity restrict liver regeneration.

PubMed

Viswanathan, Preeti; Sharma, Yogeshwar; Gupta, Priya; Gupta, Sanjeev

2018-03-05

Acetaminophen hepatotoxicity is a leading cause of hepatic failure with impairments in liver regeneration producing significant mortality. Multiple intracellular events, including oxidative stress, mitochondrial damage, inflammation, etc., signify acetaminophen toxicity, although how these may alter cell cycle controls has been unknown and was studied for its significance in liver regeneration. Assays were performed in HuH-7 human hepatocellular carcinoma cells, primary human hepatocytes and tissue samples from people with acetaminophen-induced acute liver failure. Cellular oxidative stress, DNA damage and cell proliferation events were investigated by mitochondrial membrane potential assays, flow cytometry, fluorescence staining, comet assays and spotted arrays for protein expression after acetaminophen exposures. In experimental groups with acetaminophen toxicity, impaired mitochondrial viability and substantial DNA damage were observed with rapid loss of cells in S and G2/M and cell cycle restrictions or even exit in the remainder. This resulted from altered expression of the DNA damage regulator, ATM and downstream transducers, which imposed G1/S checkpoint arrest, delayed entry into S and restricted G2 transit. Tissues from people with acute liver failure confirmed hepatic DNA damage and cell cycle-related lesions, including restrictions of hepatocytes in aneuploid states. Remarkably, treatment of cells with a cytoprotective cytokine reversed acetaminophen-induced restrictions to restore cycling. Cell cycle lesions following mitochondrial and DNA damage led to failure of hepatic regeneration in acetaminophen toxicity but their reversibility offers molecular targets for treating acute liver failure. © 2018 John Wiley & Sons Ltd.

BORIS up-regulates OCT4 via histone methylation to promote cancer stem cell-like properties in human liver cancer cells.

PubMed

Liu, Qiuying; Chen, Kefei; Liu, Zhongjian; Huang, Yuan; Zhao, Rongce; Wei, Ling; Yu, Xiaoqin; He, Jingyang; Liu, Jun; Qi, Jianguo; Qin, Yang; Li, Bo

2017-09-10

Accumulating evidence has revealed the importance of cancer stem cells (CSCs) in chemoresistance and recurrence. BORIS, a testes-specific CTCF paralog, has been shown to be associated with stemness traits of embryonic cancer cells and epithelial CSCs. We previously reported that BORIS is correlated with the expression of the CSC marker CD90 in hepatocellular carcinoma (HCC). These results encourage us to wonder whether BORIS exerts functions on CSC-like traits of human liver cancer cells. Here, we report that BORIS was enriched in HCC tissues. Exogenous overexpression of BORIS promoted CSC-like properties, including self-renewal, chemoresistance, migration and invasion in Huh7 and HCCLM3 cells. Conversely, BORIS knockdown suppressed CSC-like properties in SMMC-7721 and HepG2 cells and inhibited tumorigenicity in SMMC-7721 cells. Moreover, BORIS alteration did not affect the DNA methylation status of the minimal promoter and exon 1 region of OCT4. However, BORIS overexpression enhanced the amount of BORIS bound on the OCT4 promoter and increased H3K4me2, while reducing H3K27me3; BORIS depletion decreased BORIS and H3K4me2 on the OCT4 promoter, while increasing H3K27me3. These results revealed that BORIS is associated with the CSC-like traits of human liver cancer cells through the epigenetic regulation of OCT4. Copyright © 2017 Elsevier B.V. All rights reserved.

The combination effect of sodium butyrate and 5-Aza-2'-deoxycytidine on radiosensitivity in RKO colorectal cancer and MCF-7 breast cancer cell lines.

PubMed

Cho, Hang Joo; Kim, Sin Young; Kim, Kee Hwan; Kang, Won Kyung; Kim, Ji Il; Oh, Seong Tack; Kim, Jeong Soo; An, Chang Hyeok

2009-05-21

The overall level of chromatin compaction is an important mechanism of radiosensitivity, and modification of DNA methylation and histone deacetylation may increase radiosensitivity by altering chromatin compaction. In this study, we investigated the effect of a demethylating agent, a histone deacetylase(HDAC) inhibitor, and the two agents combined on radiosensitivity in human colon and breast cancer cell lines. In this study, we used RKO colorectal cancer cell line and MCF-7 breast cancer cell lines and normal colon cell lines. On each of the cell lines, we used three different agents: the HDAC inhibitor sodium butyrate(SB), the demethylating agent 5-Aza-2'-deoxycytidine(5-aza-DC), and radiation. We then estimated the percentage of the cell survival using the XTT method and experimented to determine if there was an augmentation in the therapeutic effect by using different combinations of the two or three of the treatment methods. After treatment of each cell lines with 5-aza-DC, SB and 6 grays of radiation, we observed that the survival fraction was lower after the treatment with 5-aza-DC or SB than with radiation alone in RKO and MCF-7 cell lines(p < 0.001). The survival fraction was lowest when the two agents, 5-aza-DC and SB were combined with radiation in both RKO and MCF-cell lines. In conclusion, 5-aza-DC and SB can enhance radiosensitivity in both MCF-7 and RKO cell lines. The combination effect of a demethylating agent and an HDAC inhibitor is more effective than that of single agent treatment in both breast and colon cancer cell lines.

Epigenetic Alterations Associated With CCCTC-Binding Factor Deregulation in Prostate Cancer

DTIC Science & Technology

2011-07-01

HPV16 E6 and/or E7 prostate cell lines. We have established stable cell lines containing inducible CTCF shRNA in pTRIPZ vector in PPC-1, LNCaPs, 293T...and non-tumorigenic HPV16 E6 and/or E7 prostate cell lines. We are in process of conducting CTCF knockdown experiments using transient transfection...which express high levels of endogenous CTCF and in non- tumorigenic HPV16 E6 and/or E7 prostate cell lines. We see efficient knockdown of CTCF

17β-estradiol exerts anticancer effects in anoikis-resistant hepatocellular carcinoma cell lines by targeting IL-6/STAT3 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Seulki, E-mail: sl10f@naver.com; Lee, Minjong, E-mail: minjonglee2@naver.com; Kim, Jong Bin, E-mail: kkimjp@hanmail.net

17β-Estradiol (E2) has been proven to exert protective effects against HCC; however, its mechanism on HCC proliferation and suppression of invasion remains to be further explored. Because HCC up-regulates serum Interleukin-6 (IL-6) levels and Signal Transducer and Activator of Transcription 3 (STAT3), molecular agents that attenuate IL-6/STAT3 signaling can potentially suppress HCC development. In this study, we examined involvement of E2 in anoikis resistance that induces invasion capacities and chemo-resistance. Huh-BAT and HepG2 cells grown under anchorage-independent condition were selected. The anoikis-resistant (AR) cells showed stronger chemo-resistance against sorafenib, doxorubicin, 5-fluorouracil and cisplatin compared to adherent HCC cells. AR HCCmore » cells exhibited decreased expression of E-cadherin and increased expression of the N-cadherin and vimentin compared to adherent HCC cells. We then demonstrated that E2 suppressed cell proliferation in AR HCC cells. IL-6 treatment enhanced invasive characteristics, and E2 reversed it. Regarding mechanism of E2, it decreased in the phosphorylation of STAT3 that overexpressed on AR HCC cells. The inhibitory effect of E2 on cell growth was accompanied with cell cycle arrest at G2/M phase and caspase-3/9/PARP activation through c-Jun N-terminal Kinase (JNK) phosphorylation. Taken together, these findings suggested that E2 inhibited the proliferation of AR HCC cells through down-regulation of IL-6/STAT3 signaling. Thus, E2 can be a potential therapeutic drug for treatment of metastatic or chemo-resistant HCC. -- Highlights: •Anoikis-resistant HCC cells characterized chemo-resistant and metastatic potentials. •17β-Estradiol down-regulated IL-6/STAT3 signaling in anoikis-resistant HCC cells. •17β-Estradiol suppressed cell proliferation by inducing G2/M phase arrest and apoptosis though JNK phosphorylation.« less

MCF-7 cells--changing the course of breast cancer research and care for 45 years.

PubMed

Lee, Adrian V; Oesterreich, Steffi; Davidson, Nancy E

2015-07-01

It is 45 years since a pleural effusion from a patient with metastatic breast cancer led to the generation of the MCF-7 breast cancer cell line. MCF-7 is the most studied human breast cancer cell line in the world, and results from this cell line have had a fundamental impact upon breast cancer research and patient outcomes. But of the authors for the nearly 25000 scientific publications that used this cell line, how many know the unique story of its isolation and development? In this commentary we will review the past, present, and future of research using MCF-7 breast cancer cells. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Computational Discovery of Niclosamide Ethanolamine, a Repurposed Drug Candidate That Reduces Growth of Hepatocellular Carcinoma Cells In Vitro and in Mice by Inhibiting Cell Division Cycle 37 Signaling.

PubMed

Chen, Bin; Wei, Wei; Ma, Li; Yang, Bin; Gill, Ryan M; Chua, Mei-Sze; Butte, Atul J; So, Samuel

2017-06-01

Drug repositioning offers a shorter approval process than new drug development. We therefore searched large public datasets of drug-induced gene expression signatures to identify agents that might be effective against hepatocellular carcinoma (HCC). We searched public databases of messenger RNA expression patterns reported from HCC specimens from patients, HCC cell lines, and cells exposed to various drugs. We identified drugs that might specifically increase expression of genes that are down-regulated in HCCs and reduce expression of genes up-regulated in HCCs using a nonparametric, rank-based pattern-matching strategy based on the Kolmogorov-Smirnov statistic. We evaluated the anti-tumor activity of niclosamide and its ethanolamine salt (NEN) in HCC cell lines (HepG2, Huh7, Hep3B, Hep40, and PLC/PRF/5), primary human hepatocytes, and 2 mouse models of HCC. In one model of HCC, liver tumor development was induced by hydrodynamic delivery of a sleeping beauty transposon expressing an activated form of Ras (v12) and truncated β-catenin (N90). In another mouse model, patient-derived xenografts were established by implanting HCC cells from patients into livers of immunocompromised mice. Tumor growth was monitored by bioluminescence imaging. Tumor-bearing mice were fed a regular chow diet or a chow diet containing niclosamide or NEN. In a separate experiment using patient-derived xenografts, tumor-bearing mice were given sorafenib (the standard of care for patients with advanced HCC), NEN, or niclosamide alone; a combination of sorafenib and NEN; or a combination sorafenib and niclosamide in their drinking water, or regular water (control), and tumor growth was monitored. Based on gene expression signatures, we identified 3 anthelmintics that significantly altered the expression of genes that are up- or down-regulated in HCCs. Niclosamide and NEN specifically reduced the viability of HCC cells: the agents were at least 7-fold more cytotoxic to HCCs than primary hepatocytes. Oral administration of NEN to mice significantly slowed growth of genetically induced liver tumors and patient-derived xenografts, whereas niclosamide did not, coinciding with the observed greater bioavailability of NEN compared with niclosamide. The combination of NEN and sorafenib was more effective at slowing growth of patient-derived xenografts than either agent alone. In HepG2 cells and in patient-derived xenografts, administration of niclosamide or NEN increased expression of 20 genes down-regulated in HCC and reduced expression of 29 genes up-regulated in the 274-gene HCC signature. Administration of NEN to mice with patient-derived xenografts reduced expression of proteins in the Wnt-β-catenin, signal transducer and activator of transcription 3, AKT-mechanistic target of rapamycin, epidermal growth factor receptor-Ras-Raf signaling pathways. Using immunoprecipitation assays, we found NEN to bind cell division cycle 37 protein and disrupt its interaction with heat shock protein 90. In a bioinformatics search for agents that alter the HCC-specific gene expression pattern, we identified the anthelmintic niclosamide as a potential anti-tumor agent. Its ethanolamine salt, with greater bioavailability, was more effective than niclosamide at slowing the growth of genetically induced liver tumors and patient-derived xenografts in mice. Both agents disrupted interaction between cell division cycle 37 and heat shock protein 90 in HCC cells, with concomitant inhibition of their downstream signaling pathways. NEN might be effective for treatment of patients with HCC. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Identification of cellular and viral factors related to anti-hepatitis C virus activity of cyclophilin inhibitor.

PubMed

Goto, Kaku; Watashi, Koichi; Inoue, Daisuke; Hijikata, Makoto; Shimotohno, Kunitada

2009-10-01

We have so far reported that an immunosuppressant cyclosporin A (CsA), a well-known cyclophilin (CyP) inhibitor (CPI), strongly suppressed hepatitis C virus (HCV) replication in cell culture, and that CyPB was a cellular cofactor for viral replication. To further investigate antiviral mechanisms of CPI, we here developed cells carrying CsA-resistant HCV replicons, by culturing the HCV subgenomic replicon cells for 4 weeks in the presence of CsA with G418. Transfection of total RNA from the isolated CsA-resistant cells to naïve Huh7 cells conferred CsA resistance, suggesting that the replicon RNA itself was responsible for the resistant phenotype. Of the identified amino acid mutations, D320E in NS5A conferred the CsA resistance. The replicon carrying the D320E mutation was sensitive to interferon-alpha, but was resistant to CsA and other CPIs including NIM811 and sanglifehrin A. Knockdown of individual CyP subtypes revealed CyP40, in addition to CyPA and CyPB, contributed to viral replication, and CsA-resistant replicons acquired independence from CyPA for efficient replication. These data provide important evidence on the mechanisms underlying the regulation of HCV replication by CyP and for designing novel and specific anti-HCV strategies with CPIs.

Acetonic Extract of Buxus sempervirens Induces Cell Cycle Arrest, Apoptosis and Autophagy in Breast Cancer Cells

PubMed Central

Ait-Mohamed, Ouardia; Battisti, Valentine; Joliot, Véronique; Fritsch, Lauriane; Pontis, Julien; Medjkane, Souhila; Redeuilh, Catherine; Lamouri, Aazdine; Fahy, Christine; Rholam, Mohamed; Atmani, Djebbar; Ait-Si-Ali, Slimane

2011-01-01

Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC50 ranging from 7.74 µg/ml to 12.5 µg/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC50 of 19.24 µg/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC50 did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer. PMID:21935420

HBeAg-induced miR-106b promotes cell growth by targeting the retinoblastoma gene.

PubMed

Samal, Jasmine; Kandpal, Manish; Vivekanandan, Perumal

2017-10-30

Chronic HBV infection is a major cause of hepatocellular carcinoma (HCC). The association between hepatitis B "e" antigen (HBeAg) and HCC is well-established by epidemiological studies. Nonetheless, the biological role of HBeAg in HCC remains enigmatic. We investigate the role of HBeAg in HBV-related HCC. Our findings suggest that HBeAg enhances cell proliferation and accelerates progression from G0/G1 phase to the S phase of the cell cycle in Huh7 cells. Examination of host gene expression and miRNA expression profiles reveals a total of 21 host genes and 12 host miRNAs that were differentially regulated in cells expressing HBeAg. Importantly, HBeAg induced the expression of miR-106b, an oncogenic miRNA. Interestingly, HBeAg-expression results in a significant reduction in the expression of retinoblastoma (Rb) gene, an experimentally validated target of miR-106b. Inhibition of miR-106b significantly increased the expression of the Rb gene, resulting in reduced cell proliferation and slowing of cell cycle progression from the G0/G1 phase to S phase. These observations suggest that the up-regulation of miR-106b by HBeAg contributes to the pathogenesis of HBV-related HCC by down-regulating the Rb gene. Our results highlight a role for HBeAg in HCC and provide a novel perspective on the molecular mechanisms underlying HBV-related HCC.

Cytotoxic components of Pereskia bleo (Kunth) DC. (Cactaceae) leaves.

PubMed

Malek, Sri Nurestri Abdul; Shin, Sim Kae; Wahab, Norhanom Abdul; Yaacob, Hashim

2009-05-06

Dihydroactinidiolide (1) and a mixture of sterols [campesterol (2), stigmasterol (3) and beta-sitosterol (4)], together with the previously isolated individual compounds beta-sitosterol (4), 2,4-di-tert-butylphenol (5), alpha-tocopherol (6), phytol (7) were isolated from the active ethyl acetate fraction of Pereskia bleo (Kunth) DC. (Cactaceae) leaves. Cytotoxic activities of the above mentioned compounds against five human carcinoma cell lines, namely the human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), human hormone-dependent breast carcinoma cell line (MCF7) and human lung carcinoma cell line (A549); and non-cancer human fibroblast cell line (MRC-5) were investigated. Compound 5 possessed very remarkable cytotoxic activity against KB cells, with an IC(50 )value of 0.81microg/mL. This is the first report on the cytotoxic activities of the compounds isolated from Pereskia bleo.

3D-printed gelatin scaffolds of differing pore geometry modulate hepatocyte function and gene expression.

PubMed

Lewis, Phillip L; Green, Richard M; Shah, Ramille N

2018-03-15

Three dimensional (3D) printing is highly amenable to the fabrication of tissue-engineered organs of a repetitive microstructure such as the liver. The creation of uniform and geometrically repetitive tissue scaffolds can also allow for the control over cellular aggregation and nutrient diffusion. However, the effect of differing geometries, while controlling for pore size, has yet to be investigated in the context of hepatocyte function. In this study, we show the ability to precisely control pore geometry of 3D-printed gelatin scaffolds. An undifferentiated hepatocyte cell line (HUH7) demonstrated high viability and proliferation when seeded on 3D-printed scaffolds of two different geometries. However, hepatocyte specific functions (albumin secretion, CYP activity, and bile transport) increases in more interconnected 3D-printed gelatin cultures compared to a less interconnected geometry and to 2D controls. Additionally, we also illustrate the disparity between gene expression and protein function in simple 2D culture modes, and that recreation of a physiologically mimetic 3D environment is necessary to induce both expression and function of cultured hepatocytes. Three dimensional (3D) printing provides tissue engineers the ability spatially pattern cells and materials in precise geometries, however the biological effects of scaffold geometry on soft tissues such as the liver have not been rigorously investigated. In this manuscript, we describe a method to 3D print gelatin into well-defined repetitive geometries that show clear differences in biological effects on seeded hepatocytes. We show that a relatively simple and widely used biomaterial, such as gelatin, can significantly modulate biological processes when fabricated into specific 3D geometries. Furthermore, this study expands upon past research into hepatocyte aggregation by demonstrating how it can be manipulated to enhance protein function, and how function and expression may not precisely correlate in 2D models. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Host scavenger receptor SR-BI plays a dual role in the establishment of malaria parasite liver infection.

PubMed

Rodrigues, Cristina D; Hannus, Michael; Prudêncio, Miguel; Martin, Cécilie; Gonçalves, Lígia A; Portugal, Sílvia; Epiphanio, Sabrina; Akinc, Akin; Hadwiger, Philipp; Jahn-Hofmann, Kerstin; Röhl, Ingo; van Gemert, Geert-Jan; Franetich, Jean-François; Luty, Adrian J F; Sauerwein, Robert; Mazier, Dominique; Koteliansky, Victor; Vornlocher, Hans-Peter; Echeverri, Christophe J; Mota, Maria M

2008-09-11

An obligatory step of malaria parasite infection is Plasmodium sporozoite invasion of host hepatocytes, and host lipoprotein clearance pathways have been linked to Plasmodium liver infection. By using RNA interference to screen lipoprotein-related host factors, we show here that the class B, type I scavenger receptor (SR-BI) is the strongest regulator of Plasmodium infection among these factors. Inhibition of SR-BI function reduced P. berghei infection in Huh7 cells, and overexpression of SR-BI led to increased infection. In vivo silencing of liver SR-BI expression in mice and inhibition of SR-BI activity in human primary hepatocytes reduced infection by P. berghei and by P. falciparum, respectively. Heterozygous SR-BI(+/-) mice displayed reduced P. berghei infection rates correlating with liver SR-BI expression levels. Additional analyses revealed that SR-BI plays a dual role in Plasmodium infection, affecting both sporozoite invasion and intracellular parasite development, and may therefore constitute a good target for malaria prophylaxis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chan, Shih-Ching; Lo, Shih-Yen; Graduate Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan

Research highlights: {yields} Lipid rafts are known to play an important role in virus entry and virus assembly of many viruses. {yields} However, HCV is the first example of the association of lipid raft with viral RNA replication. {yields} Our results in this manuscript demonstrate that purified HCV RCs with associated lipid raft membrane appeared as distinct particles of around 0.7 um under EM and AFM. {yields} Knockdown of proteins associated with lipid raft suppressed the HCV replication and reduced the number of these particles. {yields} To our knowledge, structures of HCV RCs were demonstrated at its first time inmore » this manuscript. -- Abstract: Hepatitis C viral RNA synthesis has been demonstrated to occur on a lipid raft membrane structure. Lipid raft membrane fraction purified by membrane flotation analysis was observed using transmission electron microscopy and atomic force microscopy. Particles around 0.7 um in size were found in lipid raft membrane fraction purified from hepatitis C virus (HCV) replicon but not their parental HuH7 cells. HCV NS5A protein was associated with these specialized particles. After several cycles of freezing-thawing, these particles would fuse into larger sizes up to 10 um. Knockdown of seven proteins associated with lipid raft (VAPA, COPG, RAB18, COMT, CDC42, DPP4, and KDELR2) of HCV replicon cells reduced the observed number of these particles and suppressed the HCV replication. Results in this study indicated that HCV replication complexes with associated lipid raft membrane form distinct particle structures of around 0.7 um as observed from transmission electron microscopy and atomic force microscopy.« less

Vorticity dipoles and a theoretical model of a finite force at the moving contact line singularity

NASA Astrophysics Data System (ADS)

Zhang, Peter; Devoria, Adam; Mohseni, Kamran

2017-11-01

In the well known works of Moffatt (1964) and Huh & Scriven (1971), an infinite force was reported at the moving contact line (MCL) and attributed to a non-integrable stress along the fluid-solid boundary. In our recent investigation of the boundary driven wedge, a model of the MCL, we find that the classical solution theoretically predicts a finite force at the contact line if the forces applied by the two boundaries that make up the corner are taken into consideration. Mathematically, this force can be obtained by the complex contour integral of the holomorphic vorticity-pressure function given by G = μω + ip . Alternatively, this force can also be found using a carefully defined real integral that incorporates the two boundaries. Motivated by this discovery, we have found that the rate of change in circulation, viscous energy dissipation, and viscous energy flux is also finite per unit contact line length. The analysis presented demonstrates that despite a singular stress and a relatively simple geometry, the no-slip semi-infinite wedge is capable of capturing some physical quantities of interest. Furthermore, this result provides a foundation for other challenging topics such as dynamic contact angle.

Indole-based hydrazide-hydrazones and 4-thiazolidinones: synthesis and evaluation as antitubercular and anticancer agents.

PubMed

Cihan-Üstündağ, Gökçe; Şatana, Dilek; Özhan, Gül; Çapan, Gültaze

2016-01-01

A new series of indolylhydrazones (6) and indole-based 4-thiazolidinones (7, 8) have been designed, synthesized and screened for in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv. 4-Thiazolidinone derivatives 7g-7j, 8g, 8h and 8j displayed notable antituberculosis (anti-TB) activity showing 99% inhibition at MIC values ranging from 6.25 to 25.0 µg/ml. Compounds 7g, 7h, 7i, 8h and 8j demonstrated anti-TB activity at concentrations 10-fold lower than those cytotoxic for the mammalian cell lines. The indolylhydrazone derivative 6b has also been evaluated for antiproliferative activity against human cancer cell lines at the National Cancer Institute (USA). Compound 6b showed an interesting anticancer profile against different human tumor-derived cell lines at sub-micromolar concentrations with obvious selectivity toward colon cancer cell line COLO 205.

Spontaneous Mutation at Amino Acid 544 of the Ebola Virus Glycoprotein Potentiates Virus Entry and Selection in Tissue Culture.

PubMed

Ruedas, John B; Ladner, Jason T; Ettinger, Chelsea R; Gummuluru, Suryaram; Palacios, Gustavo; Connor, John H

2017-08-01

Ebolaviruses have a surface glycoprotein (GP 1,2 ) that is required for virus attachment and entry into cells. Mutations affecting GP 1,2 functions can alter virus growth properties. We generated a recombinant vesicular stomatitis virus encoding Ebola virus Makona variant GP 1,2 (rVSV-MAK-GP) and observed emergence of a T544I mutation in the Makona GP 1,2 gene during tissue culture passage in certain cell lines. The T544I mutation emerged within two passages when VSV-MAK-GP was grown on Vero E6, Vero, and BS-C-1 cells but not when it was passaged on Huh7 and HepG2 cells. The mutation led to a marked increase in virus growth kinetics and conferred a robust growth advantage over wild-type rVSV-MAK-GP on Vero E6 cells. Analysis of complete viral genomes collected from patients in western Africa indicated that this mutation was not found in Ebola virus clinical samples. However, we observed the emergence of T544I during serial passage of various Ebola Makona isolates on Vero E6 cells. Three independent isolates showed emergence of T544I from undetectable levels in nonpassaged virus or virus passaged once to frequencies of greater than 60% within a single passage, consistent with it being a tissue culture adaptation. Intriguingly, T544I is not found in any Sudan, Bundibugyo, or Tai Forest ebolavirus sequences. Furthermore, T544I did not emerge when we serially passaged recombinant VSV encoding GP 1,2 from these ebolaviruses. This report provides experimental evidence that the spontaneous mutation T544I is a tissue culture adaptation in certain cell lines and that it may be unique for the species Zaire ebolavirus IMPORTANCE The Ebola virus (Zaire) species is the most lethal species of all ebolaviruses in terms of mortality rate and number of deaths. Understanding how the Ebola virus surface glycoprotein functions to facilitate entry in cells is an area of intense research. Recently, three groups independently identified a polymorphism in the Ebola glycoprotein (I544) that enhanced virus entry, but they did not agree in their conclusions regarding its impact on pathogenesis. Our findings here address the origins of this polymorphism and provide experimental evidence showing that it is the result of a spontaneous mutation (T544I) specific to tissue culture conditions, suggesting that it has no role in pathogenesis. We further show that this mutation may be unique to the species Zaire ebolavirus , as it does not occur in Sudan, Bundibugyo, and Tai Forest ebolaviruses. Understanding the mechanism behind this mutation can provide insight into functional differences that exist in culture conditions and among ebolavirus glycoproteins. Copyright © 2017 American Society for Microbiology.

Thyroid cell lines in research on goitrogenesis.

PubMed

Gerber, H; Peter, H J; Asmis, L; Studer, H

1991-12-01

Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Osna, Natalia A., E-mail: nosna@UNMC.edu; Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105; White, Ronda L.

The proteasome is a multi-catalytic protein degradation enzyme that is regulated by ethanol-induced oxidative stress; such suppression is attributed to CYP2E1-generated metabolites. However, under certain conditions, it appears that in addition to oxidative stress, other mechanisms are also involved in proteasome regulation. This study investigated whether impaired protein methylation that occurs during exposure of liver cells to ethanol, may contribute to suppression of proteasome activity. We measured the chymotrypsin-like proteasome activity in Huh7CYP cells, hepatocytes, liver cytosols and nuclear extracts or purified 20S proteasome under conditions that maintain or prevent protein methylation. Reduction of proteasome activity of hepatoma cell andmore » hepatocytes by ethanol or tubercidin was prevented by simultaneous treatment with S-adenosylmethionine (SAM). Moreover, the tubercidin-induced decline in proteasome activity occurred in both nuclear and cytosolic fractions. In vitro exposure of cell cytosolic fractions or highly purified 20S proteasome to low SAM:S-adenosylhomocysteine (SAH) ratios in the buffer also suppressed proteasome function, indicating that one or more methyltransferase(s) may be associated with proteasomal subunits. Immunoblotting a purified 20S rabbit red cell proteasome preparation using methyl lysine-specific antibodies revealed a 25 kDa proteasome subunit that showed positive reactivity with anti-methyl lysine. This reactivity was modified when 20S proteasome was exposed to differential SAM:SAH ratios. We conclude that impaired methylation of proteasome subunits suppressed proteasome activity in liver cells indicating an additional, yet novel mechanism of proteasome activity regulation by ethanol.« less

Inhibition of nuclear factor-kappa B enhances the tumor growth of ovarian cancer cell line derived from a low-grade papillary serous carcinoma in p53-independent pathway.

PubMed

Xiao, Xue; Yang, Gong; Bai, Peng; Gui, Shunping; Nyuyen, Tri M Bui; Mercado-Uribe, Imelda; Yang, Mei; Zou, Juan; Li, Qintong; Xiao, Jianguo; Chang, Bin; Liu, Guangzhi; Wang, He; Liu, Jinsong

2016-08-02

NF-kB can function as an oncogene or tumor suppressor depending on cancer types. The role of NF-kB in low-grade serous ovarian cancer, however, has never been tested. We sought to elucidate the function of NF-kB in the low-grade serous ovarian cancer. The ovarian cancer cell line, HOC-7, derived from a low-grade papillary serous carcinoma. Introduction of a dominant negative mutant, IkBαM, which resulted in decrease of NF-kB function in ovarian cancer cell lines. The transcription ability, tumorigenesis, cell proliferation and apoptosis were observed in derivative cell lines in comparison with parental cells. Western blot analysis indicated increased expression of the anti-apoptotic proteins Bcl-xL and reduced expression of the pro-apoptotic proteins Bax, Bad, and Bid in HOC-7/IĸBαM cell. Further investigations validate this conclusion in KRAS wildtype cell line SKOV3. Interesting, NF-kB can exert its pro-apoptotic effect by activating mitogen-activated protein kinase (MAPK) phosphorylation in SKOV3 ovarian cancer cell, whereas opposite changes detected in p-MEK in HOC-7 ovarian cancer cell, the same as some chemoresistant ovarian cancer cell lines. In vivo animal assay performed on BALB/athymic mice showed that injection of HOC-7 induced subcutaneous tumor growth, which was completely regressed within 7 weeks. In comparison, HOC-7/IĸBαM cells caused sustained tumor growth and abrogated tumor regression, suggesting that knock-down of NF-kB by IĸBαM promoted sustained tumor growth and delayed tumor regression in HOC-7 cells. Our results demonstrated that NF-kB may function as a tumor suppressor by facilitating regression of low grade ovarian serous carcinoma through activating pro-apoptotic pathways.

Hepatitis C Virus Particle Assembly Involves Phosphorylation of NS5A by the c-Abl Tyrosine Kinase.

PubMed

Yamauchi, Shota; Takeuchi, Kenji; Chihara, Kazuyasu; Sun, Xuedong; Honjoh, Chisato; Yoshiki, Hatsumi; Hotta, Hak; Sada, Kiyonao

2015-09-04

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is thought to regulate the replication of viral RNA and the assembly of virus particles in a serine/threonine phosphorylation-dependent manner. However, the host kinases that phosphorylate NS5A have not been fully identified. Here, we show that HCV particle assembly involves the phosphorylation of NS5A by the c-Abl tyrosine kinase. Pharmacological inhibition or knockdown of c-Abl reduces the production of infectious HCV (J6/JFH1) particles in Huh-7.5 cells without markedly affecting viral RNA translation and replication. NS5A is tyrosine-phosphorylated in HCV-infected cells, and this phosphorylation is also reduced by the knockdown of c-Abl. Mutational analysis reveals that NS5A tyrosine phosphorylation is dependent, at least in part, on Tyr(330) (Tyr(2306) in polyprotein numbering). Mutation of this residue to phenylalanine reduces the production of infectious HCV particles but does not affect the replication of the JFH1 subgenomic replicon. These findings suggest that c-Abl promotes HCV particle assembly by phosphorylating NS5A at Tyr(330). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

HCV core protein induces hepatic lipid accumulation by activating SREBP1 and PPAR{gamma}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Kook Hwan; Hong, Sung Pyo; Kim, KyeongJin

2007-04-20

Hepatic steatosis is a common feature in patients with chronic hepatitis C virus (HCV) infection. HCV core protein plays an important role in the development of hepatic steatosis in HCV infection. Because SREBP1 (sterol regulatory element binding protein 1) and PPAR{gamma} (peroxisome proliferators-activated receptor {gamma}) are involved in the regulation of lipid metabolism of hepatocyte, we sought to determine whether HCV core protein may impair the expression and activity of SREBP1 and PPAR{gamma}. In this study, it was demonstrated that HCV core protein increases the gene expression of SREBP1 not only in Chang liver, Huh7, and HepG2 cells transiently transfectedmore » with HCV core protein expression plasmid, but also in Chang liver-core stable cells. Furthermore, HCV core protein enhanced the transcriptional activity of SREBP1. In addition, HCV core protein elevated PPAR{gamma} transcriptional activity. However, HCV core protein had no effect on PPAR{gamma} gene expression. Finally, we showed that HCV core protein stimulates the genes expression of lipogenic enzyme and fatty acid uptake associated protein. Therefore, our finding provides a new insight into the mechanism of hepatic steatosis by HCV infection.« less

Distinct Modes of Regulation of Transcription of Hepatitis B Virus by the Nuclear Receptors HNF4α and COUP-TF1

PubMed Central

Yu, Xianming; Mertz, Janet E.

2003-01-01

To study the effects of the nuclear receptors (NRs) HNF4α and COUP-TF1 on the life cycle of hepatitis B virus (HBV), the human hepatoma cell line Huh7 was transiently cotransfected with plasmids containing the HBV genome and encoding these two NRs. Overexpression of HNF4α and COUP-TF1 led to a 9-fold increase and a 7- to 10-fold decrease, respectively, in viral DNA synthesis. These two NRs also exhibited distinct modes of regulation of viral transcription. Overexpression of HNF4α led to a more-than-10-fold increase in synthesis of the pregenomic RNA but to only a 2- to 3-fold increase in synthesis of the pre-C and S RNAs. Moreover, the NR response element within the pre-C promoter, NRREpreC, played the major role in activation of pregenomic RNA synthesis by HNF4α. On the other hand, overexpression of COUP-TF1 led to an over-10-fold repression of synthesis of both pre-C and pregenomic RNAs mediated through either NRREpreC or NRREenhI. HNF4α and COUP-TF1 antagonized each other's effects on synthesis of pregenomic RNA and viral DNA when they were co-overexpressed. A naturally occurring HBV variant which allows for binding by HNF4α but not COUP-TF1 in its NRREpreC exhibited significantly higher levels of synthesis of pregenomic RNA and viral DNA than wild-type HBV in coexpression experiments. Last, deletion analysis revealed that non-NRRE sequences located within both the C and pre-S1 regions are also essential for maximum activation of the pregenomic promoter by HNF4α but not for repression by COUP-TF1. Thus, HNF4α and COUP-TF1 function through different mechanisms to regulate expression of the HBV genes. PMID:12551987

Distinct modes of regulation of transcription of hepatitis B virus by the nuclear receptors HNF4alpha and COUP-TF1.

PubMed

Yu, Xianming; Mertz, Janet E

2003-02-01

To study the effects of the nuclear receptors (NRs) HNF4alpha and COUP-TF1 on the life cycle of hepatitis B virus (HBV), the human hepatoma cell line Huh7 was transiently cotransfected with plasmids containing the HBV genome and encoding these two NRs. Overexpression of HNF4alpha and COUP-TF1 led to a 9-fold increase and a 7- to 10-fold decrease, respectively, in viral DNA synthesis. These two NRs also exhibited distinct modes of regulation of viral transcription. Overexpression of HNF4alpha led to a more-than-10-fold increase in synthesis of the pregenomic RNA but to only a 2- to 3-fold increase in synthesis of the pre-C and S RNAs. Moreover, the NR response element within the pre-C promoter, NRRE(preC,) played the major role in activation of pregenomic RNA synthesis by HNF4alpha. On the other hand, overexpression of COUP-TF1 led to an over-10-fold repression of synthesis of both pre-C and pregenomic RNAs mediated through either NRRE(preC) or NRRE(enhI). HNF4alpha and COUP-TF1 antagonized each other's effects on synthesis of pregenomic RNA and viral DNA when they were co-overexpressed. A naturally occurring HBV variant which allows for binding by HNF4alpha but not COUP-TF1 in its NRRE(preC) exhibited significantly higher levels of synthesis of pregenomic RNA and viral DNA than wild-type HBV in coexpression experiments. Last, deletion analysis revealed that non-NRRE sequences located within both the C and pre-S1 regions are also essential for maximum activation of the pregenomic promoter by HNF4alpha but not for repression by COUP-TF1. Thus, HNF4alpha and COUP-TF1 function through different mechanisms to regulate expression of the HBV genes.

Identification of possible genetic alterations in the breast cancer cell line MCF-7 using high-density SNP genotyping microarray

PubMed Central

Wang, Hui-Yun; Greenawalt, Danielle; Cui, Xiangfeng; Tereshchenko, Irina V; Luo, Minjie; Yang, Qifeng; Azaro, Marco A; Hu, Guohong; Chu, Yi; Li, James Y; Shen, Li; Lin, Yong; Zhang, Lianjun

2009-01-01

Context: Cancer cell lines are used extensively in various research. Knowledge of genetic alterations in these lines is important for understanding mechanisms underlying their biology. However, since paired normal tissues are usually unavailable for comparison, precisely determining genetic alterations in cancer cell lines is difficult. To address this issue, a highly efficient and reliable method is developed. Aims: Establishing a highly efficient and reliable experimental system for genetic profiling of cell lines. Materials and Methods: A widely used breast cancer cell line, MCF-7, was genetically profiled with 4,396 single nucleotide polymorphisms (SNPs) spanning 11 whole chromosomes and two other small regions using a newly developed high-throughput multiplex genotyping approach. Results: The fractions of homozygous SNPs in MCF-7 (13.3%) were significantly lower than those in the control cell line and in 24 normal human individuals (25.1% and 27.4%, respectively). Homozygous SNPs in MCF-7 were found in clusters. The sizes of these clusters were significantly larger than the expected based on random allelic combination. Fourteen such regions were found on chromosomes 1p, 1q, 2q, 6q, 13, 15q, 16q, 17q and 18p in MCF-7 and two in the small regions. Conclusions: These results are generally concordant with those obtained using different approaches but are better in defining their chromosomal positions. The used approach provides a reliable way to detecting possible genetic alterations in cancer cell lines without paired normal tissues. PMID:19439911

A homogeneous cell-based assay for measurement of endogenous paraoxonase 1 activity.

PubMed

Ahmad, Syed; Carter, Jade J; Scott, John E

2010-05-01

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that plays an important role in organophosphate detoxification and prevention of atherosclerosis. Thus, there is significant interest in identifying nutritional and pharmacological enhancers of PON1 activity. To identify such compounds, we developed a rapid homogeneous assay to detect endogenous cell-associated PON1 activity. PON1 activity was measured by the simple addition of fluorigenic PON1 substrate DEPFMU to live Huh7 cells in medium and monitoring change in fluorescence. A specific PON1 inhibitor, 2-hydroxyquinoline, was used to confirm that the observed activity was due to PON1. The assay was optimized and characterized with regard to time course, substrate and sodium chloride concentration, number of cells, and tolerance to dimethyl sulfoxide and serum. Aspirin, quercetin, and simvastatin are compounds reported to increase PON1 expression. Consistent with the literature and Western blot data, these compounds enhanced PON1 activity in this assay with comparable efficacies and potencies. A known toxic compound did not increase assay signal. This assay method also detected PON1 activity in normal hepatocytes. Thus, a novel homogeneous assay for detection of endogenous PON1 expression has been developed and is amenable to high-throughput screening for the identification of small molecules that enhance PON1 expression. 2010 Elsevier Inc. All rights reserved.

Regulation of Akt/FoxO3a/Skp2 Axis Is Critically Involved in Berberine-Induced Cell Cycle Arrest in Hepatocellular Carcinoma Cells

PubMed Central

Li, Fanni; Dong, Xiwen; Lin, Peng; Jiang, Jianli

2018-01-01

The maintenance of ordinal cell cycle phases is a critical biological process in cancer genesis, which is a crucial target for anti-cancer drugs. As an important natural isoquinoline alkaloid from Chinese herbal medicine, Berberine (BBR) has been reported to possess anti-cancer potentiality to induce cell cycle arrest in hepatocellular carcinoma cells (HCC). However, the underlying mechanism remains to be elucidated. In our present study, G0/G1 phase cell cycle arrest was observed in berberine-treated Huh-7 and HepG2 cells. Mechanically, we observed that BBR could deactivate the Akt pathway, which consequently suppressed the S-phase kinase-associated protein 2 (Skp2) expression and enhanced the expression and translocation of Forkhead box O3a (FoxO3a) into nucleus. The translocated FoxO3a on one hand could directly promote the transcription of cyclin-dependent kinase inhibitors (CDKIs) p21Cip1 and p27Kip1, on the other hand, it could repress Skp2 expression, both of which lead to up-regulation of p21Cip1 and p27Kip1, causing G0/G1 phase cell cycle arrest in HCC. In conclusion, BBR promotes the expression of CDKIs p21Cip1 and p27Kip1 via regulating the Akt/FoxO3a/Skp2 axis and further induces HCC G0/G1 phase cell cycle arrest. This research uncovered a new mechanism of an anti-cancer effect of BBR. PMID:29360760

Aspirin suppresses the abnormal lipid metabolism in liver cancer cells via disrupting an NFκB-ACSL1 signaling.

PubMed

Yang, Guang; Wang, Yuan; Feng, Jinyan; Liu, Yunxia; Wang, Tianjiao; Zhao, Man; Ye, Lihong; Zhang, Xiaodong

2017-05-06

Abnormal lipid metabolism is a hallmark of tumorigenesis. Hence, the alterations of metabolism enhance the development of hepatocellular carcinoma (HCC). Aspirin is able to inhibit the growth of cancers through targeting nuclear factor κB (NF-κB). However, the role of aspirin in disrupting abnormal lipid metabolism in HCC remains poorly understood. In this study, we report that aspirin can suppress the abnormal lipid metabolism of HCC cells through inhibiting acyl-CoA synthetase long-chain family member 1 (ACSL1), a lipid metabolism-related enzyme. Interestingly, oil red O staining showed that aspirin suppressed lipogenesis in HepG2 cells and Huh7 cells in a dose-dependent manner. In addition, aspirin attenuated the levels of triglyceride and cholesterol in the cells, respectively. Strikingly, we identified that aspirin was able to down-regulate ACSL1 at the levels of mRNA and protein. Moreover, we validated that aspirin decreased the nuclear levels of NF-κB in HepG2 cells. Mechanically, PDTC, an inhibitor of NF-κB, could down-regulate ACSL1 at the levels of mRNA and protein in the cells. Functionally, PDTC reduced the levels of lipid droplets, triglyceride and cholesterol in HepG2 cells. Thus, we conclude that aspirin suppresses the abnormal lipid metabolism in HCC cells via disrupting an NFκB-ACSL1 signaling. Our finding provides new insights into the mechanism by which aspirin inhibits abnormal lipid metabolism of HCC. Therapeutically, aspirin is potentially available for HCC through controlling abnormal lipid metabolism. Copyright © 2017. Published by Elsevier Inc.

The role of IL-1beta in reduced IL-7 production by stromal and epithelial cells: a model for impaired T-cell numbers in the gut during HIV-1 infection.

PubMed

Thang, P H; Ruffin, N; Brodin, D; Rethi, B; Cam, P D; Hien, N T; Lopalco, L; Vivar, N; Chiodi, F

2010-08-01

Interleukin (IL)-7 is a key cytokine in T-cell homeostasis. Stromal cells, intestinal epithelial cells and keratinocytes are known to produce this cytokine. The mechanisms and cellular factors regulating IL-7 production are still unclear. We assessed whether IL-1beta and interferon (IFN)-gamma, cytokines produced during inflammatory conditions, may impact on IL-7 production. We used human intestinal epithelial cells (DLD-1 cell line) and bone marrow stromal cells (HS27 cell line), known to produce IL-7; IL-7 production was evaluated at the mRNA and protein levels. To assess whether treatment of HS27 cells with IL-1beta and/or IFN-gamma leads to changes in the gene expression of cytokines, Toll-like receptors (TLRs) and chemokines, we analysed gene expression profiles using the whole-genome microarray Human Gene 1.0 ST. We found that IFN-gamma enhanced the expression of IL-7 mRNA (P < 0.001) in both cell lines. IL-1beta treatment led to a significant down-regulation (P < 0.001) of IL-7 mRNA expression in both cell lines. The IL-7 concentration in supernatants collected from treated DLD-1 and HS27 cell cultures reflected the trend of IL-7 mRNA levels. The gene profiles revealed dramatic changes in expression of cytokines and their receptors (IL-7/IL-7R alpha; IL-1alpha,IL-1beta/IL-1R1; IFN-gamma/IFN-gammaR1), of IFN regulatory factors (IRF-1 and 2), of TLRs and of important chemo-attractants for T cells. The microarray results were verified by additional methods. Our results are discussed in the setting of inflammation and T-cell survival in the gut compartment during HIV-1 infection where stromal and epithelial cells may produce factors that contribute to impaired IL-7 homeostasis and homing of T cells.

Comparing Apoptosis and Necrosis Effects of Arctium Lappa Root Extract and Doxorubicin on MCF7 and MDA-MB-231 Cell Lines

PubMed Central

Ghafari, Fereshteh; Rajabi, Mohammad Reza; Mazoochi, Tahereh; Taghizadeh, Mohsen; Nikzad, Hossein; Atlasi, Mohammad Ali; Taherian, Aliakbar

2017-01-01

Objective: Breast cancer is a heterogeneous disease and very common malignancy in women worldwide. The efficacy of chemotherapy as an important part of breast cancer treatment is limited due to its side effects. While pharmaceutical companies are looking for better chemicals, research on traditional medicines that generally have fewer side effects is quite interesting. In this study, apoptosis and necrosis effect of Arctium lappa and doxorubicin was compared in MCF7, and MDA-MB-231 cell lines. Materials and Methods: MCF7 and MDA-MB-231 cells were cultured in RPMI 1640 containing 10% FBS and 100 U/ml penicillin/streptomycin. MTT assay and an annexin V/propidium iodide (AV/PI) kit were used respectively to compare the survival rate and apoptotic effects of different concentrations of doxorubicin and Arctium lappa root extract on MDA-MB-231 and MCF7 cells. Results: Arctium lappa root extract was able to reduce cell viability of the two cell lines in a dose and time dependent manner similar to doxorubicin. Flow cytometry results showed that similar to doxorubicin, Arctium Lappa root extract had a dose and time dependent apoptosis effect on both cell lines. 10µg/mL of Arctium lappa root extract and 5 µM of doxorubicin showed the highest anti-proliferative and apoptosis effect in MCF7 and MDA231 cells. Conclusion: The MCF7 (ER/PR-) and MDA-MB-231 (ER/PR+) cell lines represent two major breast cancer subtypes. The similar anti-proliferative and apoptotic effects of Arctium lappa root extract and doxorubicin (which is a conventional chemotherapy drug) on two different breast cancer cell lines strongly suggests its anticancer effects and further studies. PMID:28441789

Comparing Apoptosis and Necrosis Effects of Arctium Lappa Root Extract and Doxorubicin on MCF7 and MDA-MB-231 Cell Lines

PubMed

Ghafari, Fereshteh; Rajabi, Mohammad Reza; Mazoochi, Tahereh; Taghizadeh, Mohsen; Nikzad, Hossein; Atlasi, Mohammad Ali; Taherian, Aliakbar

2017-03-01

Objective: Breast cancer is a heterogeneous disease and very common malignancy in women worldwide. The efficacy of chemotherapy as an important part of breast cancer treatment is limited due to its side effects. While pharmaceutical companies are looking for better chemicals, research on traditional medicines that generally have fewer side effects is quite interesting. In this study, apoptosis and necrosis effect of Arctium lappa and doxorubicin was compared in MCF7, and MDA-MB-231 cell lines. Materials and Methods: MCF7 and MDA-MB-231 cells were cultured in RPMI 1640 containing 10% FBS and 100 U/ml penicillin/streptomycin. MTT assay and an annexin V/propidium iodide (AV/PI) kit were used respectively to compare the survival rate and apoptotic effects of different concentrations of doxorubicin and Arctium lappa root extract on MDA-MB-231 and MCF7 cells. Results: Arctium lappa root extract was able to reduce cell viability of the two cell lines in a dose and time dependent manner similar to doxorubicin. Flow cytometry results showed that similar to doxorubicin, Arctium Lappa root extract had a dose and time dependent apoptosis effect on both cell lines. 10μg/mL of Arctium lappa root extract and 5 μM of doxorubicin showed the highest anti-proliferative and apoptosis effect in MCF7 and MDA231 cells. Conclusion: The MCF7 (ER/PR-) and MDA-MB-231 (ER/PR+) cell lines represent two major breast cancer subtypes. The similar anti-proliferative and apoptotic effects of Arctium lappa root extract and doxorubicin (which is a conventional chemotherapy drug) on two different breast cancer cell lines strongly suggests its anticancer effects and further studies. Creative Commons Attribution License

Emodin improves lipid and glucose metabolism in high fat diet-induced obese mice through regulating SREBP pathway.

PubMed

Li, Jinmei; Ding, Lili; Song, Baoliang; Xiao, Xu; Qi, Meng; Yang, Qiaoling; Yang, Qiming; Tang, Xiaowen; Wang, Zhengtao; Yang, Li

2016-01-05

Currently, obesity has become a worldwide epidemic associated with Type 2 diabetes, dyslipidemia, cardiovascular disease and chronic metabolic diseases. Emodin is one of the active anthraquinone derivatives from Rheum palmatum and some other Chinese herbs with anti-inflammatory, anticancer and hepatoprotective properties. In the present study, we investigated the anti-obesity effects of emodin in obese mice and explore its potential pharmacological mechanisms. Male C57BL/6 mice were fed with high-fat diet for 12 weeks to induce obesity. Then the obese mice were divided into four groups randomly, HFD or emodin (40mg/kg/day and 80mg/kg/day) or lovastatin (30mg/kg/ day) for another 6 weeks. Body weight and food intake were recorded every week. At the end of the treatment, the fasting blood glucose, glucose and insulin tolerance test, serum and hepatic lipid levels were assayed. The gene expressions of liver and adipose tissues were analyzed with a quantitative PCR assay. Here, we found that emodin inhibited sterol regulatory element-binding proteins (SREBPs) transactivity in huh7 cell line. Furthermore, emodin (80mg/kg/day) treatment blocked body weight gain, decreased blood lipids, hepatic cholesterol and triglyceride content, ameliorated insulin sensitivity, and reduced the size of white and brown adipocytes. Consistently, SREBP-1 and SREBP-2 mRNA levels were significantly reduced in the liver and adipose tissue after emodin treatment. These data demonstrated that emodin could improve high-fat diet-induced obesity and associated metabolic disturbances. The underlying mechanism is probably associated with regulating SREBP pathway. Copyright © 2015 Elsevier B.V. All rights reserved.

Fabrication, optimization, and characterization of umbelliferone β-D-galactopyranoside-loaded PLGA nanoparticles in treatment of hepatocellular carcinoma: in vitro and in vivo studies

PubMed Central

Kumar, Vikas; Bhatt, Prakash Chandra; Rahman, Mahfoozur; Kaithwas, Gaurav; Choudhry, Hani; Al-Abbasi, Fahad A; Anwar, Firoz; Verma, Amita

2017-01-01

Umbelliferone β-D-galactopyranoside (UFG), isolated from plants, exhibits promising inhibitory action on numerous diseases. The present research was initiated to develop a suitable delivery system for UFG with an intention to enhance its therapeutic efficacy against diethyl nitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in Wistar rats. UFG-loaded polymeric nanoparticles prepared by sonication were scrutinized for average size, drug loading capacity, zeta potential, and drug release potency in animals. HCC cell lines HuH-7 and Hep G2 were used for in vitro cytotoxic investigation. Several hepatic, nonhepatic, antioxidant, and anti-inflammatory biochemical parameters were estimated to establish the anticancer potential of UFG nanoformulation. Microscopical and histopathological investigations were also undertaken to substantiate the results of our work. Umbelliferone β-D-galactopyranoside-loaded poly(d,l-lactide-co-glycolide) nanoparticles (UFG-PLGA-NP) with particle size of 187.1 nm and polydispersity index 0.16 were uniform in nature with 82.5% release of the total amount of drug after 48 h. Our study successfully established the development and characterization of UFG-PLGA-NP with noticeable effect against both in vivo and in vitro models. The anticancer potential of UFG-PLGA-NP was brought about by the management of DEN-induced reactive oxygen species generation, mitochondrial dysfunction, proinflammatory cytokines alteration, and induction of apoptosis. Positive zeta potential on the surface of UFG-PLGA-NP would have possibly offered higher hepatic accumulation of UFG, particularly in the electron-dense mitochondria organelles, and this was the take-home message from this study. Our results demonstrated that such polymer-loaded delivery systems of UFG can be a better option and can be further explored to improve the clinical outcomes against hepatic cancer. PMID:28932118

Inhibition of hepatitis B virus replication via HBV DNA cleavage by Cas9 from Staphylococcus aureus.

PubMed

Liu, Yu; Zhao, Miaoxian; Gong, Mingxing; Xu, Ying; Xie, Cantao; Deng, Haohui; Li, Xueying; Wu, Hongkai; Wang, Zhanhui

2018-04-01

Chronic hepatitis B virus (HBV) infection is difficult to cure due to the presence of covalently closed circular DNA (cccDNA). Accumulating evidence indicates that the CRISPR/Cas9 system effectively disrupts HBV genome, including cccDNA, in vitro and in vivo. However, efficient delivery of CRISPR/Cas9 system to the liver or hepatocytes using an adeno-associated virus (AAV) vector remains challenging due to the large size of Cas9 from Streptococcus pyogenes (Sp). The recently identified Cas9 protein from Staphylococcus aureus (Sa) is smaller than SpCas9 and thus is able to be packaged into the AAV vector. To examine the efficacy of SaCas9 system on HBV genome destruction, we designed 5 guide RNAs (gRNAs) that targeted different HBV genotypes, 3 of which were shown to be effective. The SaCas9 system significantly reduced HBV antigen expression, as well as pgRNA and cccDNA levels, in Huh7, HepG2.2.15 and HepAD38 cells. The dual expression of gRNAs/SaCas9 in these cell lines resulted in more efficient HBV genome cleavage. In the mouse model, hydrodynamic injection of gRNA/SaCas9 plasmids resulted in significantly lower levels of HBV protein expression. We also delivered the SaCas9 system into mice with persistent HBV replication using an AAV vector. Both the AAV vector and the mRNA of Cas9 could be detected in the C3H mouse liver cells. Decreased hepatitis B surface antigen (HBsAg), HBV DNA and pgRNA levels were observed when a higher titer of AAV was injected, although this decrease was not significantly different from the control. In summary, the SaCas9 system accurately and efficiently targeted the HBV genome and inhibited HBV replication both in vitro and in vivo. The system was delivered by an AAV vector and maybe used as a novel therapeutic strategy against chronic HBV infection. Copyright © 2018 Elsevier B.V. All rights reserved.

Analysing intracellular deformation of polymer capsules using structured illumination microscopy

NASA Astrophysics Data System (ADS)

Chen, Xi; Cui, Jiwei; Sun, Huanli; Müllner, Markus; Yan, Yan; Noi, Ka Fung; Ping, Yuan; Caruso, Frank

2016-06-01

Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties.Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties. Electronic supplementary information (ESI) available: Additional figures. See DOI: 10.1039/c6nr02151d

Triterpenoid Acids as Important Antiproliferative Constituents of European Elderberry Fruits.

PubMed

Gleńsk, Michał; Czapińska, Elżbieta; Woźniak, Marta; Ceremuga, Ireneusz; Włodarczyk, Maciej; Terlecki, Grzegorz; Ziółkowski, Piotr; Seweryn, Ewa

2017-01-01

In Europe, both the fruits and flowers of Sambucus nigra L. have been used against cold, as well as laxative, diaphoretic, and diuretic remedies. There are also a number of commercially available food products that contain elderberry juice, puréed or dried elderberries. Recent comprehensive literature data on pharmacology and chemistry of Sambuci fructus have encouraged us to screen extracts with different polarities from this plant material against cancer cell lines. The cytotoxic activity of the ethyl acetate and aqueous acetone extracts from elderberries as well as detected triterpenoids on human colon adenocarcinoma cell line (LoVo) and human breast cancer cell line (MCF-7) was investigated by sulforhodamine B assay. Moreover, cell migration assay was conducted for triterpenoid fraction and pure compounds. Aqueous acetone extract possessed much lower IC 50 value in cancer cell lines compared to ethyl acetate extract. The latter manifested high cytotoxicity against studied cell lines, suggesting that nonpolar compounds are responsible for the cytotoxic activity. Indeed, the phytochemical analysis revealed that ursolic and oleanolic acids are the main triterpenoids in the mentioned extract of which ursolic acid showed the highest activity with IC 50 values of 10.7 µg/mL on MCF-7 and 7.7 µg/mL on LoVo cells.

PIG7 promotes leukemia cell chemosensitivity via lysosomal membrane permeabilization

PubMed Central

Niu, Ting; Wu, Yu; Li, Jianjun; Wang, Fangfang; Zheng, Yuhuan; Liu, Ting

2016-01-01

PIG7 localizes to lysosomal membrane in leukemia cells. Our previous work has shown that transduction of pig7 into a series of leukemia cell lines did not result in either apoptosis or differentiation of most tested cell lines. Interestingly, it did significantly sensitize these cell lines to chemotherapeutic drugs. Here, we further investigated the mechanism underlying pig7-induced improved sensitivity of acute leukemia cells to chemotherapy. Our results demonstrated that the sensitization effect driven by exogenous pig7 was more effective in drug-resistant leukemia cell lines which had lower endogenous pig7 expression. Overexpression of pig7 did not directly activate the caspase apoptotic pathway, but decreased the lysosomal stability. The expression of pig7 resulted in lysosomal membrane permeabilization (LMP) and lysosomal protease (e.g. cathepsin B, D, L) release. Moreover, we also observed increased reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) induced by pig7. Some autophagy markers such as LC3I/II, ATG5 and Beclin-1, and necroptosis maker MLKL were also stimulated. However, intrinsic antagonism such as serine/cysteine protease inhibitors Spi2A and Cystatin C prevented downstream effectors from triggering leukemia cells, which were only on the “verge of apoptosis”. When combined with chemotherapy, LMP increased and more proteases were released. Once this process was beyond the limit of intrinsic antagonism, it induced programmed cell death cooperatively via caspase-independent and caspase-dependent pathways. PMID:26716897

High CHMP4B expression is associated with accelerated cell proliferation and resistance to doxorubicin in hepatocellular carcinoma.

PubMed

Hu, Baoying; Jiang, Dawei; Chen, Yuyan; Wei, Lixian; Zhang, Shusen; Zhao, Fengbo; Ni, Runzhou; Lu, Cuihua; Wan, Chunhua

2015-04-01

Charged multivesicular body protein 4B (CHMP4B), a subunit of the endosomal sorting complex required for transport (ESCRT)-III complex, plays an important part in cytokinetic membrane abscission and the late stage of mitotic cell division. In this study, we explored the prognostic significance of CHMP4B in human hepatocellular carcinoma (HCC) and its impact on the physiology of HCC cells. Western blot and immunohistochemistrical analyses showed that CHMP4B was significantly upregulated in HCC tissues, compared with adjacent non-tumorous tissues. Meanwhile, clinicopathological analysis revealed that high CHMP4B expression was correlated with multiple clinicopathological variables, including AFP, cirrhosis, AJCC stage, Ki-67 expression, and poor prognosis. More importantly, univariate and multivariate survival analyses demonstrated that CHMP4B served as an independent prognostic factor for survival of HCC patients. Using HCC cell cultures, we found that the expression of CHMP4B was progressively upregulated after the release from serum starvation. To verify whether CHMP4B could regulate the proliferation of HCC cells, CHMP4B was knocked down through the transfection of CHMP4B-siRNA oligos. Flow cytometry and CCK-8 assays indicated that interference of CHMP4B led to cell cycle arrest and proliferative impairment of HCC cells. Additionally, depletion of CHMP4B expression could increase the sensitivity to doxorubicin in HepG2 and Huh7 cells. Taken together, our results implied that CHMP4B could be a promising prognostic biomarker as well as a potential therapeutic target of HCC.

Establishment and culture optimization of a new type of pituitary immortalized cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kokubu, Yuko; Asashima, Makoto; Life Science Center of TARA, The University of Tsukuba, Ibaraki-ken 305-8577

The pituitary gland is a center of the endocrine system that controls homeostasis in an organism by secreting various hormones. The glandular anterior pituitary consists of five different cell types, each expressing specific hormones. However, their regulation and the appropriate conditions for their in vitro culture are not well defined. Here, we report the immortalization of mouse pituitary cells by introducing TERT, E6, and E7 transgenes. The immortalized cell lines mainly expressed a thyrotroph-specific thyroid stimulating hormone beta (Tshb). After optimization of the culture conditions, these immortalized cells proliferated and maintained morphological characteristics similar to those of primary pituitary cells undermore » sphere culture conditions in DMEM/F12 medium supplemented with N2, B27, basic FGF, and EGF. These cell lines responded to PKA or PKC pathway activators and induced the expression of Tshb mRNA. Moreover, transplantation of the immortalized cell line into subcutaneous regions and kidney capsules of mice further increased Tshb expression. These results suggest that immortalization of pituitary cells with TERT, E6, and E7 transgenes is a useful method for generating proliferating cells for the in vitro analysis of pituitary regulatory mechanisms. - Highlights: • Mouse pituitary cell lines were immortalized by introducing TERT, E6, and E7. • The immortalized cell lines mainly expressed thyroid stimulating hormone beta. • The cell lines responded to PKA or PKC pathway activators, and induced Tshb.« less

Autophagic Cell Death, Polyploidy and Senescence Induced in Breast Tumor Cells by the Substituted Pyrrole JG-03-14, a Novel Microtubule Poison

PubMed Central

Arthur, Christopher R.; Gupton, John T.; Kellogg, Glen E.; Yeudall, W. Andrew; Cabot, Myles C.; Newsham, Irene; Gewirtz, David A.

2007-01-01

JG-03-14, a substituted pyrrole that inhibits microtubule polymerization, was screened against MCF-7 (p53 wild type), MDA-MB 231 (p53 mutant), MCF-7/caspase 3 and MCF-7/ADR (multidrug resistant) breast tumor cell lines. Cell viability and growth inhibition were assessed by the crystal violet dye assay. Apoptosis was evaluated by the TUNEL assay, cell cycle distribution by flow cytometry, autophagy by acridine orange staining of vesicle formation, and senescence based on β-galactosidase staining and cell morphology. Our studies indicate that exposure to JG-03-14, at a concentration of 500 nM, induces time dependent cell death in the MCF-7 and MDA-MB 231 cell lines. In MCF-7 cells, a residual surviving cell population was found to be senescent; in contrast, there was no surviving senescent population in treated MDA-MB 231 cells. No proliferative recovery was detected over a period of 15 days post-treatment in either cell line. Both the TUNEL assay and FLOW cytometry indicated a relatively limited degree of apoptosis (< 10%) in response to drug treatment in MCF-7 cells with more extensive apoptosis (but < 20%) in MDA-MB231 cells; acidic vacuole formation indicative of autophagic cell death was relatively extensive in both MCF-7 and MDA-MB231 cells. In addition, JG-03-14 induced the formation of a large hyperdiploid cell population in MDA-MB231 cells. JG-03-14 also demonstrated pronounced anti-proliferative activity in MCF-7/caspase 3 cells and in the MCF-7/ADR cell line. The observation that JG-03-14 promotes autophagic cell death and also retains activity in tumor cells expressing the multidrug resistance pump indicates that novel microtubule poisons of the substituted pyrroles class may hold promise in the treatment of breast cancer. PMID:17692290

Synthesis of novel forskolin isoxazole derivatives with potent anti-cancer activity against breast cancer cell lines.

PubMed

Burra, Srinivas; Voora, Vani; Rao, Ch Prasad; Vijay Kumar, P; Kancha, Rama Krishna; David Krupadanam, G L

2017-09-15

Forskolin C 1 -isoxazole derivatives (3,5-regioisomers) (11a-e, 14, 15a-h and 15, 16a-g) were synthesized regioselectively by adopting 1,3-dipolar cycloadditions. These derivatives were tested using estrogen receptor positive breast cancer cell lines MCF-7 and BT-474. Majority of the compounds exhibited activity against the p53-positive MCF-7 breast cancer cells but not against the p53-negative BT-474 breast cancer cells. Among forskolin derivatives, compounds 11a, 11c, 14a, 14f, 14g, 14h, 15b, 16g and 17b exhibited higher anti-cancer activity against MCF-7 cell line with an IC 50 ≤1µM. The derivative 14f exhibited highest activity in both p53-positive (MCF-7) and p53-negative (BT-474) breast cancer cell lines with an IC 50 of 0.5µM. Copyright © 2017. Published by Elsevier Ltd.

Histone deacetylase inhibitor (HDACI) PCI-24781 enhances chemotherapy induced apoptosis in multidrug resistant sarcoma cell lines

PubMed Central

Yang, Cao; Choy, Edwin; Hornicek, Francis J.; Wood, Kirkham B; Schwab, Joseph H; Liu, Xianzhe; Mankin, Henry; Duan, Zhenfeng

2013-01-01

The anti-tumor activity of histone deacetylase inhibitors (HDACI) on multi-drug resistant sarcoma cell lines has never been previously described. Four multidrug resistant sarcoma cell lines treated with HDACI PCI-24781 resulted in dose-dependent accumulation of acetylated histones, p21 and PARP cleavage products. Growth of these cell lines was inhibited by PCI-24781 at IC50 of 0.43 to 2.7. When we looked for synergy of PCI-24781 with chemotherapeutic agents, we found that PCI-24781 reverses drug resistance in all four multidrug resistant sarcoma cell lines and synergizes with chemotherapeutic agents to enhance caspase-3/7 activity. Expression of RAD51 (a marker for DNA double-strand break repair) was inhibited and the expression of GADD45α (a marker for growth arrest and DNA-damage) was induced by PCI-24781 in multidrug resistant sarcoma cell lines. In conclusion, HDACI PCI-24781 synergizes with chemotherapeutic drugs to induce apoptosis and reverses drug resistance in multidrug resistant sarcoma cell lines. PMID:21508354

Heterologous Expression of the Carrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

PubMed

Ko, Eunhye; Kim, Minhye; Park, Yunho; Ahn, Yeh-Jin

2017-08-01

In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter-carrot Hsp17.7 gene-Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.

Oncolytic effects of adenovirus mutant capable of replicating in hypoxic and normoxic regions of solid tumor.

PubMed

Cho, Won-Kyung; Seong, Young Rim; Lee, Yeune Hee; Kim, Min Ji; Hwang, Kyung-Sun; Yoo, Jinsang; Choi, Seeyoung; Jung, Cho-Rok; Im, Dong-Soo

2004-11-01

Solid tumors contain normoxic and hypoxic regions depending on the distance from the capillary. Normal cells may also be exposed to hypoxia under certain physiological conditions. Tumor hypoxia has been shown to associate strongly with tumor propagation and malignant progression. Hypoxia-inducible factor (HIF)-1alpha is stable under hypoxia and induces transcription of target genes by binding to the hypoxia-response element (HRE). Here we investigated the oncolytic effects of a novel adenovirus mutant with a deleted E1B55 gene (Ad.Delta55.HRE), in which the expression of E1A, which is essential for adenoviral replication, is regulated under the control of an HRE-expression system. Ad.Delta55.HRE expressed E1A under normoxia and more E1A under hypoxia and exhibited oncolytic effects on various cultured tumor cells, but its cytotoxic effect is relatively attenuated in normal fibroblast cells under normoxic and hypoxic conditions. Ad.Delta55.HRE lysed Huh-7 hepatoma cells stably expressing HIF-1alpha more effectively compared to parental cells. Ad.Delta55.HRE treatment exhibited significant antitumor activity in PC-3 prostate- and MDA-MB-435 breast tumor-bearing nude mice in which HIF-1alpha protein was immunohistochemically detected. The E1A and hexon proteins of adenovirus were immunostained in MDA-MB-435 xenografts after Ad.Delta55.HRE treatment, suggestive of viral replication. Our results suggest that Ad.Delta55.HRE may be useful for the treatment of solid tumors.

Down-regulation of NTCP expression by cyclin D1 in hepatitis B virus-related hepatocellular carcinoma has clinical significance

PubMed Central

Kang, Jingting; Wang, Jie; Cheng, Jin; Cao, Zhiliang; Chen, Ran; Li, Huiyu; Liu, Shuang; Chen, Xiangmei; Sui, Jianhua; Lu, Fengmin

2017-01-01

The sodium-dependent taurocholate cotransporter polypeptide (NTCP) has been identified as a liver specific functional receptor for the hepatitis B virus (HBV). Previous studies indicated that the expression of NTCP may be associated with the proliferation status of hepatocytes. However, the involvement of NTCP in hepatocellular carcinoma (HCC) cells proliferation remains unclear. In this study, we confirmed that NTCP was down-regulated in HCC tumor tissues compared with that in the adjacent non-tumor tissues (P < 0.0001). Clinically, lower expression of NTCP was correlated with poor post-surgery survival rate (P = 0.0009) and larger tumor tissue mass (P = 0.003) of HCC patients. This was supported by the finding that ectopic expression of NTCP in both HepG2 and Huh-7 cells could significantly suppress hepatocytes growth by arresting cells in G0/G1 phase. We also discovered that cyclin D1 could transcriptionally suppress NTCP expression by inhibiting the activity of NTCP promoter, while arresting HCC cells in G0/G1 phase by serum starvation could upregulate NTCP mRNA levels. This is the first study to report that the transcriptional inhibition of NTCP expression during cell cycle progression was mediated by cyclin D1. The down-regulated NTCP expression was associated with poor prognosis and lower HBV cccDNA level in HCC patients. Therefore, NTCP expression levels might serve as a novel prognostic predictive marker for post-surgery survival rate of HCC patients. PMID:28915572

Down-regulation of NTCP expression by cyclin D1 in hepatitis B virus-related hepatocellular carcinoma has clinical significance.

PubMed

Kang, Jingting; Wang, Jie; Cheng, Jin; Cao, Zhiliang; Chen, Ran; Li, Huiyu; Liu, Shuang; Chen, Xiangmei; Sui, Jianhua; Lu, Fengmin

2017-08-22

The sodium-dependent taurocholate cotransporter polypeptide (NTCP) has been identified as a liver specific functional receptor for the hepatitis B virus (HBV). Previous studies indicated that the expression of NTCP may be associated with the proliferation status of hepatocytes. However, the involvement of NTCP in hepatocellular carcinoma (HCC) cells proliferation remains unclear. In this study, we confirmed that NTCP was down-regulated in HCC tumor tissues compared with that in the adjacent non-tumor tissues ( P < 0.0001). Clinically, lower expression of NTCP was correlated with poor post-surgery survival rate ( P = 0.0009) and larger tumor tissue mass ( P = 0.003) of HCC patients. This was supported by the finding that ectopic expression of NTCP in both HepG2 and Huh-7 cells could significantly suppress hepatocytes growth by arresting cells in G0/G1 phase. We also discovered that cyclin D1 could transcriptionally suppress NTCP expression by inhibiting the activity of NTCP promoter, while arresting HCC cells in G0/G1 phase by serum starvation could upregulate NTCP mRNA levels. This is the first study to report that the transcriptional inhibition of NTCP expression during cell cycle progression was mediated by cyclin D1. The down-regulated NTCP expression was associated with poor prognosis and lower HBV cccDNA level in HCC patients. Therefore, NTCP expression levels might serve as a novel prognostic predictive marker for post-surgery survival rate of HCC patients.

Ras/Mitogen-activated Protein Kinase (MAPK) Signaling Modulates Protein Stability and Cell Surface Expression of Scavenger Receptor SR-BI*

PubMed Central

Wood, Peta; Mulay, Vishwaroop; Darabi, Masoud; Chan, Karen Cecilia; Heeren, Joerg; Pol, Albert; Lambert, Gilles; Rye, Kerry-Anne; Enrich, Carlos; Grewal, Thomas

2011-01-01

The mitogen-activated protein kinase (MAPK) Erk1/2 has been implicated to modulate the activity of nuclear receptors, including peroxisome proliferator activator receptors (PPARs) and liver X receptor, to alter the ability of cells to export cholesterol. Here, we investigated if the Ras-Raf-Mek-Erk1/2 signaling cascade could affect reverse cholesterol transport via modulation of scavenger receptor class BI (SR-BI) levels. We demonstrate that in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells, Mek1/2 inhibition reduces PPARα-inducible SR-BI protein expression and activity, as judged by reduced efflux onto high density lipoprotein (HDL). Ectopic expression of constitutively active H-Ras and Mek1 increases SR-BI protein levels, which correlates with elevated PPARα Ser-21 phosphorylation and increased cholesterol efflux. In contrast, SR-BI levels are insensitive to Mek1/2 inhibitors in PPARα-depleted cells. Most strikingly, Mek1/2 inhibition promotes SR-BI degradation in SR-BI-overexpressing CHO cells and human HuH7 hepatocytes, which is associated with reduced uptake of radiolabeled and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyane-labeled HDL. Loss of Mek1/2 kinase activity reduces SR-BI expression in the presence of bafilomycin, an inhibitor of lysosomal degradation, indicating down-regulation of SR-BI via proteasomal pathways. In conclusion, Mek1/2 inhibition enhances the PPARα-dependent degradation of SR-BI in hepatocytes. PMID:21525007

Inhibition of protease-inhibitor resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

PubMed Central

Gal-Tanamy, Meital; Zemel, Romy; Bachmatov, Larissa; Jangra, Rohit K.; Shapira, Assaf; Villanueva, Rodrigo; Yi, MinKyung; Lemon, Stanley M.; Benhar, Itai; Tur-Kaspa, Ran

2015-01-01

Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing. PMID:20705106

Anti-proliferative activity of 2,6-dichloro-9- or 7-(ethoxycarbonylmethyl)-9H- or 7H-purines against several human solid tumour cell lines.

PubMed

Morales, Fátima; Ramírez, Alberto; Conejo-García, Ana; Morata, Cynthia; Marchal, Juan A; Campos, Joaquín M

2014-04-09

As leads we took several benzo-fused seven- and six-membered scaffolds linked to the pyrimidine or purine moieties with notable anti-proliferative activity against human breast, colon and melanoma cancerous cell lines. We then decided to maintain the double-ringed nitrogenous bases and change the other components to the ethyl acetate moiety. This way six purine and two 5-fluorouracil derivatives were obtained and evaluated against the MCF-7, HCT-116, A-375 and G-361 cancer cell lines. Two QSARs are obtained between the anti-proliferative IC₅₀ values for compounds 26-33 and the clog P against the melanoma cell lines A-375 and G-361. Our results show that two of the analogues [ethyl 2-(2,6-dichloro-9H- or 7H-purine-9- or 7-yl)acetates (30 and 33, respectively)] are potent cytotoxic agents against all the tumour cell lines assayed, showing single-digit micromolar IC₅₀ values. This exemplifies the potential of our previously reported purine compounds to qualify as lead structures for medicinal chemistry campaigns, affording simplified analogues easy to synthesize and with a noteworthy bioactivity. The selective activity of 30 and 33 against the melanoma cell line A-375, via apoptosis, supposes a great advantage for a future therapeutic use. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Caffeic acid, a coffee-related organic acid, inhibits infection by severe fever with thrombocytopenia syndrome virus in vitro.

PubMed

Ogawa, Motohiko; Shirasago, Yoshitaka; Ando, Shuji; Shimojima, Masayuki; Saijo, Masayuki; Fukasawa, Masayoshi

2018-04-05

Severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) causes tick-borne hemorrhagic fever in East Asia. The disease is characterized by high morbidity and mortality. Here, we evaluated the effects of caffeic acid (CA), a coffee-related organic acid with antiviral effects, against SFTSV infection. CA dose-dependently inhibited SFTSV infection in permissive human hepatoma Huh7.5.1-8 cells when SFTSV was added into the culture medium with CA. However, quinic acid (QA), another coffee-related organic acid, did not inhibit SFTSV infection. The 50% inhibitory concentration (IC 50 ) of CA against SFTSV was 0.048 mM, whereas its 50% cytotoxic concentration was 7.6 mM. The selectivity index (SI) was 158. Pre-incubation of SFTSV with CA for 4 h resulted in a greater inhibition of SFTSV infection (IC 50  = 0.019 mM; SI = 400). The pre-incubation substantially decreased viral attachment to the cells. CA treatment of the SFTSV-infected cells also inhibited the infection, albeit less effectively. CA activity after cell infection with SFTSV was more pronounced at a low multiplicity of infection (MOI) of 0.01 per cell (IC 50  = 0.18 mM) than at a high MOI of 1 per cell (IC 50  > 1 mM). Thus, CA inhibited virus spread by acting directly on the virus rather than on the infected cells. In conclusion, CA acted on SFTSV and inhibited viral infection and spread, mainly by inhibiting the binding of SFTSV to the cells. We therefore demonstrated CA to be a potential anti-SFTSV drug for preventing and treating SFTS. Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Antiproliferative activity and induction of apoptotic by ethanolic extract of Alpinia galanga rhizhome in human breast carcinoma cell line.

PubMed

Samarghandian, Saeed; Hadjzadeh, Mousa-Al-Reza; Afshari, Jalil Tavakkol; Hosseini, Mohadeseh

2014-06-17

We investigated the potential of galangal rhizomes to induce cytotoxic and apoptotic effects in the cultured human breast carcinoma cell line, (MCF-7) in compare with the non-malignant (MRC-5) cells. Both cells were cultured in DMEM medium and treated with galangal rhizomes for three consecutive days. The percentage of apoptotic cells was determined by flow cytometry using Annexin-V fluorescein isothiocyanate. The results showed that the ethanolic extract of galangal rhizomes decreased cell viability in the malignant cells as a concentration- and time- dependent manner. The IC50 values against MCF-7 were determined at 400.0 ± 11.7 and 170.0 ± 5.9 μg/ml after 48 and 72 h respectively. The morphology of MCF-7 cells treated with the ethanolic extract confirmed the cell proliferation assay results. Alpinia galanga induced apoptosis in MCF-7 cells, as determined by flow cytometry. We concluded that the extract of Alpinia galanga exerts pro-apoptotic effects in a breast cancer-derived cell line and could be considered as a potential chemotherapeutic agent in breast cancer.

C22-bronchial and T7-alveolar epithelial cell lines of the immortomouse are excellent murine cell culture model systems to study pulmonary peroxisome biology and metabolism.

PubMed

Karnati, Srikanth; Palaniswamy, Saranya; Alam, Mohammad Rashedul; Oruqaj, Gani; Stamme, Cordula; Baumgart-Vogt, Eveline

2016-03-01

In pulmonary research, temperature-sensitive immortalized cell lines derived from the lung of the "immortomouse" (H-2k(b)-tsA58 transgenic mouse), such as C22 club cells and T7 alveolar epithelial cells type II (AECII), are frequently used cell culture models to study CC10 metabolism and surfactant synthesis. Even though peroxisomes are highly abundant in club cells and AECII and might fulfill important metabolic functions therein, these organelles have never been investigated in C22 and T7 cells. Therefore, we have characterized the peroxisomal compartment and its associated gene transcription in these cell lines. Our results show that peroxisomes are highly abundant in C22 and T7 cells, harboring a common set of enzymes, however, exhibiting specific differences in protein composition and gene expression patterns, similar to the ones observed in club cells and AECII in situ in the lung. C22 cells contain a lower number of larger peroxisomes, whereas T7 cells possess more numerous tubular peroxisomes, reflected also by higher levels of PEX11 proteins. Moreover, C22 cells harbor relatively higher amounts of catalase and antioxidative enzymes in distinct subcellular compartments, whereas T7 cells exhibit higher levels of ABCD3 and plasmalogen synthesizing enzymes as well as nuclear receptors of the PPAR family. This study suggest that the C22 and T7 cell lines of the immortomouse lung are useful models to study the regulation and metabolic function of the peroxisomal compartment and its alterations by paracrine factors in club cells and AECII.

Involvement of overexpressed wild-type BRAF in the growth of malignant melanoma cell lines.

PubMed

Tanami, Hideaki; Imoto, Issei; Hirasawa, Akira; Yuki, Yasuhiro; Sonoda, Itaru; Inoue, Jun; Yasui, Kohichiro; Misawa-Furihata, Akiko; Kawakami, Yutaka; Inazawa, Johji

2004-11-18

Comparative genomic hybridization (CGH) using 40 cell lines derived from malignant melanomas (MMs) revealed frequent amplification at 7q33-q34 containing BRAF gene, which often is mutated in MM. We found this gene to be amplified to a remarkable degree in the MM cell lines that exhibited high-level gains at 7q33-q34 in CGH. Among 40 cell lines, the eight lines that revealed neither BRAF nor NRAS mutations showed even higher levels of BRAF mRNA expression than the 32 mutated lines, although DNA amplification at 7q33-q34 was not detected in every lines overexpressing BRAF. MM cells that carried wild-type BRAF and NRAS showed constitutive overexpression of B-Raf protein and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), even after serum starvation. Not only downregulation of the endogenously overexpressed wild-type B-Raf by antisense oligonucleotide but also a treatment with an inhibitor of mitogen-activated protein kinase kinase (MAPKK, MEK) reduced phosphorylated ERK1/2 and cell growth, whereas the exogenously expressed wild-type B-Raf promoted cell growth in MM cells. Our results provide the evidence that overexpression of wild-type B-Raf, in part but not always as a result of gene amplification, is one of the mechanisms underlying constitutive activation of the MAPK pathway that stimulates growth of MM cells.

Comparison of aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase induction by polycyclic aromatic compounds in human and mouse cell lines.

PubMed

Jaiswal, A K; Nebert, D W; Eisen, H W

1985-08-01

The human MCF-7 and the mouse Hepa-1 cell culture lines were compared for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]anthracene (BA) and TCDD- and BA-specific binding in the cytosol and nucleus. The effective concentration of BA in the growth medium required to induce either enzyme to 50% of its maximally inducible activity (EC50) was the same (5-11 microM) in both MCF-7 and Hepa-1 cells. On the other hand, the EC50 for TCDD in MCF-7 cells (5-25 nM) was more than 40-fold greater than that in Hepa-1 cells (0.4 to 0.6 nM). P1-450- and P3-450-specific mouse cDNA probes were used to quantitate mRNA induction in the Hepa-1 cell line. P1-450 mRNA was induced markedly by TCDD and benzo[a] anthracene, whereas P3-450 mRNA was induced negligibly. A P1-450-specific human cDNA probe was used to quantitate P1-450 mRNA induction in the MCF-7 cell line. Aryl hydrocarbon hydroxylase inducibility by TCDD or BA always paralleled P1-450 mRNA inducibility in either the mouse or human line. Although the cytosolic Ah receptor in Hepa-1 cells was easily detected by sucrose density gradient centrifugation, gel permeation chromatography, and anion-exchange high-performance liquid chromatography, the cytosolic receptor cannot be detected in MCF-7 cells. Following in vivo exposure of cultures to radiolabeled TCDD, the intranuclear concentration of inducer-receptor complex was at least fifty times greater in Hepa-1 than MCF-7 cultures. The complete lack of measurable cytosolic receptor and almost totally absent inducer-receptor complex in the nucleus of MCF-7 cells was, therefore, out of proportion to its capacity for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility. This MCF-7 line should provide an interesting model for a better understanding of the mechanisms of drug-metabolizing enzyme induction by polycyclic aromatic compounds, including the Ah receptor-mediated mechanism.

Identification of Novel Prognostic Genetic Markers in Prostate Cancer

DTIC Science & Technology

2000-02-01

alterations in two normal- and three malignant-derived prostate epithelial cell lines immortalized with the E6 and E7 transforming genes of human papilloma virus (HPV...malignant-derived prostate epithelial cell lines immortalized with the E6 and E7 transforming genes of human papilloma virus (HPV) 16. These studies...transforming genes of human papilloma virus (HPV) 16 (13). The cell lines demonstrated several numerical and structural chromosomal alterations

Rare sugar D-allose induces specific up-regulation of TXNIP and subsequent G1 cell cycle arrest in hepatocellular carcinoma cells by stabilization of p27kip1.

PubMed

Yamaguchi, Fuminori; Takata, Maki; Kamitori, Kazuyo; Nonaka, Machiko; Dong, Youyi; Sui, Li; Tokuda, Masaaki

2008-02-01

'Rare sugars' are defined as monosaccharides that exist in nature but are only present in limited quantities. The development of mass production method of rare sugars revealed some interesting physiological effects of these on animal cells, but the mechanisms have not been well studied. We examined the effect of D-allose on the proliferation of cancer cells and the underlying molecular mechanism of the action. The HuH-7 hepatocellular carcinoma cells were treated with various monosaccharides for 48 h and D-allose was shown to inhibit cell growth by 40% in a dose-dependent manner. D-allose induced G1 cell cycle arrest but not apoptosis. The microarray analysis revealed that D-allose significantly up-regulated thioredoxin interacting protein (TXNIP) gene expression, which is often suppressed in tumor cells and western blot analysis confirmed its increase at protein level. The overexpression of TXNIP also induced G1 cell cycle arrest. Analysis of cell cycle regulatory genes showed p27kip1, a key regulator of G1/S cell cycle transition, to be increased at the protein but not the transcriptional level. Protein interaction between TXNIP and jab1, and p27kip1 and jab1, was observed, suggesting stabilization of p27kip1 protein by the competitive inhibition of jab1-mediated nuclear export of p27kip1 by TXNIP. In addition, increased interaction and nuclear localization of TXNIP and p27kip1 were apparent after D-allose treatment. Our findings surprisingly suggest that D-allose, a simple monosaccharide, may act as a novel anticancer agent via unique TXNIP induction and p27kip1 protein stabilization.

F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells

PubMed Central

Wu, Bo; Liu, Zhen-Yu; Cui, Jian; Yang, Xiang-Min; Jing, Lin; Zhou, Yang; Chen, Zhi-Nan; Jiang, Jian-Li

2017-01-01

Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD) prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells. PMID:28117675

F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells.

PubMed

Wu, Bo; Liu, Zhen-Yu; Cui, Jian; Yang, Xiang-Min; Jing, Lin; Zhou, Yang; Chen, Zhi-Nan; Jiang, Jian-Li

2017-01-20

Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD) prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells.

Selenium Nanoparticles Induce the Chemo-Sensitivity of Fluorouracil Nanoparticles in Breast and Colon Cancer Cells.

PubMed

Abd-Rabou, Ahmed A; Shalby, Aziza B; Ahmed, Hanaa H

2018-05-11

Drug resistance is a major challenge of breast and colon cancer therapies leading to treatment failure. The main objective of the current study is to investigate whether selenium nanoparticles (nano-Se) can induce the chemo-sensitivity of 5-fluorouracil (FU)-encapsulated poly (D, L-lactide-co-glycolide) nanoparticles (nano-FU) in breast and colon cancer cell lines. Nano-Se and nano-FU were synthesized and characterized, then applied individually or in combination upon MCF7, MDA-MB-231, HCT 116, and Caco-2 cancerous cell lines. Cytotoxicity, cellular glucose uptake, and apoptosis, as well as malondialdehyde (MDA), nitric oxide (NO), and zinc (Zn) levels, were investigated upon the different treatments. We have resulted that nano-FU induced cell death in MCF7 and Caco-2 more effectively than MDA-MB-231 and HCT 116 cell lines. Moreover, nano-FU plus nano-Se potentiate MCF7 and Caco-2 chemo-sensitivity were higher than MDA-MB-231 and HCT 116 cancerous cell lines. It is relevant to note that Se and FU nano-formulations inhibited cancer cell bioenergetics via glucose uptake slight blockage. Furthermore, nano-FU increased the levels of NO and MDA in media over cancer cells, while their combinations with nano-Se rebalance the redox status with Zn increment. We noticed that MCF7 cell line is sensitive, while MDA-MB-231 cell line is resistant to Se and nano-Se. This novel approach could be of great potential to enhance the chemo-sensitivity in breast and colon cancer cells.

Autophagy prevention sensitizes AKTi-1/2-induced anti-hepatocellular carcinoma cell activity in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Qi; Yang, Manyi; Qu, Zhan

Molecule-targeted therapy has become the research focus for hepatocellular carcinoma (HCC). Persistent PI3K-AKT activation is often detected in HCC, representing a valuable oncotarget for treatment. Here, we tested the anti-HCC activity by a potent AKT inhibitor: AKT inhibitor 1/2 (AKTi-1/2). In both established (HepG2 and Huh-7) and primary human HCC cells, treatment with AKTi-1/2 inhibited cell survival and proliferation, but induced cell apoptosis. AKTi-1/2 blocked AKT-mTOR activation, yet simultaneously provoked cytoprotective autophagy in HCC cells. The latter was evidenced by ATG-5 and Beclin-1 upregulation, p62 downregulation as well as LC3B-GFP puncta formation. Autophagy inhibition, via pharmacological inhibitors (3-methyladenine, ammonium chloride,more » and bafilomycin A1) or Beclin-1 siRNA knockdown, significantly potentiated AKTi-1/2-induced HepG2 cell death and apoptosis. In nude mice, AKTi-1/2 intraperitoneal injection inhibited HepG2 tumor growth. Significantly, its anti-tumor activity in vivo was further sensitized when combined with Beclin-1 shRNA knockdown in HepG2 tumors. Together, these results demonstrate that autophagy activation serves as a main resistance factor of AKTi-1/2 in HCC cells. Autophagy prevention therefore sensitizes AKTi-1/2-induced anti-HCC activity in vitro and in vivo. - Highlights: • AKTi-1/2 inhibits human HCC cells in vitro. • Autophagy inhibitors sensitize AKTi-1/2-induced HCC cell death and apoptosis. • Beclin-1 siRNA potentiates AKTi-1/2-induced HepG2 cell death and apoptosis. • Beclin-1 knockdown augments AKTi-1/2-induced anti-HepG2 tumor activity in vivo.« less

Control of Foaming by Adding Known Mixtures of Pure Chemicals

DTIC Science & Technology

1949-04-01

Sodium lauryl sulfate — do -- „NACA^ 12 FACA TN No. I8k2 TABLE VIII,- AEROSOL OT HUH DIEFEBBHT, SÜBSTABCE3 IK AEBOSHELL 120...80 34.2 Nacconal NRSF 400 —_—-<-(Lo ■— 80 49.6 Sodium lauryl sulfonate 300 do —— - 90 49.6 Sapamine MS 400 do- 80 52.1 Lauryl sulfonic acid...400 do-- 80 ^9.8 Sodium heptadecyl sulfate 400 do — - — 80 7.6 Sodium octyl sulfate 4oo —do - 80 46.8 --.— Xylidines 10,000 51.5

[Experimental study on carcinogenesis by human papillomavirus type 8 E7 gene].

PubMed

Nishikawa, T

1994-05-01

Human papillomavirus (HPV) 5 and HPV8 are often detected in skin cancers developed in patients suffering from epidermodysplasia verruciformis, as well as in skin cancers developed in immunosuppressed patients. In the present study, in order to examine the transforming activity of the HPV8E7 gene, the HPV8E7 and HPV8E6/E7 genes were cloned into the expression vector (pcD2-Y), under the SV40 enhancer/promoter to construct pcD2-8E7 and pcD2-8E6/E7, respectively. The E7 and E6/E7 genes of genital high-risk HPV16 were also cloned into pcD2-Y to construct pcD2-16E7 and pcD2-16E6/E7, respectively. They were tested for their ability to collaboratively transform primary rat embryo fibroblasts (REFs) with activated H-ras gene. Transfection experiments of REFs having an activated H-ras gene revealed that pcD2-8E7, as well as pcD2-16E7 and pcD2-16E6/E7, induced transformation of cells in G418-resistant colonies at efficiencies of 11.9%, 43.0% and 53.0%, respectively. Transformed cell lines induced by activated H-ras gene and pcD2-8E7 or pcD2-16E7 were named 8RE and 16RE cell lines, respectively. Tumor induction in syngeneic newborn rats by injected the 8RE cells was higher than that of the 16RE cells. In cytological and histological examination, the 8RE cell lines and their induced tumors were different from the 16RE cell lines and their induced tumors. The 8RE cell lines showed the characteristic transformation with efficient growth ability on plastic and colony formation in 0.3% soft agar. These results support the hypothesis that the HPV8E7 gene plays an important role in the carcinogenesis of skin cancers.

High-content screening identifies Src family kinases as potential regulators of AR-V7 expression and androgen-independent cell growth

PubMed Central

Szafran, Adam T.; Stephan, Cliff; Bolt, Michael; Mancini, Maureen G.; Marcelli, Marco; Mancini, Michael A.

2018-01-01

Background AR-V7 is an androgen receptor (AR) splice variant that lacks the ligand-binding domain and is isolated from prostate cancer cell lines. Increased expression of AR-V7 is associated with the transition from hormone-sensitive prostate cancer to more advanced castration-resistant prostate cancer (CRPC). Due to the loss of the ligand-binding domain, AR-V7 is not responsive to traditional AR-targeted therapies, and the mechanisms that regulate AR-V7 are still incompletely understood. Therefore, we aimed to explore existing classes of small molecules that may regulate AR-V7 expression and intracellular localization and their potential therapeutic role in CRPC. Methods We used AR high-content analysis (AR-HCA) to characterize the effects of a focused library of well-characterized clinical compounds on AR-V7 expression at the single-cell level in PC3 prostate cancer cells stably expressing green fluorescent protein (GFP)-AR-V7 (GFP-AR-V7:PC3). In parallel, an orthogonal AR-HCA screen of a small interfering (si)RNA library targeting 635 protein kinases was performed in GFP-AR-V7:PC3. The effect of the Src-Abl inhibitor PD 180970 was further characterized using cell-proliferation assays, quantitative PCR, and western blot analysis in multiple hormone-sensitive and CRPC cell lines. Results Compounds that tended to target Akt, Abl, and Src family kinases (SFKs) decreased overall AR-V7 expression, nuclear translocation, absolute nuclear level, and/or altered nuclear distribution. We identified 20 protein kinases that, when knocked down, either decreased nuclear GFP-AR-V7 levels or altered AR-V7 nuclear distribution, a set that included the SFKs Src and Fyn. The Src-Abl dual kinase inhibitor PD180970 decreased expression of AR-V7 by greater than 46% and decreased ligand-independent transcription of AR target genes in the 22RV1 human prostate carcinoma cell line. Further, PD180970 inhibited androgen-independent cell proliferation in endogenous–AR-V7–expressing prostate cancer cell lines and also overcame bicalutamide resistance observed in the 22RV1 cell line. Conclusions SFKs, especially Src and Fyn, may be important upstream regulators of AR-V7 expression and represent promising targets in a subset of CRPCs expressing high levels of AR-V7. PMID:27699828

Urtica dioica inhibits cell growth and induces apoptosis by targeting Ornithine decarboxylase and Adenosine deaminase as key regulatory enzymes in adenosine and polyamines homeostasis in human breast cancer cell lines.

PubMed

Fattahi, Sadegh; Ghadami, Elham; Asouri, Mohsen; Motevalizadeh Ardekanid, Ali; Akhavan-Niaki, Haleh

2018-02-28

Breast cancer is a heterogeneous and multifactorial disease with variable disease progression risk, and treatment response. Urtica dioica is a traditional herb used as an adjuvant therapeutic agent in cancer. In the present study, we have evaluated the effects of the aqueous extract of Urtica dioica on Adenosine deaminase (ADA) and Ornithine decarboxylase (ODC1) gene expression in MCF-7, MDA-MB-231, two breast cancer cell lines being estrogen receptor positive and estrogen receptor negative, respectively.  Cell lines were cultured in suitable media. After 24 h, different concentrations of the extract were added and after 72 h, ADA and ODC1 gene expression as well as BCL2 and BAX apoptotic genes were assessed by Taqman real time PCR assay. Cells viability was assessed by MTT assay, and apoptosis was also evaluated at cellular level. The intra and extracellular levels of ODC1 and ADA enzymes were evaluated by ELISA. Results showed differential expression of ADA and ODC1 genes in cancer cell lines. In MCF-7 cell line, the expression level of ADA was upregulated in a dose-dependent manner but its expression did not change in MDA-MB cell line. ODC1 expression was increased in both examined cell lines. Also, increased level of the apoptotic BAX/BCL-2 ratio was detected in MCF-7 cells. These results demonstrated that Urtica dioica induces apoptosis in breast cancer cells by influencing ODC1 and ADA genes expression, and estrogen receptors. The different responses observed with these cell lines could be due to the interaction of Urtica dioica as a phytoestrogen with the estrogen receptor.

Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer.

PubMed

Liu, Jun; Luo, Yan; Zheng, Liming; Liu, Qingqing; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

2013-10-01

This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P < 0.05). After transferring cloned embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.

Design, synthesis and biological evaluation of 1H-1,2,3-Triazole-Linked-1H‑Dibenzo[b,h]xanthenes as Inductors of ROS-Mediated Apoptosis in the Breast Cancer Cell Line MCF-7.

PubMed

Bortolot, Carolina S; da S M Forezi, Luana; Marra, Roberta K F; Reis, Marcelo I P; Sa, Barbara V F E; Filho, Ricardo Imbroisi; Ghasemishahrestani, Zeinab; Sola-Penna, Mauro; Zancan, Patricia; Ferreira, Vitor F; de C da Silva, Fernando

2018-05-23

Low molecular weight 1,2,3-triazoles and naphthoquinones are endowed with various types of biological activity, such as against cancer, HIV and bacteria. However, in some cases, the conjugation of these two nuclei considerably increases their biological activities Objective: In this work, we decided to study the synthesis and screening of bis-naphthoquinones and xanthenes tethered to 1,2,3-triazoles against cancer cell lines, specifically the human breast cancer cell line MCF-7. Starting from lawsone and aryl-1H-1,2,3-triazole-4-carbaldehydes (10a-h) several new 7-(1-aryl-1H-1,2,3-triazol-4-yl)-6H-dibenzo[b,h]xanthene-5,6,8,13(7H)-tetraones (12a-h) and 3,3'-((1-aryl-1H-1,2,3-triazol-4-yl)methylene)bis(2-hydroxynaphthalene-1,4-diones) 11a-h were synthesized and evaluated for their cytotoxic activities using the human breast cancer cell line MCF-7 and the non-tumor cell line MCF10A as control. We performed test of cell viability, cell proliferation, intracellular ATP content and cell cytometry to determine reactive oxygen species (ROS) formation. Based on these results, we found that compound 12a promote ROS production, interfering with energy metabolism, cell viability and proliferation, and thus promoting an whole cell damage. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

[Expression of Chemokine receptor CXCR6 and its significance in breast cancer cell lines].

PubMed

Cheng, Hao; Chen, Nian-yong

2014-05-01

To detect the expression of Chemokine receptor CXCR6 in invasive breast cancer cell lines and normal mammary epithelial cell line, and assess the relationship between CXCR6 expression and malignant behavior of breast cancer cells. Expression level of CXCR6 in different invasive breast cancer cell lines (SK-BR-3, MCF-7, MDA-MB-231) and normal mammary epithelial cell line (MCF-10A)was detected by real time reverse transcription-polymerase chain reaction (real time-PCR) and Western blot. Lentivirus was employed to interfere CXCR6 expression in MDA-MB-231. MTT assay and transwell chamber were used to study proliferative and invasive ability of those cells respectively. Vascular enothelial growth factor (VEGF) expression was detected to study the role of CXCR6 in angiogenesis. At both mRNA level and protein level, normal mammary epithelial cell line MCF-10A showed the weakest CXCR6 expression. The breast cancer cell lines expressed CXCR6 in different levels, the expression level of CXCR6 in highly invasive cell line MDA-MB-231 was significantly higher than that in two low-invasive cell lines SK-BR-3 and MCF-7 (P < 0.05). Silencing CXCR6 gene by Lentivirus-mediated RNA interference in MDA-MB-231 inhibited its proliferation ability, invasion ability and angiogenesis ability in vitro (P < 0.05). Different invasive breast cancer cell lines express CXCR6 at different levels, positively correlated with its invasive ability.

Vesicle-associated membrane protein 7 (VAMP-7) is essential for target cell killing in a natural killer cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcet-Palacios, Marcelo; Odemuyiwa, Solomon O.; Coughlin, Jason J.

2008-02-15

Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Ourmore » data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24 h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1 ng/mL of granzyme B, compared to 1.5-2.5 {mu}g/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo.« less

7 Methyl indole ethyl isothiocyanate causes ROS mediated apoptosis and cell cycle arrest in endometrial cancer cells.

PubMed

Kristjansdottir, Katrin; Kim, Kyukwang; Choi, Joong Sub; Horan, Timothy C; Brard, Laurent; Moore, Richard G; Singh, Rakesh K

2012-08-01

Chemotherapy options for advanced endometrial cancer are limited and newer therapeutic agents are urgently needed. This study describes the therapeutic potential of 7 Methyl-indole ethyl isothiocyanate (7Me-IEITC) in endometrial cancer cell lines. 7Me-IEITC was synthesized in our laboratory. The cell viability of 7Me-IEITC treated ECC-1 and KLE endometrial cancer cell was determined by MTS assay. Morphology and apoptosis were further confirmed by DAPI-staining and TUNEL assay. The measurement of reactive oxygen species (ROS), mitochondrial transmembrane depolarization potential (ΔΨm) and cell cycle phase was determined by FACS analysis. Expression of proteins involved in apoptosis, survival and cell-cycle progression was analyzed by Western blotting. 7Me-IEITC reduced the viability of the ECC-1 and KLE cancer cell-lines (IC(50)~2.5-10 μM) in a dose dependent fashion. 7Me-IEITC treatment caused mitochondrial transmembrane potential reduction, elevated the production of ROS, leading to activation of apoptosis in endometrial cancer KLE and ECC-1 cells. 7Me-IEITC treatment activated Bad, suppressed Bcl2 phosphorylation followed by PARP-1 deactivation and caspase 3 and 7 activation. 7Me-IEITC treatment arrested the progression of KLE cells in S-phase and caused CDC25 and cyclin-D1 downregulation. Pre-treatment with ascorbic acid abrogated 7Me-IEITC induced apoptosis in ECC-1 and KLE cells, suggesting that 7Me-IEITC mediated cytotoxicity is primarily through ROS production. 7Me-IEITC demonstrated promising cytotoxic effects in endometrial cancer cell line model. Copyright © 2012 Elsevier Inc. All rights reserved.

Re-analysis of the cell line NALM-1 karyotype by GTG-banding, spectral karyotyping, and whole chromosome painting.

PubMed

Pelz, Antje-Friederike; Weilepp, Gisela; Wieacker, Peter F

2005-01-01

Chronic myelogenous leukemia (CML) is a clonal bone marrow disease with progression from a chronic phase to an aggressive blast crisis. The cell line NALM-1 was originally established by Minowada and coworkers from the peripheral blood of a patient in CML blastic crisis. A karyotype analysis of the NALM-1 cell line was performed in the 1970s. To the best of our knowledge, this karyotype was not re-analyzed by molecular cytogenetic techniques, although this cell line is the source of many molecular investigations including expression studies. To establish this cell line as a CML control in our own laboratory, NALM-1 was analyzed by GTG banding, fluorescence in situ hybridization, and spectral karyotyping. Our results differ from the original publication of Sonta and coworkers. We describe for the first time the karyotype of the NALM-1 cell line: 44,X,-X,der(7)t(7;9;15)(q10;?;q15),der(9)t(9;9)(p24;q33 approximately q34)t(9;22)(q34;q11),der(15)t(7;9;15) (?;?;q15),der(22)t(9;22)(q34;q11).

Establishment of paclitaxel-resistant breast cancer cell line and nude mice models, and underlying multidrug resistance mechanisms in vitro and in vivo.

PubMed

Chen, Si-Ying; Hu, Sa-Sa; Dong, Qian; Cai, Jiang-Xia; Zhang, Wei-Peng; Sun, Jin-Yao; Wang, Tao-Tao; Xie, Jiao; He, Hai-Rong; Xing, Jian-Feng; Lu, Jun; Dong, Ya-Lin

2013-01-01

Breast cancer is a common malignant tumor which affects health of women and multidrug resistance (MDR) is one of the main factors leading to failure of chemotherapy. This study was conducted to establish paclitaxel-resistant breast cancer cell line and nude mice models to explore underlying mechanisms of MDR. The breast cancer drug-sensitive cell line MCF-7 (MCF-7/S) was exposed in stepwise escalating paclitaxel (TAX) to induce a resistant cell line MCF-7/TAX. Cell sensitivity to drugs and growth curves were measured by MTT assay. Changes of cell morphology and ultrastructure were examined by optical and electron microscopy. The cell cycle distribution was determined by flow cytometry. Furthermore, expression of proteins related to breast cancer occurrence and MDR was tested by immunocytochemistry. In Vivo, nude mice were injected with MCF-7/S and MCF-7/TAX cells and weights and tumor sizes were observed after paclitaxel treatment. In addition, proteins involved breast cancer and MDR were detected by immunohistochemistry. Compared to MCF-7/S, MCF-7/TAX cells had a higher resistance to paclitaxel, cross-resistance and prolonged doubling time. Moreover, MCF-7/TAX showed obvious alterations of ultrastructure. Estrogen receptor (ER) expression was low in drug resistant cells and tumors while expression of human epidermal growth factor receptor 2 (HER2) and Ki-67 was up-regulated. P-glycoprotein (P-gp), lung resistance-related protein (LRP) and glutathione-S-transferase-π (GST-π) involved in the MDR phenotype of resistant cells and tumors were all overexpressed. The underlying MDR mechanism of breast cancer may involve increased expression of P-gp, LRP and GST-π.

Active RNA replication of hepatitis C virus downregulates CD81 expression.

PubMed

Ke, Po-Yuan; Chen, Steve S-L

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

Active RNA Replication of Hepatitis C Virus Downregulates CD81 Expression

PubMed Central

Ke, Po-Yuan; Chen, Steve S.-L.

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81. PMID:23349980

Activation of the N-Ras-PI3K-Akt-mTOR Pathway by Hepatitis C Virus: Control of Cell Survival and Viral Replication

PubMed Central

Mannová, Petra; Beretta, Laura

2005-01-01

The hepatitis C virus (HCV) replication complex is localized within detergent-resistant membranes or lipid rafts. We analyzed the protein contents of detergent-resistant fractions isolated from Huh7 cells expressing a self-replicating full-length HCV-1b genome. Using two-dimensional gel electrophoresis followed by mass spectrometry, we identified N-Ras as one of the proteins in which expression was increased in the detergent-resistant fractions from HCV genomic replicon clones compared to control cells. N-Ras is an activator of the phosphatidylinositol-3-kinase (PI3K)-Akt pathway. We found that the activities of PI3K and Akt, as well as the activity of their downstream target, mTOR, in the HCV-replicating cells were increased. Both PI3K-Akt- and mTOR-dependent pathways have been shown to promote cell survival. In agreement with this, HCV replicon cells were resistant to serum starvation-induced apoptosis. We also characterized the role of this pathway in HCV replication. Reduction of N-Ras expression by transfection of N-Ras small interfering RNA (siRNA) resulted in increased replication of HCV. We observed a similar increase in HCV replication in cells treated with the PI3K inhibitor LY294002 and in cells transfected with mTOR siRNA. Taken together, these data suggest that increased N-Ras levels in subcellular sites of HCV replication and stimulation of the prosurvival PI3K-Akt pathway and mTOR by HCV not only protect cells against apoptosis but also contribute to the maintenance of steady-state levels of HCV replication. These effects may contribute to the establishment of persistent infection by HCV. PMID:15994768

Novel role of TRPV2 in promoting the cytotoxicity of H2O2-mediated oxidative stress in human hepatoma cells.

PubMed

Ma, Wenbo; Li, Caiyue; Yin, Shikui; Liu, Jingxin; Gao, Chao; Lin, Zuoxian; Huang, Rongqi; Huang, Jufang; Li, Zhiyuan

2015-12-01

Oxidative stress is important for the initiation and progression of cancers, which confers the cells with a survival advantage by inducing oxidative adaption and drug resistance. Therefore, developing strategies to promote oxidative stress-induced cytotoxicity could be important for cancer therapy. Herein, we found that H2O2-mediated oxidative stress increases TRPV2 expression in human hepatoma (HepG2 and Huh-7) cells. This occurred at the mRNA and protein levels in a dose-dependent manner. The significance of TRPV2 in promoting H2O2-induced cell death was demonstrated in gain and loss of function studies with overexpression and knockdown of TRPV2, respectively. Mechanistically, H2O2-induced cell death involves inhibition of pro-survival signaling proteins (Akt, Nrf2) and activation of pro-death signaling proteins (p38, JNK1). Overexpression of TRPV2 in H2O2-treated hepatoma cells aggravates the inhibition of Akt and Nrf2, while it enhances the activation of p38 and JNK1 at the early stage of cell death. Interestingly, increased expression of TRPV2 in HepG2 cells improved the efficacy of stress-associated chemicals to induce cell death. Our findings suggest that TRPV2 acts as an important enhancer for H2O2-induced cytotoxicity. This process occurred by the inhibition of Akt and Nrf2 as well as the early activation of p38 and JNK1. These findings have important implications for inhibition of oxidative adaption and drug resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

Alpha-mangostin inhibits both dengue virus production and cytokine/chemokine expression.

PubMed

Tarasuk, Mayuri; Songprakhon, Pucharee; Chimma, Pattamawan; Sratongno, Panudda; Na-Bangchang, Kesara; Yenchitsomanus, Pa-Thai

2017-08-15

Since severe dengue virus (DENV) infection in humans associates with both high viral load and massive cytokine production - referred to as "cytokine storm", an ideal drug for treatment of DENV infection should efficiently inhibit both virus production and cytokine expression. In searching for such an ideal drug, we discovered that α-mangostin (α-MG), a major bioactive compound purified from the pericarp of the mangosteen fruit (Garcinia mangostana Linn), which has been used in traditional medicine for several conditions including trauma, diarrhea, wound infection, pain, fever, and convulsion, inhibits both DENV production in cultured hepatocellular carcinoma HepG2 and Huh-7 cells, and cytokine/chemokine expression in HepG2 cells. α-MG could also efficiently inhibit all four serotypes of DENV. Treatment of DENV-infected cells with α-MG (20μM) significantly reduced the infection rates of four DENV serotypes by 47-55%. α-MG completely inhibited production of DENV-1 and DENV-3, and markedly reduced production of DENV-2 and DENV-4 by 100 folds. Furthermore, it could markedly reduce cytokine (IL-6 and TNF-α) and chemokine (RANTES, MIP-1β, and IP-10) transcription. These actions of α-MG are more potent than those of antiviral agent (ribavirin) and anti-inflammatory drug (dexamethasone). Thus, α-MG is potential to be further developed as therapeutic agent for DENV infection. Copyright © 2017 Elsevier B.V. All rights reserved.

SUMO1 depletion prevents lipid droplet accumulation and HCV replication.

PubMed

Akil, Abdellah; Wedeh, Ghaith; Zahid Mustafa, Mohammad; Gassama-Diagne, Ama

2016-01-01

Infection by hepatitis C virus (HCV) is a major public-health problem. Chronic infection often leads to cirrhosis, steatosis, and hepatocellular carcinoma. The life cycle of HCV depends on the host cell machinery and involves intimate interaction between viral and host proteins. However, the role of host proteins in the life cycle of HCV remains poorly understood. Here, we identify the small ubiquitin-related modifier (SUMO1) as a key host factor required for HCV replication. We performed a series of cell biology and biochemistry experiments using the HCV JFH-1 (Japanese fulminate hepatitis 1) genotype 2a strain, which produces infectious particles and recapitulates all the steps of the HCV life cycle. We observed that SUMO1 is upregulated in Huh7.5 infected cells. Reciprocally, SUMO1 was found to regulate the expression of viral core protein. Moreover, knockdown of SUMO1 using specific siRNA influenced the accumulation of lipid droplets and reduced HCV replication as measured by qRT-PCR. Thus, we identify SUMO1 as a key host factor required for HCV replication. To our knowledge, this is the first report showing that SUMO1 regulates lipid droplets in the context of viral infection. Our report provides a meaningful insight into how HCV replicates and interacts with host proteins and is of significant importance for the field of HCV and RNA viruses.

Targeting ceramide metabolic pathway induces apoptosis in human breast cancer cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vethakanraj, Helen Shiphrah; Babu, Thabraz Ahmed; Sudarsanan, Ganesh Babu

2015-08-28

The sphingolipid ceramide is a pro apoptotic molecule of ceramide metabolic pathway and is hydrolyzed to proliferative metabolite, sphingosine 1 phosphate by the action of acid ceramidase. Being upregulated in the tumors of breast, acid ceramidase acts as a potential target for breast cancer therapy. We aimed at targeting this enzyme with a small molecule acid ceramidase inhibitor, Ceranib 2 in human breast cancer cell lines MCF 7 and MDA MB 231. Ceranib 2 effectively inhibited the growth of both the cell lines in dose and time dependant manner. Morphological apoptotic hallmarks such as chromatin condensation, fragmented chromatin were observedmore » in AO/EtBr staining. Moreover, ladder pattern of fragmented DNA observed in DNA gel electrophoresis proved the apoptotic activity of Ceranib 2 in breast cancer cell lines. The apoptotic events were associated with significant increase in the expression of pro-apoptotic genes (Bad, Bax and Bid) and down regulation of anti-apoptotic gene (Bcl 2). Interestingly, increase in sub G1 population of cell cycle phase analysis and elevated Annexin V positive cells after Ceranib 2 treatment substantiated its apoptotic activity in MCF 7 and MDA MB 231 cell lines. Thus, we report Ceranib 2 as a potent therapeutic agent against both ER{sup +} and ER{sup −} breast cancer cell lines. - Highlights: • Acid Ceramidase inhibitor, Ceranib 2 induced apoptosis in Breast cancer cell lines (MCF 7 and MDA MB 231 cell lines). • Apoptosis is mediated by DNA fragmentation and cell cycle arrest. • Ceranib 2 upregulated the expression of pro-apoptotic genes and down regulated anti-apoptotic gene expression. • More potent compared to the standard drug Tamoxifen.« less

Mass spectrometry (LC-MS/MS) identified proteomic biosignatures of breast cancer in proximal fluid.

PubMed

Whelan, Stephen A; He, Jianbo; Lu, Ming; Souda, Puneet; Saxton, Romaine E; Faull, Kym F; Whitelegge, Julian P; Chang, Helena R

2012-10-05

We have begun an early phase of biomarker discovery in three clinically important types of breast cancer using a panel of human cell lines: HER2 positive, hormone receptor positive and HER2 negative, and triple negative (HER2-, ER-, PR-). We identified and characterized the most abundant secreted, sloughed, or leaked proteins released into serum free media from these breast cancer cell lines using a combination of protein fractionation methods before LC-MS/MS mass spectrometry analysis. A total of 249 proteins were detected in the proximal fluid of 7 breast cancer cell lines. The expression of a selected group of high abundance and/or breast cancer-specific potential biomarkers including thromobospondin 1, galectin-3 binding protein, cathepsin D, vimentin, zinc-α2-glycoprotein, CD44, and EGFR from the breast cancer cell lines and in their culture media were further validated by Western blot analysis. Interestingly, mass spectrometry identified a cathepsin D protein single-nucleotide polymorphism (SNP) by alanine to valine replacement from the MCF-7 breast cancer cell line. Comparison of each cell line media proteome displayed unique and consistent biosignatures regardless of the individual group classifications, demonstrating the potential for stratification of breast cancer. On the basis of the cell line media proteome, predictive Tree software was able to categorize each cell line as HER2 positive, HER2 negative, and hormone receptor positive and triple negative based on only two proteins, muscle fructose 1,6-bisphosphate aldolase and keratin 19. In addition, the predictive Tree software clearly identified MCF-7 cell line overexpresing the HER2 receptor with the SNP cathepsin D biomarker.

Antiproliferative activity and induction of apoptotic by ethanolic extract of Alpinia galanga rhizhome in human breast carcinoma cell line

PubMed Central

2014-01-01

Background We investigated the potential of galangal rhizomes to induce cytotoxic and apoptotic effects in the cultured human breast carcinoma cell line, (MCF-7) in compare with the non-malignant (MRC-5) cells. Methods Both cells were cultured in DMEM medium and treated with galangal rhizomes for three consecutive days. The percentage of apoptotic cells was determined by flow cytometry using Annexin-V fluorescein isothiocyanate. Results The results showed that the ethanolic extract of galangal rhizomes decreased cell viability in the malignant cells as a concentration- and time- dependent manner. The IC50 values against MCF-7 were determined at 400.0 ± 11.7 and 170.0 ± 5.9 μg/ml after 48 and 72 h respectively. The morphology of MCF-7 cells treated with the ethanolic extract confirmed the cell proliferation assay results. Alpinia galanga induced apoptosis in MCF-7 cells, as determined by flow cytometry. Conclusions We concluded that the extract of Alpinia galanga exerts pro-apoptotic effects in a breast cancer-derived cell line and could be considered as a potential chemotherapeutic agent in breast cancer. PMID:24935101

Electrophysiological characteristics of embryonic stem cell-derived cardiomyocytes are cell line-dependent.

PubMed

Hannes, Tobias; Wolff, Marie; Doss, Michael Xavier; Pfannkuche, Kurt; Haustein, Moritz; Müller-Ehmsen, Jochen; Sachinidis, Agapios; Hescheler, Jürgen; Khalil, Markus; Halbach, Marcel

2015-01-01

Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs) requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs) of different murine ESC lines. Two wild-type (D3 and R1) and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7) were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP) and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC) promoter. Action potentials (APs) were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Spontaneous AP frequency and AP duration (APD) as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully. © 2015 S. Karger AG, Basel.

Biomedical applications of SPION@APTES@PEG-folic acid@carboxylated quercetin nanodrug on various cancer cells

NASA Astrophysics Data System (ADS)

Akal, Z. Ü.; Alpsoy, L.; Baykal, A.

2016-08-01

In this study, carboxylated quercetin (CQ) was conjugated to superparamagnetic iron oxide nanoparticles (SPIONs) which were modified by (3-aminopropyl) triethoxysilane (APTES), Folic acid (FA) and carboxylated Polyethylene glycol (PEG); (SPION@APTES@FA-PEG@CQ), nanodrug has been synthesized via polyol and accompanying by various chemical synthesis routes. The characterization of the final product was done via X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Thermal gravimetric analysis (TGA), Transmission electron spectroscopy (TEM) and Vibrating sample magnetometer (VSM). Its cytotoxic and apoptotic activities on over expressed folic acid receptor (FR +) (MCF-7, HeLa) and none expressed folic acid receptor (FR-) (A549) cancer cell lines were determined by using MTT assay, Real-Time Cell Analysis, TUNEL assay, Annexin assay and RT-PCR analysis for Caspase3/7 respectively. SPION@APTES@FA-PEG@CQ nanodrug showed higher cytotoxicity against HeLa and MCF-7 cell lines as compared with A549 cell line. Moreover, SPION@APTES@FA-PEG@CQ nanodrug also caused higher apoptotic and necrotic effects in 100 μg/mL HeLa and MCF-7 cells than A549 cells. The findings showed that SPION@APTES@FA-PEG@CQ nanodrug has cytotoxic, apoptotic and necrotic effects on HeLa and MCF-7 which are FR over expressed cell lines and can be potentially used for the delivery of quercetin to cervical and breast cancer cells.

Inositol Pyrophosphate Profiling of Two HCT116 Cell Lines Uncovers Variation in InsP8 Levels

PubMed Central

Gu, Chunfang; Wilson, Miranda S. C.; Jessen, Henning J.; Saiardi, Adolfo; Shears, Stephen B.

2016-01-01

The HCT116 cell line, which has a pseudo-diploid karotype, is a popular model in the fields of cancer cell biology, intestinal immunity, and inflammation. In the current study, we describe two batches of diverged HCT116 cells, which we designate as HCT116NIH and HCT116UCL. Using both gel electrophoresis and HPLC, we show that HCT116UCL cells contain 6-fold higher levels of InsP8 than HCT116NIH cells. This observation is significant because InsP8 is one of a group of molecules collectively known as ‘inositol pyrophosphates’ (PP-InsPs)—highly ‘energetic’ and conserved regulators of cellular and organismal metabolism. Variability in the cellular levels of InsP8 within divergent HCT116 cell lines could have impacted the phenotypic data obtained in previous studies. This difference in InsP8 levels is more remarkable for being specific; levels of other inositol phosphates, and notably InsP6 and 5-InsP7, are very similar in both HCT116NIH and HCT116UCL lines. We also developed a new HPLC procedure to record 1-InsP7 levels directly (for the first time in any mammalian cell line); 1-InsP7 comprised <2% of total InsP7 in HCT116NIH and HCT116UCL lines. The elevated levels of InsP8 in the HCT116UCL lines were not due to an increase in expression of the PP-InsP kinases (IP6Ks and PPIP5Ks), nor to a decrease in the capacity to dephosphorylate InsP8. We discuss how the divergent PP-InsP profiles of the newly-designated HCT116NIH and HCT116UCL lines should be considered an important research opportunity: future studies using these two lines may uncover new features that regulate InsP8 turnover, and may also yield new directions for studying InsP8 function. PMID:27788189

Functional characterization of cell hybrids generated by induced fusion of primary porcine mesenchymal stem cells with an immortal murine cell line.

PubMed

Islam, M Q; Ringe, J; Reichmann, E; Migotti, R; Sittinger, M; da S Meirelles, L; Nardi, N B; Magnusson, P; Islam, K

2006-10-01

Bone marrow mesenchymal stem cells (MSC) integrate into various organs and contribute to the regeneration of diverse tissues. However, the mechanistic basis of the plasticity of MSC is not fully understood. The change of cell fate has been suggested to occur through cell fusion. We have generated hybrid cell lines by polyethylene-glycol-mediated cell fusion of primary porcine MSC with the immortal murine fibroblast cell line F7, a derivative of the GM05267 cell line. The hybrid cell lines display fibroblastic morphology and proliferate like immortal cells. They contain tetraploid to hexaploid porcine chromosomes accompanied by hypo-diploid murine chromosomes. Interestingly, many hybrid cell lines also express high levels of tissue-nonspecific alkaline phosphatase, which is considered to be a marker of undifferentiated embryonic stem cells. All tested hybrid cell lines retain osteogenic differentiation, a few of them also retain adipogenic potential, but none retain chondrogenic differentiation. Conditioned media from hybrid cells enhance the proliferation of both early-passage and late-passage porcine MSC, indicating that the hybrid cells secrete diffusible growth stimulatory factors. Murine F7 cells thus have the unique property of generating immortal cell hybrids containing unusually high numbers of chromosomes derived from normal cells. These hybrid cells can be employed in various studies to improve our understanding of regenerative biology. This is the first report, to our knowledge, describing the generation of experimentally induced cell hybrids by using normal primary MSC.

5-aminolevulinic acid-mediated photodynamic therapy on Hep-2 and MCF-7c3 cells.

PubMed

Alvarez, María Gabriela; Lacelli, M S; Rivarola, Viviana; Batlle, Alcira; Fukuda, Haydée

2007-01-01

The cytotoxic effect of 5-aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX) on two human carcinoma cell lines, MCF-7c3 cells and Hep 2 cells, was studied. In both cell lines, PPIX content depends on the ALA concentration and incubation time. The maximal PPIX content was higher in the MCF-7c3 cells, reaching a value of 8 microg/10(6) cells, compared to the Hep-2 cells, which accumulated 3.2 microg/10(6) cells. Treatment of cells with the iron chelator desferrioxamine prior to ALA exposure enhances the amount of PPIX, consequently diminishing enzymatic activity of ferroquelatase. Photo sensitization of the cells was in correlation with the PPIX content; therefore, conditions leading to 80% cell death in the MCF-7c3 cells provoke a 50% cell death in the Hep 2 cells. Using fluorescence microscopy, cell morphology was analyzed after incubation with 1 mM ALA during 5 hr and irradiation with 54 Jcm(-2); 24 hr post-PDT, MCF-7c3 cells revealed the typical morphological changes of necrosis. Under the same conditions, Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. PPIX accumulation was observed to occur in a perinuclear region in the MCF-7c3 cells; while in Hep-2 cells, it was localized in lysosomes. Different mechanisms of cell death were observed in both cell lines, depending on the different intracellular localization of PPIX.

[Cytotoxicity of the secondary metabolites of Marine Mangrove Fungus Paecilomyces sp. tree 1-7 on human hepatoma cell line HepG2].

PubMed

Cai, Xiao-Ling; Gao, Jun-Ping; Li, Qing; Wen, Lu; She, Zhi-Gang; Lin, Yong-Cheng

2008-06-01

To study the cytotoxicity of the secondary metabolites of Marine Mangrove Fungus Paecilomyces sp. Tree 1-7 on human hepatoma cell line HepG2 cultured in vitro. Three groups were divided: compounds group, 5-Fu group and control group. The cytotoxicity was measured by MTT method when HepG2 cells were treated by different concentration of the secondary metabolites of Paecilomyces sp. Tree 1-7. Secalonic acid A, tenellic acid A and alternin inhibited the growth of human hepatoma cell line HepG2, the IC50 separately were 2.0, 62.1 and 7.0 microg/ml. Secalonic acid A and alternin have strong cytotoxicity on HepG2 cultured in vitro.

Expression of estrogenicity genes in a lineage cell culture model of human breast cancer progression

PubMed Central

Fu, Jiaqi; Weise, Amy M.; Falany, Josie L.; Falany, Charles N.; Thibodeau, Bryan J.; Miller, Fred R.; Kocarek, Thomas A.

2013-01-01

TaqMan Gene Expression assays were used to profile the mRNA expression of estrogen receptor (ERα and ERβ) and estrogen metabolism enzymes including cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1, and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17β-hydroxysteroid dehydrogenases (17βHSD1 and 2), CYP1B1, and catechol-O-methyltransferase (COMT) in an MCF10A-derived lineage cell culture model for basal-like human breast cancer progression and in ERα-positive luminal MCF7 breast cancer cells. Low levels of ERα and ERβ mRNA were present in MCF10A-derived cell lines. SULT1E1 mRNA was more abundant in confluent relative to subconfluent MCF10A cells, a non-tumorigenic proliferative breast disease cell line. SULT1E1 was also expressed in preneoplastic MCF10AT1 and MCF10AT1K.cl2 cells, but was markedly repressed in neoplastic MCF10A-derived cell lines as well as in MCF7 cells. Steroid-metabolizing enzymes SULT1A1 and SULT2B1 were only expressed in MCF7 cells. STS and COMT were widely detected across cell lines. Pro-estrogenic 17βHSD1 mRNA was most abundant in neoplastic MCF10CA1a and MCF10DCIS.com cells, while 17βHSD2 mRNA was more prominent in parental MCF10A cells. CYP1B1 mRNA was most abundant in MCF7 cells. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) induced SULT1E1 and CYP19 mRNA but suppressed CYP1B1, STS, COMT, 17βHSD1, and 17βHSD2 mRNA in MCF10A lineage cell lines. In MCF7 cells, TSA treatment suppressed ERα, CYP1B1, STS, COMT, SULT1A1, and SULT2B1 but induced ERβ, CYP19 and SULT2A1 mRNA expression. The results indicate that relative to the MCF7 breast cancer cell line, key determinants of breast estrogen metabolism are differentially regulated in the MCF10A-derived lineage model for breast cancer progression. PMID:19308726

Activity of Saponins from Medicago species Against HeLa and MCF-7 Cell Lines and their Capacity to Potentiate Cisplatin Effect.

PubMed

Avato, Pinarosa; Migoni, Danilo; Argentieri, Mariapia; Fanizzi, Francesco P; Tava, Aldo

2017-11-24

Saponins from Medicago species display several biological activities, among them apoptotic effects against plant cells have been evidenced. In contrast, their cytotoxic and antitumor activity against animal cells have not been studied in great details. To explore the cytotoxic properties of saponin from Medicago species against animal cells and their effect in combination with the antitumoral drug cisplatin. Cytotoxic activity of saponin mixtures from M. arabica (tops and roots), M. arborea (tops) and M. sativa (tops, roots and seeds) and related prosapogenins from M. arborea and M. sativa (tops) against HeLa and MCF-7 cell lines is described. In addition, cytotoxicity of soyasaponin I and purified saponins (1-8) of hederagenin, medicagenic and zanhic acid is also presented. Combination experiments with cisplatin have been also conducted. Saponins from M. arabica tops and roots (mainly monodesmosides of hederagenin and bayogenin) were the most effective to reduce proliferation of HeLa and MCF-7 cell lines. Among the purified saponins, the most cytotoxic was saponin 1, 3-O-ß-D-glucopyranosyl(1→2)-α-L-arabinopyranosyl hederagenin. When saponins, derived prosapogenins and pure saponins were used in combination with cisplatin, they all, to different extent, were able to potentiate cisplatin activity against HeLa cells but not against MCF-7 cell lines. Moreover uptake of cisplatin in these cell lines was significantly reduced. Overall results showed that specific molecular types of saponins (hederagenin glycosides) have potential as anti-cancer agents or as leads for anti-cancer agents. Moreover saponins from Medicago species have evidenced interesting properties to mediate cisplatin effects in tumor cell lines. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Synthesis, stereochemistry determination, pharmacological studies and quantum chemical analyses of bisthiazolidinone derivative

NASA Astrophysics Data System (ADS)

Mushtaque, Md.; Avecilla, Fernando; Hafeez, Zubair Bin; Jahan, Meriyam; Khan, Md. Shahzad; Rizvi, M. Moshahid A.; Khan, Mohd. Shahid; Srivastava, Anurag; Mallik, Anwesha; Verma, Saurabh

2017-01-01

A new compound (3) bisthaizolidinone derivative was synthesized by Knoevenagel condensation reaction. The structure of synthesized compound was elucidated by different spectral techniques and X-ray diffraction studies. The stereochemistry of the compound (3) was determined by 1Hsbnd 1H NOESY, 1Hsbnd 1H NMR COSY and single crystal X-ray diffraction studies as (Z, Z)-configuration. The computational quantum chemical studies of compound(3) like, IR, UV, NBO analysis were performed by DFT with Becke-3-Lee-Yang-Parr (B3LYP) exchange-correlation functional in combination with 6-311++G(d,p) basis sets. The DNA-binding of compound (3) exhibited a moderate binding constant (Kb = 1 × 105 Lmol-1) with hypochromic shift. The molecular docking displayed good binding affinity -7.18 kcal/mol. The MTT assay of compound (3) was screened against different cancerous cell lines, HepG2, Siha, Hela and MCF-7. Studies against these cell lines depicted that the screened compound (3) showed potent inhibitory activity against HepG2 cell (IC50 = 7.5 μM) followed by MCF-7 (IC50 = 52.0 μM), Siha (IC50 = 66.98 μM), Hela (IC50 = 74.83 μM) cell lines, and non-toxic effect against non-cancerous HEK-293 cells (IC50 = 287.89 μM) at the concentration range (0-300) μM. Furthermore, cell cycle perturbation was performed on HepG2 & Siha cell lines and observed that cells were arrested in G2/M in HepG2, and G0/G1 in Siha cell lines with respect to untreated control. Hence, compound (3) possesses potent anti-cancerous activity against HepG2 cell line.

Chrysin, Abundant in Morinda citrifolia Fruit Water-EtOAc Extracts, Combined with Apigenin Synergistically Induced Apoptosis and Inhibited Migration in Human Breast and Liver Cancer Cells.

PubMed

Huang, Cheng; Wei, Yu-Xuan; Shen, Ma-Ching; Tu, Yu-Hsuan; Wang, Chia-Chi; Huang, Hsiu-Chen

2016-06-01

The composition of Morinda citrifolia (M. citrifolia) was determined using high-performance liquid chromatography (HPLC), and the anticancer effects of M. citrifolia extract evaluated in HepG2, Huh7, and MDA-MB-231 cancer cells. M. citrifolia fruit extracts were obtained by using five different organic solvents, including hexane (Hex), methanol (MeOH), ethyl acetate (EtOAc), chloroform (CHCl3), and ethanol (EtOH). The water-EtOAc extracts from M. citrifolia fruits was found to have the highest anticancer activity. HPLC data revealed the predominance of chrysin in water-EtOAc extracts of M. citrifolia fruit. Furthermore, the combined effects of cotreatment with apigenin and chrysin on liver and breast cancer were investigated. Treatment with apigenin plus chrysin for 72-96 h reduced HepG2 and MDA-MB-231 cell viability and induced apoptosis through down-regulation of S-phase kinase-associated protein-2 (Skp2) and low-density lipoprotein receptor-related protein 6 (LRP6) expression. However, the combination treatment for 36 h synergistically decreased MDA-MB-231 cell motility but not cell viability through down-regulation of MMP2, MMP9, fibronectin, and snail in MDA-MB-231 cells. Additionally, chrysin combined with apigenin also suppressed tumor growth in human MDA-MB-231 breast cancer cells xenograft through down-regulation of ki-67 and Skp2 protein. The experimental results showed that chrysin combined with apigenin can reduce HepG2 and MDA-MB-231 proliferation and cell motility and induce apoptosis. It also offers opportunities for exploring new drug targets, and further investigations are underway in this regard.

Water extract from Pleurotus pulmonarius with antioxidant activity exerts in vivo chemoprophylaxis and chemosensitization for liver cancer.

PubMed

Xu, Wen Wen; Li, Bin; Lai, Emily Tsz Ching; Chen, Lei; Huang, Jim Jun Hui; Cheung, Annie Lai Man; Cheung, Peter Chi Keung

2014-01-01

Chemoprophylaxis and chemosensitization are promising strategies to combat human cancers. Natural antioxidant agents show great promise in cancer therapy, and the use of edible mushrooms against cancer is receiving more interest globally. In this study, the radical scavenging activities including diphenyl-1-picrylhydrazyl, superoxide anion radical, hydroxyl radical, and hydrogen peroxide were compared among hot water extracts from 3 edible mushrooms, among which Pleurotus pulmonarius (Pp) possessed the highest antioxidant potential. Oral administration of Pp 2 wk in advance could markedly inhibit the incidence and size of tumor (Huh7 liver cancer cells) with an inhibition rate of 93.1% in nude mice. No obvious side effect was observed in the Pp-treated mice as indicated by their body weight and histological analysis of major organs. The cancer prevention by Pp treatment might be explained by the inhibition of cancer cell proliferation indicated by reduction of ki-67 staining and the inactivation of phosphoinositide 3-kinase (PI3K)/AKT signaling pathway in the Pp-treated mice. Furthermore, a significant synergistic effect was observed when the mice were treated with a combination of low dose of cisplatin and Pp. Taken together, these results suggest the potential application of Pp as an adjuvant in the chemotherapy of liver cancer.

HCV Core Protein Uses Multiple Mechanisms to Induce Oxidative Stress in Human Hepatoma Huh7 Cells

PubMed Central

Ivanov, Alexander V.; Smirnova, Olga A.; Petrushanko, Irina Y.; Ivanova, Olga N.; Karpenko, Inna L.; Alekseeva, Ekaterina; Sominskaya, Irina; Makarov, Alexander A.; Bartosch, Birke; Kochetkov, Sergey N.; Isaguliants, Maria G.

2015-01-01

Hepatitis C virus (HCV) infection is accompanied by the induction of oxidative stress, mediated by several virus proteins, the most prominent being the nucleocapsid protein (HCV core). Here, using the truncated forms of HCV core, we have delineated several mechanisms by which it induces the oxidative stress. The N-terminal 36 amino acids of HCV core induced TGFβ1-dependent expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 1 and 4, both of which independently contributed to the production of reactive oxygen species (ROS). The same fragment also induced the expression of cyclo-oxygenase 2, which, however, made no input into ROS production. Amino acids 37–191 of HCV core up-regulated the transcription of a ROS generating enzyme cytochrome P450 2E1. Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1α. The latter triggered efflux of Ca2+ from ER to mitochondria via mitochondrial Ca2+ uniporter, leading to generation of superoxide anions, and possibly also H2O2. Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production. Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein. PMID:26035647

Oxygen-dependent secretion of a bioactive hepcidin-GFP chimera.

PubMed

Chachami, Georgia; Lyberopoulou, Aggeliki; Kalousi, Alkmini; Paraskeva, Efrosyni; Pantopoulos, Kostas; Simos, George

2013-06-14

Hepcidin, a hepatic hormone, regulates serum iron levels by controlling both intestinal iron absorption and iron release from macrophages. Although transcription of hepcidin is controlled by diverse stimuli, it remains elusive if post-transcriptional steps of its production are also regulated. To address this issue, GFP was fused to the C-terminus of hepcidin and the chimeric hepcidin-GFP protein was expressed in hepatoma Huh7 cells. Expression and secretion of hepcidin-GFP were analyzed by fluorescence microscopy or western blotting and its activity was assessed by in vitro biological assays. Transient over-expression of hepcidin-GFP resulted in production and secretion of premature forms. On the other hand, stable low-level expression led to synthesis and secretion of a properly matured hepcidin-GFP. This form was biologically active since it affected appropriately the levels of IRP2 and ferritin in human THP1 monocytes and targeted ferroportin in mouse J774 macrophages. Treatment of hepcidin-GFP expressing cells with hypoxia (0.1% O2) altered the subcellular distribution of pro-hepcidin-GFP and significantly reduced the secretion of mature hepcidin-GFP. Our hepcidin-GFP expression system allows the investigation of post-transcriptional processing of hepcidin and implicates hypoxia in its secretion control. Copyright © 2013 Elsevier Inc. All rights reserved.

Anticancer Effects of Extracts from the Fruit of Morinda Citrifolia (Noni) in Breast Cancer Cell Lines.

PubMed

Sharma, K; Pachauri, S D; Khandelwal, K; Ahmad, H; Arya, A; Biala, P; Agrawal, S; Pandey, R R; Srivastava, A; Srivastav, A; Saxena, J K; Dwivedi, A K

2016-03-01

Morinda citrifolia L. (NONI) fruits have been used for thousands of years for the treatment of many health problems including cancer, cold, diabetes, flu, hypertension, and pain. Plant extracts have reported several therapeutic benefits, but extraction of individual compound from the extract often exhibits limited clinical utility as the synergistic effect of various natural ingredients gets lost. They generally constitute polyphenols and flavonoids. Studies have suggested that these phytochemicals, especially polyphenols, display high antioxidant properties, which help to reduce the risk of degenerative diseases, such as cancer and cardiovascular diseases. Several in-vitro and in-vivo studies have shown that Noni fruits have antioxidant, anti-inflammatory, anti-dementia, liver-protective, anticancer, analgesic, and immunomodulatory effects. Till date about 7 in vitro cancer studies have been done, but a detailed in vitro study including cell cycle and caspase activation assay on breast cancer cell line has not been done. In the present study different Noni fruit fractions have tested on cancer cell lines MCF-7, MDA-MB-231 (breast adenocarcinoma) and one non-cancer cell line HEK-293 (Human embryonic kidney). Out of which ethylacetate extract showed a higher order of in vitro anticancer activity profile. The ethylacetate extract strongly inhibited the proliferation of MCF-7, MDA-MB-231 and HEK-293 cell lines with IC50 values of 25, 35, 60 µg/ml respectively. The extract showed increase in apoptotic cells in MCF-7 and MDA-MB-231 cells and arrested the cell cycle in the G1/S phase in MCF-7 and G0/G1 phase in MDA-MB-231 cells. Noni extract also decreases the intracellular ROS generation and mitochondrial membrane potential. © Georg Thieme Verlag KG Stuttgart · New York.

Antiproliferative activity of flavonoids on several cancer cell lines.

PubMed

Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M

1999-05-01

Twenty-seven Citrus flavonoids were examined for their antiproliferative activities against several tumor and normal human cell lines. As a result, 7 flavonoids were judged to be active against the tumor cell lines, while they had weak antiproliferative activity against the normal human cell lines. The rank order of potency was luteolin, natsudaidain, quercetin, tangeretin, eriodictyol, nobiletin, and 3,3',4',5,6,7,8-heptamethoxyflavone. The structure-activity relationship established from comparison among these flavones and flavanones showed that the ortho-catechol moiety in ring B and a C2-C3 double bond were important for the antiproliferative activity. As to polymethoxylated flavones, C-3 hydroxyl and C-8 methoxyl groups were essential for high activity.

Utilization of RNA polymerase I promoter and terminator sequences to develop a DNA transfection system for the study of hepatitis C virus internal ribosomal entry site-dependent translation.

PubMed

Oem, Jae-Ku; Xiang, Zhonghua; Zhou, Yan; Babiuk, Lorne A; Liu, Qiang

2007-09-01

Hepatitis C virus (HCV) causes severe liver diseases in a large population worldwide. HCV protein translation is controlled by an internal ribosomal entry site (IRES) within the 5'-untranslated region (UTR). HCV IRES-dependent translation is critical for HCV-associated pathogenesis. To develop a plasmid DNA transfection system by using RNA polymerase I promoter and terminator sequences for studying HCV IRES-dependent translation. A gene cassette containing HCV 5'-UTR, Renilla luciferase reporter gene, and HCV 3'-UTR was inserted between RNA polymerase I promoter and terminator sequences. HCV IRES-directed translation was determined by luciferase assay after transfection. Transfection of the RNA polymerase I-HCV IRES plasmid into human hepatoma Huh-7 and HepG2 cells resulted in luciferase gene expression. Deletion of the IIIf domain in HCV IRES dramatically reduced luciferase activity. Our results indicated that the plasmid vector system-based on RNA polymerase I promoter and terminator sequences represents an effective approach for the study of HCV IRES-dependent translation.

Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

PubMed

Zong, Li; Qin, Yanli; Jia, Haodi; Ye, Lei; Wang, Yongxiang; Zhang, Jiming; Wands, Jack R; Tong, Shuping; Li, Jisu

2017-05-01

Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Biodegradable human serum albumin nanoparticles as contrast agents for the detection of hepatocellular carcinoma by magnetic resonance imaging.

PubMed

Watcharin, Waralee; Schmithals, Christian; Pleli, Thomas; Köberle, Verena; Korkusuz, Hüdayi; Huebner, Frank; Zeuzem, Stefan; Korf, Hans W; Vogl, Thomas J; Rittmeyer, Claudia; Terfort, Andreas; Piiper, Albrecht; Gelperina, Svetlana; Kreuter, Jörg

2014-05-01

Tumor visualization by magnetic resonance imaging (MRI) and nanoparticle-based contrast agents may improve the imaging of solid tumors such as hepatocellular carcinoma (HCC). In particular, human serum albumin (HSA) nanoparticles appear to be a suitable carrier due to their safety and feasibility of functionalization. In the present study HSA nanoparticles were conjugated with gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) using carbodiimide chemistry. The nanoparticles had a uniform spherical shape and a diameter of 235±19nm. For better optical visualization in vitro and in vivo, the HSA-Gd nanoparticles were additionally labeled with rhodamine 123. As shown by confocal microscopy and flow cytometry analysis, the fluorescent nanoparticles were readily taken up by Huh-7 hepatocellular carcinoma cells. After 24h incubation in blood serum, less than 5% of the Gd(III) was released from the particles, which suggests that this nanoparticulate system may be stable in vivo and, therefore, may serve as potentially safe T1 MRI contrast agent for MRI of hepatocellular carcinoma. Copyright © 2013 Elsevier B.V. All rights reserved.

DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination.

PubMed

Korch, Christopher; Spillman, Monique A; Jackson, Twila A; Jacobsen, Britta M; Murphy, Susan K; Lessey, Bruce A; Jordan, V Craig; Bradford, Andrew P

2012-10-01

Cell lines derived from human ovarian and endometrial cancers, and their immortalized non-malignant counterparts, are critical tools to investigate and characterize molecular mechanisms underlying gynecologic tumorigenesis, and facilitate development of novel therapeutics. To determine the extent of misidentification, contamination and redundancy, with evident consequences for the validity of research based upon these models, we undertook a systematic analysis and cataloging of endometrial and ovarian cell lines. Profiling of cell lines by analysis of DNA microsatellite short tandem repeats (STR), p53 nucleotide polymorphisms and microsatellite instability was performed. Fifty-one ovarian cancer lines were profiled with ten found to be redundant and five (A2008, OV2008, C13, SK-OV-4 and SK-OV-6) identified as cervical cancer cells. Ten endometrial cell lines were analyzed, with RL-92, HEC-1A, HEC-1B, HEC-50, KLE, and AN3CA all exhibiting unique, uncontaminated STR profiles. Multiple variants of Ishikawa and ECC-1 endometrial cancer cell lines were genotyped and analyzed by sequencing of mutations in the p53 gene. The profile of ECC-1 cells did not match the EnCa-101 tumor, from which it was reportedly derived, and all ECC-1 isolates were genotyped as Ishikawa cells, MCF-7 breast cancer cells, or a combination thereof. Two normal, immortalized endometrial epithelial cell lines, HES cells and the hTERT-EEC line, were identified as HeLa cervical carcinoma and MCF-7 breast cancer cells, respectively. Results demonstrate significant misidentification, duplication, and loss of integrity of endometrial and ovarian cancer cell lines. Authentication by STR DNA profiling is a simple and economical method to verify and validate studies undertaken with these models. Copyright © 2012 Elsevier Inc. All rights reserved.

Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress

PubMed Central

Gonzalez, Yanira; Aryal, Baikuntha; Chehab, Leena; Rao, V. Ashutosh

2014-01-01

The interplay between oxidative stress and autophagy is critical for determining the fate of cancer cells exposed to redox-active and cytotoxic chemotherapeutic agents. Mitoquinone (MitoQ), a mitochondrially-targeted redox-active ubiquinone conjugate, selectively kills breast cancer cells over healthy mammary epithelial cells. We reported previously that MitoQ, although a derivative of the antioxidant ubiquinone, can generate excess ROS and trigger the Keap1-Nrf2 antioxidant response in the MDA-MB-231 cell line. Following MitoQ treatment, a greater number of cells underwent autophagy than apoptosis. However, the relationship between MitoQ-induced oxidative stress and autophagy as a primary cellular response was unclear. In this report, we demonstrate that MitoQ induces autophagy related gene 7 (Atg7)-dependent, yet Beclin-1-independent, autophagy marked by an increase in LC3-II. Both the ATG7-deficient human MDA-MB-231 cells and Atg7-knockout mouse embryonic fibroblasts exhibited lower levels of autophagy following MitoQ treatment than their respective wild-type counterparts. Increased apoptosis was confirmed in these autophagy-deficient isogenic cell line pairs, indicating that autophagy was attempted for survival in wild type cell lines. Furthermore, we observed higher levels of ROS in Atg7-deficient cells, as measured by hydroethidine oxidation. In Atg7-deficient cells, redox-sensitive Keap1 degradation was decreased, suggesting autophagy- and Atg7-dependent degradation of Keap1. Conversely, downregulation of Keap1 decreased autophagy levels, increased Nrf2 activation, upregulated cytoprotective antioxidant gene expression, and caused accumulation of p62, suggesting a feedback loop between ROS-regulated Keap1-Nrf2 and Atg7-regulated autophagy. Our data indicate that excessive ROS causes the upregulation of autophagy, and autophagy acts as an antioxidant feedback response triggered by cytotoxic levels of MitoQ. PMID:24681637

Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress.

PubMed

Gonzalez, Yanira; Aryal, Baikuntha; Chehab, Leena; Rao, V Ashutosh

2014-03-30

The interplay between oxidative stress and autophagy is critical for determining the fate of cancer cells exposed to redox-active and cytotoxic chemotherapeutic agents. Mitoquinone (MitoQ), a mitochondrially-targeted redox-active ubiquinone conjugate, selectively kills breast cancer cells over healthy mammary epithelial cells. We reported previously that MitoQ, although a derivative of the antioxidant ubiquinone, can generate excess ROS and trigger the Keap1-Nrf2 antioxidant response in the MDA-MB-231 cell line. Following MitoQ treatment, a greater number of cells underwent autophagy than apoptosis. However, the relationship between MitoQ-induced oxidative stress and autophagy as a primary cellular response was unclear. In this report, we demonstrate that MitoQ induces autophagy related gene 7 (Atg7)-dependent, yet Beclin-1-independent, autophagy marked by an increase in LC3-II. Both the ATG7-deficient human MDA-MB-231 cells and Atg7-knockout mouse embryonic fibroblasts exhibited lower levels of autophagy following MitoQ treatment than their respective wild-type counterparts. Increased apoptosis was confirmed in these autophagy-deficient isogenic cell line pairs, indicating that autophagy was attempted for survival in wild type cell lines. Furthermore, we observed higher levels of ROS in Atg7-deficient cells, as measured by hydroethidine oxidation. In Atg7-deficient cells, redox-sensitive Keap1 degradation was decreased, suggesting autophagy- and Atg7-dependent degradation of Keap1. Conversely, downregulation of Keap1 decreased autophagy levels, increased Nrf2 activation, upregulated cytoprotective antioxidant gene expression, and caused accumulation of p62, suggesting a feedback loop between ROS-regulated Keap1-Nrf2 and Atg7-regulated autophagy. Our data indicate that excessive ROS causes the upregulation of autophagy, and autophagy acts as an antioxidant feedback response triggered by cytotoxic levels of MitoQ.

High-Content Screening Identifies Src Family Kinases as Potential Regulators of AR-V7 Expression and Androgen-Independent Cell Growth.

PubMed

Szafran, Adam T; Stephan, Cliff; Bolt, Michael; Mancini, Maureen G; Marcelli, Marco; Mancini, Michael A

2017-01-01

AR-V7 is an androgen receptor (AR) splice variant that lacks the ligand-binding domain and is isolated from prostate cancer cell lines. Increased expression of AR-V7 is associated with the transition from hormone-sensitive prostate cancer to more advanced castration-resistant prostate cancer (CRPC). Due to the loss of the ligand-binding domain, AR-V7 is not responsive to traditional AR-targeted therapies, and the mechanisms that regulate AR-V7 are still incompletely understood. Therefore, we aimed to explore existing classes of small molecules that may regulate AR-V7 expression and intracellular localization and their potential therapeutic role in CRPC. We used AR high-content analysis (AR-HCA) to characterize the effects of a focused library of well-characterized clinical compounds on AR-V7 expression at the single-cell level in PC3 prostate cancer cells stably expressing green fluorescent protein (GFP)-AR-V7 (GFP-AR-V7:PC3). In parallel, an orthogonal AR-HCA screen of a small interfering (si)RNA library targeting 635 protein kinases was performed in GFP-AR-V7:PC3. The effect of the Src-Abl inhibitor PD 180970 was further characterized using cell-proliferation assays, quantitative PCR, and western blot analysis in multiple hormone-sensitive and CRPC cell lines. Compounds that tended to target Akt, Abl, and Src family kinases (SFKs) decreased overall AR-V7 expression, nuclear translocation, absolute nuclear level, and/or altered nuclear distribution. We identified 20 protein kinases that, when knocked down, either decreased nuclear GFP-AR-V7 levels or altered AR-V7 nuclear distribution, a set that included the SFKs Src and Fyn. The Src-Abl dual kinase inhibitor PD180970 decreased expression of AR-V7 by greater than 46% and decreased ligand-independent transcription of AR target genes in the 22RV1 human prostate carcinoma cell line. Further, PD180970 inhibited androgen-independent cell proliferation in endogenous-AR-V7-expressing prostate cancer cell lines and also overcame bicalutamide resistance observed in the 22RV1 cell line. SFKs, especially Src and Fyn, may be important upstream regulators of AR-V7 expression and represent promising targets in a subset of CRPCs expressing high levels of AR-V7. Prostate 77:82-93, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Synthesis, characterization and cytotoxic studies of water soluble [(η5-C5H5)2Mo(thionucleobase/thionucleoside)]Cl complexes in breast and colon cancer cell lines

PubMed Central

Acevedo-Acevedo, Débora; Matta, Jaime; Meléndez, Enrique

2010-01-01

Four new water soluble molybdenocene complexes were synthesized in aqueous solution at pH 7.0. The new species, [(η5-C5H5)2Mo(L)]Cl (L= 6-mercaptopurine, 2-amino-6-mercaptopurine, (-)-2-amino-6-mercaptopurine ribose and 6-mercaptopurine ribose), were characterized by spectroscopic methods. NMR spectroscopic data showed the presence of two coordination isomers, S(6), N(7) and S(6), N(1), in aqueous solution, being S(6), N(7) the most stable. The antiproliferative activities of the new species were investigated in HT-29 colon and MCF-7 breast cancer cell lines. The incorporation of molybdenocene (Cp2Mo2+) into the thionucleobases/thionucleosides decreases their cytotoxic activities in HT-29 colon cancer cell line. In contrast, in the MCF-7 cell line, [Cp2Mo(2-amino-6-mercaptopurine)]Cl showed a high cytotoxic activity. This is most likely a consequence of the enhanced lipophilic character on the thionucleobase combined with synergism between Cp2Mo2+ and the thionucleobase ligand. PMID:21399723

The Effects of Allicin, a Reactive Sulfur Species from Garlic, on a Selection of Mammalian Cell Lines

PubMed Central

Gruhlke, Martin C. H.; Nicco, Carole; Batteux, Frederic; Slusarenko, Alan J.

2016-01-01

Garlic (Allium sativum L.) has been used as a spice and medicinal plant since ancient times. Garlic produces the thiol-reactive defence substance, allicin, upon wounding. The effects of allicin on human lung epithelium carcinoma (A549), mouse fibroblast (3T3), human umbilical vein endothelial cell (HUVEC), human colon carcinoma (HT29) and human breast cancer (MCF7) cell lines were tested. To estimate toxic effects of allicin, we used a standard MTT-test (methylthiazoltetrazolium) for cell viability and 3H-thymidine incorporation for cell proliferation. The glutathione pool was measured using monobromobimane and the formation of reactive species was identified using 2′,7′-dichlorofluoresceine-diacetate. The YO-PRO-1 iodide staining procedure was used to estimate apoptosis. Allicin reduced cell viability and cell proliferation in a concentration dependent manner. In the bimane test, it was observed that cells treated with allicin showed reduced fluorescence, suggesting glutathione oxidation. The cell lines tested differed in sensitivity to allicin in regard to viability, cell proliferation and glutathione oxidation. The 3T3 and MCF-7 cells showed a higher proportion of apoptosis compared to the other cell types. These data show that mammalian cell lines differ in their sensitivity and responses to allicin. PMID:28035949

Investigation of SP94 Peptide as a Specific Probe for Hepatocellular Carcinoma Imaging and Therapy

PubMed Central

Li, Yanli; Hu, Yan; Xiao, Jie; Liu, Guobing; Li, Xiao; Zhao, Yanzhao; Tan, Hui; Shi, Hongcheng; Cheng, Dengfeng

2016-01-01

SP94 (SFSIIHTPILPL), a novel peptide, has shown specific binding to hepatocellular carcinoma (HCC) cells. We aimed to investigate the capability of SP94 as a targeting probe for HCC imaging and therapy following labeling with technetium-99m (99mTc) and rhenium-188 (188Re). HYNIC-SP94 was prepared by solid phase synthesis and then labeled with 99mTc. Cell competitive binding, internalization assay, in vitro and in vivo stability, biodistribution and micro-single photon emission computed tomography /computed tomography (SPECT/CT) imaging studies were performed to investigate the capability of 99mTc tricine-EDDA/HYNIC-SP94 as a specific HCC imaging probe. Initial promising targeting results inspired evaluation of its therapeutic effect when labeled by 188Re. HYNIC-SP94 was then labeled again with 188Re to perform cell apoptosis, microSPECT/CT imaging evaluation and immunohistochemistry. Huh-7 cells exhibited typical apoptotic changes after 188Re irradiation. According to 99mTc tricine-EDDA/HYNIC-SP94 microSPECT/CT imaging, tumor uptake was significantly decreased compared with that of pre-treatment with 188Re-HYNIC-SP94. The immunohistochemistry also displayed obvious necrosis and apoptosis as well as inhibition of proliferation in the 188Re-HYNIC-SP94 treatment group. The results supported that 99mTc tricine-EDDA/HYNIC-SP94 is able to target HCC cells and 188Re-HYNIC- SP94 holds potential as a therapeutic agent for HCC, making 99mTc/188Re-HYNIC-SP94 a promising targeting probe for HCC imaging and therapy. PMID:27649935

A novel 3p22.3 gene CMTM7 represses oncogenic EGFR signaling and inhibits cancer cell growth.

PubMed

Li, H; Li, J; Su, Y; Fan, Y; Guo, X; Li, L; Su, X; Rong, R; Ying, J; Mo, X; Liu, K; Zhang, Z; Yang, F; Jiang, G; Wang, J; Zhang, Y; Ma, D; Tao, Q; Han, W

2014-06-12

Deletion of 3p12-22 is frequent in multiple cancer types, indicating the presence of critical tumor-suppressor genes (TSGs) at this region. We studied a novel candidate TSG, CMTM7, located at the 3p22.3 CMTM-gene cluster, for its tumor-suppressive functions and related mechanisms. The three CMTM genes, CMTM6, 7 and 8, are broadly expressed in human normal adult tissues and normal epithelial cell lines. Only CMTM7 is frequently silenced or downregulated in esophageal and nasopharyngeal cell lines, but uncommon in other carcinoma cell lines. Immunostaining of tissue microarrays for CMTM7 protein showed its downregulation or absence in esophageal, gastric, pancreatic, liver, lung and cervix tumor tissues. Promoter CpG methylation and loss of heterozygosity were both found contributing to CMTM7 downregulation. Ectopic expression of CMTM7 in carcinoma cells inhibits cell proliferation, motility and tumor formation in nude mice, but not in immortalized normal cells, suggesting a tumor inhibitory role of CMTM7. The tumor-suppressive function of CMTM7 is associated with its role in G1/S cell cycle arrest, through upregulating p27 and downregulating cyclin-dependent kinase 2 (CDK2) and 6 (CDK6). Moreover, CMTM7 could promote epidermal growth factor receptor (EGFR) internalization, and further suppress AKT signaling pathway. Thus, our findings suggest that CMTM7 is a novel 3p22 tumor suppressor regulating G1/S transition and EGFR/AKT signaling during tumor pathogenesis.

Effect of the oncolytic ECHO-7 virus Rigvir® on the viability of cell lines of human origin in vitro.

PubMed

Tilgase, Andra; Patetko, Liene; Blāķe, Ilze; Ramata-Stunda, Anna; Borodušķis, Mārtiņš; Alberts, Pēteris

2018-01-01

Background: The role of oncolytic viruses in cancer treatment is increasingly studied. The first oncolytic virus (Rigvir®, ECHO-7) was registered in Latvia over a decade ago. In a recent retrospective study Rigvir® decreased mortality 4.39-6.57-fold in stage IB-IIC melanoma patients. The aims of the present study are to test the effect of Rigvir® on cell line viability in vitro and to visualize the cellular presence of Rigvir® by immunocytochemistry. Methods: The cytolytic effect of Rigvir® on the viability of FM-9, RD, AGS, A549, HDFa, HPAF‑II, MSC, MCF7, HaCaT, and Sk-Mel-28 cell lines was measured using live cell imaging. PBMC viability was measured using flow cytometry. The presence of ECHO-7 virus was visualized using immunocytochemistry. Statistical difference between treatment groups was calculated using two-way ANOVA. Results: Rigvir® (10%, volume/volume) reduced cell viability in FM-9, RD, AGS, A549, HDFa, HPAF‑II and MSC cell lines by 67-100%. HaCaT cell viability was partly affected while Rigvir® had no effect on MCF7, Sk-Mel-28 and PBMC viability. Detection of ECHO-7 by immunocytochemistry in FM-9, RD, AGS, A549, HDFa, HPAF-II and Sk-Mel-28 cell lines suggests that the presence of Rigvir® in the cells preceded or coincided with the time of reduction of cell viability. Rigvir® (10%) had no effect on live PBMC count. Conclusions: The results suggest that Rigvir® in vitro reduces the viability of cells of human melanoma, rhabdomyosarcoma, gastric adenocarcinoma, lung carcinoma, pancreas adenocarcinoma but not in PBMC. The presence of Rigvir® in the sensitive cells was confirmed using anti-ECHO-7 antibodies. The present results suggest that a mechanism of action for the clinical benefit of Rigvir® is its cytolytic properties. The present results suggest that the effect of Rigvir® could be tested in other cancers besides melanoma. Further studies of possible Rigvir® entry receptors are needed.

Increased Growth of a Newly Established Mouse Epithelial Cell Line Transformed with HPV-16 E7 in Diabetic Mice.

PubMed

He, Lan; Law, Priscilla T Y; Boon, Siaw Shi; Zhang, Chuqing; Ho, Wendy C S; Banks, Lawrence; Wong, C K; Chan, Juliana C N; Chan, Paul K S

2016-01-01

Epidemiological evidence supports that infection with high-risk types of human papillomavirus (HPV) can interact with host and environmental risk factors to contribute to the development of cervical, oropharyngeal, and other anogenital cancers. In this study, we established a mouse epithelial cancer cell line, designated as Chinese University Papillomavirus-1 (CUP-1), from C57BL/KsJ mice through persistent expression of HPV-16 E7 oncogene. After continuous culturing of up to 200 days with over 60 passages, we showed that CUP-1 became an immortalized and transformed epithelial cell line with continuous E7 expression and persistent reduction of retinoblastoma protein (a known target of E7). This model allowed in-vivo study of interaction between HPV and co-factors of tumorigenesis in syngeneic mice. Diabetes has been shown to increase HPV pathogenicity in different pathological context. Herein, with this newly-established cell line, we uncovered that diabetes promoted CUP-1 xenograft growth in syngeneic db/db mice. In sum, we successfully established a HPV-16 E7 transformed mouse epithelial cell line, which allowed subsequent studies of co-factors in multistep HPV carcinogenesis in an immunocompetent host. More importantly, this study is the very first to demonstrate the promoting effect of diabetes on HPV-associated carcinogenesis in vivo, implicating the importance of cancer surveillance in diabetic environment.

The 5-HT{sub 2A} serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sonier, Brigitte; Arseneault, Madeleine; Institut National de la Recherche Scientifique-Institut Armand-Frappier, Montreal, Que.

2006-05-19

Serotonin (5-hydroxytryptamine, 5-HT) has been described as a mitogen in a variety of cell types and carcinomas. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Certain studies suggest that some selective serotonin re-uptake inhibitors promote breast cancer in animals and humans. This study attempts to clarify the role of serotonin in promoting the growth of neoplastic mammary cells. Expression of the 5-HT{sub 2A} serotoninergic receptor subtype in MCF-7 cells was determined by RT-PCR, Western blotting, and immunofluorescence analysis. The mitogenic effect of 5-HT on MCF-7 cells was determined by means of the MTTmore » proliferation assay. We have demonstrated that the 5-HT{sub 2A} receptor subtype is fully expressed in the MCF-7 human breast cancer cell line, in terms of encoding mRNA and receptor protein. Automated sequencing has confirmed that the 5-HT{sub 2A} receptor present in this cell line is identical to the 5-HT{sub 2A} receptor found in human platelets and in human cerebral cortex. Furthermore, this receptor was found by immunofluorescence to be on the plasma membrane. MTT proliferation assays revealed that 5-HT and DOI, a selective 5-HT{sub 2A} receptor subtype agonist, stimulated MCF-7 cell. These results indicate that 5-HT plays a mitogenic role in neoplastic mammary cells. Our data also indicate that 5-HT exerts this positive growth effect on MCF-7 cells through, in part, the 5-HT{sub 2A} receptor subtype, which is fully expressed in this cell line.« less

Synthesis of 1,2,4-triazole-linked urea/thiourea conjugates as cytotoxic and apoptosis inducing agents.

PubMed

Tokala, Ramya; Bale, Swarna; Janrao, Ingle Pavan; Vennela, Aluri; Kumar, Niggula Praveen; Senwar, Kishna Ram; Godugu, Chandraiah; Shankaraiah, Nagula

2018-06-01

A new series of 1,2,4-triazole-linked urea and thiourea conjugates have been synthesized and evaluated for their in vitro cytotoxicity against selected human cancer cell lines namely, breast (MCF-7, MDA-MB-231), lung (A549) prostate (DU145) and one mouse melanoma (B16-F10) cell line and compared with reference drug. The compound 5t showed significant cytotoxicity on MCF-7 breast cancer cell line with a IC 50 value of 7.22 ± 0.47 µM among all the tested compounds. Notably, induction of apoptosis by compound 5t on MCF-7 cells was evaluated using different staining techniques such as acridine orange/ethidium bromide (AO/EB), annexin V-FITC/PI, and DAPI. Further, clonogenic assay indicates the inhibition of colony formation on MCF-7 cells by compound 5t. Moreover, the flow-cytometric analysis also revealed that compound 5t caused the arrest of cells at G0/G1 phase of cell cycle. In addition, the compounds when tested on normal human cells (L-132) were found to be safer with low cytotoxicity profile. Copyright © 2018 Elsevier Ltd. All rights reserved.

Sulfamic and succinic acid derivatives of 25-OH-PPD and their activities to MCF-7, A-549, HCT-116, and BGC-823 cell lines.

PubMed

Zhou, Wu-Xi; Cao, Jia-Qing; Wang, Xu-De; Guo, Jun-Hui; Zhao, Yu-Qing

2017-02-15

In the search for new anti-tumor agents with higher potency than our previously identified compound 1 (25-OH-PPD, 25-hydroxyprotopanaxadiol), 12 novel sulfamic and succinic acid derivatives that could improve water solubility and contribute to good drug potency and pharmacokinetic profiles were designed and synthesized. Their in vitro anti-tumor activities in MCF-7, A-549, HCT-116, and BGC-823 cell lines and one normal cell line were tested by standard MTT assay. Results showed that compared with compound 1, compounds 2, 3, and 7 exhibited higher cytotoxic activity on A-549 and BGC-823 cell lines, together with lower toxicity in the normal cell. In particular, compound 2 exhibited the best anti-tumor activity in the in vitro assays, which may provide valuable data for the research and development of new anti-tumor agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tumorigenic risk of human induced pluripotent stem cell explants cultured on mouse SNL76/7 feeder cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamada, Mizuna; Mitsui, Youji, E-mail: y-mitsui8310@hb.tp1.jp; Kumazaki, Tsutomu

2014-10-24

Highlights: • hiPS cell explants formed malignant tumors when SNL76/7 feeder cells were used. • Multi type tumors developed by interaction of SNL76/7 feeder cells with hiPS cells. • Tumorigenic risk occurs by co-culture of hiPS cells with SNL76/7 feeder cells. - Abstract: The potential for tumor formation from transplanted human induced pluripotent stem cell (hiPSC) derivatives represents a high risk in their application to regenerative medicine. We examined the genetic origin and characteristics of tumors, that were formed when 13 hiPSC lines, established by ourselves, and 201B7 hiPSC from Kyoto University were transplanted into severe combined immune-deficient (SCID) mice.more » Though teratomas formed in 58% of mice, five angiosarcomas, one malignant solitary fibrous tumor and one undifferentiated pleomorphic sarcoma formed in the remaining mice. Three malignant cell lines were established from the tumors, which were derived from mitomycin C (MMC)-treated SNL76/7 (MMC-SNL) feeder cells, as tumor development from fusion cells between MMC-SNL and hiPSCs was negative by genetic analysis. While parent SNL76/7 cells produced malignant tumors, neither MMC-SNL nor MMC-treated mouse embryo fibroblast (MEF) produced malignant tumors. When MMC-SNL feeder cells were co-cultured with hiPSCs, growing cell lines were generated, that expressed genes similar to the parent SNL76/7 cells. Thus, hiPSCs grown on MMC-SNL feeder cells have a high risk of generating feeder-derived malignant tumors. The possible mechanism(s) of growth restoration and the formation of multiple tumor types are discussed with respect of the interactions between MMC-SNL and hiPSC.« less

Generation and validation of PAX7 reporter lines from human iPS cells using CRISPR/Cas9 technology.

PubMed

Wu, Jianbo; Hunt, Samuel D; Xue, Haipeng; Liu, Ying; Darabi, Radbod

2016-03-01

Directed differentiation of iPS cells toward various tissue progenitors has been the focus of recent research. Therefore, generation of tissue-specific reporter iPS cell lines provides better understanding of developmental stages in iPS cells. This technical report describes an efficient strategy for generation and validation of knock-in reporter lines in human iPS cells using the Cas9-nickase system. Here, we have generated a knock-in human iPS cell line for the early myogenic lineage specification gene of PAX7. By introduction of site-specific double-stranded breaks (DSB) in the genomic locus of PAX7 using CRISPR/Cas9 nickase pairs, a 2A-GFP reporter with selection markers has been incorporated before the stop codon of the PAX7 gene at the last exon. After positive and negative selection, single cell-derived human iPS clones have been isolated and sequenced for in-frame positioning of the reporter construct. Finally, by using a nuclease-dead Cas9 activator (dCas9-VP160) system, the promoter region of PAX7 has been targeted for transient gene induction to validate the GFP reporter activity. This was confirmed by flow cytometry analysis and immunostaining for PAX7 and GFP. This technical report provides a practical guideline for generation and validation of knock-in reporters using CRISPR/Cas9 system. Published by Elsevier B.V.

Decreased hepatocyte membrane potential differences and GABAA-beta3 expression in human hepatocellular carcinoma.

PubMed

Minuk, Gerald Y; Zhang, Manna; Gong, Yuewen; Minuk, Leonard; Dienes, Hans; Pettigrew, Norman; Kew, Michael; Lipschitz, Jeremy; Sun, Dongfeng

2007-03-01

To determine whether hepatocyte membrane potential differences (PDs) are depolarized in human HCC and whether depolarization is associated with changes in GABAA receptor expression, hepatocyte PDs and gamma-aminobutyric acid (GABA)A receptor messenger RNA (mRNA) and protein expression were documented in HCC tissues via microelectrode impalement, real-time reverse-transcriptase polymerase chain reaction, and Western blot analysis, respectively. HCC tissues were significantly depolarized (-19.8+/-1.3 versus -25.9+/-3.2 mV, respectively [P<0.05]), and GABAA-beta3 expression was down-regulated (GABAA-beta3 mRNA and protein expression in HCC; 5,693+/-1,385 and 0.29+/-0.11 versus 11,046+/-4,979 copies/100 mg RNA and 0.62+/-0.16 optical density in adjacent tumor tissues, respectively [P=0.002 and P<0.0001, respectively]) when compared with adjacent nontumor tissues. To determine the physiological relevance of the down-regulation, human malignant hepatocytes deficient in GABAA-beta3 receptor expression (Huh-7 cells) were transfected with GABAA-beta3 complementary DNA (cDNA) or vector alone and injected into nu/nu nude mice (n=16-17 group). Tumors developed after a mean (+/-SD) of 51+/-6 days (range: 41-60 days) in 7/16 (44%) mice injected with vector-transfected cells and 70+/-12 days (range: 59-86 days) in 4/17 (24%) mice injected with GABAA-beta3 cDNA-transfected cells (P<0.005). The results of this study indicate that (1) human HCC tissues are depolarized compared with adjacent nontumor tissues, (2) hepatic GABAA-beta3 receptor expression is down-regulated in human HCC, and (3) restoration of GABAA-beta3 receptor expression results in attenuated in vivo tumor growth in nude mice.

TM6SF2 is a regulator of liver fat metabolism influencing triglyceride secretion and hepatic lipid droplet content

PubMed Central

Mahdessian, Hovsep; Taxiarchis, Apostolos; Popov, Sergej; Silveira, Angela; Franco-Cereceda, Anders; Hamsten, Anders; Eriksson, Per; van't Hooft, Ferdinand

2014-01-01

Genome-wide association studies have identified a locus on chromosome 19 associated with plasma triglyceride (TG) concentration and nonalcoholic fatty liver disease. However, the identity and functional role of the gene(s) responsible for these associations remain unknown. Of 19 expressed genes contained in this locus, none has previously been implicated in lipid metabolism. We performed gene expression studies and expression quantitative trait locus analysis in 206 human liver samples to identify the putative causal gene. Transmembrane 6 superfamily member 2 (TM6SF2), a gene with hitherto unknown function, expressed predominantly in liver and intestine, was identified as the putative causal gene. TM6SF2 encodes a protein of 351 amino acids with 7–10 predicted transmembrane domains. Otherwise, no other protein features were identified which could help to elucidate the function of TM6SF2. Protein subcellular localization studies with confocal microscopy demonstrated that TM6SF2 is localized in the endoplasmic reticulum and the ER-Golgi intermediate compartment of human liver cells. Functional studies for secretion of TG-rich lipoproteins (TRLs) and lipid droplet content were performed in human hepatoma Huh7 and HepG2 cells using confocal microscopy and siRNA inhibition and overexpression techniques. In agreement with the genome-wide association data, it was found that TM6SF2 siRNA inhibition was associated with reduced secretion of TRLs and increased cellular TG concentration and lipid droplet content, whereas TM6SF2 overexpression reduced liver cell steatosis. We conclude that TM6SF2 is a regulator of liver fat metabolism with opposing effects on the secretion of TRLs and hepatic lipid droplet content. PMID:24927523

Upregulation of erythropoietin receptor in UT-7/EPO cells inhibits simulated microgravity-induced cell apoptosis

NASA Astrophysics Data System (ADS)

Zou, Li-xue; Cui, Shao-yan; Zhong, Jian; Yi, Zong-chun; Sun, Yan; Fan, Yu-bo; Zhuang, Feng-yuan

2011-07-01

Hematopoietic progenitor cell proliferation can be altered in either spaceflight or under simulated microgravity experiments on the ground, however, the underlying mechanism remains unknown. Our previous study showed that exposure of the human erythropoietin (EPO)-dependent leukemia cell line UT-7/EPO to conditions of simulated microgravity significantly inhibited the cellular proliferation rate and induced cell apoptosis. We postulated that the downregulation of the erythropoietin receptor (EPOR) expression in UT-7/EPO cells under simulated microgravity may be a possible reason for microgravity triggered apoptosis. In this paper, a human EPOR gene was transferred into UT-7/EPO cells and the resulting expression of EPOR on the surface of UT-7/EPO cells increased approximately 61% ( p < 0.05) as selected by the antibiotic G418. It was also shown through cytometry assays and morphological observations that microgravity-induced apoptosis markedly decreased in these UT-7/EPO-EPOR cells. Thus, we concluded that upregulation of EPOR in UT-7/EPO cells could inhibit the simulated microgravity-induced cell apoptosis in this EPO dependent cell line.

Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) ligands inhibit growth of UACC903 and MCF7 human cancer cell lines

PubMed Central

Girroir, Elizabeth E.; Hollingshead, Holly E.; Billin, Andrew N.; Willson, Timothy M.; Robertson, Gavin P.; Sharma, Arun K.; Amin, Shantu; Gonzalez, Frank J.; Peters, Jeffrey M.

2008-01-01

The development of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) ligands for the treatment of diseases including metabolic syndrome, diabetes and obesity has been hampered due to contradictory findings on their potential safety. For example, while some reports show that ligand activation of PPARβ/δ promotes the induction of terminal differentiation and inhibition of cell growth, other reports suggest that PPARβ/δ ligands potentiate tumorigenesis by increasing cell proliferation. Some of the contradictory findings could be due in part to differences in the ligand examined, the presence or absence of serum in cell cultures, differences in cell lines, or differences in the method used to quantify cell growth. For these reasons, this study examined the effect of ligand activation of PPARβ/δ on cell growth of two human cancer cell lines, MCF7 (breast cancer) and UACC903 (melanoma) in the presence or absence of serum using two highly specific PPARβ/δ ligands, GW0742 or GW501516. Culturing cells in the presence of either GW0742 or GW501516 caused upregulation of the known PPARβ/δ target gene angiopoetin-like protein 4 (ANGPTL4). Inhibition of cell growth was observed in both cell lines cultured in the presence of either GW0742 or GW501516, and the presence or absence of serum had little influence on this inhibition. Results from the present studies demonstrate that ligand activation of PPARβ/δ inhibits the growth of both MCF7 and UACC903 cell lines and provide further evidence that PPARβ/δ ligands are not mitogenic in human cancer cell lines. PMID:18054822

The defensin from avocado (Persea americana var. drymifolia) PaDef induces apoptosis in the human breast cancer cell line MCF-7.

PubMed

Guzmán-Rodríguez, Jaquelina Julia; López-Gómez, Rodolfo; Salgado-Garciglia, Rafael; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

2016-08-01

Antimicrobial peptides (AMPs) are cytotoxic to cancer cells; however, mainly the effects of AMPs from animals have been evaluated. In this work, we assessed the cytotoxicity of PaDef defensin from avocado (Persea americana var. drymifolia) on the MCF-7 cancer cell line (a breast cancer cell line) and evaluated its mechanism of action. PaDef inhibited the viability of MCF-7 cells in a concentration-dependent manner, with an IC50=141.62μg/ml. The viability of normal peripheral blood mononuclear cells was unaffected by this AMP. Additionally, PaDef induced apoptosis in MCF-7 cells in a time-dependent manner, but did not affect the membrane potential or calcium flow. In addition, PaDef IC50 induced the expression of cytochrome c, Apaf-1, and the caspase 7 and 9 genes. Likewise, this defensin induced the loss of mitochondrial Δψm and increased the phosphorylation of MAPK p38, which may lead to MCF-7 apoptosis by the intrinsic pathway. This is the first report of an avocado defensin inducing intrinsic apoptosis in cancer cells, which suggests that it could be a potential therapeutic molecule in the treatment of cancer. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Characterization of protein marker expression, tumorigenicity, and doxorubicin chemoresistance in two new canine mammary tumor cell lines.

PubMed

Hsiao, Yen-Ling; Hsieh, Tai-Zu; Liou, Chian-Jiun; Cheng, Yeong-Hsiang; Lin, Chung-Tien; Chang, Chi-Yao; Lai, Yu-Shen

2014-09-30

Canine mammary tumors (CMTs) are the most common type of cancer found in female dogs. Establishment and evaluation of tumor cell lines can facilitate investigations of the biological mechanisms of cancer. Different cell models are used to investigate genetic, epigenetic, and cellular pathways, cancer progression, and cancer therapeutics. Establishment of new cell models will greatly facilitate research in this field. In the present study, we established and characterized two new CMT cell lines derived from a single CMT. We established two cell lines from a single malignant CMT specimen: DTK-E and DTK-SME. Morphologically, the DTK-E cells were large, flat, and epithelial-like, whereas DTK-SME cells were round and epithelial-like. Doubling times were 24 h for DTK-E and 18 h for DTK-SME. On western blots, both cell lines expressed cytokeratin AE1, vimentin, cytokeratin 7 (CK7), and heat shock protein 27 (HSP27). Moreover, investigation of chemoresistance revealed that DTK-SME was more resistant to doxorubicin-induced apoptosis than DTK-E was. After xenotransplantation, both DTK-E and DTK-SME tumors appeared within 14 days, but the average size of DTK-SME tumors was greater than that of DTK-E tumors after 56 days. We established two new cell lines from a single CMT, which exhibit significant diversity in cell morphology, protein marker expression, tumorigenicity, and chemoresistance. The results of this study revealed that the DTK-SME cell line was more resistant to doxorubicin-induced apoptosis and exhibited higher tumorigenicity in vivo than the DTK-E cell line. We anticipate that the two novel CMT cell lines established in this study will be useful for investigating the tumorigenesis of mammary carcinomas and for screening anticancer drugs.

Antiproliferative Activity of Xanthones Isolated from Artocarpus obtusus

PubMed Central

Hashim, Najihah Mohd; Rahmani, Mawardi; Ee, Gwendoline Cheng Lian; Sukari, Mohd Aspollah; Yahayu, Maizatulakmal; Oktima, Winda; Ali, Abd Manaf; Go, Rusea

2012-01-01

An investigation of the chemical constituents in Artocarpus obtusus species led to the isolation of three new xanthones, pyranocycloartobiloxanthone A (1), dihydroartoindonesianin C (2), and pyranocycloartobiloxanthone B (3). The compounds were subjected to antiproliferative assay against human promyelocytic leukemia (HL60), human chronic myeloid leukemia (K562), and human estrogen receptor (ER+) positive breast cancer (MCF7) cell lines. Pyranocycloartobiloxanthone A (1) consistently showed strong cytotoxic activity against the three cell lines compared to the other two with IC50 values of 0.5, 2.0 and 5.0 μg/mL, respectively. Compound (1) was also observed to exert antiproliferative activity and apoptotic promoter towards HL60 and MCF7 cell lines at respective IC50 values. The compound (1) was not toxic towards normal cell lines human nontumorigenic breast cell line (MCF10A) and human peripheral blood mononuclear cells (PBMCs) with IC50 values of more than 30 μg/mL. PMID:21960741

Flow Line, Durafill VS, and Dycal toxicity to dental pulp cells: effects of growth factors

PubMed Central

Furey, Alyssa; Hjelmhaug, Julie; Lobner, Doug

2010-01-01

Introduction The objective was to determine the effects of growth factor treatment on dental pulp cell sensitivity to toxicity of two composite restoration materials, Flow Line and Durafill VS, and a calcium hydroxide pulp capping material, Dycal. Methods Toxicity of the dental materials to cultures of primary dental pulp cells was determined by the MTT metabolism assay. The ability of six different growth factors to influence the toxicity was tested. Results A 24 hour exposure to either Flow Line or Durafill VS caused approximately 40% cell death, while Dycal exposure caused approximately 80% cell death. The toxicity of Flow Line and Durafill VS was mediated by oxidative stress. Four of the growth factors tested (BMP-2, BMP-7, EGF, and TGF-β) decreased the basal MTT values while making the cells resistant to Flow Line and Durafill VS toxicity, except BMP-2 which made the cells more sensitive to Flow Line. Treatment with FGF-2 caused no change in basal MTT metabolism, prevented the toxicity of Durafill VS, but increased the toxicity of Flow Line. Treatment with IGF-I increased basal MTT metabolism and made the cells resistant to Flow Line and Durafill VS toxicity. None of the growth factors made the cells resistant to Dycal toxicity. Conclusions The results indicate that growth factors can be used to alter the sensitivity of dental pulp cells to commonly used restoration materials. The growth factors BMP-7, EGF, TGF-β, and IGF-I provided the best profile of effects, making the cells resistant to both Flow Line and Durafill VS toxicity. PMID:20630288