[Effect of tagalsin on p53 and Bcl-2 expression in hepatoma H(22) tumor-bearing mice].

PubMed

Song, Xiu-qi; Guo, Yun-liang; Wang, Bing-gao; Sun, Shao-jie; Yao, Ru-yong

2011-07-01

To explore the effect and mechanism of tagalsin on hepatoma cells. The animal models were established by transplanting H(22) mouse hepatoma cells to mouse liver, and ten days later the mice were randomly divided into five groups: blank group, carmofur positive group and tagalsin groups, including low-dose, middle-dose and high-dose groups. Then medicine or oil was given to the mice by gastric gavage in consecutive 5 days with a 2-days interval as a course of treatment, two courses in all. All mice were killed at 24 hours after medication, and the survival period, ascites conditions, aggressive conditions intra- or extra-liver, weight changes, tumor volume and spleen index of the tumor-bearing mice were observed. Pathological changes of the tumors were examined. Apoptotic factors p53 and Bcl-2 protien and mRNA were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). tagalsin inhibited the hepatoma growth effectively without influencing spleen index to some extent. The tumor inhibition rate of tagalsin low, middle and high dose groups were 17.9%, 63.1% and 71.8%, respectively. Immunohistochemical results showed that the p53 and Bcl-2 protein positive cell counts of the positive control and experimental groups were significantly lower than those of the blank group (P < 0.01). RT-PCR results showed that the p53 mRNA expression was significantly enhanced and Bcl-2 mRNA expression was decreased in the positive control groups and tagalsin treatment groups, especially in the high dose group, compared with those of the blank group (P < 0.05). tagalsin can inhibit the growth of mouse hepatoma cells significantly. The mechanism of its anti-tumor effect may work via up-regulating the wild type p53 gene expression and down-regulating Bcl-2 gene expression and thus regulating tumor cell apoptosis.

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

Effects of hypoxia-inducible factor-1α silencing on the proliferation of CBRH-7919 hepatoma cells

PubMed Central

Xu, Lin-Feng; Ni, Jia-Yan; Sun, Hong-Liang; Chen, Yao-Ting; Wu, Yu-Dan

2013-01-01

AIM: To study the effects of hypoxia-inducible factor-1α (HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells. METHODS: The CBRH-7919 rat hepatoma cell line was used in this study and the hypoxic model was constructed using CoCl2. The HIF-1α-specific RNAi sequences were designed according to the gene coding sequence of rat HIF-1α obtained from GeneBank. The secondary structure of the HIF-1α gene sequence was analyzed using RNA draw software. The small interfering RNA (siRNA) transfection mixture was produced by mixing the siRNA and Lipofectamine2000TM, and transfected into the hypoxic hepatoma cells. Real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assay were used to detect the expression levels of mRNA and protein. HIF-1α and vascular endothelial growth factor (VEGF) mRNA was determined using real time RT-PCR; the protein expression levels of AKT, p-AKT, p21 and cyclinD1 were determined using Western blotting. The proliferation of hepatoma cells was observed using the methyl thiazolyl tetrazolium (MTT) assay and the bromodeoxyuridine (BrdU) incorporation cell proliferation assay. RESULTS: Under induced hypoxia, the viability of the hepatoma cells reached a minimum at 800 μmol/L CoCl2; the viability of the cells was relatively high at CoCl2 concentrations between 100 μmol/L and 200 μmol/L. Under hypoxia, the mRNA and protein expression levels of HIF-1α and VEGF were significantly higher than that of hepatoma cells that were cultured in normaxia. HIF-1α-specific RNAi sequences were successfully transfected into hepatoma cells. The transfection of specific siRNAs significantly inhibited the mRNA and protein expression levels of HIF-1α and VEGF, along with the protein expression levels of p-AKT and cyclinD1; the protein expression of p21 was significantly increased, and there was no significant difference in the expression of AKT. The MTT assay showed that the amount of hepatoma cells in S

Epigenetic Targeting of Granulin in Hepatoma Cells by Synthetic CRISPR dCas9 Epi-suppressors.

PubMed

Wang, Hong; Guo, Rui; Du, Zhonghua; Bai, Ling; Li, Lingyu; Cui, Jiuwei; Li, Wei; Hoffman, Andrew R; Hu, Ji-Fan

2018-06-01

The CRISPR-associated Cas9 system can modulate disease-causing alleles both in vivo and ex vivo, raising the possibility of therapeutic genome editing. In addition to gene targeting, epigenetic modulation by the catalytically inactive dCas9 may also be a potential form of cancer therapy. Granulin (GRN), a potent pluripotent mitogen and growth factor that promotes cancer progression by maintaining self-renewal of hepatic stem cancer cells, is upregulated in hepatoma tissues and is associated with decreased tumor survival in patients with hepatoma. We synthesized a group of dCas9 epi-suppressors to target GRN by tethering the C terminus of dCas9 with three epigenetic suppressor genes: DNMT3a (DNA methyltransferase), EZH2 (histone 3 lysine 27 methyltransferase), and KRAB (the Krüppel-associated box transcriptional repression domain). In conjunction with guide RNAs (gRNAs), the dCas9 epi-suppressors caused significant decreases in GRN mRNA abundance in Hep3B hepatoma cells. These dCas9 epi-suppressors initiated de novo CpG DNA methylation in the GRN promoter, and they produced histone codes that favor gene suppression, including decreased H3K4 methylation, increased H3K9 methylation, and enhanced HP1a binding. Epigenetic knockdown of GRN led to the inhibition of cell proliferation, decreased tumor sphere formation, and reduced cell invasion. These changes were achieved at least partially through the MMP/TIMP pathway. This study thus demonstrates the potential utility of using dCas9 epi-suppressors in the development of epigenetic targeting against tumors. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Retinoic acid-related orphan receptor alpha reprograms glucose metabolism in glutamine-deficient hepatoma cells.

PubMed

Byun, Jun-Kyu; Choi, Yeon-Kyung; Kang, Yu Na; Jang, Byoung Kuk; Kang, Koo Jeong; Jeon, Yong Hyun; Lee, Ho-Won; Jeon, Jae-Han; Koo, Seung-Hoi; Jeong, Won-Il; Harris, Robert A; Lee, In-Kyu; Park, Keun-Gyu

2015-03-01

The metabolism of glutamine and glucose is recognized as a promising therapeutic target for the treatment of cancer; however, targeted molecules that mediate glutamine and glucose metabolism in cancer cells have not been addressed. Here, we show that restricting the supply of glutamine in hepatoma cells, including HepG2 and Hep3B cells, markedly increased the expression of retinoic acid-related orphan receptor alpha (RORα). Up-regulation of RORα in glutamine-deficient hepatoma cells resulted from an increase in the level of cellular reactive oxygen species and in the nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate reduced (NADP+ /NADPH) ratio, which was consistent with a reduction in the glutathione/glutathione disulfide (GSH/GSSG) ratio. Adenovirus (Ad)-mediated overexpression of RORα (Ad-RORα) or treatment with the RORα activator, SR1078, reduced aerobic glycolysis and down-regulated biosynthetic pathways in hepatoma cells. Ad-RORα and SR1078 reduced the expression of pyruvate dehydrogenase kinase 2 (PDK2) and inhibited the phosphorylation of pyruvate dehydrogenase and subsequently shifted pyruvate to complete oxidation. The RORα-mediated decrease in PDK2 levels was caused by up-regulation of p21, rather than p53. Furthermore, RORα inhibited hepatoma growth both in vitro and in a xenograft model in vivo. We also found that suppression of PDK2 inhibited hepatoma growth in a xenograft model. These findings mimic the altered glucose utilization and hepatoma growth caused by glutamine deprivation. Finally, tumor tissue from 187 hepatocellular carcinoma patients expressed lower levels of RORα than adjacent nontumor tissue, supporting a potential beneficial effect of RORα activation in the treatment of liver cancer. RORα mediates reprogramming of glucose metabolism in hepatoma cells in response to glutamine deficiency. The relationships established here between glutamine metabolism, RORα expression and signaling, and

Metabolism of the tropine indole-3-carboxylate ICS 205-930 by differentiated rat and human hepatoma cells.

PubMed

Fischer, V; Baldeck, J P; Wiebel, F J

The metabolism of the tropine indole-3-carboxylate ICS 205-930 (ICS), a highly potent and selective antagonist of 5-HT3 receptors, was investigated in continuous cell lines derived from rat or human liver and compared to the in vivo metabolism in rat and human. The well-differentiated rat hepatoma line 2sFou extensively metabolized ICS by hydroxylation of the indole moiety and subsequent conjugation to form the corresponding glucuronides and sulfates. The 2sFou cells also oxidized ICS at the tropinyl moiety to form both N-demethyl and N-oxide derivatives. The relative amount of the various metabolites was dependent on the substrate concentration. Pretreatment of the cells with dexamethasone increased the rate of metabolism for all pathways, while benz[a]anthracene caused an increase in hydroxylation at the indole moiety at the expense of N-oxidation. Phenobarbital pretreatment had no effect on ICS metabolism. The pattern of metabolites formed in 2sFou cells was qualitatively similar to that formed in rat urine. The human hepatoma line HepG2 metabolized ICS only to a small extent. The HepG2 cells failed to form detectable amounts of ICS conjugates found in human urine. The N-oxide-ICS was not found in HepG2 cells or in human urine. Virtually no ICS metabolites were found in human lung adenocarcinoma lines NCI-H358 or NCI-H322. The results suggest that continuous cell lines such as the differentiated rat hepatoma cells 2sFou might be used to mimic the metabolism of xenobiotics in rat and to clarify their complex metabolic pathways.

Liver-enriched transcription factors uncoupled from expression of hepatic functions in hepatoma cell lines.

PubMed Central

Chaya, D; Fougère-Deschatrette, C; Weiss, M C

1997-01-01

Among the liver-enriched transcription factors identified to date, only expression of hepatocyte nuclear factor 4 (HNF4) and hepatocyte nuclear factor 1 (HNF1) is in strict correlation with hepatic differentiation in cultured rat hepatoma cells. Indeed, differentiated hepatoma cells that stably express an extensive set of adult hepatic functions express liver-enriched transcription factors, while dedifferentiated cells that have lost expression of all these hepatic functions no longer express HNF4 and HNF1. We describe a new heritable phenotype, designated as uncoupled, in which there is a spontaneous dissociation between the expression of these transcription factors and that of the hepatic functions. Cells presenting this phenotype, isolated from differentiated hepatoma cells, cease to accumulate all transcripts coding for hepatic functions but nevertheless maintain expression of HNF4 and HNF1. Transitory transfection experiments indicate that these two factors present in these cells have transcriptional activity similar to that of differentiated hepatoma cells. Characterization of the appropriate intertypic cell hybrids demonstrates that this new phenotype is recessive to the dedifferentiated state and fails to be complemented by differentiated cells. These results indicate the existence of mechanisms that inhibit transcription of genes coding for hepatocyte functions in spite of the presence of functional HNF4 and HNF1. Cells of the uncoupled phenotype present certain properties of oval cells described for pathological states of the liver. PMID:9343392

Liver-enriched transcription factors uncoupled from expression of hepatic functions in hepatoma cell lines.

PubMed

Chaya, D; Fougère-Deschatrette, C; Weiss, M C

1997-11-01

Among the liver-enriched transcription factors identified to date, only expression of hepatocyte nuclear factor 4 (HNF4) and hepatocyte nuclear factor 1 (HNF1) is in strict correlation with hepatic differentiation in cultured rat hepatoma cells. Indeed, differentiated hepatoma cells that stably express an extensive set of adult hepatic functions express liver-enriched transcription factors, while dedifferentiated cells that have lost expression of all these hepatic functions no longer express HNF4 and HNF1. We describe a new heritable phenotype, designated as uncoupled, in which there is a spontaneous dissociation between the expression of these transcription factors and that of the hepatic functions. Cells presenting this phenotype, isolated from differentiated hepatoma cells, cease to accumulate all transcripts coding for hepatic functions but nevertheless maintain expression of HNF4 and HNF1. Transitory transfection experiments indicate that these two factors present in these cells have transcriptional activity similar to that of differentiated hepatoma cells. Characterization of the appropriate intertypic cell hybrids demonstrates that this new phenotype is recessive to the dedifferentiated state and fails to be complemented by differentiated cells. These results indicate the existence of mechanisms that inhibit transcription of genes coding for hepatocyte functions in spite of the presence of functional HNF4 and HNF1. Cells of the uncoupled phenotype present certain properties of oval cells described for pathological states of the liver.

Trichloroethylene toxicity in a human hepatoma cell line

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

Thevenin, E.; McMillian, J.

1994-12-31

The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

Cytotoxicity of nitric oxide in Fu5 rat hepatoma cells: evidence for co-operative action with hydrogen peroxide.

PubMed Central

Ioannidis, I; de Groot, H

1993-01-01

The NO-releasing compounds 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1), sodium nitroprusside (SNP) and S-nitroso-N-acetyl-DL-penicillamine (SNAP) mediated a rapid loss of viability of Fu5 rat hepatoma cells. SIN-1 in addition to NO also released the superoxide anion radical (O2-.). Its cytotoxicity, however, was not affected by superoxide dismutase. In contrast, the H2O2-converting enzyme catalase significantly, but not completely, diminished cell damage, indicating participation of H2O2 in the tumoricidal activity of SIN-1. Glucose oxidase (5 m-units/ml), producing similar amounts of H2O2 to 5 mM SIN-1, had no effect on cell viability. When 5 m-units/ml glucose oxidase was added to incubations with 5 mM SNP, which alone initiated cell injury of about 40%, cell damage was significantly increased up to 95%. Similar results were observed with 1 mM SNAP and 20 m-units/ml xanthine oxidase, which mediated cytotoxicity of about 90% when both compounds were added together, compared with 35% and 55% cell injury, respectively, induced by the single compounds. The results indicate that a co-operative action with H2O2 enhances the tumoricidal activity of NO in Fu5 cells. No evidence for an interplay of NO with O2-. in cytotoxicity, e.g. via the peroxynitrite anion (ONOO-), was found. PMID:8257422

Cell surface GRP78 facilitates hepatoma cells proliferation and migration by activating IGF-IR.

PubMed

Yin, Yancun; Chen, Chen; Chen, Jinliang; Zhan, Renhui; Zhang, Qiang; Xu, Xiaoyan; Li, Defang; Li, Minjing

2017-07-01

The 78kDa glucose regulated protein (GRP78) is a multifunctional chaperone that is involved in a variety of cellular processes. Insulin like growth factor I receptor (IGF-IR) often aberrant expresses in many types of tumor cells. The IGF-IR signaling plays key roles in carcinogenesis and maintenance of the malignant phenotype. The crosstalk between GRP78 and IGF-IR molecules has not well been illuminated. Here, we demonstrated a reciprocal regulation of GRP78 expression and IGF-IR pathway activation. IGF-I induced GRP78 expression in hepatoma cells. IGF-IR knockdown or IGF-IR inhibitor repressed GRP78 expression. Both phosphatidylinositol 3-kianase (PI3K) and mitogen-activated protein kinase (MAPK) pathways involved in IGF-I induction of GRP78 expression. Interestingly, treatment of hepatoma cells with IGF-I re-distributes GRP78 from endoplasmic reticulum (ER) to cell surface and promotes its physical interaction with IGF-IR. Also, GRP78 promotes IGF-IR phosphorylation and activation. Blocked of GRP78 by small interfering RNA or inhibition of GRP78 function by (-)-epigallocatechin gallate (EGCG) blocks IGF-I induced IGF-IR phosphorylation and its downstream signaling. Further, blocked cell surface GRP78 with antibody inhibits IGF-I stimulated cellular proliferation and migration. These data reveal an essential role for the molecular chaperone GRP78 in IGF-IR signaling and implicate the use of GRP78 inhibitors in blocking IGF-IR signaling in hepatoma cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Enhanced proliferation of human hepatoma cells by PAR-2 agonists via the ERK/AP-1 pathway.

PubMed

Xie, Liqun; Zheng, Yanmin; Li, Xuan; Zhao, Junyan; Chen, Xiaoyi; Chen, Li; Zhou, Jing; Hai, Ou; Li, Fei

2012-11-01

To investigate the expression and role of PAR-2 in the proliferation of the human hepatoma cell line HepG2, PAR-2 protein and mRNA expression were evaluated by immuno-histochemistry, immunofluorescence and RT-PCR analysis. The signaling pathways downstream of PAR-2 activation that lead to hepatoma cell proliferation were analyzed. The results showed that PAR-2 is expressed in human hepatoma cells and PAR-2 mRNA expression was found to be upregulated in cells treated with trypsin or SLIGKV-NH2 (P<0.001). The proliferation rate of HepG2 cells treated with trypsin or SLIGKV-NH2 was significantly increased (P<0.001). The percentage of S phase, G2/M phase and the proliferation index (PI) of HepG2 cells treated with trypsin or SLIGKV-NH2 were significantly elevated (P<0.001). The proliferative responses of HepG2 to trypsin and SLIGKV-NH2 were associated with the upregulation of c-fos and PCNA, which were significantly blocked by PD98059 pretreatment. In conclusion, our results indicate that PAR-2 enhances proliferation of human hepatoma cells possibly via the ERK/AP-1 pathway.

[IL-12 induces autophagy via AKT/mTOR/STAT3 signaling pathway in human hepatoma cells].

PubMed

Liu, Cuiying; Xie, Changli; Lin, Yan; Wu, Bitao; Wang, Qin; Li, Ziwei; Tu, Zhiguang

2016-07-01

Objective To investigate the effect of IL-12 on autophagy and the relative possible mechanism in HepG2 and SMMC-7721 human hepatoma cells. Methods The hepatoma cells were treated with IL-12 (10 ng/mL) for 6 hours. Western blotting was applied to detect the expressions of microtubule-associated protein 1 light chain 3 (LC-3), Beclin 1 and the phosphorylated levels of protein kinase B (AKT), mammalian target of rapamycin (mTOR), signal transducer and activator of transcription 3 (STAT3); immunofluorescence assay (IFA) and transmission electron microscopy (TEM) were used to observe the formation of autophagosome. After STAT3 was inhibited by STATTIC or siSTAT3 and AKT was activated by insulin-like growth factor (IGF-1), Western blotting and IFA were performed again to analyze the change of IL-12-induced autophagy. After the cells were treated with IL-12 (10 ng/mL) for 1, 2, 3, 4, 5 days, CCK-8 assay was used to determine the growth ability. After the hepatoma cells were treated with IL-12 (10 ng/mL) for 48 hours, trypan blue staining was used to detect the death rate of the cells. After cell autophagy was inhibit by siBeclin 1, CCK-8 assay and trypan blue staining were performed again to study the effect of IL-12 on the proliferation and death of human hepatoma cells. Results IL-12 induced autophagy and inhibited cell growth in the hepatoma cells. Silencing Beclin 1 gene enhanced IL-12-mediated growth inhibition and cell death. Furthermore, IL-12 treatment also decreased the expressions of p-AKT, p-mTOR and p-STAT3. The pretreatment of siSTAT3 or STATTIC inhibited STAT3-enhanced IL-12-induced autophagy. Accordingly, activation of AKT with IGF-1 decreased IL-12-induced autophagy. Conclusion IL-12 could induce autophagy through AKT/mTOR/STAT3 signaling pathways and the induction of autophagy attenuates the growth-inhibitory effect of IL-12 on hepatoma cells.

Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability

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

Marchissio, Maria Julia; Francés, Daniel Eleazar Antonio; Carnovale, Cristina Ester

Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H{sub 2}O{sub 2} across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H{sub 2}O{sub 2} release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72 h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p < 0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H{sub 2}O{sub 2} release, assessedmore » by Amplex Red, was reduced by about 45% (p < 0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+ 120%, p < 0.05) and loss of mitochondrial membrane potential (− 80%, p < 0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H{sub 2}O{sub 2} release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death. -- Highlights: ► Aquaporin-8 is expressed in mitochondria of human hepatoma HepG2 cells. ► Aquaporin-8 knockdown impairs mitochondrial H{sub 2}O{sub 2} release and increases ROS. �

Esculetin-induced protection of human hepatoma HepG2 cells against hydrogen peroxide is associated with the Nrf2-dependent induction of the NAD(P)H: Quinone oxidoreductase 1 gene

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

Subramaniam, Sudhakar R.; Ellis, Elizabeth M., E-mail: elizabeth.ellis@strath.ac.uk

Esculetin (6,7-dihydroxy coumarin), is a potent antioxidant that is present in several plant species. The aim of this study was to investigate the mechanism of protection of esculetin in human hepatoma HepG2 cells against reactive oxygen species (ROS) induced by hydrogen peroxide. Cell viability, cell integrity, intracellular glutathione levels, generation of reactive oxygen species and expression of antioxidant enzymes were used as markers to measure cellular oxidative stress and response to ROS. The protective effect of esculetin was compared to a well-characterized chemoprotective compound quercetin. Pre-treatment of HepG2 cells with sub-lethal (10-25 {mu}M) esculetin for 8 h prevented cell deathmore » and maintained cell integrity following exposure to 0.9 mM hydrogen peroxide. An increase in the generation of ROS following hydrogen peroxide treatment was significantly attenuated by 8 h pre-treatment with esculetin. In addition, esculetin ameliorated the decrease in intracellular glutathione caused by hydrogen peroxide exposure. Moreover, treatment with 25 {mu}M esculetin for 8 h increased the expression of NAD(P)H: quinone oxidoreductase (NQO1) at both protein and mRNA levels significantly, by 12-fold and 15-fold, respectively. Esculetin treatment also increased nuclear accumulation of Nrf2 by 8-fold indicating that increased NQO1 expression is Nrf2-mediated. These results indicate that esculetin protects human hepatoma HepG2 cells from hydrogen peroxide induced oxidative injury and that this protection is provided through the induction of protective enzymes as part of an adaptive response mediated by Nrf2 nuclear accumulation.« less

NDRG2 overexpression suppresses hepatoma cells survival during metabolic stress through disturbing the activation of fatty acid oxidation

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

Pan, Tao; Xi'an Medical University, Xi'an, Shaanxi Province; Zhang, Mei

Because of the high nutrient consumption and inadequate vascularization, solid tumor constantly undergoes metabolic stress during tumor development. Oncogenes and tumor suppressor genes participated in cancer cells' metabolic reprogramming. N-Myc downstream regulated gene 2 (NDRG2) is a recently identified tumor suppressor gene, but its function in cancer metabolism, particularly during metabolic stress, remains unclear. In this study, we found that NDRG2 overexpression significantly reduced hepatoma cell proliferation and enhanced cell apoptosis under glucose limitation. Moreover, NDRG2 overexpression aggravated energy imbalance and oxidative stress by decreasing the intracellular ATP and NADPH generation and increasing ROS levels. Strikingly, NDRG2 inhibited the activationmore » of fatty acid oxidation (FAO), which preserves ATP and NADPH purveyance in the absence of glucose. Finally, mechanistic investigation showed that NDRG2 overexpression suppressed the glucose-deprivation induced AMPK/ACC pathway activation in hepatoma cells, whereas the expression of a constitutively active form of AMPK abrogated glucose-deprivation induced AMPK activation and cell apoptosis. Thus, as a negative regulator of AMPK, NDRG2 disturbs the induction of FAO genes by glucose limitation, leading to dysregulation of ATP and NADPH, and thus reduces the tolerance of hepatoma cells to glucose limitation. - Highlights: • NDRG2 overexpression reduces the tolerance of hepatoma cells to glucose limitation. • NDRG2 overexpression aggravates energy imbalance and oxidative stress under glucose deprivation. • NDRG2 overexpression disturbs the activation of FAO in hepatoma cells under glucose limitation. • NDRG2 overexpression inhibits the activation of AMPK/ACC pathway in hepatoma cells during glucose starvation.« less

Coordinated pH/redox dual-sensitive and hepatoma-targeted multifunctional polymeric micelle system for stimuli-triggered doxorubicin release: Synthesis, characterization and in vitro evaluation.

PubMed

Wang, Lele; Tian, Baocheng; Zhang, Jing; Li, Keke; Liang, Yan; Sun, Yujie; Ding, Yuanyuan; Han, Jingtian

2016-03-30

Multifunctional polymeric micelles self-assembled from a DOX-conjugated methoxypolyethylene glycols-b-poly (6-O-methacryloyl-D-galactopyranose)-disulfide bond-DOX (mPEG-b-PMAGP-SS-DOX) copolymer were prepared as an antitumor carrier for doxorubicin delivery, of which the chemical modification with disulfide bonds and hydrazone bonds allowed micelles to release doxorubicin (DOX) selectively at acidic pH and high redox conditions. The resulting micelles exhibited coordinated pH/redox dual-sensitive and hepatoma-targeted multifunction with sustaining stability in aqueous media. The multifunctional micelles showed spherical shapes with a mean diameter of 93 ± 2.08 nm, a low polydispersity index (PDI) of 0.21, a low CMC value of 0.095 mg/mL, a high drug grafting degree of 56.9% and a drug content of 39.0%. Remarkably, in vitro drug release studies clearly exhibited a pH and redox dual-sensitive drug release profile with significantly accelerated drug release treated with pH 5.0 and 10mM GSH (88.4% in 72 h) without drug burst release. The tumor proliferation assays indicated that DOX-grafted micelles, along with low cytotoxicity and well biocompatibility to normal cells up to a concentration of 10 μg/mL, inhibited the proliferation of HepG2 cells in a formulation-, time- and concentration-dependent manner in comparison with MCF-7 cells which was similar to free DOX. Anticancer activity releaved that the disulfide-modified micelles possessed much higher anti-hepatoma activity with a low IC50 value of 1.1 μg/mL following a 72 h incubation. Furthermore, the intracellular uptake tested by CLSM and FCM demonstrated that multifunctional polymeric micelles could be more efficiently taken up by HepG2 cells compared with MCF-7 cells, agreed well with MTT assays, suggesting these well-defined micelles provide a potential drug delivery system for dual-responsive controlled drug release and enhanced anti-hepatoma therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

Bioactive chemical constituents of Curcuma longa L. rhizomes extract inhibit the growth of human hepatoma cell line (HepG2).

PubMed

Abdel-Lateef, Ezzat; Mahmoud, Faten; Hammam, Olfat; El-Ahwany, Eman; El-Wakil, Eman; Kandil, Sherihan; Abu Taleb, Hoda; El-Sayed, Mortada; Hassenein, Hanaa

2016-09-01

The present study was designed to identify the chemical constituents of the methanolic extract of Curcuma longa L. rhizomes and their inhibitory effect on a hepatoma cell line. The methanolic extract was subjected to GC-MS analysis to identify the volatile constituents and the other part of the same extract was subjected to liquid column chromatographic separation to isolate curcumin. The inhibition of cell growth in the hepatoma cell line and the cytopathological changes were studied. GC-MS analysis showed the presence of fifty compounds in the methanolic extract of C. longa. The major compounds were ar-turmerone (20.50 %), β-sesquiphellandrene (5.20 %) and curcumenol (5.11 %). Curcumin was identified using IR, 1H and 13C NMR. The inhibition of cell growth by curcumin (IC50 = 41.69 ± 2.87 μg mL-1) was much more effective than that of methanolic extract (IC50 = 196.12 ± 5.25 μg mL-1). Degenerative and apoptotic changes were more evident in curcumin- treated hepatoma cells than in those treated with the methanol extract. Antitumor potential of the methanolic extract may be attributed to the presence of sesquiterpenes and phenolic constituents including curcumin (0.051 %, 511.39 μg g-1 dried methanol extract) in C. longa rhizomes.

H32, a non-quinone sulfone analog of vitamin K3, inhibits human hepatoma cell growth by inhibiting Cdc25 and activating ERK.

PubMed

Kar, Siddhartha; Wang, Meifang; Ham, Seung Wook; Carr, Brian I

2006-10-01

We previously synthesized a K-vitamin derivative, Cpd 5, which was a potent growth inhibitor of human tumor cells, including Hep3B hepatoma cells. However, being a quinone compound, Cpd 5 has the potential for generating toxic reactive oxygen species (ROS). We therefore synthesized a nonquinone sulfone derivative, H32, which has a sufone group substituting the quinone. The IC50 of H32 for Hep3B cells was found to be 2.5 microM, which was 2.5 and 3.2 times more potent than Cpd 5 and vitamin K3 respectively. It induced apoptosis in Hep3B cells but did not generate ROS when compared to Cpd 5. Interestingly, under similar culture conditions, normal rat hepatocytes were 14-fold more and 7-fold more resistant to the growth inhibitory effects of H32 than Hep3B and PLC/PRF5 cells respectively. H32 preferentially inhibited the activities of the cell cycle controlling Cdc25A phosphatase likely by binding to its catalytic cysteine. As a consequence, it induced inhibitory tyrosine phosphorylation of the Cdc25 substrate kinases Cdk2 and Cdk4 in Hep3B cells and the cells undergo an arrest in the G1 phase of the cell cycle. H32 also induced persistent phosphorylation of the MAPK protein ERK1/2, but marginal JNK1/2 and p38 phosphorylation. The ERK inhibitor U0126, added at least 30 min prior to H32, antagonized the growth inhibition induced by H32. However, the JNK and p38 inhibitors, JNKI-II and SB203580, were not able to antagonize H32 induced growth inhibition. Thus, H32 differentially inhibited growth of normal and liver tumor cells by preferentially inhibiting the actions of Cdc25 phosphatases and inducing persistent ERK phosphorylation.

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

[Ursodeoxycholic acid induced apoptosis of human hepatoma cells HepG2 and SMMC-7721 bymitochondrial-mediated pathway].

PubMed

Wu, Duan; Zhou, Jianyin; Yin, Zhenyu; Liu, Pingguo; Zhao, Yilin; Liu, Jianming; Wang, Xiaomin

2014-12-02

To explore the effects and underlying mechanisms of ursodeoxycholic acid on human hepatoma cells. HepG2 and SMMC-7721 HCC cell lines were respectively treated with ursodeoxycholic acid. And cell proliferation, apoptosis and the expression of Bax/Bcl-2 gene were detected by methyl thiazolyl tetrazolium (MTT), inverted microscopy, fluorescent microscopy, flow cytometry and Western blot. Ursodeoxycholic acid significantly inhibited the proliferation of human hepatoma cells in a concentration- and time-dependent manner. The half maximal inhibitory concentrations (IC50) of HepG2 and SMMC-7721 were 397.3 and 387.7 µg/ml respectively after a 48-hour treatment of 400 µg /ml ursodeoxycholic acid. And it also induced the apoptosis of HepG2 and SMMC-7721 cells, up-regulated Bax gene and down-regulated Bcl-2 gene. Ursodeoxycholic acid inhibits the proliferation of hepatoma cells and induce apoptosis by mitochondrial-mediated pathway.

Effects of JS-K, a novel anti-cancer nitric oxide prodrug, on gene expression in human hepatoma Hep3B cells.

PubMed

Dong, Ray; Wang, Xueqian; Wang, Huan; Liu, Zhengyun; Liu, Jie; Saavedra, Joseph E

2017-04-01

JS-K is a novel anticancer nitric oxide (NO) prodrug effective against a variety of cancer cells, including the inhibition of AM-1 hepatoma cell growth in rats. To further evaluate anticancer effects of JS-K, human hepatoma Hep3B cells were treated with JS-K and the compound control JS-43-126 at various concentrations (0-100μM) for 24h, and cytotoxicity was determined by the MTS assay. The compound control JS-43-126 was not cytotoxic to Hep3B cells at concentrations up to 100μM, while the LC 50 for JS-K was about 10μM. To examine the molecular mechanisms of antitumor effects of JS-K, Hep3B cells were treated with 1-10μM of JS-K for 24h, and then subjected to gene expression analysis via real time RT-PCR and protein immunostain via confocal images. JS-K is a GST-α targeting NO prodrug, and decreased immunostaining for GST-α was associated with JS-K treatment. JS-K activated apoptosis pathways in Hep3B cells, including induction of caspase-3, caspase-9, Bax, TNF-α, and IL-1β, and immunostaining for caspase-3 was intensified. The expressions of thrombospondin-1 (TSP-1) and the tissue inhibitors of metalloproteinase-1 (TIMP-1) were increased by JS-K at both transcript and protein levels. JS-K treatment also increased the expression of differentiation-related genes CD14 and CD11b, and depressed the expression of c-myc in Hep3B cells. Thus, multiple molecular events appear to be associated with anticancer effects of JS-K in human hepatoma Hep3B cells, including activation of genes related to apoptosis and induction of genes involved in antiangiogenesis and tumor cell migration. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

[Effect of ERK/AP-1 signaling pathway on proliferation of hepatoma cells induced by PAR-2 agonists].

PubMed

Zheng, Yan-min; Xie, Li-qun; Li, Xuan; Zhao, Jun-yan; Chen, Xiao-yi; Chen, Li; Zhou, Jing; Li, Fei

2009-12-01

To investigate the expression of protease activated receptor-2 (PAR-2) in human HepG2 hepatoma cells and elucidate the effects of trypsin and PAR-2 agonist peptide SLIGKV-NH(2) upon the proliferation of hepatoma cells and its intracellular signaling mechanism. PAR-2 protein and mRNA expression were detected by immunofluorescence and RT-PCR. The cells were treated with SLIGKV-NH(2), trypsin, reverse PAR-2 agonist peptide VKGILS-NH(2) or PD98059. The changes of cell cycle distribution were evaluated by flow cytometry. The proliferative potential of HepG2 cells was estimated by MTT. The changes of PAR-2, c-fos and PCNA mRNA expression were detected by RT-PCR. The changes of c-fos and PCNA protein expression were detected by Western blotting. PAR-2 protein and mRNA were expressed in HepG2 cells. PAR-2 mRNA expression (PAR-2/beta-actin) were 0.70 +/- 0.04 and 0.99 +/- 0.05 respectively in cells treated with trypsin and SLIGKV-NH(2). They were both significantly higher than that in the control group (0.35 +/- 0.05, F = 135.534, P < 0.01). Percent G(0)/G(1) phase of HepG2 cells treated with trypsin or SLIGKV-NH(2) were significantly lower than those in the control group [(56.11 +/- 0.85)%, (57.85 +/- 0.46)% vs (79.12 +/- 0.67)%, both P < 0.01] Percent S phase, G(2)/M phase and proliferation index (PI) of HepG2 cells treated with trypsin or SLIGKV-NH(2) were significantly elevated (P < 0.01). The proliferation-enhancing effects and the up-regulation of mRNA and protein of c-fos and PCNA induced by trypsin or SLIGKV-NH(2) were significantly blocked by pretreatment with PD98059 (P < 0.01). There was no statistical significance in proliferation of HepG2 cells between the reverse PAR-2 agonist peptide VKGILS-NH(2) and control group (P > 0.05). PAR-2 is expressed in HepG2 hepatoma cells. PAR-2 activation induced by trypsin or SLIGKV-NH(2) promotes the proliferation of HepG2 cells partially via the ERK/AP-1 pathway.

[Transformation of attached cells derived from fetal umbilical cord blood induced by conditional medium of hepatoma cells].

PubMed

Zhang, Xiao-Dong; Cai, Na; Wang, Hong-Hui; Guo, Shi-Yi; Ye, Li-Hong

2006-01-01

Stem cells derived from fetal umbilical cord blood are of undifferentiated at early stage. They are sensitive to stimulations from the environment, and may be transformed under the effects of carcinogenic factors. This study was to explore the sensitivity of stem cells derived from fetal umbilical cord blood to carcinogenic factors. Mononuclear cells were isolated from fetal umbilical cord blood, and the attached cells were cultured in the medium containing 10% conditional medium of HepG2 hepatoma cells. A new cell line was gained, termed H-UCB. The biological features of H-UCB cells were detected by electron microscopy, karyotype analysis, cell cytometry, Western blot, and colony formation assay. H-UCB cells proliferated faster after passage 3. The cells were fibroblast-like and hepatocyte-like, with the ratio of nucleus to cytoplasm increased. Under electron microscope, many microvilli on the surface and numbers of vacuoles in the cytoplasm of the cells were observed, the nuclei were large and irregular, endocytosis phenomena were noticed. Karyotype analysis indicated that the cells were heteroploid, and the number of chromosomes was between 50 and 70. Flow cytometry data indicated that the proliferation period was 22.9 h, and the karyotype was between diploid and tetraploid. Western blot showed that c-Myc protein and proliferating cell nuclear antigen (PCNA) were overexpressed in H-UCB cells. According to flow cytometry, the positive rates of surface markers of H-UCB cells were 79.0% for CD105, 1.2% for CD34, and 12.2% for CD106; those of control HepG2 cells were 15.0% for CO105, 9.8% for CD34, and 1.4% for CD106. The colony formation rate of H-UCB cells in soft agar was (13.2+/-2.6)%. H-UCB cells are derived from endothelial cells, and are transformed as malignant cells with tumor cell characteristics.

Recent advances in live cell imaging of hepatoma cells

PubMed Central

2014-01-01

Live cell imaging enables the study of dynamic processes of living cells in real time by use of suitable reporter proteins and the staining of specific cellular structures and/or organelles. With the availability of advanced optical devices and improved cell culture protocols it has become a rapidly growing research methodology. The success of this technique relies mainly on the selection of suitable reporter proteins, construction of recombinant plasmids possessing cell type specific promoters as well as reliable methods of gene transfer. This review aims to provide an overview of the recent developments in the field of marker proteins (bioluminescence and fluorescent) and methodologies (fluorescent resonance energy transfer, fluorescent recovery after photobleaching and proximity ligation assay) employed as to achieve an improved imaging of biological processes in hepatoma cells. Moreover, different expression systems of marker proteins and the modes of gene transfer are discussed with emphasis on the study of lipid droplet formation in hepatocytes as an example. PMID:25005127

Spontaneous apoptosis in a rat hepatoma cell line and its inhibition by dexamethasone.

PubMed

Chaya, D; Fougère-Deschatrette, C; Weiss, M C

1996-01-01

Cultures of dedifferentiated rat hepatoma Rab1-5-1 cells exhibit spontaneously a high level of mortality during the exponential growth phase. We demonstrate that these cells die by apoptosis, showing chromatin condensation and internucleosomal DNA fragmentation. Cells of the original H4II cell line and of its differentiated and dedifferentiated derivatives also die by apoptosis, but only in heavily confluent cultures. We evaluated mortality with time in Rab1-5-1 cultures by establishing growth curves, including quantification of floating cells, and conclude that up to half of the cells in a culture are lost to apoptosis. The production of apoptotic cells is abolished by the presence of 10(-6) M dexamethasone and this inhibition is reversible in 48 hours. Rab1-5-1 cells that spontaneously die by apoptosis with high frequency represent a novel model to investigate factors that regulate the spontaneous frequency of death, and to study the nature and the kinetics of commitment to the apoptotic pathway.

Inhibition of Hepatitis B Virus and Induction of Hepatoma Cell Apoptosis by ASGPR-Directed Delivery of shRNAs

PubMed Central

Yao, Xinxin; Shi, Chuan; Sun, Lifang; Yuan, Lu; Lei, Ping; Zhu, Huifen; Liu, Hongbo; Wu, Xiongwen; Ning, Qin; Zhou, Chun; Shen, Guanxin

2012-01-01

Hepatitis B virus (HBV) infection is a worldwide liver disease and nearly 25% of chronic HBV infections terminate in hepatocellular carcinoma (HCC). Currently, there is no effective therapy to inhibit HBV replication and to eliminate hepatoma cells, making it highly desired to develop novel therapies for these two stages of the HBV-caused detrimental disease. Recently, short hairpin RNA (shRNA) has emerged as a potential therapy for virus-infected disease and cancer. Here, we have generated a shRNA, pGenesil-siHBV4, which effectively inhibits HBV replication in the human hepatoma cell line HepG2.2.15. The inhibitory effects of pGenesil-siHBV4 are manifested by the decrease of both the HBV mRNA level and the protein levels of the secreted HBV surface antigen (HBsAg) and HBV e antigen (HBeAg), and by the reduction of secreted HBV DNA. Using mouse hydrodynamic tail vein injection, we demonstrate that pGenesil-siHBV4 is effective in inhibiting HBV replication in vivo. Because survivin plays a key role in cancer cell escape from apoptosis, we further generated pGenesil-siSurvivin, a survivin-silencing shRNA, and showed its effect of triggering apoptosis of HBV-containing hepatoma cells. To develop targeted shRNA therapy, we have identified that as a specific binder of the asialoglycoprotein receptor (ASGPR), jetPEI-Hepatocyte delivers pGenesil-siHBV4 and pGenesil-siSurvivin specifically to hepatocytes, not other types of cells. Finally, co-transfection of pGenesil-siHBV4 and pGenesil-siSurvivin exerts synergistic effects in inducing hepatoma cell apoptosis, a novel approach to eliminate hepatoma by downregulating survivin via multiple mechanisms. The application of these novel shRNAs with the jetPEI-Hepatocyte targeting strategy demonstrates the proof-of-principle for a promising approach to inhibit HBV replication and eliminate hepatoma cells with high specificity. PMID:23094023

Ethanolic Extract of Agaricus blazei Fermentation Product Inhibits the Growth and Invasion of Human Hepatoma HA22T/VGH and SK-Hep-1 Cells

PubMed Central

Tung, Yen-Chen; Su, Zheng-Yuan; Kuo, Min-Liang; Sheen, Lee-Yan

2012-01-01

Hepatoma is a leading cause of death in the world. SK-Hep-1 and HA22T/VGH cells are poorly differentiated human hepatocellular carcinoma cell lines with invasive and migratory abilities. Agaricus blazei (AB) is a mushroom with many biological effects and active ingredients, and the ethanolic extract of AB fermentation product (AB-pE) was demonstrated to inhibit the growth of hepatoma Hep3B and HepG2 cells in our previous study. In this study, we further investigated the anticancer and anti-invasive abctivities of the AB-pE. Results showed that the AB-pE inhibited the growth of SK-Hep1 and HA22T/VGH cells (with IC50 values of 26.8 and 28.7 μg/mL, respectively) and led cells toward apoptosis after 48 h of treatment. Activation of caspase-3 by AB-pE (12.5~200 μg/mL) in a dose-dependent manner was observed in both cell lines using fluorescence microscopy and flow cytometry. The apoptosis triggered by the AB-pE was regulated by the increased expression of Bax, the activation of caspase-3, caspase-9, and PARP, and the decreased expression of Bcl-2. Additionally, the AB-pE showed the potential ability to inhibit invasion of SK-Hep1 and HA22T/VGH cells according to the results of a Matrigel invasion assay. Our results suggested that the AB-pE may be a further developed for its potential against hepatoma due to its antiproliferative (via apoptosis) and anti-invasive activities in hepatoma cells. PMID:24716127

Effects of β-caryophyllene and Murraya paniculata essential oil in the murine hepatoma cells and in the bacteria and fungi 24-h time-kill curve studies.

PubMed

Selestino Neta, Maria Cipriano; Vittorazzi, Catia; Guimarães, Aline Cristina; Martins, João Damasceno Lopes; Fronza, Marcio; Endringer, Denise Coutinho; Scherer, Rodrigo

2017-12-01

Orange Jessamine [Murraya paniculata L. (Rutaceae)] has been used worldwide in folk medicine as an anti-inflammatory, antibiotic and analgesic. The objective of this study is to investigate the in vitro antioxidant, cytotoxic, antibacterial and antifungal activity and the time-kill curve studies of orange jessamine essential oil and β-caryophyllene, as well as the chemical composition of the essential oil. The cytotoxic activity of M. paniculata and β-caryophyllene (7.8-500 μg/mL) was evaluated using the MTT assay on normal fibroblasts and hepatoma cells. The minimal inhibitory concentration and time-kill curves (24 h) were evaluated against those of Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Enterococcus faecallis, Aspergillus (niger, fumigates and parasiticum) and F. solani by the broth microdilution method. The antioxidant activity was measured by the DPPH and ABTS assays. Chemical composition was evaluated by GC/MS analyses. GC/MS analyses identified 13 compounds, with β-caryophyllene as the major compound. The oil exhibited moderate antibacterial activity (MIC <1.0 mg/mL) and strong antifungal activity. Time-kill curve studies showed that either the essential oil or β-caryophyllene presented rapid bacterial killing (4 h for S. aureus) and fungicidal effect (2-4 h for F. solani); however, both displayed weak free radical scavenger capacity. The cytotoxic activity exhibited a prominent selective effect against hepatoma cancer cells (IC 50 value =63.7 μg/mL) compared with normal fibroblasts (IC 50 value =195.0 μg/mL), whereas the β-caryophyllene showed low cytotoxicity. The experimental data suggest that the activities of M. paniculata essential oil are due to the synergistic action among its components.

Knockdown of hepatoma-derived growth factor-related protein-3 induces apoptosis of H1299 cells via ROS-dependent and p53-independent NF-κB activation

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

Yun, Hong Shik; Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791; Baek, Jeong-Hwa

2014-07-11

Highlights: • HRP-3 is a radiation- and anticancer drug-responsive protein in H1299 cells. • Depletion of HRP-3 induces apoptosis of radio- and chemoresistant H1299 cells. • Depletion of HRP-3 promotes ROS generation via inhibition of the Nrf2/HO-1 pathway. • ROS generation enhances NF-κB activity, which acts as an upstream signal in the c-Myc/Noxa apoptotic pathway. - Abstract: We previously identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistant biomarker in p53 wild-type A549 cells and found that p53-dependent induction of the PUMA pathway was a critical event in regulating the radioresistant phenotype. Here, we found that HRP-3 knockdown regulates themore » radioresistance of p53-null H1299 cells through a distinctly different molecular mechanism. HRP-3 depletion was sufficient to cause apoptosis of H1299 cells by generating substantial levels of reactive oxygen species (ROS) through inhibition of the Nrf2/HO-1 antioxidant pathway. Subsequent, ROS-dependent and p53-independent NF-κB activation stimulated expression of c-Myc and Noxa proteins, thereby inducing the apoptotic machinery. Our results thus extend the range of targets for the development of new drugs to treat both p53 wild-type or p53-null radioresistant lung cancer cells.« less

Cell damage of hepatoma-22 cells exposed to continuous wave ultrasound.

PubMed

Wang, Pan; Wang, Xiaobing; Liu, Quanhong

2012-01-01

The cellular response of hepatoma-22 cells to ultrasonic irradiation and the potential cause for the action were evaluated. Hepatoma-22 cells were subjected to ultrasound irradiation at a frequency of 2.17 MHz and a spatial average intensity of 1.6 W/cm2 for variable periods of time, and several biological parameters were analyzed. The terephthalic acid (TA) dosimetry method was used to evaluate the efficacies of irradiation parameters on the acoustic cavitation activity by monitoring hydroxyl radical (OH) production. Lactate dehydrogenase (LDH) leakage was assayed to investigate cell membrane integrity. The polarization value of fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was measured to monitor plasma membrane fluidity. The malonaldehyde content in cells was determined to reflect lipid peroxidation. Trypan blue exclusion was used to detect cell viability. Additionally, electron microscopy was used to observe morphological changes. The generation of intracellular reactive oxygen species, mitochondria swelling and the loss of mitochondria membrane potential were also investigated. The results showed that 1) the concentration of ·OH production by ultrasonic irradiation in air-saturated cell suspensions increased as ultrasound exposure time increased; 2) compared with control, lactate dehydrogenase leakage, the polarization value of 1,6-diphenyl-1,3,5-hexatriene, malonaldehyde content and cell lysis were significantly elevated when cells were treated by ultrasound for 60 s; 3) cytotoxicity by ultrasound irradiation was also accompanied by an increase in production of intracellular reactive oxygen species and dissipation of mitochondria membrane potential as well as by mitochondria swelling. Presently available information indicates that the plasma membrane and mitochondria are the main targets for ultrasound treatment, and free radicals formation such as ·OH due to ultrasound cavitation may play an important role in mediating these cellular response

Berberine Suppresses Cyclin D1 Expression through Proteasomal Degradation in Human Hepatoma Cells.

PubMed

Wang, Ning; Wang, Xuanbin; Tan, Hor-Yue; Li, Sha; Tsang, Chi Man; Tsao, Sai-Wah; Feng, Yibin

2016-11-15

The aim of this study is to explore the underlying mechanism on berberine-induced Cyclin D1 degradation in human hepatic carcinoma. We observed that berberine could suppress both in vitro and in vivo expression of Cyclin D1 in hepatoma cells. Berberine exhibits dose- and time-dependent inhibition on Cyclin D1 expression in human hepatoma cell HepG2. Berberine increases the phosphorylation of Cyclin D1 at Thr286 site and potentiates Cyclin D1 nuclear export to cytoplasm for proteasomal degradation. In addition, berberine recruits the Skp, Cullin, F-box containing complex-β-Transducin Repeat Containing Protein (SCF β-TrCP ) complex to facilitate Cyclin D1 ubiquitin-proteasome dependent proteolysis. Knockdown of β-TrCP blocks Cyclin D1 turnover induced by berberine; blocking the protein degradation induced by berberine in HepG2 cells increases tumor cell resistance to berberine. Our results shed light on berberine's potential as an anti-tumor agent for clinical cancer therapy.

Berberine Suppresses Cyclin D1 Expression through Proteasomal Degradation in Human Hepatoma Cells

PubMed Central

Wang, Ning; Wang, Xuanbin; Tan, Hor-Yue; Li, Sha; Tsang, Chi Man; Tsao, Sai-Wah; Feng, Yibin

2016-01-01

The aim of this study is to explore the underlying mechanism on berberine-induced Cyclin D1 degradation in human hepatic carcinoma. We observed that berberine could suppress both in vitro and in vivo expression of Cyclin D1 in hepatoma cells. Berberine exhibits dose- and time-dependent inhibition on Cyclin D1 expression in human hepatoma cell HepG2. Berberine increases the phosphorylation of Cyclin D1 at Thr286 site and potentiates Cyclin D1 nuclear export to cytoplasm for proteasomal degradation. In addition, berberine recruits the Skp, Cullin, F-box containing complex-β-Transducin Repeat Containing Protein (SCFβ-TrCP) complex to facilitate Cyclin D1 ubiquitin-proteasome dependent proteolysis. Knockdown of β-TrCP blocks Cyclin D1 turnover induced by berberine; blocking the protein degradation induced by berberine in HepG2 cells increases tumor cell resistance to berberine. Our results shed light on berberine′s potential as an anti-tumor agent for clinical cancer therapy. PMID:27854312

Galactose-functionalized multi-responsive nanogels for hepatoma-targeted drug delivery

NASA Astrophysics Data System (ADS)

Lou, Shaofeng; Gao, Shan; Wang, Weiwei; Zhang, Mingming; Zhang, Ju; Wang, Chun; Li, Chen; Kong, Deling; Zhao, Qiang

2015-02-01

We report here a hepatoma-targeting multi-responsive biodegradable crosslinked nanogel, poly(6-O-vinyladipoyl-d-galactose-ss-N-vinylcaprolactam-ss-methacrylic acid) P(ODGal-VCL-MAA), using a combination of enzymatic transesterification and emulsion copolymerization for intracellular drug delivery. The nanogel exhibited redox, pH and temperature-responsive properties, which can be adjusted by varying the monomer feeding ratio. Furthermore, the volume phase transition temperature (VPTT) of the nanogels was close to body temperature and can result in rapid thermal gelation at 37 °C. Scanning electron microscopy also revealed that the P(ODGal-VCL-MAA) nanogel showed uniform spherical monodispersion. With pyrene as a probe, the fluorescence excitation spectra demonstrated nanogel degradation in response to glutathione (GSH). X-ray diffraction (XRD) showed an amorphous property of DOX within the nanogel, which was used in this study as a model anti-cancer drug. Drug-releasing characteristics of the nanogel were examined in vitro. The results showed multi-responsiveness of DOX release by the variation of environmental pH values, temperature or the availability of GSH, a biological reductase. An in vitro cytotoxicity assay showed a higher anti-tumor activity of the galactose-functionalized DOX-loaded nanogels against human hepatoma HepG2 cells, which was, at least in part, due to specific binding between the galactose segments and the asialoglycoprotein receptors (ASGP-Rs) in hepatic cells. Confocal laser scanning microscopy (CLSM) and flow cytometric profiles further confirmed elevated cellular uptake of DOX by the galactose-functionalised nanogels. Thus, we report here a multi-responsive P(ODGal-VCL-MAA) nanogel with a hepatoma-specific targeting ability for anti-cancer drug delivery.We report here a hepatoma-targeting multi-responsive biodegradable crosslinked nanogel, poly(6-O-vinyladipoyl-d-galactose-ss-N-vinylcaprolactam-ss-methacrylic acid) P(ODGal-VCL-MAA), using

Inhibitory effects of α-pinene on hepatoma carcinoma cell proliferation.

PubMed

Chen, Wei-Qiang; Xu, Bin; Mao, Jian-Wen; Wei, Feng-Xiang; Li, Ming; Liu, Tao; Jin, Xiao-Bao; Zhang, Li-Rong

2014-01-01

Pine needle oil from crude extract of pine needles has anti-tumor effects, but the effective component is not known. In the present study, compounds from a steam distillation extract of pine needles were isolated and characterized. Alpha-pinene was identified as an active anti-proliferative compound on hepatoma carcinoma BEL-7402 cells using the MTT assay. Further experiments showed that α-pinene inhibited BEL-7402 cells by arresting cell growth in the G2/M phase of the cell cycle, downregulating Cdc25C mRNA and protein expression, and reducing cycle dependence on kinase 1(CDK1) activity. Taken together, these findings indicate that α-pinene may be useful as a potential anti-tumor drug.

Characterization of genetically engineered mouse hepatoma cells with inducible liver functions by overexpression of liver-enriched transcription factors.

PubMed

Yamamoto, Hideaki; Tonello, Jane Marie; Sambuichi, Takanori; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

2018-01-01

New cell sources for the research and therapy of organ failure could significantly alleviate the shortage of donor livers that are available to patients who suffer from liver disease. Liver carcinoma derived cells, or hepatoma cells, are the ideal cells for developing bioartificial liver systems. Such cancerous liver cells are easy to prepare in large quantities and can be maintained over long periods under standard culture conditions, unlike primary hepatocytes. However, hepatoma cells possess only a fraction of the functions of primary hepatocytes. In a previous study, by transducing cells with liver-enriched transcription factors that could be inducibly overexpressed-hepatocyte nuclear factor (HNF)1α, HNF1β, HNF3β [FOXA2], HNF4α, HNF6, CCAAT/enhancer binding protein (C/EBP)α, C/EBPβ and C/EBPγ-we created mouse hepatoma cells with high liver-specific gene expression called the Hepa/8F5 cell line. In the present study, we performed functional and genetic analyses to characterize the Hepa/8F5 cell line. Further, in three-dimensional cultures, the function of these cells improved significantly compared to parental cells. Ultimately, these cells might become a new resource that can be used in basic and applied hepatic research. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Fucoxanthin Enhances Cisplatin-Induced Cytotoxicity via NFκB-Mediated Pathway and Downregulates DNA Repair Gene Expression in Human Hepatoma HepG2 Cells

PubMed Central

Liu, Cheng-Ling; Lim, Yun-Ping; Hu, Miao-Lin

2013-01-01

Cisplain, a platinum-containing anticancer drug, has been shown to enhance DNA repair and to inhibit cell apoptosis, leading to drug resistance. Thus, the combination of anticancer drugs with nutritional factors is a potential strategy for improving the efficacy of cisplatin chemotherapy. In this study, we investigated the anti-proliferative effects of a combination of fucoxanthin, the major non-provitamin A carotenoid found in Undaria Pinnatifida, and cisplatin in human hepatoma HepG2 cells. We found that fucoxanthin (1–10 μΜ) pretreatment for 24 h followed by cisplatin (10 μΜ) for 24 h significantly decreased cell proliferation, as compared with cisplatin treatment alone. Mechanistically, we showed that fucoxanthin attenuated cisplatin-induced NFκB expression and enhanced the NFκB-regulated Bax/Bcl-2 mRNA ratio. Cisplatin alone induced mRNA expression of excision repair cross complementation 1 (ERCC1) and thymidine phosphorylase (TP) through phosphorylation of ERK, p38 and PI3K/AKT pathways. However, fucoxanthin pretreatment significantly attenuated cisplatin-induced ERCC1 and TP mRNA expression, leading to improvement of chemotherapeutic efficacy of cisplatin. The results suggest that a combined treatment with fucoxanthin and cisplatin could lead to a potentially important new therapeutic strategy against human hepatoma cells. PMID:23299493

Caveolin-1 Confers Resistance of Hepatoma Cells to Anoikis by Activating IGF-1 Pathway.

PubMed

Tang, Wenqing; Feng, Xuemei; Zhang, Si; Ren, Zhenggang; Liu, Yinkun; Yang, Biwei; lv, Bei; Cai, Yu; Xia, Jinglin; Ge, Ningling

2015-01-01

Anoikis resistance is a prerequisite for hepatocellular carcinoma (HCC) metastasis. The role of Caveolin-1 (CAV1) in anoikis resistance of HCC remains unclear. The oncogenic effect of CAV1 on anchor-independent growth and anoikis resistance was investigated by overexpression and knockdown of CAV1 in hepatoma cells. IGF-1 pathway and its downstream signals were detected by immunoblot analysis. Caveolae invagination and IGF-1R internalization was studied by electron microscopy and (125)I-IGF1 internalization assay, respectively. The role of IGF-1R and tyrosine-14 residue (Y-14) of CAV1 was explored by deletion experiment and mutation experiment, respectively. The correlation of CAV1 and IGF-1R was further examined by immunochemical analysis in 120 HCC specimens. CAV1 could promote anchor-independent growth and anoikis resistance in hepatoma cells. CAV1-overexpression increased the expression of IGF-1R and subsequently activated PI3K/Akt and RAF/MEK/ERK pathway, while CAV1 knockdown showed the opposite effect. The mechanism study revealed that CAV1 facilitated caveolae invagination and (125)I-IGF1 internalization. IGF-1R deletion or Y-14 mutation reversed CAV1 mediated anchor-independent growth and anoikis resistance. In addition, CAV1 expression was positively related to IGF-1R expression in human HCC tissues. CAV1 confers resistance of hepatoma cells to anoikis by activating IGF-1 pathway, providing a potential therapeutic target for HCC metastasis. © 2015 S. Karger AG, Basel.

Analysis of the cytotoxicity of carbon-based nanoparticles, diamond and graphite, in human glioblastoma and hepatoma cell lines.

PubMed

Zakrzewska, Karolina Ewa; Samluk, Anna; Wierzbicki, Mateusz; Jaworski, Sławomir; Kutwin, Marta; Sawosz, Ewa; Chwalibog, André; Pijanowska, Dorota Genowefa; Pluta, Krzysztof Dariusz

2015-01-01

Nanoparticles have attracted a great deal of attention as carriers for drug delivery to cancer cells. However, reports on their potential cytotoxicity raise questions of their safety and this matter needs attentive consideration. In this paper, for the first time, the cytotoxic effects of two carbon based nanoparticles, diamond and graphite, on glioblastoma and hepatoma cells were compared. First, we confirmed previous results that diamond nanoparticles are practically nontoxic. Second, graphite nanoparticles exhibited a negative impact on glioblastoma, but not on hepatoma cells. The studied carbon nanoparticles could be a potentially useful tool for therapeutics delivery to the brain tissue with minimal side effects on the hepatocytes. Furthermore, we showed the influence of the nanoparticles on the stable, fluorescently labeled tumor cell lines and concluded that the labeled cells are suitable for drug cytotoxicity tests.

Hypoxia/hepatoma dual specific suicide gene expression plasmid delivery using bio-reducible polymer for hepatocellular carcinoma therapy.

PubMed

Kim, Hyun Ah; Nam, Kihoon; Lee, Minhyung; Kim, Sung Wan

2013-10-10

Gene therapy is suggested as a promising alternative strategy of hepatocellular carcinoma (HCC, also called hepatoma) therapy. To achieve a successful and safe gene therapy, tight regulation of gene expression is required to minimize side-effects in normal tissues. In this study, we developed a novel hypoxia and hepatoma dual specific gene expression vector. The constructed vectors were transfected into various cell lines using bio-reducible polymer, PAM-ABP. First, pAFPS-Luc or pAFPL-Luc vector was constructed with the alpha-fectoprotein (AFP) promoter and enhancer for hepatoma tissue specific gene expression. Then, pEpo-AFPL-Luc was constructed by insertion of the erythropoietin (Epo) enhancer for hypoxic cancer specific gene expression. In vitro transfection assay showed that pEpo-AFPL-Luc transfected hepatoma cell increased gene expression under hypoxic condition. To confirm the therapeutic effect of dual specific vector, herpes simplex virus thymidine kinase (HSV-TK) gene was introduced for cancer cell killing. The pEpo-AFPL-TK was transfected into hepatoma cell lines in the presence of ganciclovir (GCV) pro-drug. Caspase-3/7, MTT and TUNEL assays elucidated that pEpo-AFPL-TK transfected cells showed significant increasing of death rate in hypoxic hepatoma cells compared to controls. Therefore, the hypoxia/hepatoma dual specific gene expression vector with the Epo enhancer and AFP promoter may be useful for hepatoma specific gene therapy. © 2013.

BWTG3 hepatoma cells can acquire phenylalanine hydroxylase, cystathionine synthase and CPS-I without genetic manipulation, but activation of the silent OTC gene requires cell fusion with hepatocytes.

PubMed

Farmer, A A; Goss, S J

1991-04-01

The mouse hepatoma BWTG3 has been tested for its ability to grow in three different media that select for traits normally expressed in adult liver: homocysteine medium to select for cystathionine synthase (CS), tyrosine-free medium for phenylalanine hydroxylase (PH), and ornithine medium for carbamylphosphate synthetase-I (CPS-I) and ornithine transcarbamylase (OTC). In no case were the cells immediately capable of bulk growth, showing that all these traits were in some degree deficient. However, the cultures in homocysteine medium and in tyrosine-free medium both gave rise, spontaneously, to growing clones with frequencies of approximately 10(-3) and 10(-5), respectively. The deficiencies of CS and PH were accordingly excluded from further study, in view of their inherent instability. In contrast, no colonies ever formed in ornithine medium. Though neither CPS-I nor OTC were detectable in stock BWTG3 cells, it was found that CPS-I was readily inducible by hormones. The deficiency of OTC, however, appeared to be totally stable showing no reversion in response either to hormones or to azacytidine treatment. This deficiency was investigated by fusing the hepatoma to OTC+ liver cells prepared from normal or sparse-fur (spf) mice. Sparse-fur mice were used because their OTC is mutant and has a distinctive pH-dependence. OTC+ hybrids were readily produced, without the need for any specific selection for OTC, and, in one case at least, with only minimal chromosome segregation. In all the OTC+ hybrids made with spf cells, there was clear reactivation of the wild-type, hepatoma-derived OTC gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the Cytotoxicity of Carbon-Based Nanoparticles, Diamond and Graphite, in Human Glioblastoma and Hepatoma Cell Lines

PubMed Central

Wierzbicki, Mateusz; Jaworski, Sławomir; Kutwin, Marta; Sawosz, Ewa; Chwalibog, André; Pijanowska, Dorota Genowefa; Pluta, Krzysztof Dariusz

2015-01-01

Nanoparticles have attracted a great deal of attention as carriers for drug delivery to cancer cells. However, reports on their potential cytotoxicity raise questions of their safety and this matter needs attentive consideration. In this paper, for the first time, the cytotoxic effects of two carbon based nanoparticles, diamond and graphite, on glioblastoma and hepatoma cells were compared. First, we confirmed previous results that diamond nanoparticles are practically nontoxic. Second, graphite nanoparticles exhibited a negative impact on glioblastoma, but not on hepatoma cells. The studied carbon nanoparticles could be a potentially useful tool for therapeutics delivery to the brain tissue with minimal side effects on the hepatocytes. Furthermore, we showed the influence of the nanoparticles on the stable, fluorescently labeled tumor cell lines and concluded that the labeled cells are suitable for drug cytotoxicity tests. PMID:25816103

Release of overexpressed CypB activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress.

PubMed

Kim, Kiyoon; Kim, Hunsung; Jeong, Kwon; Jung, Min Hyung; Hahn, Bum-Soo; Yoon, Kyung-Sik; Jin, Byung Kwan; Jahng, Geon-Ho; Kang, Insug; Ha, Joohun; Choe, Wonchae

2012-08-01

Cyclophilin, a cytosolic receptor for the immunosuppressive drug cyclosporin A, plays a role in diverse pathophysiologies along with its receptor, CD147. Although the interaction between cyclophilin A and CD147 is well established in inflammatory disease, that of cyclophilin B (CypB) with CD147 has not been fully explored, especially in cancer cell biology, and the exact molecular mechanism underlying such an association is poorly understood. In this study, we first identified high expression levels of CypB in 54 % of hepatocellular carcinoma patient tissues but in only 12.5 % of normal liver tissues. Then, we demonstrated that CypB overexpression protects human hepatoma cells against oxidative stress through its binding to CD147; this protective effect depends on the peptidyl prolyl isomerase activity of CypB. siRNA-mediated knockdown of CypB expression rendered hepatoma cells more vulnerable to ROS-mediated apoptosis. Furthermore, we also determined that a direct interaction between secreted CypB and CD147 regulates the extracellular signal-regulated kinase intracellular signaling pathway and is indispensible for the protective functions of CypB. For the first time, we demonstrated that CypB has an essential function in protecting hepatoma cells against oxidative stress through binding to CD147 and regulating the ERK pathway.

Synergistic effects of acyclic retinoid and OSI-461 on growth inhibition and gene expression in human hepatoma cells.

PubMed

Shimizu, Masahito; Suzui, Masumi; Deguchi, Atsuko; Lim, Jin T E; Xiao, Danhua; Hayes, Julia H; Papadopoulos, Kyriakos P; Weinstein, I Bernard

2004-10-01

Hepatoma is one of the most frequently occurring cancers worldwide. However, effective chemotherapeutic agents for this disease have not been developed. Acyclic retinoid, a novel synthetic retinoid, can reduce the incidence of postsurgical recurrence of hepatoma and improve the survival rate. OSI-461, a potent derivative of exisulind, can increase intracellular levels of cyclic GMP, which leads to activation of protein kinase G and induction of apoptosis in cancer cells. In the present study, we examined the combined effects of acyclic retinoid plus OSI-461 in the HepG2 human hepatoma cell line. We found that the combination of as little as 1.0 micromol/L acyclic retinoid and 0.01 micromol/L OSI-461 exerted synergistic inhibition of the growth of HepG2 cells. Combined treatment with low concentrations of these two agents also acted synergistically to induce apoptosis in HepG2 cells through induction of Bax and Apaf-1, reduction of Bcl-2 and Bcl-xL, and activation of caspase-3, -8, and -9. OSI-461 enhanced the G0-G1 arrest caused by acyclic retinoid, and the combination of these agents caused a synergistic decrease in the levels of expression of cyclin D1 protein and mRNA, inhibited cyclin D1 promoter activity, decreased the level of hyperphosphorylated forms of the Rb protein, induced increased cellular levels of the p21(CIP1) protein and mRNA, and stimulated p21(CIP1) promoter activity. Moreover, OSI-461 enhanced the ability of acyclic retinoid to induce increased cellular levels of retinoic acid receptor beta and to stimulate retinoic acid response element-chloramphenicol acetyltransferase activity. A hypothetical model involving concerted effects on p21(CIP1) and retinoic acid receptor beta expression is proposed to explain these synergistic effects. Our results suggest that the combination of acyclic retinoid plus OSI-461 might be an effective regimen for the chemoprevention and chemotherapy of human hepatoma and possibly other malignancies.

[Effect of polychromatic visible light combined with infrared radiation on tumorigenicity of murine hepatoma cells and their sensitivity to lytic activity of natural killers].

PubMed

Filatova, N A; Kniazev, N A; Kosheverova, V V; Shatrova, A N; Samoĭlova, K A

2013-01-01

Tumorigenicity of murine hepatoma cells (MH22a) and their sensitivity to lysis by natural killers (NKs) have been studied after exposure to polychromatic visible and infrared light (VIS-IR, 480-3400 nm, 40 mW/cm2), similar to the terrestrial solar spectrum without its minor UV component, in order to elucidate the involvement of this important environmental and physiotherapeutic factor in regulation of the anti-tumor defense system. The MH22 cells were in vitro exposed to VIS-IR light and their sensitivity to lytic activity of NKs was evaluated. We found that sensitivity of MH22a cells to lysis by NKs after exposure to VIS-IR light at a dose of 4.8 J/cm2 increased 1.5-2 times, while it did not change after exposure to a dose of 9.6 J/cm2 at all ratios (1 : 5-1 : 50) of the number of NKs (effectors) to that of hepatoma cells (targets). The increase in the sensitivity of hepatoma cells to NKs was accompanied by structural changes of cell surface: the capability of supramembraneous glycoproteins (glycocalix) to sorb the vital dye alcian blue (AB) was significantly lower as compared with the unexposed cells of control group. However, no changes in AB sorption was revealed in hepatoma cells exposed to the light at a dose 9.6 J/cm2. Tumorigenicity of photo-irradiated MH22a cells has been studied in the in vivo experiments. Light-exposed (4.8 and 9.6 J/cm2) and intact hepatoma cells were transplanted into syngenic mice C3HA. Tumor volumes 25 days after transplantation proved to be smaller after exposure to the light at both doses than in the control group (4-4.5 times and 2.5-4 times, respectively), which correlated with the increase in the sensitivity to lisys by NKs and decrease in the AB sorption only after light exposure at dose 4.8 J/cm2. Using the flow cytometry method we could show that VIS-IR light at the applied doses did not interfere with the distribution of hepatoma cells over the cycle phases and thus deceleration of the tumor growth was not associated with

Enhanced migration of tissue inhibitor of metalloproteinase overexpressing hepatoma cells is attributed to gelatinases: Relevance to intracellular signaling pathways

PubMed Central

Roeb, Elke; Bosserhoff, Anja-Katrin; Hamacher, Sabine; Jansen, Bettina; Dahmen, Judith; Wagner, Sandra; Matern, Siegfried

2005-01-01

AIM: To study the effect of gelatinases (especially MMP-9) on migration of tissue inhibitor of metalloproteinase (TIMP-1) overexpressing hepatoma cells. METHODS: Wild type HepG2 cells, cells stably transfected with TIMP-1 and TIMP-1 antagonist (MMP-9-H401A, a catalytically inactive matrix metalloproteinase (MMP) which still binds and neutralizes TIMP-1) were incubated in Boyden chambers either with or without Galardin (a synthetic inhibitor of MMP-1, -2, -3, -8, -9) or a specific inhibitor of gelatinases. RESULTS: Compared to wild type HepG2 cells, the cells overexpressing TIMP-1 showed 115% migration (P<0.05) and the cells overexpressing MMP-9-H401A showed 62% migration (P<0.01). Galardin reduced cell migration dose dependently in all cases. The gelatinase inhibitor reduced migration in TIMP-1 overexpressing cells predominantly. Furthermore, we examined intracellular signal transduction pathways of TIMP-1-dependent HepG2 cells. TIMP-1 deactivates cell signaling pathways of MMP-2 and MMP-9 involving p38 mitogen-activated protein kinase. Specific blockade of the ERK pathway suppresses gelatinase expression either in the presence or absence of TIMP-1. CONCLUSION: Overexpressing functional TIMP-1- enhanced migration of HepG2-TIMP-1 cells depends on enhanced MMP-activity, especially MMP-9. PMID:15754388

Molecular switch of Cre/loxP for radiation modulated gene therapy on hepatoma

NASA Astrophysics Data System (ADS)

Hsieh, Ya-Ju; Chen, Fu-Du; Wang, Fu Hui; Ke, Chien Chih; Wang, Hsin-Ell; Liu, Ren-Shyan

2007-02-01

For the purpose of enhancement of AFP promoter for the use of radiation modulated gene therapy for hepatocellular carcinoma (HCC), we combined hepatitis B virus (HBV) enhancer II with AFP promoter which shows the selectivity to the target cells to control the Cre/loxP system. Different gene constructs, pE4luc, pE4Tk, EIIAPA-Cre, E4CMV-STOP-Tk and chimeric promoters combined with HBV enhancer were constructed and transfected into HepG2, HeLa and NIH-3T3 cell lines. Cell experiments revealed that E4 enhancer responses to radiation best after 60 h irradiation at a dose range of 5-7 Gy in HepG2 stable clone. The EIIAPA promoter provided high specificity to hepatoma and activated the Cre downstream and removed the stop cassette only in hepatoma cells. After removal of the stop cassette, the E4 response to radiation could encode more Tk protein and kill more tumor cells. In summary, the chimeric EIIAPA promoter can stringently control the expression of Cre recombinase only in HCC. The radiation effect of the EIIAPA-Cre and E4CMV-STOP-Tk system shows promising results in terms of cell survival of HCC.

Dioxin-like activity of brominated dioxins as individual compounds or mixtures in in vitro reporter gene assays with rat and mouse hepatoma cell lines.

PubMed

Suzuki, G; Nakamura, M; Michinaka, C; Tue, N M; Handa, H; Takigami, H

2017-10-01

In vitro reporter gene assays detecting dioxin-like compounds have been developed and validated since the middle 1990's, and applied to the determination of dioxin-like activities in various samples for their risk management. Data on characterizing the potency of individual brominated dioxins and their activity in mixture with chlorinated dioxins are still limited on the cell-based assay. This study characterized the dioxin-like activities of the 32 brominated dioxins, such as polybrominated dibenzo-p-dioxins, polybrominated dibenzofurans (PBDFs), coplanar polybrominated biphenyls, mixed halogenated dibenzo-p-dioxins and dibenzofurans (PXDFs), as a sole component or in a mixture by DR-CALUX (dioxin-responsive chemically activated luciferase expression) using the rat hepatoma H4IIE cell line and XDS-CALUX (xenobiotic detection systems-chemically activated luciferase expression) assays using the mouse hepatoma H1L6.1 cell line. The 2,3,7,8-TCDD-relative potencies (REPs) of most of the brominated dioxins were within a factor of 10 of the WHO toxicity equivalency factor (WHO-TEF) for the chlorinated analogues. The REPs of a few PXDFs were an order of magnitude higher than the corresponding WHO-TEFs, indicating their toxicological importance. Results with reconstituted mixtures suggest that the activity of brominated and chlorinated dioxins in both CALUX assays was dose-additive. Thus, obtained results indicated the applicability of the CALUX assays as screening tools of brominated dioxins together with their chlorinated analogues. Copyright © 2017 Elsevier Ltd. All rights reserved.

AMF/PGI transactivates the MMP-3 gene through the activation of Src-RhoA-phosphatidylinositol 3-kinase signaling to induce hepatoma cell migration.

PubMed

Shih, Wen-Ling; Liao, Ming-Huei; Yu, Feng-Ling; Lin, Ping-Yuan; Hsu, Hsue-Yin; Chiu, Shu-Jun

2008-11-08

We have previously shown that AMF/PGI induces hepatoma cell migration through the induction of MMP-3. This work investigates how AMF/PGI activates the MMP-3 gene. We demonstrated that AMF/PGI transactivates the MMP-3 gene promoter through AP-1. The transactivation and induction of cell migration effect of AMF/PGI directly correlates with its enzymatic activity. Various analyses showed that AMF/PGI stimulated the Src-RhoA-PI3-kinase signaling pathway, and these three signaling molecules could form a complex. Our results demonstrate a new mechanism of AMF/PGI-induced cell migration and a link between Src-RhoA-PI3-kinase, AP-1, MMP-3 and hepatoma cell migration.

[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.

Interaction of tributyltin with 3,3',4,4',5-pentachlorobiphenyl-induced ethoxyresorufin O-deethylase activity in rat hepatoma cells.

PubMed

Kannan, K; Villeneuve, D L; Blankenship, A L; Giesy, J P

1998-11-13

Interaction of tributyltin (TBT) with 3,3',4,4',5-pentachlorobiphenyl (PCB-126)-induced ethoxyresorufin O-deethylase (EROD) activity was examined in vitro using H4IIE rat hepatoma cells. H4IIE cells were exposed to TBT and PCB-126, individually or in combination, at different concentrations. TBT was cytotoxic at concentrations greater than 98 nM. PCB-126 was not cytotoxic in the concentration range of 49 to 3140 pM. At concentrations greater than 49 nM, PCB-126 enhanced the cytotoxicity of TBT in the 24-98 nM range. In the absence of inducers of EROD activity, TBT significantly inhibited constitutive EROD activity in H4IIE cells in a concentration-dependent manner. EROD activity in H4IIE cells was significantly increased by exposure to PCB-126 alone. This effect was potentiated by coexposure to low, noncytotoxic concentrations of TBT. The induction of cytochrome P-4501A (CYP1A) activity in the presence of both an inducer (PCB-126) and low concentrations of an inhibitor (TBT) indicates that TBT does not interfere with the Ah receptor binding, but acts at the transcriptional level. Potentiation of EROD activity and cytotoxicity as a consequence of coexposure to PCB-126 and TBT is of considerable toxicological significance, given their coaccumulation in a variety of marine organisms.

Establishment of a rat hepatoma-derived cell line proliferating in D-phenylalanine medium and expressing D-amino-acid oxidase.

PubMed

Yoda, N; Konno, R; Nagashima, S

2001-01-01

A cell line (R-Y121B.DF) has been established from a cell line (R-Y121B) derived from a rat hepatoma line (H4-II-E). The R-Y121B.DF cells have been continuously cultured in a serum-free modified Eagle's minimum essential medium in which L-phenylalanine was replaced by D-phenylalanine. They had D-amino-acid oxidase (DAO) activity which is essential for the growth in the medium containing D-amino acids. The enzyme activity of the R-Y121B.DF cells was approximately one-fourth of that of the rat liver. Northern hybridization using a DAO cDNA probe detected a hybridizing signal in the R-Y121B.DF cells and the rat liver but not in the parental R-Y121B and H4-II-E cells. Reverse transcription-polymerase chain reaction using DAO-specific primers amplified a DNA fragment of the expected size in the R-Y121B.DF cells but not in the R-Y121B and H4-II-E cells. This fragment was confirmed to be DAO cDNA by nucleotide sequencing. Western blotting showed that DAO protein was present in the R-Y121B.DF cells and the rat liver but not in the R-Y121B and H4-II-E cells. Southern hybridization showed that the DAO gene structure was not different among the R-Y121B.DF cells, R-Y121B cells, H4-II-E cells, and the rat liver. These results indicate that the R-Y121B.DF is a unique cell line which proliferates in the medium containing D-phenylalanine and explicitly expresses DAO. This line is useful for the study of DAO in vitro.

INDUCTION OF AN ESTROGEN-RESPONSIVE REPORTER GENE IN RAINBOW TROUT HEPATOMA CELLS (RTH 149) AT 11 OR 18 DEGREES C

EPA Science Inventory

A reporter gene assay in a cultured rainbow trout cell line was used to determine the influence of temperature on the expression of an estrogen-responsive gene. Rainbow trout hepatoma cells (RTH 149) incubated at 11 or 18 degrees C were co-transfected with an estrogen-responsive ...

Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line.

PubMed Central

Takahashi, Shigeru; Matsuura, Naomi; Kurokawa, Takako; Takahashi, Yuji; Miura, Takashi

2002-01-01

We reported previously that the 5'-flanking region (nucleotides -1976 to -1655) of the human haem oxygenase-1 ( hHO-1 ) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231-235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50 bp fragment (-1793 to -1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to -1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells. PMID:12133007

Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line.

PubMed

Takahashi, Shigeru; Matsuura, Naomi; Kurokawa, Takako; Takahashi, Yuji; Miura, Takashi

2002-11-01

We reported previously that the 5'-flanking region (nucleotides -1976 to -1655) of the human haem oxygenase-1 ( hHO-1 ) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231-235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50 bp fragment (-1793 to -1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to -1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells.

Knock down of GCN5 histone acetyltransferase by siRNA decreases ethanol-induced histone acetylation and affects differential expression of genes in human hepatoma cells.

PubMed

Choudhury, Mahua; Pandey, Ravi S; Clemens, Dahn L; Davis, Justin Wade; Lim, Robert W; Shukla, Shivendra D

2011-06-01

We have investigated whether Gcn5, a histone acetyltransferase (HAT), is involved in ethanol-induced acetylation of histone H3 at lysine 9 (H3AcK9) and has any effect on the gene expression. Human hepatoma HepG2 cells transfected with ethanol-metabolizing enzyme alcohol dehydrogenase 1 (VA 13 cells) were used. Knock down of Gcn5 by siRNA silencing decreased mRNA and protein levels of general control nondepressible 5 (GCN5), HAT activity, and also attenuated ethanol-induced H3AcK9 in VA13 cells. Illumina gene microarray analysis using total RNA showed 940 transcripts affected by GCN5 silencing or ethanol. Silencing caused differential expression of 891 transcripts (≥1.5-fold upregulated or downregulated). Among these, 492 transcripts were upregulated and 399 were downregulated compared with their respective controls. Using a more stringent threshold (≥2.5-fold), the array data from GCN5-silenced samples showed 57 genes differentially expressed (39 upregulated and 18 downregulated). Likewise, ethanol caused differential regulation of 57 transcripts with ≥1.5-fold change (35 gene upregulated and 22 downregulated). Further analysis showed that eight genes were differentially regulated that were common for both ethanol treatment and GCN5 silencing. Among these, SLC44A2 (a putative choline transporter) was strikingly upregulated by ethanol (three fold), and GCN5 silencing downregulated it (1.5-fold). The quantitative real-time polymerase chain reaction profile corroborated the array findings. This report demonstrates for the first time that (1) GCN5 differentially affects expression of multiple genes, (2) ethanol-induced histone H3-lysine 9 acetylation is mediated via GCN5, and (3) GCN5 is involved in ethanol-induced expression of the putative choline transporter SLC44A2. Copyright © 2011 Elsevier Inc. All rights reserved.

Inhibition of glutathione synthesis eliminates the adaptive response of ascitic hepatoma 22 cells to nedaplatin that targets thioredoxin reductase

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

Wang, Yijun; Lu, Hongjuan; Wang, Dongxu

2012-12-15

Thioredoxin reductase (TrxR) is a target for cancer therapy and the anticancer mechanism of cisplatin involves TrxR inhibition. We hypothesize that the anticancer drug nedaplatin (NDP), an analogue of cisplatin and a second-generation platinum complex, also targets TrxR. Furthermore, we investigate whether the therapeutic efficacy of NDP can be enhanced by simultaneous modulation of 1) TrxR, via NDP, and 2) glutathione (GSH), via the GSH synthesis inhibitor buthionine sulfoximine (BSO). Mice bearing ascitic hepatoma 22 (H22) cells were treated with NDP alone or NDP plus BSO. TrxR activity of H22 cells was inhibited by NDP in a dose-dependent manner. Amore » high correlation between the inhibition of TrxR activity at 6 h and the inhibition of ascitic fluid volume at 72 h was established (r = 0.978, p < 0.01). As an adaptive response, the viable ascitic cancer cells after NDP treatment displayed an enlarged cell phenotype, assembled with several-fold more antioxidant enzymes and GSH-predominant non-protein free thiols. This adaptive response was largely eliminated when BSO was co-administered with NDP, leading to the decimation of the H22 cell population without enhancing renal toxicity, since at this dose, NDP did not inhibit renal TrxR activity. In conclusion, the pharmacological effect of NDP involves TrxR inhibition, and the adaptive response of NDP-treated ascitic H22 cells can be efficiently counteracted by BSO. Simultaneous modulation of TrxR and GSH on ascitic H22 cells using NDP plus BSO greatly enhances therapeutic efficacy as compared with the single modulation of TrxR using NDP alone. -- Highlights: ► Nedaplatin at a pharmacological dose inhibits TrxR in cancer cells but not in kidney. ► The nedaplatin-treated cancer cells exhibit adaptive response. ► Buthionine sulfoximine inhibits glutathione in both cancer cells and kidney. ► Buthionine sulfoximine counteracts the adaptive response to the nedaplatin treatment. ► Buthionine sulfoximine does

Species-specific differences in peroxisome proliferation, catalase, and SOD2 upregulation as well as toxicity in human, mouse, and rat hepatoma cells induced by the explosive and environmental pollutant 2,4,6-trinitrotoluene.

PubMed

Naumenko, Ekaterina Anatolevna; Ahlemeyer, Barbara; Baumgart-Vogt, Eveline

2017-03-01

2,4,6-Trinitrotoluene (TNT) has been widely used as an explosive substance and its toxicity is still of interest as it persisted in polluted areas. TNT is metabolized in hepatocytes which are prone to its toxicity. Since analysis of the human liver or hepatocytes is restricted due to ethical reasons, we investigated the effects of TNT on cell viability, reactive oxygen species (ROS) production, peroxisome proliferation, and antioxidative enzymes in human (HepG2), mouse (Hepa 1-6), and rat (H4IIEC3) hepatoma cell lines. Under control conditions, hepatoma cells of all three species were highly comparable exhibiting identical proliferation rates and distribution of their cell cycle phases. However, we found strong differences in TNT toxicity with the lowest IC 50 values (highest cell death rate) for rat cells, whereas human and mouse cells were three to sevenfold less sensitive. Moreover, a strong decrease in cellular dehydrogenase activity (MTT assay) and increased ROS levels were noted. TNT caused peroxisome proliferation with rat hepatoma cells being most responsive followed by those from mouse and human. Under control conditions, rat cells contained fivefold higher peroxisomal catalase and mitochondrial SOD2 activities and a twofold higher capacity to reduce MTT than human and mouse cells. TNT treatment caused an increase in catalase and SOD2 mRNA and protein levels in human and mouse, but not in rat cells. Similarly, human and mouse cells upregulated SOD2 activity, whereas rat cells failed therein. We conclude that TNT induced oxidative stress, peroxisome proliferation and mitochondrial damage which are highest in rat cells rendering them most susceptible toward TNT. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 989-1006, 2017. © 2016 Wiley Periodicals, Inc.

Hepatoma targeting peptide conjugated bio-reducible polymer complexed with oncolytic adenovirus for cancer gene therapy

PubMed Central

Choi, Joung-Woo; Kim, Hyun Ah; Nam, Kihoon; Na, Youjin; Yun, Chae-Ok; Kim, SungWan

2015-01-01

Despite adenovirus (Ad) vector’s numerous advantages for cancer gene therapy, such as high ability of endosomal escape, efficient nuclear entry mechanism, and high transduction, and therapeutic efficacy, tumor specific targeting and antiviral immune response still remain as a critical challenge in clinical setting. To overcome these obstacles and achieve cancer-specific targeting, we constructed tumor targeting bioreducible polymer, an arginine grafted bio-reducible polymer (ABP)-PEG-HCBP1, by conjugating PEGylated ABP with HCBP1 peptides which has high affinity and selectivity towards hepatoma. The ABP-PEG-HCBP1-conjugated replication incompetent GFP-expressing ad, (Ad/GFP)-ABP-PEG-HCBP1, showed a hepatoma cancer specific uptake and transduction compared to either naked Ad/GFP or Ad/GFP-ABP. Competition assays demonstrated that Ad/GFP-ABP-PEG-HCBP1-mediated transduction was specifically inhibited by HCBP1 peptide rather than coxsackie and adenovirus receptor specific antibody. In addition, ABP-PEG-HCBP1 can protect biological activity of Ad against serum, and considerably reduced both innate and adaptive immune response against Ad. shMet-expressing oncolytic Ad (oAd; RdB/shMet) complexed with ABP-PEG-HCBP1 delivered oAd efficiently into hepatoma cancer cells. The oAd/ABP-PEG-HCBP1 demonstrated enhanced cancer cell killing efficacy in comparison to oAd/ABP complex. Furthermore, Huh7 and HT1080 cancer cells treated with oAd/shMet-ABP-PEG-HCBP1 complex had significantly decreased Met and VEGF expression in hepatoma cancer, but not in non-hepatoma cancer. In sum, these results suggest that HCBP1-conjugated bioreducible polymer could be used to deliver oncolytic Ad safely and efficiently to treat hepatoma. PMID:26437261

Hepatic Arterial Infusion of Low-Density Lipoprotein Docosahexaenoic Acid Nanoparticles Selectively Disrupts Redox Balance in Hepatoma cells and Reduces Growth of Orthotopic Liver Tumors in Rats

PubMed Central

Wen, Xiaodong; Reynolds, Lacy; Mulik, Rohit S.; Kim, Soo Young; Van Treuren, Tim; Nguyen, Liem H.; Zhu, Hao; Corbin, Ian R.

2015-01-01

Background & Aims Dietary intake of the natural omega-3 fatty acid docosahexaenoic acid (DHA) has been implicated in protecting patients with viral hepatitis B or C from developing hepatocellular carcinoma (HCC). Little is known about the effects of DHA on established solid tumors. Herein, we describe a low-density lipoprotein (LDL)-based nanoparticle that acts as a transporter for unesterified DHA (LDL–DHA) and demonstrates selective cytotoxicity towards HCC cells. We investigated the ability of LDL–DHA to reduce growth of orthotopic hepatomas in rats. Methods ACI rats were given intrahepatic injections of rat hepatoma cells (H4IIE); 24 tumor-bearing rats (mean tumor diameter, ~1 cm) were subject to a single hepatic artery injection of LDL nanoparticles (2 mg/kg) loaded with DHA (LDL–DHA), triolein (LDL–TO) or sham surgery controls. Tumor growth was measured by magnetic resonance imaging and other methods; tumor, liver and serum samples were collected and assessed by histochemical, immunofluorescence, biochemical and immunoblot analyses. Results Three days after administration of LDL–TO or sham surgery, the control rats had large, highly vascularized tumors that contained proliferating cells. However, rats given LDL–DHA had smaller, pale tumors that were devoid of vascular supply and greater than 80% of the tumor tissue was necrotic. Four to 6 days after injection of LDL–DHA, the tumors were 3-fold smaller than those of control rats. The liver tissue that surrounded the tumors showed no histologic or biochemical evidence of injury. Injection of LDL–DHA into the hepatic artery of rats selectively deregulated redox reactions in tumor tissues by: increasing levels of reactive oxygen species and lipid peroxidation, depleting and oxidizing glutathione and nicotinamide adenine dinucleotide phosphate, and significantly downregulating the antioxidant enzyme glutathione peroxidase-4. Remarkably, the redox balance in the surrounding liver was not disrupted

Hepatic Arterial Infusion of Low-Density Lipoprotein Docosahexaenoic Acid Nanoparticles Selectively Disrupts Redox Balance in Hepatoma Cells and Reduces Growth of Orthotopic Liver Tumors in Rats.

PubMed

Wen, Xiaodong; Reynolds, Lacy; Mulik, Rohit S; Kim, Soo Young; Van Treuren, Tim; Nguyen, Liem H; Zhu, Hao; Corbin, Ian R

2016-02-01

Dietary intake of the natural omega-3 fatty acid docosahexaenoic acid (DHA) has been implicated in protecting patients with viral hepatitis B or C from developing hepatocellular carcinoma (HCC). Little is known about the effects of DHA on established solid tumors. Here we describe a low-density lipoprotein-based nanoparticle that acts as a transporter for unesterified DHA (LDL-DHA) and demonstrates selective cytotoxicity toward HCC cells. We investigated the ability of LDL-DHA to reduce growth of orthotopic hepatomas in rats. AxC-Irish (ACI) rats were given intrahepatic injections of rat hepatoma cells (H4IIE); 24 tumor-bearing rats (mean tumor diameter, ∼1 cm) were subject to a single hepatic artery injection of LDL nanoparticles (2 mg/kg) loaded with DHA (LDL-DHA), triolein (LDL-TO), or sham surgery controls. Tumor growth was measured by magnetic resonance imaging and other methods; tumor, liver, and serum samples were collected and assessed by histochemical, immunofluorescence, biochemical, and immunoblot analyses. Three days after administration of LDL-TO or sham surgery, the control rats had large, highly vascularized tumors that contained proliferating cells. However, rats given LDL-DHA had smaller, pale tumors that were devoid of vascular supply and >80% of the tumor tissue was necrotic. Four to 6 days after injection of LDL-DHA, the tumors were 3-fold smaller than those of control rats. The liver tissue that surrounded the tumors showed no histologic or biochemical evidence of injury. Injection of LDL-DHA into the hepatic artery of rats selectively deregulated redox reactions in tumor tissues by increasing levels of reactive oxygen species and lipid peroxidation, depleting and oxidizing glutathione and nicotinamide adenine dinucleotide phosphate, and significantly down-regulating the antioxidant enzyme glutathione peroxidase-4. Remarkably, the redox balance in the surrounding liver was not disrupted. LDL-DHA nanoparticle selectively kills hepatoma cells

The oncoprotein HBXIP suppresses gluconeogenesis through modulating PCK1 to enhance the growth of hepatoma cells.

PubMed

Shi, Hui; Fang, Runping; Li, Yinghui; Li, Leilei; Zhang, Weiying; Wang, Huawei; Chen, Fuquan; Zhang, Shuqin; Zhang, Xiaodong; Ye, Lihong

2016-11-28

Hepatitis B X-interacting protein (HBXIP) as an oncoprotein plays crucial roles in the development of cancer, involving glucose metabolism reprogramming. In this study, we are interested in whether the oncoprotein HBXIP is involved in the modulation of gluconeogenesis in liver cancer. Here, we showed that the expression level of phosphoenolpyruvate carboxykinase (PCK1), a key enzyme of gluconeogenesis, was lower in clinical hepatocellular carcinoma (HCC) tissues than that in normal tissues. Mechanistically, HBXIP inhibited the expression of PCK1 through down-regulating transcription factor FOXO1 in hepatoma cells, and up-regulated miR-135a targeting the 3'UTR of FOXO1 mRNA in the cells. In addition, HBXIP increased the phosphorylation levels of FOXO1 protein by activating PI3K/Akt pathway, leading to the export of FOXO1 from nucleus to cytoplasm. Strikingly, over-expression of PCK1 could abolish the HBXIP-promoted growth of hepatoma cells in vitro and in vivo. Thus, we conclude that the oncoprotein HBXIP is able to depress the gluconeogenesis through suppressing PCK1 to promote hepatocarcinogenesis, involving miR-135a/FOXO1 axis and PI3K/Akt/p-FOXO1 pathway. Our finding provides new insights into the mechanism by which oncoprotein HBXIP modulates glucose metabolism reprogramming in HCC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Hepatitis C virus Core overcomes all-trans retinoic acid-induced apoptosis in human hepatoma cells by inhibiting p14 expression via DNA methylation

PubMed Central

Kwak, Juri; Choi, Jung-Hye; Jang, Kyung Lib

2017-01-01

All-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, is known to induce p14 expression via promoter hypomethylation to activate the p14-MDM2-p53 pathway, which leads to activation of the p53-dependent apoptotic pathway and subsequent induction of apoptosis in human hepatoma cells. In the present study, we found that hepatitis C virus (HCV) Core derived from ectopic expression or HCV infection overcomes ATRA-induced apoptosis in p53-positive hepatoma cells. For this effect, HCV Core upregulated both protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b and thereby repressed p14 expression via promoter hypermethylation, resulting in inactivation of the pathway leading to p53 accumulation in the presence of ATRA. As a result, HCV Core prevented ATRA from activating several apoptosis-related molecules, including Bax, p53 upregulated modulator of apoptosis, caspase-9, caspase-3, and poly (ADP-ribose) polymerase. In addition, complementation of p14 in the Core-expressing cells by either ectopic expression or treatment with 5-Aza-2′dC almost completely abolished the potential of HCV Core to suppress ATRA-induced apoptosis. Based on these observations, we conclude that HCV Core executes its oncogenic potential by suppressing the p53-dependent apoptosis induced by ATRA in human hepatoma cells. PMID:29156743

Hepatoma targeting peptide conjugated bio-reducible polymer complexed with oncolytic adenovirus for cancer gene therapy.

PubMed

Choi, Joung-Woo; Kim, Hyun Ah; Nam, Kihoon; Na, Youjin; Yun, Chae-Ok; Kim, SungWan

2015-12-28

Despite adenovirus (Ad) vector's numerous advantages for cancer gene therapy, such as high ability of endosomal escape, efficient nuclear entry mechanism, and high transduction, and therapeutic efficacy, tumor specific targeting and antiviral immune response still remain as a critical challenge in clinical setting. To overcome these obstacles and achieve cancer-specific targeting, we constructed tumor targeting bioreducible polymer, an arginine grafted bio-reducible polymer (ABP)-PEG-HCBP1, by conjugating PEGylated ABP with HCBP1 peptides which has high affinity and selectivity towards hepatoma. The ABP-PEG-HCBP1-conjugated replication incompetent GFP-expressing ad, (Ad/GFP)-ABP-PEG-HCBP1, showed a hepatoma cancer specific uptake and transduction compared to either naked Ad/GFP or Ad/GFP-ABP. Competition assays demonstrated that Ad/GFP-ABP-PEG-HCBP1-mediated transduction was specifically inhibited by HCBP1 peptide rather than coxsackie and adenovirus receptor specific antibody. In addition, ABP-PEG-HCBP1 can protect biological activity of Ad against serum, and considerably reduced both innate and adaptive immune response against Ad. shMet-expressing oncolytic Ad (oAd; RdB/shMet) complexed with ABP-PEG-HCBP1 delivered oAd efficiently into hepatoma cancer cells. The oAd/ABP-PEG-HCBP1 demonstrated enhanced cancer cell killing efficacy in comparison to oAd/ABP complex. Furthermore, Huh7 and HT1080 cancer cells treated with oAd/shMet-ABP-PEG-HCBP1 complex had significantly decreased Met and VEGF expression in hepatoma cancer, but not in non-hepatoma cancer. In sum, these results suggest that HCBP1-conjugated bioreducible polymer could be used to deliver oncolytic Ad safely and efficiently to treat hepatoma. Copyright © 2015 Elsevier B.V. All rights reserved.

Coordinated modulation of albumin synthesis and mRNA levels in cultured hepatoma cells by hydrocortisone and cyclic AMP analogs

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

Brown, P.C.; Papaconstantinou, J.

The treatment of Hepa-2 cells, a permanent mouse hepatoma cell line, for 72 h with hydrocortisone (10/sup -6/ M), N/sup 6/,O/sup 2/-dibutyryl cyclic AMP (10/sup -3/ M), or 8-bromo cyclic AMP(10/sup -3/ M) results in a 2-, 3-, or 4-fold increase, respectively, in rates of synthesis and secretion of mouse serum albumin. Simultaneous treatment with hydrocortisone and N/sup 6/,O/sup 2/-dibutyryl cyclic AMP results in a 10-fold stimulation in these parameters, an effect that is significantly more than additive for the two compounds tested. The number of albumin mRNA sequences, determined by hybridization of total cell RNA to albumin complementary DNA,more » was increased in direct proportion to the increases in albumin synthesis in all experiments. The relative rate of albumin synthesis approaches in vivo levels in cells treated simultaneously with hydrocortisone and N/sup 6/,O/sup 2/-dibutyryl cyclic AMP. We propose that these factors may be necessary to maintain the maximal level of differentiated function in the continuous culture of Hepa-2 cells.« less

CD147 stimulates hepatoma cells escaping from immune surveillance of T cells by interaction with Cyclophilin A.

PubMed

Ren, Yi-Xin; Wang, Shu-Jing; Fan, Jian-Hui; Sun, Shi-Jie; Li, Xia; Padhiar, Arshad Ahmed; Zhang, Jia-Ning

2016-05-01

T cells play an important role in tumor immune surveillance. CD147 is a member of immunoglobulin superfamily present on the surface of many tumor cells and mediates malignant cell behaviors. Cyclophilin A (CypA) is an intracellular protein promoting inflammation when released from cells. CypA is a natural ligand for CD147. In this study, CD147 specific short hairpin RNAs (shRNA) were transfected into murine hepatocellular carcinoma Hepa1-6 cells to assess the effects of CD147 on hepatoma cells escaping from immune surveillance of T cells. We found extracellular CypA stimulated cell proliferation through CD147 by activating ERK1/2 signaling pathway. Downregulation of CD147 expression on Hepa1-6 cells significantly suppressed tumor progression in vivo, and decreased cell viability when co-cultured with T cells in vitro. Importantly, knockdown of CD147 on Hepa1-6 cells resulted in significantly increased T cells chemotaxis induced by CypA both in vivo and in vitro. These findings provide novel mechanisms how tumor cells escaping from immune surveillance of T cells. We provide a potential therapy for hepatocellular carcinoma by targeting CD147 or CD147-CypA interactions. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Pokemon silencing leads to Bim-mediated anoikis of human hepatoma cell QGY7703.

PubMed

Liu, Kun; Liu, Feng; Zhang, Nannan; Liu, Shiying; Jiang, Yuyang

2012-01-01

Pokemon is an important proto-oncogene that plays a critical role in cellular oncogenic transformation and tumorigenesis. Anoikis, which is regulated by Bim-mediated apoptosis, is critical to cancer cell invasion and metastasis. We investigated the role of Pokemon in anoikis, and our results show that Pokemon renders liver cells resistant to anoikis via suppression of Bim transcription. We knocked-down Pokemon in human hepatoma cells QGY7703 with small interfering RNAs (siRNA). Knockdown of Pokemon alone did not significantly affect the growth and survival of QGY7703 cells but notably enhanced their sensitivity to apoptotic stress due to the presence of chemical agents or cell detachment, thereby inducing anoikis, as evidenced by flow cytometry and caspase-3 activity assays. In contrast, ectopic expression of Pokemon in HL7702 cells led to resistance to anoikis. Dual-luciferase reporter and ChIP assays illustrated that Pokemon suppressed Bim transcription via direct binding to its promoter. Our results suggest that Pokemon prevents anoikis through the suppression of Bim expression, which facilitates tumor cell invasion and metastasis. This Pokemon-Bim pathway may be an effective target for therapeutic intervention for cancer.

Pokemon Silencing Leads to Bim-Mediated Anoikis of Human Hepatoma Cell QGY7703

PubMed Central

Liu, Kun; Liu, Feng; Zhang, Nannan; Liu, Shiying; Jiang, Yuyang

2012-01-01

Pokemon is an important proto-oncogene that plays a critical role in cellular oncogenic transformation and tumorigenesis. Anoikis, which is regulated by Bim-mediated apoptosis, is critical to cancer cell invasion and metastasis. We investigated the role of Pokemon in anoikis, and our results show that Pokemon renders liver cells resistant to anoikis via suppression of Bim transcription. We knocked-down Pokemon in human hepatoma cells QGY7703 with small interfering RNAs (siRNA). Knockdown of Pokemon alone did not significantly affect the growth and survival of QGY7703 cells but notably enhanced their sensitivity to apoptotic stress due to the presence of chemical agents or cell detachment, thereby inducing anoikis, as evidenced by flow cytometry and caspase-3 activity assays. In contrast, ectopic expression of Pokemon in HL7702 cells led to resistance to anoikis. Dual-luciferase reporter and ChIP assays illustrated that Pokemon suppressed Bim transcription via direct binding to its promoter. Our results suggest that Pokemon prevents anoikis through the suppression of Bim expression, which facilitates tumor cell invasion and metastasis. This Pokemon-Bim pathway may be an effective target for therapeutic intervention for cancer. PMID:22754333

Synergistic cytotoxicity and mechanism of caffeine and lysozyme on hepatoma cell line HepG2

NASA Astrophysics Data System (ADS)

Yang, Hongchao; Li, Jingjuan; Cui, Lin; Ren, Yanqing; Niu, Liying; Wang, Xinguo; Huang, Yun; Cui, Lijian

2018-03-01

The influences of caffeine, lysozyme and the joint application of them on the hepatoma cell line HepG2 proliferation inhibition and cell apoptosis were observed by 3-(4, 5-dimethyl-2-thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and Hoechst 33342, which showed the proliferation inhibition rate of the joint application on HepG2 cells was 47.21%, significantly higher than caffeine or lysozyme, and the joint application promoted the apoptosis of HepG2 cells obviously. Van't Hoff classical thermodynamics formula, the Föster theory of non-radiation energy transfer and fluorescence phase diagram were used to manifest that the process of lysozyme binding to caffeine followed a two-state model, which was spontaneous at low temperature driven by enthalpy change, and the predominant intermolecular force was hydrogen bonding or Van der Waals force to stabilize caffeine-lysozyme complex with the distance 5.86 nm. The attenuated total reflection-Fourier transform infrared spectra indicated that caffeine decreased the relative contents of α-helix and β-turn, which inferred the structure of lysozyme tended to be "loose". Synchronous fluorescence spectra and ultraviolet spectra supported the above conclusion. The amino acid residues in the cleft of lysozyme were exposed and electropositivity was increased attributing to the loose structure, which were conducive to increasing caffeine concentration on the HepG2 cell surface by electrostatic interaction to show synergistic effect. The great quantities of microvilli on the liver cancer cell membrane surface, is beneficial for the lysozyme-caffeine compound to aggregate on cell surface to increase the concentration of caffeine to play stronger physiological role by electrostatic effect.

Zinc Mesoporphyrin Induces Rapid Proteasomal Degradation of Hepatitis C Nonstructural 5A Protein in Human Hepatoma Cells

PubMed Central

Hou, Weihong; Tian, Qing; Zheng, Jianyu; Bonkovsky, Herbert L.

2009-01-01

Background & Aims The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV), plays a critical role in HCV replication and is an attractive target for the therapy of HCV infection. So far, little is known about the post-translational regulation of NS5A protein and its precise role in HCV RNA replication. Our objectives were to elucidate the down-regulation of NS5A protein and HCV RNA replication by zinc mesoporphyrin (ZnMP), and the mechanism by which this process occurs. Methods Human hepatoma cells expressing HCV proteins were used to investigate the post-translational regulation of ZnMP on NS5A protein by Western blots (WB) and immunoprecipitation (IP). Quantitative RT-PCR (qRT-PCR) was used to determine the effects of ZnMP on HCV RNA replication. Results ZnMP selectively and markedly down-regulated NS5A protein levels by increasing degradation of NS5A protein [half life fell from 18.7 h to 2.7 h]. The proteasome inhibitors, epoxomicin and MG132, significantly abrogated degradation of NS5A protein by ZnMP without affecting levels of NS5A in the absence of ZnMP. Analysis of immunoprecipitates with an anti-ubiquitin antibody revealed polyubiquitination of NS5A, suggesting that ZnMP induces ubiquitination of NS5A protein. In addition, 10 μM of ZnMP reduced HCV replication by ~63% in the Con1 replicon cells, ~70% in J6/JFH1 HCV transfected cells, and ~90% in J6/JFH1 HCV infected cells without affecting cell viability. Conclusions ZnMP produces a rapid and profound down-regulation of the NS5A protein by enhancing its polyubiquitination and proteasome-dependent catabolism. Zinc mesoporphyrin may hold promise as a novel agent to treat HCV infection. PMID:19909748

Antioxidative and apoptotic properties of polyphenolic extracts from edible part of artichoke (Cynara scolymus L.) on cultured rat hepatocytes and on human hepatoma cells.

PubMed

Miccadei, Stefania; Di Venere, Donato; Cardinali, Angela; Romano, Ferdinando; Durazzo, Alessandra; Foddai, Maria Stella; Fraioli, Rocco; Mobarhan, Sohrab; Maiani, Giuseppe

2008-01-01

Cultured rat hepatocytes and human hepatoma HepG2 cells were used to evaluate the hepatoprotective properties of polyphenolic extracts from the edible part of artichoke (AE). The hepatocytes were exposed to H2O2generated in situ by glucose oxidase and were treated with either AE, or pure chlorogenic acid (ChA) or with the well known antioxidant, N, N'-diphenyl-p-phenilenediamine (DPPD). Addition of glucose oxidase to the culture medium caused depletion of intracellular glutathione (GSH) content, accumulation of malondialdehyde (MDA) in the cultures, as a lipid peroxidation indicator, and cell death. These results demonstrated that AE protected cells from the oxidative stress caused by glucose oxidase, comparable to DPPD. Furthermore, AE, as well as ChA, prevented the loss of total GSH and the accumulation of MDA. Treatment of HepG2 cells for 24 h with AE reduced cell viability in a dose-dependent manner, however, ChA had no prominent effects on the cell death rate. Similarly, AE rather than ChA induced apoptosis, measured by flow cytometric analysis of annexin and by activation of caspase-3, in HepG2 cells. Our findings indicate that AE had a marked antioxidative potential that protects hepatocytes from an oxidative stress. Furthermore, AE reduced cell viability and had an apoptotic activity on a human liver cancer cell line.

Vinclozolin, a widely used fungizide, enhanced BaP-induced micronucleus formation in human derived hepatoma cells by increasing CYP1A1 expression.

PubMed

Wu, Xin-Jiang; Lu, Wen-qing; Roos, Peter H; Mersch-Sundermann, Volker

2005-10-15

Vinclozolin, a widely used fungicide, can be identified as a residue in numerous vegetable and fruit samples. To get insight in its genetic toxicity, we investigated the genotoxic effect of vinclozolin in the human derived hepatoma cell line HepG2 using the micronucleus (MN) assay. Additionally, to evaluate the co- or anti-mutagenic potency of vinclozolin, we treated HepG2 cells with different concentrations of vinclozolin for 24 h. Subsequently, the cells were exposed to benzo[a]pyrene (BaP) for 1h. Exposure of HepG2 cells to 50-400 microM vinclozolin alone did not cause any induction of micronuclei. However, a pronounced co-mutagenic effect was observed. MN frequencies caused by BaP increased by 30.6%, 52.8% and 65.3% after pretreatment of the cell cultures with 50, 100 and 200 microM vinclozolin, respectively. The highest concentration (400 microM) of vinclozolin tested caused cytotoxicity. Therefore, micronuclei were not considered for that concentration. To clarify the mechanism of cogenotoxicity, we assayed cytochrome P450 1A1 (CYP1A1), which plays a pivotal role in activation of BaP. Cells exposed to vinclozolin led to significant increase of CYP1A1 expression in Western blot. The result suggested that induction of CYP1A1 by vinclozolin account for its enhancing effect on genotoxicity caused by BaP.

Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine-guanine phosphoribosyltransferase gene.

PubMed

Graf, L H; McRoberts, J A; Harrison, T M; Martin, D W

1976-07-01

Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6-mercaptopurine (6-MP) or 6-thioguanine (6-ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities. The cells from one of these clones, 1020/12, posses 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme. PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild-type cells. The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP "sparing" effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction. We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.

Antitumor activity of Type I and Type III interferons in BNL hepatoma model

PubMed Central

Abushahba, Walid; Balan, Murugabaskar; Castaneda, Ismael; Yuan, Yao; Reuhl, Kenneth; Raveche, Elizabeth; de la Torre, Andrew

2015-01-01

Hepatocellular carcinoma (HCC) occurs most commonly secondary to cirrhosis due to chronic hepatitis C or B virus (HCV/HBV) infections. Type I interferon (IFN-α) treatment of chronic HCV/HBV infections reduces the incidence of HCC in cirrhotic patients. However, IFN-α toxicity limits its tolerability and efficacy highlighting a need for better therapeutic treatments. A recently discovered type III IFN (IFN-λ) has been shown to possess antiviral properties against HCV and HBV in vitro. In phase I clinical trials, IFN-λ treatment did not cause significant adverse reactions. Using a gene therapy approach, we compared the antitumor properties of IFN-α and IFN-λ in a transplantable hepatoma model of HCC. BALB/c mice were inoculated with syngeneic BNL hepatoma cells, or BNL cells expressing IFN-λ (BNL.IFN-λ cells) or IFN-α (BNL.IFN-α cells). Despite the lack of antiproliferative activity of IFNs on BNL cells, both BNL.IFN-λ and BNL.IFN-α cells displayed retarded growth kinetics in vivo. Depletion of NK cells from splenocytes inhibited splenocyte-mediated cytotoxicity, demonstrating that NK cells play a role in IFN-induced antitumor responses. However, isolated NK cells did not respond directly to IFN-λ. There was also a marked NK cell infiltration in IFN-λ producing tumors. In addition, IFN-λ and, to a lesser extent, IFN-α enhanced immunocytotoxicity of splenocytes primed with irradiated BNL cells. Splenocyte cytotoxicity against BNL cells was dependent on IL-12 and IFN-λ, and mediated by dendritic cells. In contrast to NK cells, isolated from spleen CD1 1c+ and mPDCA+ dendritic cells responded directly to IFN-λ. The antitumor activities of IFN-λ against hepatoma, in combination with HCV and HBV antiviral activities warrant further investigation into the clinical use of IFN-λ to prevent HCC in HCV/HBV-infected cirrhotic patients, as well as to treat liver cancer. PMID:20217081

Antitumor activity of type I and type III interferons in BNL hepatoma model.

PubMed

Abushahba, Walid; Balan, Murugabaskar; Castaneda, Ismael; Yuan, Yao; Reuhl, Kenneth; Raveche, Elizabeth; de la Torre, Andrew; Lasfar, Ahmed; Kotenko, Sergei V

2010-07-01

Hepatocellular carcinoma (HCC) occurs most commonly secondary to cirrhosis due to chronic hepatitis C or B virus (HCV/HBV) infections. Type I interferon (IFN-alpha) treatment of chronic HCV/HBV infections reduces the incidence of HCC in cirrhotic patients. However, IFN-alpha toxicity limits its tolerability and efficacy highlighting a need for better therapeutic treatments. A recently discovered type III IFN (IFN-lambda) has been shown to possess antiviral properties against HCV and HBV in vitro. In phase I clinical trials, IFN-lambda treatment did not cause significant adverse reactions. Using a gene therapy approach, we compared the antitumor properties of IFN-alpha and IFN-lambda in a transplantable hepatoma model of HCC. BALB/c mice were inoculated with syngeneic BNL hepatoma cells, or BNL cells expressing IFN-lambda (BNL.IFN-lambda cells) or IFN-alpha (BNL.IFN-alpha cells). Despite the lack of antiproliferative activity of IFNs on BNL cells, both BNL.IFN-lambda and BNL.IFN-alpha cells displayed retarded growth kinetics in vivo. Depletion of NK cells from splenocytes inhibited splenocyte-mediated cytotoxicity, demonstrating that NK cells play a role in IFN-induced antitumor responses. However, isolated NK cells did not respond directly to IFN-lambda. There was also a marked NK cell infiltration in IFN-lambda producing tumors. In addition, IFN-lambda and, to a lesser extent, IFN-alpha enhanced immunocytotoxicity of splenocytes primed with irradiated BNL cells. Splenocyte cytotoxicity against BNL cells was dependent on IL-12 and IFN-gamma, and mediated by dendritic cells. In contrast to NK cells, isolated from spleen CD11c+ and mPDCA+ dendritic cells responded directly to IFN-lambda. The antitumor activities of IFN-lambda against hepatoma, in combination with HCV and HBV antiviral activities warrant further investigation into the clinical use of IFN-lambda to prevent HCC in HCV/HBV-infected cirrhotic patients, as well as to treat liver cancer.

Identification of Genes Potentially Regulated by Human Polynucleotide Phosphorylase (hPNPaseold-35) Using Melanoma as a Model

PubMed Central

Sokhi, Upneet K.; Bacolod, Manny D.; Dasgupta, Santanu; Emdad, Luni; Das, Swadesh K.; Dumur, Catherine I.; Miles, Michael F.; Sarkar, Devanand; Fisher, Paul B.

2013-01-01

Human Polynucleotide Phosphorylase (hPNPaseold-35 or PNPT1) is an evolutionarily conserved 3′→5′ exoribonuclease implicated in the regulation of numerous physiological processes including maintenance of mitochondrial homeostasis, mtRNA import and aging-associated inflammation. From an RNase perspective, little is known about the RNA or miRNA species it targets for degradation or whose expression it regulates; except for c-myc and miR-221. To further elucidate the functional implications of hPNPaseold-35 in cellular physiology, we knocked-down and overexpressed hPNPaseold-35 in human melanoma cells and performed gene expression analyses to identify differentially expressed transcripts. Ingenuity Pathway Analysis indicated that knockdown of hPNPaseold-35 resulted in significant gene expression changes associated with mitochondrial dysfunction and cholesterol biosynthesis; whereas overexpression of hPNPaseold-35 caused global changes in cell-cycle related functions. Additionally, comparative gene expression analyses between our hPNPaseold-35 knockdown and overexpression datasets allowed us to identify 77 potential “direct” and 61 potential “indirect” targets of hPNPaseold-35 which formed correlated networks enriched for cell-cycle and wound healing functional association, respectively. These results provide a comprehensive database of genes responsive to hPNPaseold-35 expression levels; along with the identification new potential candidate genes offering fresh insight into cellular pathways regulated by PNPT1 and which may be used in the future for possible therapeutic intervention in mitochondrial- or inflammation-associated disease phenotypes. PMID:24143183

Combined inhibitors of angiogenesis and histone deacetylase: efficacy in rat hepatoma.

PubMed

Ganslmayer, Marion; Zimmermann, Annette; Zopf, Steffen; Herold, Christoph

2011-08-21

To evaluate the antitumoral effect of combined inhibitors of angiogenesis and histone deacetylases in an experimental rat hepatoma model. MH7777A hepatoma cells were injected into the liver of male Buffalo rats. After 7 d treatment with the vascular endothelial growth factor receptor antagonist PTK787/ZK222584 (PTK/ZK), the histone deacetylase inhibitor MS-275, tamoxifen (TAM) and/or retinoic acid was initiated (n ≥ 8 animals/group). Natural tumor development was shown in untreated control groups (control 1 with n = 12, control 2 with n = 8). The control groups were initiated at different time points to demonstrate the stability of the hepatoma model. For documentation of possible side effects, we documented any change in body weight, loss of fur and diarrhea. After 21 d treatment, the rats were euthanized. Main target parameters were tumor size and metastasis rate. Additionally, immunohistochemistry for the proliferating cell nuclear antigen (PCNA) and TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay were performed. The control groups developed large tumor nodules with extrahepatic tumor burden in the lung and abdominal organs (control 1: 6.18 cm(3) ± 4.14 cm(3) and control 2: 8.0 cm(3) ± 4.44 cm(3) 28 d after tumor cell injection). The tumor volume did not differ significantly in the control groups (P = 0.13). As single agents MS-275 and PTK/ZK reduced tumor volume by 58.6% ± 2.6% and 48.7% ± 3.2% vs control group 1, which was significant only for MS-275 (P = 0.025). The combination of MS-275 and PTK/ZK induced a nearly complete and highly significant tumor shrinkage by 90.3% ± 1% (P = 0.005). Addition of TAM showed no further efficacy, while quadruple therapy with retinoic acid increased antitumoral efficacy (tumor reduction by 93 ± 1%) and side effects. PCNA positive cells were not significantly reduced by the single agents, while dual therapy (MS-275 and PTK/ZK) and quadruple therapy reduced the PCNA-positive cell fraction significantly by 9

Evaluation of the anticancer potential of six herbs against a hepatoma cell line

PubMed Central

2012-01-01

Background Six herbs in the Plant Genetics Conservation Project that have been used as complementary medicines were chosen on the basis of their medicinal value, namely Terminalia mucronata, Diospyros winitii, Bridelia insulana, Artabotrys harmandii, Terminallia triptera, and Croton oblongifolius. This study aims to evaluate the potential anticancer activity of 50% ethanol-water extracts of these six herbs. Methods Fifty percent ethanol-water crude extracts of the six herbs were prepared. The cytotoxicity of the herbal extracts relative to that of melphalan was evaluated using a hepatoma cell line (HepG2), and examined by neutral red assays and apoptosis induction by gel electrophoresis and flow cytometry after 24 h. Results A significant difference was found between the cytotoxicity of the 50% ethanol-water crude extracts and melphalan (P = 0.000). The 50% ethanol-water crude extracts of all six herbs exhibited cytotoxicity against HepG2 cells, with IC50 values ranging from 100 to 500 μg/mL. The extract of T. triptera showed the highest cytotoxicity with an IC50 of 148.7 ± 12.3 μg/mL, while melphalan had an IC50 of 39.79 ± 7.62 μg/mL. The 50% ethanol-water crude extracts of D. winitii and T. triptera, but not A. harmandii, produced a DNA ladder. The 50% ethanol-water crude extracts of D. winitii, T. triptera, and A. harmandii induced apoptosis detected by flow cytometry. Conclusion The 50% ethanol-water crude extracts of D. winitii, T. triptera, and A. harmandii showed anticancer activity in vitro. PMID:22682026

Genotoxicity and induction of DNA damage responsive genes by food-borne heterocyclic aromatic amines in human hepatoma HepG2 cells.

PubMed

Pezdirc, Marko; Žegura, Bojana; Filipič, Metka

2013-09-01

Heterocyclic aromatic amines (HAAs) are potential human carcinogens formed in well-done meats and fish. The most abundant are 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-Amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-Amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ). HAAs exert genotoxic activity after metabolic transformation by CYP1A enzymes, that is well characterized, however the genomic and intervening responses are not well explored. We have examined cellular and genomic responses of human hepatoma HepG2 cells after 24h exposure to HAAs. Comet assay revealed increase in formation of DNA strand breaks by PhIP, MeIQx and IQ but not 4,8-DiMeIQx, whereas increased formation of micronuclei was not observed. The four HAAs up-regulated expression of genes encoding metabolic enzymes CYP1A1, CYP1A2 and UGT1A1 and expression of TP53 and its downstream regulated genes CDKN1A, GADD45α and BAX. Consistent with the up-regulation of CDKN1A and GADD45α the cell-cycle analysis showed arrest in S-phase by PhIP and IQ, and in G1-phase by 4,8-DiMeIQx and MeIQx. The results indicate that upon exposure to HAAs the cells respond with the cell-cycle arrest, which enables cells to repair the damage or eliminate them by apoptosis. However, elevated expression of BCL2 and down-regulation of BAX may indicate that HAAs could suppress apoptosis meaning higher probability of damaged cells to survive and mutate. Copyright © 2013 Elsevier Ltd. All rights reserved.

Chemopreventive Activities of Sulforaphane and Its Metabolites in Human Hepatoma HepG2 Cells.

PubMed

Liu, Peng; Wang, Wei; Zhou, Zhigang; Smith, Andrew J O; Bowater, Richard P; Wormstone, Ian Michael; Chen, Yuqiong; Bao, Yongping

2018-05-09

Sulforaphane (SFN) exhibits chemopreventive effects through various mechanisms. However, few studies have focused on the bioactivities of its metabolites. Here, three metabolites derived from SFN were studied, known as sulforaphane glutathione, sulforaphane cysteine and sulforaphane- N -acetylcysteine. Their effects on cell viability, DNA damage, tumorigenicity, cell migration and adhesion were measured in human hepatoma HepG2 cells, and their anti-angiogenetic effects were determined in a 3D co-culture model of human umbilical vein endothelial cells (HUVECs) and pericytes. Results indicated that these metabolites at high doses decreased cancer cell viability, induced DNA damage and inhibited motility, and impaired endothelial cell migration and tube formation. Additionally, pre-treatment with low doses of SFN metabolites protected against H₂O₂ challenge. The activation of the nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway and the induction of intracellular glutathione (GSH) played an important role in the cytoprotective effects of SFN metabolites. In conclusion, SFN metabolites exhibited similar cytotoxic and cytoprotective effects to SFN, which proves the necessity to study the mechanisms of action of not only SFN but also of its metabolites. Based on the different tissue distribution profiles of these metabolites, the most relevant chemical forms can be selected for targeted chemoprevention.

Specific gene transfer mediated by galactosylated poly-L-lysine into hepatoma cells.

PubMed

Han, J; Il Yeom, Y

2000-07-20

Plasmid DNA/galactosylated poly-L-lysine(GalPLL) complex was used to transfer luciferase reporter gene in vitro into human hepatoma cells by a receptor-mediated endocytosis process. DNA was combined with galPLL via charge interaction (DNA:GalPLL:fusogenic peptide, 1:0.4:5, w/w/w) and the resulting complex was characterized by dynamic light scattering, gel retardation assay and zeta potential analyzer to determine the particle size, electrostatic charge interaction, and apparent surface charge. The complex was tested for the efficiency of gene transfer in cultured human hepatoblastoma cell line Hep G2 and fibroblast cells NIH/3T3 in vitro. The mean diameter of the complex (DNA:GalPLL=1:0.4, w/w) was 256+/-34.8 nm, and at this ratio, it was positively charged (zeta potential of this complex was 10.1 mV). Hep G2 cells, which express a galactose specific membrane lectin, were efficiently and selectively transfected with the RSV Luc/GalPLL complex in a sugar-dependent manner. NIH/3T3 cells, which do not express the galactose-specific membrane lectin, showed only a marginal level of gene expression. The transfection efficiency of GalPLL-conjugated DNA complex into Hep G2 cells was greatly enhanced in the presence of fusogenic peptide that can disrupt endosomes, where the GalPLL-DNA complex is entrapped with the fusogenic peptide. With the fusogenic peptide KALA, the luciferase activity in Hep G2 cells was ten-fold higher than that of cells transfected in the absence of the fusogenic peptide. Our gene transfer formulation may find potential application for the gene therapy of liver diseases.

Bupivacaine-induced apoptosis independently of WDR35 expression in mouse neuroblastoma Neuro2a cells

PubMed Central

2012-01-01

Background Bupivacaine-induced neurotoxicity has been shown to occur through apoptosis. Recently, bupivacaine was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in a human neuroblastoma cell line. We have reported that WDR35, a WD40-repeat protein, may mediate apoptosis through caspase-3 activation. The present study was undertaken to test whether bupivacaine induces apoptosis in mouse neuroblastoma Neuro2a cells and to determine whether ROS, p38 MAPK, and WDR35 are involved. Results Our results showed that bupivacaine induced ROS generation and p38 MAPK activation in Neuro2a cells, resulting in apoptosis. Bupivacaine also increased WDR35 expression in a dose- and time-dependent manner. Hydrogen peroxide (H2O2) also increased WDR35 expression in Neuro2a cells. Antioxidant (EUK-8) and p38 MAPK inhibitor (SB202190) treatment attenuated the increase in caspase-3 activity, cell death and WDR35 expression induced by bupivacaine or H2O2. Although transfection of Neuro2a cells with WDR35 siRNA attenuated the bupivacaine- or H2O2-induced increase in expression of WDR35 mRNA and protein, in contrast to our previous studies, it did not inhibit the increase in caspase-3 activity in bupivacaine- or H2O2-treated cells. Conclusions In summary, our results indicated that bupivacaine induced apoptosis in Neuro2a cells. Bupivacaine induced ROS generation and p38 MAPK activation, resulting in an increase in WDR35 expression, in these cells. However, the increase in WDR35 expression may not be essential for the bupivacaine-induced apoptosis in Neuro2a cells. These results may suggest the existence of another mechanism of bupivacaine-induced apoptosis independent from WDR35 expression in Neuro2a cells. PMID:23227925

Hydroquinone stimulates cell invasion through activator protein-1-dependent induction of MMP-9 in HepG2 human hepatoma cells.

PubMed

Yu, Mi-Hee; Lee, Syng-Ook

2016-03-01

Hydroquinone (HQ) is a well-known environmental carcinogen and exposure of humans to HQ can also occur through plant foods, cosmetics, and tobacco products. Although liver is a major organ metabolizing HQ and susceptible to its toxicity, role of HQ in metastatic progression of human hepatocellular carcinoma (HCC) remains unclear. In this study, we examined the effect of HQ on the invasion of HCC cells and its underlying molecular mechanisms. HQ strongly induced matrix metalloproteinase-9 (MMP-9) expression and secretion in HepG2 human hepatoma cells, which were well correlated with increased cell invasion. Mechanistic studies further demonstrated that HQ induced transcriptional activity of MMP-9 gene by activating activator protein-1 (AP-1), the well-known key element mediating MMP-9 gene expression, via MAP kinase (MAPK) signaling pathways. These results suggest that HQ may promote metastatic progression of HCC, although data on in vivo hydroquinone exposure and risk for HCC are contradictory. Copyright © 2016 Elsevier Ltd. All rights reserved.

Acetyl-CoA carboxylase in Reuber hepatoma cells: variation in enzyme activity, insulin regulation, and cellular lipid content.

PubMed

Bianchi, A; Evans, J L; Nordlund, A C; Watts, T D; Witters, L A

1992-01-01

Reuber hepatoma cells are useful cultured lines for the study of insulin action, lipid and lipoprotein metabolism, and the regulation of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid biosynthesis. During investigations in different clonal lines of these cells, we have uncovered marked intercellular variability in the activity, enzyme content, and insulin regulation of ACC paralleled by differences in cellular neutral lipid (triglyceride) content. Two contrasting clonal lines, Fao and H356A-1, have been studied in detail. Several features distinguish these two lines, including differences in ACC activity and enzyme kinetics, the content of the two major hepatic ACC isozymes (Mr 280,000 and 265,000 Da) and their heteroisozymic complex, the extent of ACC phosphorylation, and the ability of ACC to be activated on stimulation by insulin and insulinomimetic agonists. As studied by Nile Red staining and fluorescence-activated cell sorting, these two lines also display marked differences in neutral lipid content, which correlates with both basal levels of ACC activity and inhibition of ACC by the fatty acid analog, 5-(tetradecyloxy)-2-furoic acid (TOFA). These results emphasize the importance of characterization of any particular clonal line of Reuber cells for studies of enzyme regulation, substrate metabolism, and hormone action. With respect to ACC, studies in contrasting clonal lines of Reuber cells could provide valuable clues to understanding both the complex mechanisms of intracellular ACC regulation in the absence and presence of hormones and its regulatory role(s) in overall hepatic lipid metabolism.

In Vitro Generation of IL-35-expressing Human Wharton's Jelly-derived Mesenchymal Stem Cells Using Lentiviral Vector.

PubMed

Amari, Afshin; Ebtekar, Massoumeh; Moazzeni, Seyed Mohammad; Soleimani, Masoud; Mohammadi Amirabad, Leila; Tahoori, Mohammad Taher; Massumi, Mohammad

2015-08-01

Human Wharton's Jelly-derived Mesenchymal Stem Cells (hWJ-MSCs) are easily available cells without transplant rejection problems or ethical concerns compared to bone-marrow-derived MSCs for prospective clinical applications. These cells display immunosuppressive properties and may be able to play an important role in autoimmune disorders. Regulatory T-cells (Treg) are important to prevent autoimmune disease development. Interleukin 35 (IL-35) induces the proliferation of Treg cell populations and reduces the activity of T helper 17 (Th17) and T helper 1 (Th1) cells, which play a central role in initiation of inflammation and autoimmune disease. Recent studies identified IL-35 as a new inhibitory cytokine required for the suppressive function of Treg cells. We created IL-35-producing hWJ-MSCs as a good vehicle for reduction of inflammation and autoimmune diseases. We isolated hWJ-MSCs based on explant culture. HWJ-MSCs were transduced at MOI=50 (Multiplicity of Infection) with lentiviral particles harboring murine Interleukin 35 (mIL-35). Expression of IL-35 in hWJ-MSCs was quantified by an IL-35 ELISA kit. IL-35 bioactivity was analyzed by inhibiting the proliferation of mouse splenocytes using CFSE cell proliferation kit. Frequency of CD4+CD25+CD127 low/neg Foxp3+ Treg cells was measured by flow cytometry. There was an up to 85% GFP positive transduction rate, and the cells successfully released a high level of mIL-35 protein (750 ng/ml). IL-35 managed to inhibit CD4+ T cell proliferation with PHA, and improved the frequency of Treg cells. Our data suggest that transduced hWJ-MSCs overexpressing IL-35 may provide a useful approach for basic research on gene therapy for autoimmune disorders.

Complete replication of hepatitis B virus and hepatitis C virus in a newly developed hepatoma cell line.

PubMed

Yang, Darong; Zuo, Chaohui; Wang, Xiaohong; Meng, Xianghe; Xue, Binbin; Liu, Nianli; Yu, Rong; Qin, Yuwen; Gao, Yimin; Wang, Qiuping; Hu, Jun; Wang, Ling; Zhou, Zebin; Liu, Bing; Tan, Deming; Guan, Yang; Zhu, Haizhen

2014-04-01

The absence of a robust cell culture system for hepatitis B virus (HBV) and hepatitis C virus (HCV) infection has limited the analysis of the virus lifecycle and drug discovery. We have established a hepatoma cell line, HLCZ01, the first cell line, to the authors' knowledge, supporting the entire lifecycle of both HBV and HCV. HBV surface antigen (HBsAg)-positive particles can be observed in the supernatant and the lumen of the endoplasmic reticulum of the cells via electron microscopy. Interestingly, HBV and HCV clinical isolates propagate in HLCZ01 cells. Both viruses replicate in the cells without evidence of overt interference. HBV and HCV entry are blocked by antibodies against HBsAg and human CD81, respectively, and the replication of HBV and HCV is inhibited by antivirals. HLCZ01 cells mount an innate immune response to virus infection. The cell line provides a powerful tool for exploring the mechanisms of virus entry and replication and the interaction between host and virus, facilitating the development of novel antiviral agents and vaccines.

Solvent-Free Addition of Indole to Aldehydes: Unexpected Synthesis of Novel 1-[1-(1H-Indol-3-yl) Alkyl]-1H-Indoles and Preliminary Evaluation of Their Cytotoxicity in Hepatocarcinoma Cells.

PubMed

Tocco, Graziella; Zedda, Gloria; Casu, Mariano; Simbula, Gabriella; Begala, Michela

2017-10-17

New 1-[1-(1 H -indol-3-yl) alkyl]-1 H -indoles, surprisingly, have been obtained from the addition of indole to a variety of aldehydes under neat conditions. CaO, present in excess, was fundamental for carrying out the reaction with paraformaldehyde. Under the same reaction conditions, aromatic and heteroaromatic aldehydes afforded only classical bis (indolyl) aryl indoles. In this paper, the role of CaO, together with the regiochemistry and the mechanism of the reaction, are discussed in detail. The effect of some selected 3,3'- and 1,3'-diindolyl methane derivatives on cell proliferation of the hepatoma cell line FaO was also evaluated.

Effects of the peroxisome proliferator clofibric acid on superoxide dismutase expression in the human HepG2 hepatoma cell line.

PubMed

Bécuwe, P; Bianchi, A; Keller, J M; Dauça, M

1999-09-15

We examined the effects of clofibric acid, a peroxisome proliferator, on the production of superoxide radicals, on the levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), and on the expression of superoxide dismutases (SODs) in the human HepG2 hepatoma cell line. To this end, HepG2 cells were treated for 1 or 5 days with 0.25, 0.50, or 0.75 mM clofibric acid. The production of superoxide radicals was only enhanced in HepG2 cells exposed for 5 days to the different clofibric acid concentrations. However, this overproduction of superoxide radicals was not accompanied by increased rates of lipid peroxidation, as the MDA and 4-HNE levels did not change significantly. Manganese (Mn) SOD activity was increased when HepG2 cells were treated for 1 day with 0.50 or 0.75 mM clofibric acid. For this duration of treatment, no change was observed in total SOD and copper/zinc (Cu/Zn) SOD activities. For a 5-day treatment, total SOD and MnSOD activities as well as the enzyme apoprotein and MnSOD mRNA levels increased whatever the clofibric acid concentration used. This transcriptional induction of the MnSOD gene was correlated with an activation of the activator protein-1 transcription factor for 1 and 5 days of treatment, but was independent of nuclear factor-kappa B and of peroxisome proliferator-activated receptor. On the other hand, the PP exerted very little effect if any on Cu,ZnSOD expression. In contrast to rodent data, PP treatment of human hepatoma cells induces MnSOD expression.

Saponins isolated from Asparagus induce apoptosis in human hepatoma cell line HepG2 through a mitochondrial-mediated pathway

PubMed Central

Ji, Y.; Ji, C.; Yue, L.; Xu, H.

2012-01-01

Objective Many scientific studies have shown that Asparagus officinalis has an antitumour effect and enhances human immunity, but the active components and the antitumour mechanisms are unclear. We investigated the effects of saponins isolated from Asparagus on proliferation and apoptosis in the human hepatoma cell line HepG2. Methods HepG2 cells were treated with varying concentrations of Asparagus saponins at various times. Using mtt and flow cytometry assays, we evaluated the effects of Asparagus saponins on the growth and apoptosis of HepG2 cells. Transmission electron microscopy was used to observe the morphology of cell apoptosis. Confocal laser scanning microscopy was used to analyze intracellular calcium ion concentration, mitochondrial permeability transition pore (mptp), and mitochondrial membrane potential (mmp). Spectrophotometry was applied to quantify the activity of caspase-9 and caspase-3. Flow cytometry was used to investigate the levels of reactive oxygen species (ros) and pH, and the expressions of Bcl2, Bax, CytC, and caspase-3, in HepG2 cells. Results Asparagus saponins inhibited the growth of HepG2 cells in a dose-dependent manner. The median inhibitory concentration (IC50) was 101.15 mg/L at 72 hours. The apoptosis morphology at 72 hours of treatment was obvious, showing cell protuberance, concentrated cytoplasm, and apoptotic bodies. The apoptotic rates at 72 hours were 30.9%, 51.7%, and 62.1% (for saponin concentrations of 50 mg/L, 100 mg/L, 200 mg/L). Treatment with Asparagus saponins for 24 hours increased the intracellular level of ros and Ca2+, lowered the pH, activated intracellular mptp, and decreased mmp in a dose-dependent manner. Treatment also increased the activity of caspase-9 and caspase-3, downregulated the expression of Bcl2, upregulated the expression of Bax, and induced release of CytC and activation of caspase-3. Conclusions Asparagus saponins induce apoptosis in HepG2 cells through a mitochondrial-mediated and caspase

Phosphorus NMR of isolated perfused morris hepatomas

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

Graham, R.A.; Meyer, R.A.; Brown, T.R.

1986-03-05

The authors are developing techniques for the study of perfused solid tumors by NMR. Tissue-isolated solid hepatomas were grown to 1-2 cm diameter as described previously. The arterial supply was isolated and the tumors perfused (0.5 - 1.0 ml/min) in vitro at 25 C with a 15% suspension of red blood cells in Krebs-Henseliet solution. /sup 31/P-NMR spectra were acquired at 162 MHz in a specially-designed NMR probe using a solenoidal coil. Intracellular pH (monitored from the chemical shift of inorganic phosphate) and ATP levels were stable for up to 6 hrs during perfusion. During 30 min of global ischemia,more » ATP decreased by 75% and pH fell from 7.0 to 6.7. These changes were reversed by 1 hr reperfusion. In addition to ATP and phosphate, the spectra included a large resonance due to phosphomonoesters, as well as peaks consistent with glycerylphosphocholine, glyceryl-phosphoethanolamine, phosphocreatine, NAD, and UDPG. However, the most novel feature of the spectra was the presence of an unidentified peak in the phosphonate region (+ 16.9 ppm). The peak was not present in spectra of muscle, liver, brain, kidney, or fat tissues excised from the same animals. They are presently attempting to identify the compound that gives rise to this peak and to establish its metabolic origin.« less

Genotoxic effect of 6-gingerol on human hepatoma G2 cells.

PubMed

Yang, Guang; Zhong, Laifu; Jiang, Liping; Geng, Chengyan; Cao, Jun; Sun, Xiance; Ma, Yufang

2010-04-15

6-gingerol, a major component of ginger, has antioxidant, anti-apoptotic, and anti-inflammatory activities. However, some dietary phytochemicals possess pro-oxidant effects as well, and the risk of adverse effects is increased by raising the use of doses. The aim of this study was to assess the genotoxic effects of 6-gingerol and to clarify the mechanisms, using human hepatoma G2 (HepG2) cells. Exposure of the cells to 6-gingerol caused significant increase of DNA migration in comet assay, increase of micronuclei frequencies at high concentrations at 20-80 and 20-40 microM, respectively. These results indicate that 6-gingerol caused DNA strand breaks and chromosome damage. To further elucidate the underlying mechanisms, we tested lysosomal membrane stability, mitochondrial membrane potential, the intracellular generation of reactive oxygen species (ROS) and reduced glutathione (GSH). In addition, the level of oxidative DNA damage was evaluated by immunocytochemical analysis on 8-hydroxydeoxyguanosine (8-OHdG). Results showed that lysosomal membrane stability was reduced after treatment by 6-gingerol (20-80 microM) for 40 min, mitochondrial membrane potential decreased after treatment for 50 min, GSH and ROS levels were significantly increased after treatment for 60 min. These suggest 6-gingerol induces genotoxicity probably by oxidative stress; lysosomal and mitochondrial damage were observed in 6-gingerol-induced toxicity. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Cytotoxic and genotoxic potential of geraniol in peripheral blood mononuclear cells and human hepatoma cell line (HepG2).

PubMed

Queiroz, T B; Santos, G F; Ventura, S C; Hiruma-Lima, C A; Gaivão, I O M; Maistro, E L

2017-09-27

Geraniol is an acyclic monoterpene alcohol present in the essential oil of many aromatic plants and is one of the most frequently used molecules by the flavor and fragrance industries. The literature also reports its therapeutic potential, highlighting itself especially as a likely molecule for the development of drugs against cancer. In view of these considerations, this study was designed to evaluate the cytotoxic and genotoxic potential of geraniol, in an in vitro protocol, using two types of human cells: one without the ability to metabolize (peripheral blood mononuclear cells - PBMC), and the other with this capability (human hepatoma cell line - HepG2) through the comet assay and the micronucleus test. Four concentrations (10, 25, 50, and 100 µg/mL) were selected for the genotoxic assessment for PBMC and three (1.25, 2.5, and 5 µg/mL) for HepG2 cells based on cytotoxicity tests (MTT assay). Results showed that geraniol did not present genotoxic or clastogenic/aneugenic effects on both cell types under the conditions studied. However, caution is advised in the use of this substance by humans, since a significant reduction in viability of HepG2 and a marked decrease in cell viability on normal PBMC were verified.

Induction apoptosis of luteolin in human hepatoma HepG2 cells involving mitochondria translocation of Bax/Bak and activation of JNK

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

Lee, H.-J.; Wang, C.-J.; Kuo, H.-C.

2005-03-01

Since hepatocellular carcinoma remains a major challenging clinical problem in many parts of the world including Eastern Asia and Southern Africa, it is imperative to develop more effective chemopreventive and chemotherapy agents. Herein, we present an investigation regarding the anticancer potential of luteolin, a natural flavonoid, and the mechanism of its action in human hepatoma HepG2 cells. Using DNA fragmentation assay and nuclear staining assay, it showed that luteolin induced apoptosis of HepG2 cells. Luteolin induced the cytosolic release of cytochrome c and activated CPP32. We found that Bax and Bak translocated to mitochondria apparently, whereas Fas ligand (FasL) wasmore » unchanged after a treatment with luteolin for 3 h. In addition, it showed that c-Jun NH{sub 2}-terminal kinase (JNK) was activated after the treatment of luteolin for 3-12 h. Further investigation showed that a specific JNK inhibitor, SP600125, reduced the activation of CPP 32, the mitochondrial translocation of Bax, as well as the cytosolic release of cytochrome c that induced by luteolin. Finally, the apoptosis induced by luteolin was suppressed by a pretreatment with SP600125 via evaluating annexin V-FITC binding assay. These data suggest that luteolin induced apoptosis via mechanisms involving mitochondria translocation of Bax/Bak and activation of JNK.« less

Let-7c overexpression inhibits dengue virus replication in human hepatoma Huh-7 cells.

PubMed

Escalera-Cueto, Manuel; Medina-Martínez, Ingrid; del Angel, Rosa M; Berumen-Campos, Jaime; Gutiérrez-Escolano, Ana Lorena; Yocupicio-Monroy, Martha

2015-01-22

MicroRNAs (miRNAs) constitute an important class of non-coding RNA implicated in gene expression regulation. More than 1900 miRNA molecules have been identified in humans and their modulation during viral infection and it is recognized to play a role in latency regulation or in establishing an antiviral state. The liver cells are targets during DENV infection, and alteration of liver functions contributes to severe disease. In this work the miRNAs expression profile of the human hepatoma cell line, Huh-7, infected with DENV-2 was determined using microarray and real-time PCR. Let-7c is one of the miRNAs up-regulated during DENV infection in the hepatic Huh-7 as well as in the macrophage-monocytic cell line U937-DC-SIGN. Let-7c overexpression down-regulates both DENV-2 and DENV-4 infection. Additionally, we found that the transcription factor BACH1, a let-7c target, is also down-regulated during DENV infection. In accordance with this finding, HO-1, the main responsive factor of BACH1 was found up-regulated. The up-regulation of HO-1 may contribute to the stress oxidative response in infected cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Genotoxicity assessment of membrane concentrates of landfill leachate treated with Fenton reagent and UV-Fenton reagent using human hepatoma cell line.

PubMed

Wang, Guifang; Lu, Gang; Yin, Pinghe; Zhao, Ling; Yu, Qiming Jimmy

2016-04-15

Membrane concentrates of landfill leachates contain organic and inorganic contaminants that could be highly toxic and carcinogenic. In this paper, the genotoxicity of membrane concentrates before and after Fenton and UV-Fenton reagent was assessed. The cytotoxicity and genotoxicity was determined by using the methods of methyltetrazolium (MTT), cytokinesis-block micronucleus (CBMN) and comet assay in human hepatoma cells. MTT assay showed a cytotoxicity of 75% after 24h of exposure to the highest tested concentration of untreated concentrates, and no cytotoxocity for UV-Fenton and Fenton treated concentrates. Both CBMN and comet assays showed increased levels of genotoxicity in cells exposed to untreated concentrates, compared to those occurred in cells exposed to UV-Fenton and Fenton reagent treated concentrates. There was no significant difference between negative control and UV-Fenton treated concentrates for micronucleus and comet assay parameters. UV-Fenton and Fenton treatment, especially the former, were effective methods for degradation of bisphenol A and nonylphenol in concentrates. These findings showed UV-Fenton and Fenton reaction were effective methods for treatment of such complex concentrates, UV-Fenton reagent provided toxicological safety of the treated effluent, and the genotoxicity assays were found to be feasible tools for assessment of toxicity risks of complex concentrates. Copyright © 2016 Elsevier B.V. All rights reserved.

Cytotoxicity of the dicarboximide fungicides, vinclozolin and iprodione, in rat hepatoma-derived Fa32 cells.

PubMed

Dierickx, Paul J

2004-10-01

Dicarboximide fungicides are widely used to control various fungal species. Their primary action is not known, due to a lack of knowledge concerning the mechanism of action of the dicarboximide group. The cytotoxicities of vinclozolin and iprodione in rat hepatoma-derived Fa32 cells were investigated. Cytotoxicity was measured by neutral red uptake inhibition after treatment for 24 hours. Iprodione was more toxic than vinclozolin. Vinclozolin was less toxic in glutathione-depleted cells than in control cells. This was also true for iprodione at lower concentrations, but iprodione became more toxic at higher concentrations. Both the fungicides increased the endogenous glutathione content by 20% after 1 hour. After 24 hours, the glutathione content was doubled by vinclozolin, but was not affected by iprodione. No effect on glutathione S-transferase activity or reactive oxygen species formation could be observed. Cytochrome P450-dependent ethoxyresorufin-O-deethylase and pentoxyresorufin-O-depentylase activities were moderately activated by iprodione and strongly activated by vinclozolin. A glutathione-related cytochrome P450-dependent metabolic attack of vinclozolin and iprodione could be responsible for their cytotoxicity in Fa32 cells. Further research is needed to fully elucidate these (or other) mechanisms.

Reversibly Switchable, pH-Dependent Peptide Ligand Binding via 3,5-Diiodotyrosine Substitutions.

PubMed

Ngambenjawong, Chayanon; Sylvestre, Meilyn; Gustafson, Heather H; Pineda, Julio Marco B; Pun, Suzie H

2018-04-20

Cell type-specific targeting ligands utilized in drug delivery applications typically recognize receptors that are overexpressed on the cells of interest. Nonetheless, these receptors may also be expressed, to varying extents, on off-target cells, contributing to unintended side effects. For the selectivity profile of targeting ligands in cancer therapy to be improved, stimuli-responsive masking of these ligands with acid-, redox-, or enzyme-cleavable molecules has been reported, whereby the targeting ligands are exposed in specific environments, e.g., acidic tumor hypoxia. One possible drawback of these systems lies in their one-time, permanent trigger, which enables the "demasked" ligands to bind off-target cells if released back into the systemic circulation. A promising strategy to address the aforementioned problem is to design ligands that show selective binding based on ionization state, which may be microenvironment-dependent. In this study, we report a systematic strategy to engineer low pH-selective targeting peptides using an M2 macrophage-targeting peptide (M2pep) as an example. 3,5-Diiodotyrosine mutagenesis into native tyrosine residues of M2pep confers pH-dependent binding behavior specific to acidic environment (pH 6) when the amino acid is protonated into the native tyrosine-like state. At physiological pH of 7.4, the hydroxyl group of 3,5-diiodotyrosine on the peptide is deprotonated leading to interruption of the peptide native binding property. Our engineered pH-responsive M2pep (Ac-Y-Î-Î) binds target M2 macrophages more selectively at pH 6 than at pH 7.4. In addition, 3,5-diiodotyrosine substitutions also improve serum stability of the peptide. Finally, we demonstrate pH-dependent reversibility in target binding via a postbinding peptide elution study. The strategy presented here should be applicable for engineering pH-dependent functionality of other targeting peptides with potential applications in physiology-dependent in vivo targeting

Altered expression of CD1d molecules and lipid accumulation in the human hepatoma cell line HepG2 after iron loading.

PubMed

Cabrita, Marisa; Pereira, Carlos F; Rodrigues, Pedro; Cardoso, Elsa M; Arosa, Fernando A

2005-01-01

Iron overload in the liver may occur in clinical conditions such as hemochromatosis and nonalcoholic steatohepatitis, and may lead to the deterioration of the normal liver architecture by mechanisms not well understood. Although a relationship between the expression of ICAM-1, and classical major histocompatibility complex (MHC) class I molecules, and iron overload has been reported, no relationship has been identified between iron overload and the expression of unconventional MHC class I molecules. Herein, we report that parameters of iron metabolism were regulated in a coordinated-fashion in a human hepatoma cell line (HepG2 cells) after iron loading, leading to increased cellular oxidative stress and growth retardation. Iron loading of HepG2 cells resulted in increased expression of Nor3.2-reactive CD1d molecules at the plasma membrane. Expression of classical MHC class I and II molecules, ICAM-1 and the epithelial CD8 ligand, gp180 was not significantly affected by iron. Considering that intracellular lipids regulate expression of CD1d at the cell surface, we examined parameters of lipid metabolism in iron-loaded HepG2 cells. Interestingly, increased expression of CD1d molecules by iron-loaded HepG2 cells was associated with increased phosphatidylserine expression in the outer leaflet of the plasma membrane and the presence of many intracellular lipid droplets. These data describe a new relationship between iron loading, lipid accumulation and altered expression of CD1d, an unconventional MHC class I molecule reported to monitor intracellular and plasma membrane lipid metabolism, in the human hepatoma cell line HepG2.

[Effect of depsides salts from Salvia miltiorrhiza on human hepatoma cell line SMMC-7721 subcutaneous xenografts in nude mice].

PubMed

Li, Xiangping; Song, Zhouye; Zhong, Haiying; Gong, Zhicheng; Yin, Tao; Zhang, Zanling; Zhou, Boting

2015-02-01

To exlpore the eff ect of depsides salts from Salvia miltiorrhiza on human hepatoma cell line SMMC-7721 xenograft tumors and the possible mechanisms. A total of 36 nude mice were divided into 6 groups: A model group, a negative control group, a positive control group, and 3 treatment groups at low, middle or high dose (n=6). The tumor model of nude mice was given depsides salts at a dose of 10, 20 or 50 mg/kg every 3 day for 16 days. Then samples of subcutaneous tumors in nude mice were collected. The morphological changes of tumor samples were observed by HE staining and the expression of vascular endothelial growth factor (VEGF) and the tumor antigen Ki67 was detected by immunohistochemical method. The tumor growth was inhibited by all doses of depsides salts. The morphology of tumors was shrinkage, broken and irregularly arranged compared with the tumors in the model group and the negative control group. Morphological changes were more obvious in tumors with treatment at high dose. Expression of VEGF and Ki67 in treatment groups and the positive control group were lower than that in the model group and the negative control group, with a significant difference (P<0.05). Depsides salts from Salvia miltiorrhiza can inhibit the growth of human hepatoma cell line SMMC-7721 tumor in nude mice, which is related to the inhibition of Ki67 and VEGF.

Icaritin inhibits the expression of alpha-fetoprotein in hepatitis B virus-infected hepatoma cell lines through post-transcriptional regulation.

PubMed

Zhang, Chao; Li, Hui; Jiang, Wei; Zhang, Xiaowei; Li, Gang

2016-12-13

Although it has showed that icaritin can apparently suppress growth of HCC by reducing the level of AFP, the intrinsic mechanism remains unclear. In this study, we explored the possible mechanism of miRNAs on post-transcriptional regulation of AFP gene, as well as the effects of HBV infection and icaritin in hepatoma cells. The results showed that miR-620, miR-1236 and miR-1270 could bind target sites in the range of 9-18 nt and 131-151 nt downstream of the stop codon in the AFP mRNA 3'-UTR to suppress the expression of AFP. Mutation of these target sites could reverse the effects of these miRNAs. Icaritin (10 μM) might reduce the stability and translational activity of AFP mRNA by increasing the expression levels of these mentioned miRNAs. HBV infection resulted in apparent decreases of these miRNAs and, consequently, increased AFP expression. The results indicated that miR-620, miR-1236 and miR-1270 are critical factors in the post-transcriptional regulation of AFP. Icaritin can counteract the effect of HBV. These findings will contribute to full understanding of the regulatory mechanism of AFP expression in hepatoma cells. And also it revealed a synergistic mechanism of HBV infection and elevation of AFP in the pathogenesis of HCC, as well as the potential clinical significance of icaritin on the therapy of HCC induced by HBV.

Cytotoxic effect induced by retinoic acid loaded into galactosyl-sphingosine containing liposomes on human hepatoma cell lines.

PubMed

Díaz, Cecilia; Vargas, Ernesto; Gätjens-Boniche, Omar

2006-11-15

Two retinoids, ATRA and 13cisRA, were incorporated into liposomes of different composition and charge and added to two hepatoma cell lines with different degree of transformation to measure cytotoxicity by MTT assay. Retinoid-free cationic liposomes were more toxic than the other kinds (anionic and made only of PC) but were also the best delivery system for retinoic acid to induce specific cytotoxic effects on these tumor hepatoma cell lines. Galactosyl-sphingosine containing cationic liposomes increased the cytotoxic effect induced by ATRA on Hep3B cells when compared to glucosyl-sphingosine cationic liposomes, but did not improve the effect induced by free retinoid or ATRA loaded into liposomes without glycolipids. This suggests that in this cell line, ATRA is being incorporated by a mechanism mediated by the asialoglycoprotein receptor, but at the same time, non-specific sugar-independent capture is also taking place as well as free diffusion of ATRA directly through the membrane. Galactose-specific effect was not observed in HepG2 cells treated with ATRA or both cell lines treated with 13cisRA. In fact, treatment of HepG2 cells with retinoids entrapped into liposomes likely induces proliferation instead of cytotoxicity, a result that interferes with the measurement of cell death by MTT. Compared to the specific effect of ATRA entrapped into cationic liposomes, vesicles made only by PC, did not mediate a specific mechanism, since differences between ATRA in galactosyl- and glucosyl-shpingosine PC-liposomes were not statistically significant. The specific mechanism was not present in the myoblastic cell line C2C12, where ATRA incorporated into galactosyl- and glucosyl-sphingosine containing cationic and PC-liposomes, was able to induce cytotoxicity at the same extent. Micelles containing ATRA and galactosyl-sphingosine had a significantly more toxic effect than the retinoid administered together with glucosyl-sphingosine, in Hep3B cells. Also, micelles containing

The spleen can influence the metastasis of AH130 hepatoma cells in rats.

PubMed

Toyonaga, M; Hiraoka, T; Tanaka, H; Miyauchi, Y

1993-06-01

The effect of pathophysiological conditions due to disturbance of the spleen is still unclear. We studied the effects of splenectomy in normal and methylcellulose-induced hypersplenic rats on the development of pulmonary metastases created by intravenous injection of ascites containing AH130 hepatoma cells from male Hos-Donryu rats. Growth of metastatic lesions in the lung was not affected by splenectomy in normal rats, but was increased by splenectomy in hypersplenic rats. Overall, there were fewer pulmonary metastases in rats with hypersplenism, but after splenectomy rats with hypersplenism had a significantly greater number of metastases than did normal rats. The metastases rate correlated somewhat with changes in the blood coagulation and T lymphocyte profile. There is a relationship between the spleen and formation of metastases in cancer. Formation of metastases in the lung was affected most by splenectomy in hypersplenism. To elucidate the mechanism by which metastases are formed in the lung under these pathologic conditions, further studies on the exact role of the spleen are required.

Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells.

PubMed

Su, Chun-Li; Huang, Lynn L H; Huang, Li-Min; Lee, Jenq-Chang; Lin, Chun-Nan; Won, Shen-Jeu

2006-05-29

Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase-8 to increase tBid, disrupted mitochondrial membrane potential (Delta psi(m)), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl-x(L) and increased Bax and Bak in mitochondria. Caspase-8 inhibitor (Z-IETD) attenuated the justicidin A-induced disruption of Delta psi(m). Growth of Hep 3B implanted in NOD-SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A-induced apoptosis in these cells proceeds via caspase-8 and is followed by mitochondrial disruption.

Human Papillomavirus Type 18 E6 and E7 Genes Integrate into Human Hepatoma Derived Cell Line Hep G2

PubMed Central

Ma, Tianzhong; Su, Zhongjing; Chen, Ling; Liu, Shuyan; Zhu, Ningxia; Wen, Lifeng; Yuan, Yan; Lv, Leili; Chen, Xiancai; Huang, Jianmin; Chen, Haibin

2012-01-01

Background and Objectives Human papillomaviruses have been linked causally to some human cancers such as cervical carcinoma, but there is very little research addressing the effect of HPV infection on human liver cells. We chose the human hepatoma derived cell line Hep G2 to investigate whether HPV gene integration took place in liver cells as well. Methods We applied PCR to detect the possible integration of HPV genes in Hep G2 cells. We also investigated the expression of the integrated E6 and E7 genes by using RT-PCR and Western blotting. Then, we silenced E6 and E7 expression and checked the cell proliferation and apoptosis in Hep G2 cells. Furthermore, we analyzed the potential genes involved in cell cycle and apoptosis regulatory pathways. Finally, we used in situ hybridization to detect HPV 16/18 in hepatocellular carcinoma samples. Results Hep G2 cell line contains integrated HPV 18 DNA, leading to the expression of the E6 and E7 oncogenic proteins. Knockdown of the E7 and E6 genes expression reduced cell proliferation, caused the cell cycle arrest at the S phase, and increased apoptosis. The human cell cycle and apoptosis real-time PCR arrays analysis demonstrated E6 and E7-mediated regulation of some genes such as Cyclin H, UBA1, E2F4, p53, p107, FASLG, NOL3 and CASP14. HPV16/18 was found in only 9% (9/100) of patients with hepatocellular carcinoma. Conclusion Our investigations showed that HPV 18 E6 and E7 genes can be integrated into the Hep G2, and we observed a low prevalence of HPV 16/18 in hepatocellular carcinoma samples. However, the precise risk of HPV as causative agent of hepatocellular carcinoma needs further study. PMID:22655088

188Re-SSS lipiodol: radiolabelling and biodistribution following injection into the hepatic artery of rats bearing hepatoma.

PubMed

Garin, Etienne; Denizot, Benoit; Noiret, Nicolas; Lepareur, Nicolas; Roux, Jerome; Moreau, Myriam; Herry, Jean-Yves; Bourguet, Patrick; Benoit, Jean-Pierre; Lejeune, Jean-Jacques

2004-10-01

Although intra-arterial radiation therapy with 131I-lipiodol is a useful therapeutic approach to the treatment of hepatocellular carcinoma, various disadvantages limit its use. To describe the development of a method for the labelling of lipiodol with 188Re-SSS (188Re (S2CPh)(S3CPh)2 complex) and to investigate its biodistribution after injection into the hepatic artery of rats with hepatoma. 188Re-SSS lipiodol was obtained after dissolving a chelating agent, previously labelled with 188Re, in cold lipiodol. The radiochemical purity (RCP) of labelling was checked immediately. The 188Re-SSS lipiodol was injected into the hepatic artery of nine rats with a Novikoff hepatoma. They were sacrificed 1, 24 and 48 h after injection, and used for ex vivo counting. Labelling of 188Re-SSS lipiodol was achieved with a yield of 97.3+/-2.1%. The immediate RCP was 94.1+/-1.7%. Ex vivo counting confirmed a predominantly hepatic uptake, with a good tumoral retention of 188Re-SSS lipiodol, a weak pulmonary uptake and a very faint digestive uptake. The 'tumour/non-tumoral liver' ratio was high at 1, 24 and 48 h after injection (2.9+/-1.5, 4.1+/-/4.1 and 4.1+/-0.7, respectively). Using the method described here, 188Re-SSS lipiodol can be obtained with a very high yield and a satisfactory RCP. The biodistribution in rats with hepatoma indicates a good tumoral retention of 188Re-SSS lipiodol associated with a predominant hepatic uptake, a weak pulmonary uptake and a very faint digestive uptake. This product should be considered for intra-arterial radiation therapy in human hepatoma.

Increased IL-35 producing Tregs and CD19+IL-35+ cells are associated with disease progression in leprosy patients.

PubMed

Tarique, Mohd; Saini, Chaman; Naqvi, Raza Ali; Khanna, Neena; Rao, D N

2017-03-01

The clinical forms of leprosy consist of a spectrum that reflects the host's immune response to the M. leprae; it provides an ideal model to study the host pathogen interaction and immunological dysregulation in humans. IL-10 and TGF-β producing Tregs are high in leprosy patients and responsible for immune suppression and M. leprae specific T cells anergy. In leprosy, involvement of IL-35 producing Tregs and Bregs remain unstudied. To study the role of IL-35 producing Tregs and Bregs in the human leprosy. Peripheral blood mononuclear cells from leprosy patients were isolated and stimulated with M. leprae antigen (MLCwA) for 48h. Intracellular cytokine IL-35 was evaluated in CD4 + CD25 + Tregs, CD19 + cells by FACS. Expression of PD-1 on CD4 + CD25 + Tregs, CD19 + cells and its ligand (PD-L1) on B cells, CD11c cells were evaluated by flow cytometry (FACS). Serum IL-35 level was estimated by ELISA. The frequency of IL-35 producing Tregs and Bregs cells were found to be high in leprosy patients (p<0.0001) as compared to healthy controls. These cells produced suppressive cytokine IL-35 which showed positive correlation with bacteriological index (BI) and TGF-β producing Tregs, indicating its suppressive nature. We found higher expression of PD-1 on Tregs, B cell and its ligand (PD-L1) on antigen presenting cells in leprosy patients. This study point out a shift in our understanding of the immunological features that mediate and regulate the immune suppression and the disease progression in leprosy patients with a new paradigm (IL-35 producing Tregs and Bregs) that is beyond TGF-β and IL-10 producing Treg cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Thyroid hormone receptor inhibits hepatoma cell migration through transcriptional activation of Dickkopf 4

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

Chi, Hsiang-Cheng; Liao, Chen-Hsin; Huang, Ya-Hui

Highlights: •T{sub 3} affects DKK4 mRNA and protein expression in HepG2-TR cells. •Regulation of DKK4 by T{sub 3} is at transcriptional level. •DKK4 overexpression suppresses hepatoma cell metastasis. -- Abstract: Triiodothyronine (T{sub 3}) is a potent form of thyroid hormone mediates several physiological processes including cellular growth, development, and differentiation via binding to the nuclear thyroid hormone receptor (TR). Recent studies have demonstrated critical roles of T{sub 3}/TR in tumor progression. Moreover, long-term hypothyroidism appears to be associated with the incidence of human hepatocellular carcinoma (HCC), independent of other major HCC risk factors. Dickkopf (DKK) 4, a secreted protein thatmore » antagonizes the canonical Wnt signaling pathway, is induced by T{sub 3} at both mRNA and protein levels in HCC cell lines. However, the mechanism underlying T{sub 3}-mediated regulation of DKK4 remains unknown. In the present study, the 5′ promoter region of DKK4 was serially deleted, and the reporter assay performed to localize the T{sub 3} response element (TRE). Consequently, we identified an atypical direct repeat TRE between nucleotides −1645 and −1629 conferring T{sub 3} responsiveness to the DKK4 gene. This region was further validated using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Stable DKK4 overexpression in SK-Hep-1 cells suppressed cell invasion and metastatic potential, both in vivo andin vitro, via reduction of matrix metalloproteinase-2 (MMP-2) expression. Our findings collectively suggest that DKK4 upregulated by T{sub 3}/TR antagonizes the Wnt signal pathway to suppress tumor cell progression, thus providing new insights into the molecular mechanism underlying thyroid hormone activity in HCC.« less

18 CFR Appendix A to Subpart H of... - Appendix A to Subpart H of Part 35

Code of Federal Regulations, 2010 CFR

2010-04-01

... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Appendix A to Subpart H of Part 35 A Appendix A to Subpart H of Part 35 Conservation of Power and Water Resources FEDERAL... Rates Pt. 35, Subpt. H, App. A Appendix A to Subpart H of Part 35 Appendix A Standard Screen Format...

18 CFR Appendix B to Subpart H of... - Appendix B to Subpart H of Part 35

Code of Federal Regulations, 2010 CFR

2010-04-01

... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Appendix B to Subpart H of Part 35 B Appendix B to Subpart H of Part 35 Conservation of Power and Water Resources FEDERAL... Rates Pt. 35, Subpt. H, App. B Appendix B to Subpart H of Part 35 This is an example of the required...

Study on relationship between expression level and molecular conformations of gene drugs targeting to hepatoma cells in vitro

PubMed Central

Yang, Dong-Ye; Lu, Fang-Gen; Tang, Xi-Xiang; Zhao, Shui-Ping; Ouyang, Chun-Hui; Wu, Xiao-Ping; Liu, Xiao-Wei; Wu, Xiao-Ying

2003-01-01

AIM: To increase exogenous gene expression level by modulating molecular conformations of targeting gene drugs. METHODS: The full length cDNAs of both P40 and P35 subunits of human interleukin 12 were amplified through polymerase chain reaction (PCR) and cloned into eukaryotic expressing vectors pcDNA3.1 (±) to construct plasmids of P (+)/IL-12, P (+)/P40 and P (-)/P35. These plasmids were combined with ASOR-PLL to form two targeting gene drugs [ASOR-PLL-P (+)/IL-12 and ASOR-PLL-P (+)/P40 + ASOR-PLL-P (-)/P35] in optimal ratios. The conformations of these two drugs at various concentrations adjuvant were examined under electron microscope (EM) and the drugs were transfected into HepG2 (ASGr+) cells. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed with total RNA extracted from the transfected cells to determine the hIL12 mRNA transcript level. The hIL12 protein in the cultured supernatant was measured with enzyme-linked immunosorbent assay (ELISA) 48 hours after transfection. RESULTS: Targeting gene drugs, whose structures were granular and circle-like and diameters ranged from 25 nm to 150 nm, had the highest hIL-12 expression level. The hIL-12 expression level in the group co-transfected with ASOR-PLL-P (+)/P40 and ASOR-PLL-P (-)/P35 was higher than that of ASOR-PLL-P (+)/IL-12 transfected group. CONCLUSION: The molecular conformations of targeting gene drugs play an important role in exogenous gene expression level, the best structures are granular and circle-like and their diameters range from 25 nm to 150 nm. The sizes and linking styles of exogenous genes also have some effects on their expression level. PMID:12970883

Transcriptional regulation of human paraoxonase 1 by PXR and GR in human hepatoma cells.

PubMed

Ponce-Ruiz, N; Rojas-García, A E; Barrón-Vivanco, B S; Elizondo, G; Bernal-Hernández, Y Y; Mejía-García, A; Medina-Díaz, I M

2015-12-25

Human paraoxonase 1 (PON1) is A-esterase synthesized in the liver and secreted into the plasma, where it associates with HDL. PON1 acts as an antioxidant preventing lipid oxidation and detoxifies a wide range of substrates, including organophosphate compounds. The variability of PON1 (enzyme activity/serum levels) has been attributed to internal and external factors. However, the molecular mechanisms involved in the transcriptional regulation of PON1 have not been well-studied. The aim of this study was to evaluate and characterize the transcriptional activation of PON1 by nuclear receptors (NR) in human hepatoma cells. In silico analysis was performed on the promoter region of PON1 to determine the response elements of NR. Real-time PCR was used to evaluate the effect of specific NR ligands on the mRNA levels of genes regulated by NR and PON1. The results indicated that NR response elements had 95% homology to pregnenolone (PXR), glucocorticoids (GR), retinoic acid (RXR) and peroxisomes proliferator-activated receptor alpha (PPARα). Treatments with Dexamethasone (GR ligand), Rifampicin (PXR ligand) and TCDD (AhR ligand) increased the mRNA levels of PON1 at 24 and 48 h. We showed that the activation of GR by Dexamethasone results in PON1 gene induction accompanied by an increase in activity levels. In conclusion, these results demonstrate that GR regulates PON1 gene transcription through directly binding to NR response elements at -95 to -628 bp of the PON1 promoter. This study suggests new molecular mechanisms for the transcriptional regulation of PON1 through a process involving the activation of PXR. Copyright © 2015 Elsevier B.V. All rights reserved.

Intrinsic anticarcinogenic effects of Piper sarmentosum ethanolic extract on a human hepatoma cell line

PubMed Central

Zainal Ariffin, Shahrul Hisham; Wan Omar, Wan Haifa Haryani; Zainal Ariffin, Zaidah; Safian, Muhd Fauzi; Senafi, Sahidan; Megat Abdul Wahab, Rohaya

2009-01-01

Background Piper sarmentosum, locally known as kaduk is belonging to the family of Piperaceae. It is our interest to evaluate their effect on human hepatoma cell line (HepG2) for the potential of anticarcinogenic activity. Results The anticarcinogenic activity of an ethanolic extract from Piper sarmentosum in HepG2 and non-malignant Chang's liver cell lines has been previously determined using (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) (MTT) assays, where the IC50 value was used as a parameter for cytotoxicity. The ethanolic extract that showed anticarcinogenic properties in HepG2 cells had an IC50 of 12.5 μg mL-1, while IC50 values in the non-malignant Chang's liver cell line were greater than 30 μg mL-1. Apoptotic morphological changes in HepG2 cells were observed using an inverted microscope and showed chromatin condensation, cell shrinkage and apoptotic bodies following May-Grunwald-Giemsa's staining. The percentage of apoptotic cells in the overall population (apoptotic index) showed a continuously significant increase (p < 0.05) in 12.5 μg mL-1 ethanolic extract-treated cells at 24, 48 and 72 hours compared to controls (untreated cells). Following acridine orange and ethidium bromide staining, treatment with 10, 12 and 14 μg mL-1 of ethanolic extracts caused typical apoptotic morphological changes in HepG2 cells. Molecular analysis of DNA fragmentation was used to examine intrinsic apoptosis induced by the ethanolic extracts. These results showed a typical intrinsic apoptotic characterisation, which included fragmentation of nuclear DNA in ethanolic extract-treated HepG2 cells. However, the non-malignant Chang's liver cell line produced no DNA fragmentation. In addition, the DNA genome was similarly intact for both the untreated non-malignant Chang's liver and HepG2 cell lines. Conclusion Therefore, our results suggest that the ethanolic extract from P. sarmentosum induced anticarcinogenic activity through an intrinsic apoptosis

Role of intracellular Ca2+ signal in the ascorbate-induced apoptosis in a human hepatoma cell line.

PubMed

Lee, Yong Soo

2004-12-01

Although ascorbate (vitamin C) has been shown to have anti-cancer actions, its effect on human hepatoma cells has not yet been investigated, and thus, the exact mechanism of this action is not fully understood. In this study, the mechanism by which ascorbate induces apoptosis using HepG2 human hepatoblastoma cells is investigated. Ascorbate induced apoptotic cell death in a dose-dependent manner in the cells, was assessed through flow cytometric analysis. Contrary to expectation, ascorbate did not alter the cellular redox status, and treatment with antioxidants (N-acetyl cysteine and N,N-diphenyl-p-phenylenediamine) had no influence on the ascorbate-induced apoptosis. However, ascorbate induced a rapid and sustained increase in intracellular Ca2+ concentration. EGTA, an extracellular Ca2+ chelator did not significantly alter the ascorbate-induced intracellular Ca2+ increase and apoptosis, whereas dantrolene, an intracellular Ca2+ release blocker, completely blocked these actions of ascorbate. In addition, phospholipase C (PLC) inhibitors (U-73122 and manoalide) significantly suppressed the intracellular Ca2+ release and apoptosis induced by ascorbate. Collectively, these results suggest that ascorbate induced apoptosis without changes in the cellular redox status in HepG2 cells, and that the PLC-coupled intracellular Ca2+ release mechanism may mediate ascorbate-induced apoptosis.

PKC-Theta is a Novel SC35 Splicing Factor Regulator in Response to T Cell Activation.

PubMed

McCuaig, Robert Duncan; Dunn, Jennifer; Li, Jasmine; Masch, Antonia; Knaute, Tobias; Schutkowski, Mike; Zerweck, Johannes; Rao, Sudha

2015-01-01

Alternative splicing of nuclear pre-mRNA is essential for generating protein diversity and regulating gene expression. While many immunologically relevant genes undergo alternative splicing, the role of regulated splicing in T cell immune responses is largely unexplored, and the signaling pathways and splicing factors that regulate alternative splicing in T cells are poorly defined. Here, we show using a combination of Jurkat T cells, human primary T cells, and ex vivo naïve and effector virus-specific T cells isolated after influenza A virus infection that SC35 phosphorylation is induced in response to stimulatory signals. We show that SC35 colocalizes with RNA polymerase II in activated T cells and spatially overlaps with H3K27ac and H3K4me3, which mark transcriptionally active genes. Interestingly, SC35 remains coupled to the active histone marks in the absence of continuing stimulatory signals. We show for the first time that nuclear PKC-θ co-exists with SC35 in the context of the chromatin template and is a key regulator of SC35 in T cells, directly phosphorylating SC35 peptide residues at RNA recognition motif and RS domains. Collectively, our findings suggest that nuclear PKC-θ is a novel regulator of the key splicing factor SC35 in T cells.

Overexpression of p35 in Min6 pancreatic beta cells induces a stressed neuron-like apoptosis.

PubMed

Zheng, Ya-Li; Hu, Ya-Fang; Zhang, Aiping; Wang, Wei; Li, Bo; Amin, Niranjana; Grant, Philip; Pant, Harish C

2010-12-15

Cdk5 activity has been implicated in brain development and the regulation of many neuronal processes. Recently, the expression of p35 and Cdk5 activity has been reported in pancreatic beta cells. Decreased Cdk5 activity enhanced glucose-stimulated insulin secretion. This suggests that Cdk5 may play an important role in the regulation of insulin secretion. To further understand how Cdk5 regulates insulin secretion in glucose-stimulated pancreatic β cells, we first confirmed the presence of a low level of p35 in pancreatic Min6 cells. Next, in a time-course experiment in high glucose (25 mM) we showed that endogenous p35 increased gradually accompanied by a 3-fold increase in Cdk5 activity by 16 h. Insulin secretion, however, doubled after 2 h followed by progressive downregulation, negatively correlated with Cdk5 activity. On the other hand, overexpression of p35 in these cells resulted in more than a three-fold increase in Cdk5 activity within 2 h coupled to a 50% reduction in insulin secretion in both high and low (3 mM) glucose. Most significantly, cells overexpressing p35, treated with high glucose for 4 h, showed induction of p25, the p35-derived truncated fragment which hyperactivates Cdk5 in neurons. As a result, insulin secretion was inhibited and cells became apoptotic. Roscovitine or co-infection of dominant negative Cdk5 (dnCdk5) with p35 increased insulin secretion and inhibited apoptosis. These results suggest that the model for deregulation and hyperactivation of Cdk5 in neurodegeneration may apply to the pathology seen in type 2 diabetes (T2DM). It is consistent with the view that Alzheimer's disease and T2DM are linked metabolically and pathologically by Cdk5 in a number of ways. Copyright © 2010. Published by Elsevier B.V.

Anti-proliferative and pro-apoptotic effect of Smilax glabra Roxb. extract on hepatoma cell lines.

PubMed

Sa, Fei; Gao, Jian-Li; Fung, Kwok-Pui; Zheng, Ying; Lee, Simon Ming-Yuen; Wang, Yi-Tao

2008-01-10

Smilax glabra Roxb. (SGR) is the root of a traditional Chinese herb, referred to as tu fu ling in Chinese medicine. It is an inexpensive traditional Chinese medicine commonly used for the treatment of liver diseases, and a few studies have indicated that SGR has anti-hepatocarcinogenic and anti-cancer growth activities. In the current study, raw SGR plant was extracted with Accelerate Solvent Extractor, and the molecular mechanism by which S. glabra Roxb. extract (SGRE) has an anti-proliferative effect on the human hepatoma cell lines, HepG2 and Hep3B, was determined. We showed that SGRE inhibited HepG2 and Hep3B cell growth by causing cell-cycle arrest at either S phase or S/G2 transition and induced apoptosis, as evidenced by a DNA fragmentation assay. SGRE-induced apoptosis by alternation of mitochondrial transmembrane depolarization, release of mitochondrial cytochrome c, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. The SGRE-mediated mitochondria-caspase dependent apoptotic pathway also involved activation of p38, JNK, and ERK mitogen-activated protein kinase signaling. Isometric compounds of astilbin (flavonoids) and smilagenin (saponin) have been identified as the main chemical constituents in SGRE by HPLC-MS/MS. These results have identified, for the first time, the biological activity of SGRE in HepG2 and Hep3B cells and should lead to further development of SGR for liver disease therapy.

Cytochrome P4501A induction, benzo[a]pyrene metabolism, and nucleotide adduct formation in fish hepatoma cells: Effect of preexposure to 3,3',4,4',5-pentachlorobiphenyl

USGS Publications Warehouse

Smeets, J.M.W.; Voormolen, A.; Tillitt, D.E.; Everaarts, J.M.; Seinen, W.; Vanden Berg, M.D.

1999-01-01

In PLHC-1 hepatoma cells, benzo[a]pyrene (B[a]P) caused a maximum induction of cytochrome P4501A (CYP1A) activity, measured as ethoxyresorufin O-deethylation (EROD), after 4 to 8 h of exposure, depending on the B[a]P concentration. The decline of EROD activity at longer exposure times was probably caused by the rapid metabolism of B[a]P in this system (57% metabolism within 4 h incubation). In subsequent experiments, PLHC-1 cells were preinduced with PCB 126 for 24 h and then received a dose of 10, 100, or 1,000 nM 3H-B[a]P. A 1-nM concentration of PCB 126 caused an 80-fold induction of CYP1A activity, resulting in an increase in B[a]P metabolism of less than 10%, except at the highest concentration of B[a]P (1,000 nM), where a 50% increase was observed. In another experiment, an 80-fold induction of CYP1A activity caused a 20% increase in the metabolism of B[a]P (100 nM), and RNA adduct formation was increased approximately twofold. These results indicate that, at exposure concentrations up to 100 nM B[a]P, CYP1A activity is not rate limiting for B[a]P metabolism. Furthermore, CYP1A seems to also he specifically involved in B[a]P activation in PLHC-1 cells. However, CYP1A induction causes only a relatively small increase in activation, probably because of the action of other enzymes involved in B[a]P activation and deactivation.

Interferon induced protein 35 exacerbates H5N1 influenza disease through the expression of IL-12p40 homodimer.

PubMed

Gounder, Anshu P; Yokoyama, Christine C; Jarjour, Nicholas N; Bricker, Traci L; Edelson, Brian T; Boon, Adrianus C M

2018-04-01

Pro-inflammatory cytokinemia is a hallmark of highly pathogenic H5N1 influenza virus (IAV) disease yet little is known about the role of host proteins in modulating a pathogenic innate immune response. The host Interferon Induced Protein 35 (Ifi35) has been implicated in increased susceptibility to H5N1-IAV infection. Here, we show that Ifi35 deficiency leads to reduced morbidity in mouse models of highly pathogenic H5N1- and pandemic H1N1-IAV infection. Reduced weight loss in Ifi35-/- mice following H5N1-IAV challenge was associated with reduced cellular infiltration and decreased production of specific cytokines and chemokines including IL-12p40. Expression of Ifi35 by the hematopoietic cell compartment in bone-marrow chimeric mice contributed to increased immune cell recruitment and IL-12p40 production. In addition, Ifi35 deficient primary macrophages produce less IL-12p40 following TLR-3, TLR-4, and TLR-7 stimulation in vitro. Decreased levels of IL-12p40 and its homodimer, IL-12p80, were found in bronchoalveolar lavage fluid of H5N1-IAV infected Ifi35 deficient mice. Specific antibody blockade of IL-12p80 ameliorated weight loss and reduced cellular infiltration following H5N1-IAV infection in wild-type mice; suggesting that increased levels of IL-12p80 alters the immune response to promote inflammation and IAV disease. These data establish a role for Ifi35 in modulating cytokine production and exacerbating inflammation during IAV infection.

Antitumor potential of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H-pyrazoles in human bladder cancer cells.

PubMed

Tessmann, Josiane Weber; Buss, Julieti; Begnini, Karine Rech; Berneira, Lucas Moraes; Paula, Favero Reisdorfer; de Pereira, Claudio Martin Pereira; Collares, Tiago; Seixas, Fabiana Kömmling

2017-10-01

Bladder cancer is a genitourinary malignant disease common worldwide. Current chemotherapy is often limited mainly due to toxicity and drug resistance. Thus, there is a continued need to discover new therapies. Recently evidences shows that pyrazoline derivatives are promising antitumor agents in many types of cancers, but there are no studies with bladder cancer. In order to find potent and novel chemotherapy drugs for bladder cancer, a series of pyrazoline derivatives 2a-2d were tested for their antitumor activity in two human bladder cancer cell lines 5647 and T24. The MTT assay showed that the compounds 1-thiocarbamoyl-3,5-diphenyl-4,5-dihydro-1H-pyrazole (2a) and 1-thiocarbamoyl-5-(4-chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole (2c) decrease the cell viability of 5637 cells. Molecular modeling indicated that these compounds had a good oral bioavailability and low toxicities. Clonogenic assay and flow cytometric analysis were used to assess colony formation, apoptosis induction and cell cycle distribution. Overall, our results suggest that pyrazoline 2a and 2c, with the substituents hydrogen and chlorine respectively, may decrease cell viability and colony formation of bladder cancer 5637 cell line by inhibition of cell cycle progression, and for pyrazoline 2a, by induction of apoptosis. As indicated by the physicochemical properties of these compounds, the steric factor influences the activity. Therefore, these pyrazoline derivatives can be considered promising anticancer agents for the treatment of bladder cancer. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Hypoxia on the Expression of Hepatoma Upregulated Protein in Prostate Cancer Cells

PubMed Central

Espinoza, Ingrid; Sakiyama, Marcelo J.; Ma, Tangeng; Fair, Logan; Zhou, Xinchun; Hassan, Mohamed; Zabaleta, Jovanny; Gomez, Christian R.

2016-01-01

Hepatoma upregulated protein (HURP) is a multifunctional protein with clinical promise. This protein has been demonstrated to be a predictive marker for the outcome in high-risk prostate cancer (PCa) patients, besides being a resistance factor in PCa. Although changes in oxygen tension (pO2) are associated with PCa aggressiveness, the role of hypoxia in the regulation of tumor progression genes such as HURP has not yet been described. We hypothesized that pO2 alteration is involved in the regulation of HURP expression in PCa cells. In the present study, PCa cells were incubated at 2% O2 (hypoxia) and 20% O2 (normoxia) conditions. Hypoxia reduced cell growth rate of PCa cells, when compared to the growth rate of cells cultured under normoxia (p < 0.05). The decrease in cell viability was accompanied by fivefold (p < 0.05) elevated rate of vascular endothelial growth factor (VEGF) release. The expression of VEGF and the hypoxia-inducible metabolic enzyme carbonic anhydrase 9 were elevated maximally nearly 61-fold and 200-fold, respectively (p < 0.05). Noted in two cell lines (LNCaP and C4-2B) and independent of the oxygen levels, HURP expression assessed at both mRNA and protein levels was reduced. However, the decrease was more pronounced in cells cultured under hypoxia (p < 0.05). Interestingly, the analysis of patients’ specimens by Western blot revealed a marked increase of HURP protein (fivefold), when compared to control (cystoprostatectomy) tissue (p < 0.05). Immunohistochemistry analysis showed an increase in the immunostaining intensity of HURP and the hypoxia-sensitive molecules, hypoxia-inducible factor 1-alpha (HIF-1α), VEGF, and heat-shock protein 60 (HSP60) in association with tumor grade. The data also suggested a redistribution of subcellular localization for HURP and HIF-1α from the nucleus to the cytoplasmic compartment in relation to increasing tumor grade. Analysis of HURP Promoter for HIF-1-binding sites revealed presence

pPKCδ activates SC35 splicing factor during H9c2 myoblastic differentiation.

PubMed

Zara, Susi; Falconi, Mirella; Rapino, Monica; Zago, Michela; Orsini, Giovanna; Mazzotti, Giovanni; Cataldi, Amelia; Teti, Gabriella

2011-01-01

Although Protein Kinase C (PKC) isoforms' role in the neonatal and adult cardiac tissue development and ageing has been widely described "in vivo", the interaction of such enzymes with specific nuclear substrates needs to be investigated. The aim of our research has been the study of the expression, localization and interaction with the splicing factor SC35 of PKC isoforms (α, δ, ε, ζ) and their potential role in modulating the transcription machinery. H9c2 cells induced to myoblast differentiation in the presence of 1% Horse Serum (HS) have represented our experimental model. The expression of PKC isoforms, their distribution and interaction with SC35 have been evaluated by western blotting, co-immunoprecipitation and double gold immunolabeling for transmission and scanning electron microscopy. Our results show PKCδ as the most expressed isoform in differentiated cells. Surprisingly, the distribution of PKCδ and SC35 does not show any significant modification between 10%FBS and 1%HS treated samples and no co-localization is observed. Moreover the interaction between the phosphorylated form of PKCδ (pPKCδ) and SC35 increases, is distributed and co-localizes within the nucleus of differentiated H9c2. These data represent reasonable evidence of pPKCδ mediated SC35 splicing factor activation, suggesting its direct effect on transcription via interaction with the transcription machinery. Furthermore, this co-localization represents a crucial event resulting in downstream changes in transcription of components which determine the morphological modifications related to cardiomyoblast differentiated phenotype.

Evaluating the potential of (188)Re-ECD/lipiodol as a therapeutic radiopharmaceutical by intratumoral injection for hepatoma treatment.

PubMed

Luo, Tsai-Yueh; Shih, Ying-Hsia; Chen, Chiung-Yu; Tang, I-Chung; Wu, Yu-Long; Kung, Hong-Chang; Lin, Wuu-Jyh; Lin, Xi-Zhang

2009-10-01

Intratumoral injection of a radiopharmaceutical is a potential modality to treat liver tumors. Rhenium-188 ((188)Re) was used to chelate with ethyl cysteinate dimer (ECD) in lipiodol solution to form (188)Re-ECD/lipiodol, which was then evaluated for its therapeutic potential in a rodent hepatoma model. Male Sprague-Dawley rats were implanted with N1-S1 hepatoma cells orthotopically and randomly divided into two groups. Group 1 (n = 29) and group 2 (n = 10) received (188)Re-ECD/lipiodol (30.4 +/- 21.8 MBq/0.1 mL) and 0.1 mL of normal saline by intratumoral injection, respectively. Three rats in group 1 were imaged by micro-single-photon emission computed tomography/computed tomography scan to evaluate the biodistribution pattern. All rats were monitored for change of tumor size and survival rate after 2 months. The in vitro stability test showed that (188)Re-ECD was well-retained in the lipiodol phase for 48 hours. The biodistribution image revealed that radioactivity was retained well in hepatomas 24 hours postinjection. Long-term studies demonstrated that rats treated with (188)Re-ECD/Lipiodol had smaller tumor volumes and a better survival rate, compared to the control group. At the end of observation, the survival rates in groups 1 and 2 were 62% and 20%, respectively (p < 0.05). (188)Re-ECD/lipiodol via direct intratumoral injection shows potential for treating hepatoma and warrants further clinical trials.

Synthesis and cytotoxic activity evaluation of some novel 1-(3-(aryl-4,5-dihydroisoxazol-5-yl)methyl)-4-trihalomethyl-1H-pyrimidin-2-ones in human cancer cells.

PubMed

Lobo, Marcio M; Viau, Cassiana M; Dos Santos, Josiane M; Bonacorso, Helio G; Martins, Marcos A P; Amaral, Simone S; Saffi, Jenifer; Zanatta, Nilo

2015-08-28

The synthesis of a series of 14 new 1-(3-(aryl-4,5-dihydroisoxazol-5-yl)methyl)-4-trihalomethyl-1H-pyrimidin-2-ones from the 1,3-dipolar cycloaddition reaction of 1-allyl-4-(trihalomethyl)pyrimidin-2(1H)-ones with aryl nitrile oxides is described. Also, the antiproliferative activity of the title compounds was tested against five human tumoral cell lines: MCF-7 breast cancer cell line, ER+ (estrogen receptor positive); HepG-2 (hepatoma); T-24 (bladder cancer); HCT-116 cell (colorectal carcinoma); and CACO-2. The preliminary results are promising, since three compounds presented IC50 values below 2 μM, as well as moderate to high selectivity. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Carrier effects of dosing the h4iie cells with 3,3′,4,4tt´etrachlorobiphenyl (PCB77) in dimethyl sulfoxide or isooctane

USGS Publications Warehouse

Yu, Kyung O.; Fisher, Jeff W.; Burton, G. Allen; Tillitt, Donald E.

1997-01-01

A rat hepatoma cell line, H4IIE serves as a bioassay tool to assess the potential toxicity of dioxin-like chemicals, including polychlorinated biphenyls (PCB) in environmental samples. PCB exposure to these cells induces cytochrome (CYP) P4501A1 activity in a dose-dependent fashion, thus allowing assessment of mixtures. The objective of this study was to determine the effect of different carriers, dimethyl sulfoxide (DMSO) and isooctane on the concentrations of PCBs in the H411E cells and induction of CYPIA1 activity as measured by ethoxyresorufm O-deethylase (EROD) activity. H4IIE cells were dosed with three micrograms of UL-14C-PCB77/ plate dissolved in DMSO or isooctane, and were harvested at sequential time periods for 4 days. PCB77 concentration and EROD activity were measured in the cells. EROD activity was greater when using DMSO as compared to isooctane, while there was no difference in the distribution of PCB77-derived radioactivities within the cell culture system based upon the carrier solvent used to deliver PCB77.

Modifications in plasma membrane lipid composition and morphological features of AH-130 hepatoma cells by polyenylphosphatidylcholine in vivo treatment.

PubMed

Cinosi, Vincenzo; Antonini, Roberto; Crateri, Pasqualina; Arancia, Giuseppe

2011-07-01

The plasma membrane lipid composition in AH-130 hepatoma cells was found to change remarkably after polyenylphosphatidylcholine (PPC) treatment. Plasma membranes from cells grown in rats treated for 7 days i.v. with 20 mg/kg/day PPC, when compared to those of control cells, did not show significantly different amounts of cholesterol or phospholipids relative to protein content, but, surprisingly, the individual phospholipid distribution inside the two membrane leaflets changed dramatically. Phosphatidylcholine (PC), the major phospholipid in the external membrane leaflet, increased ~47% (p<0.001). By contrast, phosphatidylethanolamine (PE), the most important component of the inner leaflet, decreased nearly 37% (p<0.001), while sphingomyelin (SM) also decreased ~17%, (p=0.1). Tumor cells collected from control rats at the same time interval and observed by scanning electron microscopy, exhibited a spherical shape with numerous and randomly distributed long microvilli, the same morphological and ultrastructural features displayed by the implanted cells. Conversely, tumor cells from PPC-treated rats no longer showed the roundish cell profile, and microvilli appeared shortened and enlarged, with the formation of surface blebs. Transmission electron microscopy observations confirmed the morphological and ultrastructural cell changes, mainly seen as loss of microvilli and intense cytoplasmic vacuolization. Taken together, these results indicate that the new phospholipid class distribution in the plasma membrane leaflets, modifying tumor cell viable structures, produced heavy cell damage and in many cases brought about complete cellular disintegration.

Interleukin-35 induces regulatory B cells that suppress autoimmune disease.

PubMed

Wang, Ren-Xi; Yu, Cheng-Rong; Dambuza, Ivy M; Mahdi, Rashid M; Dolinska, Monika B; Sergeev, Yuri V; Wingfield, Paul T; Kim, Sung-Hye; Egwuagu, Charles E

2014-06-01

Interleukin-10 (IL-10)-producing regulatory B (Breg) cells suppress autoimmune disease, and increased numbers of Breg cells prevent host defense to infection and promote tumor growth and metastasis by converting resting CD4(+) T cells to regulatory T (Treg) cells. The mechanisms mediating the induction and development of Breg cells remain unclear. Here we show that IL-35 induces Breg cells and promotes their conversion to a Breg subset that produces IL-35 as well as IL-10. Treatment of mice with IL-35 conferred protection from experimental autoimmune uveitis (EAU), and mice lacking IL-35 (p35 knockout (KO) mice) or defective in IL-35 signaling (IL-12Rβ2 KO mice) produced less Breg cells endogenously or after treatment with IL-35 and developed severe uveitis. Adoptive transfer of Breg cells induced by recombinant IL-35 suppressed EAU when transferred to mice with established disease, inhibiting pathogenic T helper type 17 (TH17) and TH1 cells while promoting Treg cell expansion. In B cells, IL-35 activates STAT1 and STAT3 through the IL-35 receptor comprising the IL-12Rβ2 and IL-27Rα subunits. As IL-35 also induced the conversion of human B cells into Breg cells, these findings suggest that IL-35 may be used to induce autologous Breg and IL-35(+) Breg cells and treat autoimmune and inflammatory disease.

IL-35-producing B cells in gastric cancer patients.

PubMed

Wang, Ke; Liu, Jianming; Li, Jiansheng

2018-05-01

A significant characteristic of advanced gastric cancer (GC) is immune suppression, which can promote the progression of GC. Interleukin 35 (IL-35) is an immune-suppressing cytokine, and it is generally recognized that this cytokine is secreted by regulatory T (Treg) cells. Recently, studies have found that IL-35 can also be produced by B cells in mice. However, scientific studies reporting that IL-35 is secreted by B cells in humans, specifically in cancer patients, are very rare.Blood samples were collected from 30 healthy controls (HCs) and 50 untreated GC patients, and IL-35-producing B cells in the peripheral blood were investigated. Moreover, Treg cells (CD4CD25CD127), myeloid-derived suppressor cells (MDSCs) (CD14HLA-DR) and other lymphocyte subsets (CD3, CD4, CD8 T cells, activated and memory CD4 T cells, activated CD8 T cells, CD14 monocytes, and IL-10-producing B cells) were also examined.IL-35-producing B cells were significantly upregulated in patients with advanced GC. Furthermore, the frequency of IL-35-producing B cells was positively correlated with the frequencies of Treg cells (CD4CD25CD127), MDSCs (CD14HLA-DR), IL-10-producing B cells, and CD14 monocytes in these GC patients.In summary, the frequency of IL-35-producing B cells is significantly elevated in advanced GC; this outcome implies that this group of B cells may participate in GC progression.

Overexpression of protein kinase FA/GSK-3 alpha (a proline-directed protein kinase) correlates with human hepatoma dedifferentiation/progression.

PubMed

Yang, S D; Yu, J S; Yang, C C; Lee, S C; Lee, T T; Ni, M H; Kuan, C Y; Chen, H C

1996-05-01

Computer analysis of protein phosphorylation sites sequence revealed that transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of the proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3 alpha (kinase F(A)/GSK-3 alpha) (a member of PDPK family) has been optimized for human hepatoma and used to demonstrate for the first time significantly increased (P < 0.01) activity in poorly differentiated SK-Hep-1 hepatoma (24.2 +/- 2.8 units/mg) and moderately differentiated Mahlavu hepatoma (14.5 +/- 2.2 units/mg) when compared to well differentiated Hep 3B hepatoma (8.0 +/- 2.4 units/mg). Immunoblotting analysis revealed that increased activity of kinase FA/GSK-3 alpha is due to overexpression of the protein. Elevated kinase FA/GSK-3 alpha expression in human hepatoma biopsies relative to normal liver tissue was found to be even more profound. This kinase appeared to be fivefold overexpressed in well differentiated hepatoma and 13-fold overexpressed in poorly differentiated hepatoma when compared to normal liver tissue. Taken together, the results provide initial evidence that overexpression of kinase FA/GSK-3 alpha is involved in human hepatoma dedifferentiation/progression. Since kinase FA/GSK-3 alpha is a PDPK, the results further support a potential role of this kinase in human liver tumorigenesis, especially in its dedifferentiation/progression.

A study on the thermochemotherapy effect of nanosized As2O3/MZF thermosensitive magnetoliposomes on experimental hepatoma in vitro and in vivo

NASA Astrophysics Data System (ADS)

Wang, Li; Zhang, Jia; An, Yanli; Wang, Ziyu; Liu, Jing; Li, Yutao; Zhang, Dongsheng

2011-08-01

In this paper, we describe the synthesis and characterization of a nanosized, thermosensitive magnetoliposome encapsulating magnetic nanoparticles (MZFs) and antitumor drugs (As2O3). The nanoliposomes were spherical and mostly single volume, with an average diameter of 128.2 nm. Differential scanning calorimetry (DSC) showed a liposome phase transition temperature of 42.71 °C. After that, we studied the liposomes' anti-hepatoma effect in vitro and in vivo. The antitumor effect of the nanoliposomes on human hepatoma cells, SMMC-7721, and changes in expression of apoptosis-related proteins were examined in vitro. The results show that As2O3/MZF thermosensitive magnetoliposomes combined with hyperthermia had a great impact on the Bax/Bcl-2 ratio, which increased to 1.914 and exhibited a rapid response to induce apoptosis of tumor cells. An in situ rabbit liver tumor model was established and used to evaluate the antitumor effect of combined hyperthermia and chemotherapy following transcatheter arterial embolization with As2O3/MZF thermosensitive magnetoliposomes. The results demonstrated a strong anti-hepatoma effect, with a tumor volume inhibition rate of up to 85.22%. Thus, As2O3/MZF thermosensitive magnetoliposomes may play a great role in the treatment of hepatocarcinoma.

Magnetic nanoparticles for targeted therapeutic gene delivery and magnetic-inducing heating on hepatoma

NASA Astrophysics Data System (ADS)

Yuan, Chenyan; An, Yanli; Zhang, Jia; Li, Hongbo; Zhang, Hao; Wang, Ling; Zhang, Dongsheng

2014-08-01

Gene therapy holds great promise for treating cancers, but their clinical applications are being hampered due to uncontrolled gene delivery and expression. To develop a targeted, safe and efficient tumor therapy system, we constructed a tissue-specific suicide gene delivery system by using magnetic nanoparticles (MNPs) as carriers for the combination of gene therapy and hyperthermia on hepatoma. The suicide gene was hepatoma-targeted and hypoxia-enhanced, and the MNPs possessed the ability to elevate temperature to the effective range for tumor hyperthermia as imposed on an alternating magnetic field (AMF). The tumoricidal effects of targeted gene therapy associated with hyperthermia were evaluated in vitro and in vivo. The experiment demonstrated that hyperthermia combined with a targeted gene therapy system proffer an effective tool for tumor therapy with high selectivity and the synergistic effect of hepatoma suppression.

MiR-30e suppresses proliferation of hepatoma cells via targeting prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA

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

Feng, Guoxing; Shi, Hui; Li, Jiong

Aberrant microRNA expression has been shown to be characteristic of many cancers. It has been reported that the expression levels of miR-30e are decreased in liver cancer tissues. However, the role of miR-30e in hepatocellular carcinoma remains poorly understood. In the present study, we investigated the significance of miR-30e in hepatocarcinogenesis. Bioinformatics analysis reveals a putative target site of miR-30e in the 3′-untranslated region (3′UTR) of prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA. Moreover, luciferase reporter gene assays verified that miR-30e directly targeted 3′UTR of P4HA1 mRNA. Then, we demonstrated that miR-30e was able to reduce the expression of P4HA1 atmore » the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis. Enforced expression of miR-30e suppressed proliferation of HepG2 cells by 5-ethynyl-2-deoxyuridine (EdU) assay and reduced colony formation of these cells by colony formation analysis. Conversely, anti-miR-30e enhanced the proliferation of hepatoma cells in vitro. Interestingly, the ectopic expression of P4HA1 could efficiently rescue the inhibition of cell proliferation mediated by miR-30e in HepG2 cells. Meanwhile, silencing of P4HA1 abolished the anti-miR-30e-induced proliferation of cells. Clinically, quantitative real-time PCR showed that miR-30e was down-regulated in liver tumor tissues relative to their peritumor tissues. The expression levels of miR-30e were negatively correlated to those of P4HA1 mRNA in clinical liver tumor tissues. Thus, we conclude that miR-30e suppresses proliferation of hepatoma cells through targeting P4HA1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • P4HA1 is a novel target gene of miR-30e. • P4HA1 is increased in clinical HCC tissues. • MiR-30e is negatively correlated with P4HA1 in clinical HCC tissues. • MiR-30e suppresses the proliferation of HCC cells

Cytotoxic and genotoxic potential of Cr(VI), Cr(III)-nitrate and Cr(III)-EDTA complex in human hepatoma (HepG2) cells.

PubMed

Novotnik, Breda; Ščančar, Janez; Milačič, Radmila; Filipič, Metka; Žegura, Bojana

2016-07-01

Chromium (Cr) and ethylenediaminetetraacetate (EDTA) are common environmental pollutants and can be present in high concentrations in surface waters at the same time. Therefore, chelation of Cr with EDTA can occur and thereby stable Cr(III)-EDTA complex is formed. Since there are no literature data on Cr(III)-EDTA toxicity, the aim of our work was to evaluate and compare Cr(III)-EDTA cytotoxic and genotoxic activity with those of Cr(VI) and Cr(III)-nitrate in human hepatoma (HepG2) cell line. First the effect of Cr(VI), Cr(III)-nitrate and Cr(III)-EDTA on cell viability was studied in the concentration range from 0.04 μg mL(-1) to 25 μg mL(-1) after 24 h exposure. Further the influence of non-cytotoxic concentrations of Cr(VI), Cr(III)-nitrate and Cr(III)-EDTA on DNA damage and genomic stability was determined with the comet assay and cytokinesis block micronucleus cytome assay, respectively. Cell viability was decreased only by Cr(VI) at concentrations above 1.0 μg mL(-1). Cr(VI) at ≥0.2 μg mL(-1) and Cr(III) at ≥1.0 μg mL(-1) induced DNA damage, while after Cr(III)-EDTA exposure no formation DNA strand breaks was determined. Statistically significant formation of micronuclei was induced only by Cr(VI) at ≥0.2 μg mL(-1), while no influence on the frequency of nuclear buds nor nucleoplasmic bridges was observed at any exposure. This study provides the first evidence that Cr(III)-EDTA did not induce DNA damage and had no influence on the genomic stability of HepG2 cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

[Pseudolaric acid B induces G2/M arrest and inhibits invasion and migration in HepG2 hepatoma cells].

PubMed

Li, Shuai; Guo, Lianyi

2018-01-01

Objective To investigate the mechanisms of pseudolaric acid B (PAB) blocks cell cycle and inhibits invasion and migration in human hepatoma HepG2 cells. Methods The proliferation effect of PAB on HepG2 cells was evaluated by MTT assay. The effect of PAB on the cell cycle of HepG2 cells was analyzed by flow cytometry. Immunofluorescence cytochemical staining was applied to observe the effect of PAB on the α-tubulin polymerization and expression in HepG2 cells. Transwell TM chamber invasion assay and wound healing assay were performed to detect the influence of PAB on the migration and invasion ability of HepG2 cells. Western blotting was used to determine the expressions of α-tubulin, E-cadherin and MMP-9 in HepG2 cells after treated with PAB. Results PAB inhibited the proliferation of HepG2 cells in a dose-dependent manner and blocked the cell cycle in G2/M phase. PAB significantly changed the polymerization and decreased the expression of α-tubulin. The capacities of invasion and migration of HepG2 cells treated by PAB were significantly depressed. The protein levels of α-tubulin and MMP-9 decreased while the E-cadherin protein level increased. Conclusion PAB can inhibits the proliferation of HepG2 cells by down-regulating the expression of α-tubulin and influencing its polymerization, arresting HepG2 cells in G2/M phase. Meanwhile, PAB also can inhibit the invasion and migration of HepG2 cells by lowering cytoskeleton α-tubulin and MMP-9, and increasing E-cadherin.

Determination of glutathione in apoptotic SMMC-7221 cells induced by xylitol selenite using capillary electrophoresis.

PubMed

Wu, Xue; Cao, Yu; Zhang, Jian; Lei, Ming; Deng, Xiaojie; Zahid, Kashif Rafiq; Liu, Yanli; Liu, Ke; Yang, Jihong; Xiong, Guomei; Yao, Hanchao; Qi, Chao

2016-05-01

To determine the glutathione (GSH) content in a human hepatoma cell line (SMMC-7221) treated with xylitol/selenite, providing a part of an investigation of its anti-cancer mechanisms. The nuclei of SMMC-7221 cells were stained with Hoechst 33258 in an apoptosis assay, and their morphology subsequently changed from circular to crescent shape. The calibration curve (r(2) = 0.992) was established, and GSH content markedly decreased after treated with 0.5 and 1 mg xylitol/selenite l(-1) for 12, 36 and 60 h (12 h: from 95.57 ± 19.57 to 29.09 ± 7.74 and 24.27 ± 11.15; 36 h: from 70.73 ± 11.35 to 19.54 ± 6.39 and 9.35 ± 6.69; 60 h: from 72.63 ± 16.94 to 7.432 ± 3.84 and 0). The depletion rate of GSH was more related to the concentration of xylitol/selenite than the treatment time (from 69.95 ± 1.87 to 100 % vs. 0.22 ± 0.2 to 100 %). Xylitol/selenite is a promising anti-cancer drug to induce apoptosis in SMMC-7221 cells. It may regulate the apoptosis through the co-action of multiple mechanisms related to GSH depletion.

Cells for bioartificial liver devices: the human hepatoma-derived cell line C3A produces urea but does not detoxify ammonia.

PubMed

Mavri-Damelin, Demetra; Damelin, Leonard H; Eaton, Simon; Rees, Myrddin; Selden, Clare; Hodgson, Humphrey J F

2008-02-15

Extrahepatic bioartificial liver devices should provide an intact urea cycle to detoxify ammonia. The C3A cell line, a subclone of the hepatoma-derived HepG2 cell line, is currently used in this context as it produces urea, and this has been assumed to be reflective of ammonia detoxification via a functional urea cycle. However, based on our previous findings of perturbed urea-cycle function in the non-urea producing HepG2 cell line, we hypothesized that the urea produced by C3A cells was via a urea cycle-independent mechanism, namely, due to arginase II activity, and therefore would not detoxify ammonia. Urea was quantified using (15)N-ammonium chloride metabolic labelling with gas chromatography-mass spectrometry. Gene expression was determined by real-time reverse transcriptase-PCR, protein expression by western blotting, and functional activities with radiolabelling enzyme assays. Arginase inhibition studies used N(omega)-hydroxy-nor-L-arginine. Urea was detected in C3A conditioned medium; however, (15)N-ammonium chloride-labelling indicated that (15)N-ammonia was not incorporated into (15)N-labelled urea. Further, gene expression of two urea cycle genes, ornithine transcarbamylase and arginase I, were completely absent. In contrast, arginase II mRNA and protein was expressed at high levels in C3A cells and was inhibited by N(omega)-hydroxy-nor-L-arginine, which prevented urea production, thereby indicating a urea cycle-independent pathway. The urea cycle is non-functional in C3A cells, and their urea production is solely due to the presence of arginase II, which therefore cannot provide ammonia detoxification in a bioartificial liver system. This emphasizes the continued requirement for developing a component capable of a full repertoire of liver function. (c) 2007 Wiley Periodicals, Inc.

Lipopolysaccharide stimulates HepG2 human hepatoma cells in the presence of lipopolysaccharide-binding protein via CD14.

PubMed

Nanbo, A; Nishimura, H; Muta, T; Nagasawa, S

1999-02-01

Lipopolysaccharide (LPS)-binding protein (LBP), an opsonin for activation of macrophages by bacterial LPS, is synthesized in hepatocytes and is known to be an acute phase protein. Recently, cytokine-induced production of LBP was reported to increase 10-fold in hepatocytes isolated from LPS-treated rats, compared with those from normal rats. However, the mechanism by which the LPS treatment enhances the effect of cytokines remains to be clarified. In the present study, we examined whether LPS alone or an LPS/LBP complex directly stimulates the hepatocytes, leading to acceleration of the cytokine-induced LBP production. HepG2 cells (a human hepatoma cell line) were shown to express CD14, a glycosylphosphatidylinositol-anchored LPS receptor, by both RT/PCR and flow cytometric analyses. An LPS/LBP complex was an effective stimulator for LBP and CD14 production in HepG2 cells, but stimulation of the cells with either LPS or LBP alone did not significantly accelerate the production of these proteins. The findings were confirmed by semiquantitative RT/PCR analysis of mRNA levels of LBP and CD14 in HepG2 cells after stimulation with LPS alone and an LPS/LBP complex. In addition, two monoclonal antibodies (mAbs) to CD14 (3C10 and MEM-18) inhibited LPS/LBP-induced cellular responses of HepG2 cells. Furthermore, prestimulation of HepG2 cells with LPS/LBP augmented cytokine-induced production and gene expression of LBP and CD14. All these findings suggest that an LPS/LBP complex, but not free LPS, stimulates HepG2 cells via CD14 leading to increased basal and cytokine-induced LBP and CD14 production.

18 CFR Appendix A to Subpart H of... - Appendix A to Subpart H of Part 35

Code of Federal Regulations, 2011 CFR

2011-04-01

... Rates Pt. 35, Subpt. H, App. A Appendix A to Subpart H of Part 35 Appendix A Standard Screen Format... Reference Seller and Affiliate Capacity A Installed Capacity 19,500 Workpaper. B Long-Term Firm Purchases... Capacity E Installed Capacity 8,000 Workpaper. F Long-Term Firm Purchases 500 Workpaper. G Long-Term Firm...

Chronic ecotoxic effects to Pseudomonas putida and Vibrio fischeri, and cytostatic and genotoxic effects to the hepatoma cell line (HepG2) of ofloxacin photo(cata)lytically treated solutions.

PubMed

Vasquez, M I; Garcia-Käufer, M; Hapeshi, E; Menz, J; Kostarelos, K; Fatta-Kassinos, D; Kümmerer, K

2013-04-15

Ofloxacin (OFL), a broad-spectrum and widespread-used photolabile fluoroquinolone, is frequently found in treated wastewaters, aquatic and terrestrial ecosystems leading to increasing concern during the past decades regarding its effects to the environment and human health. The elimination of OFL and other xenobiotics by the application of advanced oxidation processes using photolytic (PL) and photocatalytic (PC) treatments seems promising. However, an integrated assessment scheme is needed, in which, not only the removal of the parent compound, but also the effects of the photo-transformation products (PTPs) are investigated. For this purpose, in the present study, a chronic ecotoxic assessment using representative bacteria of marine and terrestrial ecosystems and a cytostatic and genotoxic evaluation using hepatoma cell line were performed. PL and PC treatments of OFL were applied using UV radiation. The photo-transformation of OFL during the treatments was monitored by DOC measurements and UPLC-MS/MS analysis. The chronic ecotoxicity of OFL and treated samples was evaluated using Pseudomonas putida and Vibrio fischeri; whereas the cytostasis and genotoxicity were estimated by the cytokinesis-block micronucleus assay (CBMN). The main results suggest that photo-transformation of OFL took place during these treatments since the concentration of OFL decreased when the irradiation time increased, as quantified by UPLC-MS/MS analysis, and this was not coupled with an analogous DOC removal. Furthermore, nine compounds were identified as probable PTPs formed through piperazinyl dealkylation and decarboxylation. The ecotoxicity of treated solutions to the bacteria studied decreased while the cytostasis to the hepatoma cell line remained at low levels during both treatments. However, the genotoxicity to the hepatoma cell line demonstrated a different pattern in which treated samples induced a greater number of MNi for the 4-16 min of irradiation (p<0.05) during both

Effects of drugs in subtoxic concentrations on the metabolic fluxes in human hepatoma cell line Hep G2

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

Niklas, Jens; Noor, Fozia, E-mail: fozia.noor@mx.uni-saarland.d; Heinzle, Elmar

2009-11-01

Commonly used cytotoxicity assays assess the toxicity of a compound by measuring certain parameters which directly or indirectly correlate to the viability of the cells. However, the effects of a given compound at concentrations considerably below EC{sub 50} values are usually not evaluated. These subtoxic effects are difficult to identify but may eventually cause severe and costly long term problems such as idiosyncratic hepatotoxicity. We determined the toxicity of three hepatotoxic compounds, namely amiodarone, diclofenac and tacrine on the human hepatoma cell line Hep G2 using an online kinetic respiration assay and analysed the effects of subtoxic concentrations of thesemore » drugs on the cellular metabolism by using metabolic flux analysis. Several changes in the metabolism could be detected upon exposure to subtoxic concentrations of the test compounds. Upon exposure to diclofenac and tacrine an increase in the TCA-cycle activity was observed which could be a signature of an uncoupling of the oxidative phosphorylation. The results indicate that metabolic flux analysis could serve as an invaluable novel tool for the investigation of the effects of drugs. The described methodology enables tracking the toxicity of compounds dynamically using the respiration assay in a range of concentrations and the metabolic flux analysis permits interesting insights into the changes in the central metabolism of the cell upon exposure to drugs.« less

Plectin deficiency in liver cancer cells promotes cell migration and sensitivity to sorafenib treatment.

PubMed

Cheng, Chiung-Chi; Chao, Wei-Ting; Liao, Chen-Chun; Tseng, Yu-Hui; Lai, Yen-Chang Clark; Lai, Yih-Shyong; Hsu, Yung-Hsiang; Liu, Yi-Hsiang

2018-01-02

Plectin involved in activation of kinases in cell signaling pathway and plays important role in cell morphology and migration. Plectin knockdown promotes cell migration by activating focal adhesion kinase and Rac1-GTPase activity in liver cells. Sorafenib is a multi-targeting tyrosine kinase inhibitor that improves patient survival on hepatocellular carcinoma. The aim of this study is to investigate the correlation between the expression of plectin and cell migration as well as the sensitivity of hepatoma cell lines exposing to sorafenib. Hepatoma cell lines PLC/PRF/5 and HepG2 were used to examine the level of plectin expression and cell migration in comparison with Chang liver cell line. In addition, sensitivity of the 3 cell lines to sorafenib treatment was also measured. Expression of plectin was lower in PLC/PRF/5 and HepG2 hepatoma cells than that of Chang liver cells whereas HepG2 and PLC/PRF/5 cells exhibit higher rate of cell migration in trans-well migration assay. Immunohistofluorecent staining on E-cadherin revealed the highest rate of collective cell migration in HepG2 cells and the lowest was found in Chang liver cells. Likewise, HepG2 cell line was most sensitive to sorafenib treatment and Chang liver cells exhibited the least sensitivity. The drug sensitivity to sorafenib treatment showed inverse correlation with the expression of plectin. We suggest that plectin deficiency and increased E-cadherin in hepatoma cells were associated with higher rates of cell motility, collective cell migration as well as higher drug sensitivity to sorafenib treatment.

Effect of Daytime Blue-enriched LED Light on the Nighttime Circadian Melatonin Inhibition of Hepatoma 7288CTC Warburg Effect and Progression.

PubMed

Dauchy, Robert T; Wren-Dail, Melissa A; Dupepe, Lynell M; Hill, Steven M; Xiang, Shulin; Anbalagan, Muralidharan; Belancio, Victoria P; Dauchy, Erin M; Blask, David E

2018-06-06

Liver cancer is the second leading cause of cancer death worldwide. Metabolic pathways within the liver and liver cancersare highly regulated by the central circadian clock in the suprachiasmatic nuclei (SCN). Daily light and dark cycles regulate the SCN-driven pineal production of the circadian anticancer hormone melatonin and temporally coordinate circadianrhythms of metabolism and physiology in mammals. In previous studies, we demonstrated that melatonin suppresses linoleicacid metabolism and the Warburg effect (aerobic glycolysis)in human breast cancer xenografts and that blue-enriched light(465-485 nm) from light-emitting diode lighting at daytime (bLAD) amplifies nighttime circadian melatonin levels in ratsby 7-fold over cool white fluorescent (CWF) lighting. Here we tested the hypothesis that daytime exposure of tissue-isolatedMorris hepatoma 7288CTC-bearing male rats to bLAD amplifies the nighttime melatonin signal to enhance the inhibition oftumor growth. Compared with rats housed under a 12:12-h light:dark cycle in CWF light, rats in bLAD light evinced a 7-fold higher peak plasma melatonin level at the mid-dark phase; in addition, high melatonin levels were prolonged until 4 h intothe light phase. After implantation of tissue-isolated hepatoma 7288CTC xenografts, tumor growth rates were markedly delayed,and tumor cAMP levels, LA metabolism, the Warburg effect, and growth signaling activities were decreased in rats inbLAD compared with CWF daytime lighting. These data show that the increased nighttime circadian melatonin levels dueto bLAD exposure decreases hepatoma metabolic, signaling, and proliferative activities beyond what occurs after normalmelatonin signaling under CWF light.

Isatis indigotica induces hepatocellular cancer cell death via caspase-independent apoptosis-inducing factor translocation apoptotic pathway in vitro and in vivo.

PubMed

Chung, Ying-Cheng; Tang, Feng-Yao; Liao, Jiunn-Wang; Chung, Chia-Hua; Jong, Ting-Ting; Chen, Shih-Shiung; Tsai, Ching-Hsiu; Chiang, En-Pei

2011-06-01

Isatis indigotica is a biennial herbaceous cruciferous medical herb with antipyretic, antiviral, anti-inflammatory, and anti-endotoxin activity. This study explored the chemotherapeutic potential of I indigotica on human hepatoma cells and investigated the mechanism by which metabolites from I indigotica inhibit hepatoma cell growth. Antitumor activity was discovered in dried I indigotica leaf chloroform extracts (CEDLI). In nude mice xenotransplanted with human hepatoma cells, CEDLI supplementation inhibited tumor growth by ~40% compared with nonsupplemented animals without affecting body weight/food intake. CEDLI induced sub-G1 cell cycle arrest and apoptosis in hepatoma cells. Furthermore, CEDLI activates p53 and Bax, reduces Bcl-2 expression, and causes mitochondrial stress and the release of apoptosis-inducing factor into the cytosol followed by its translocation into the nucleus, resulting in hepatoma cell apoptosis. This study provides novel in vivo evidence of I indigotica's antitumor activity. The chemotherapeutic activity against human hepatoma tumorigenesis was because of a distinguished caspase-independent apoptotic pathway.

Effects of Extracellular pH on Dental Pulp Cells In Vitro.

PubMed

Hirose, Yujiro; Yamaguchi, Masaya; Kawabata, Shigetada; Murakami, Masashi; Nakashima, Misako; Gotoh, Momokazu; Yamamoto, Tokunori

2016-05-01

The proliferation and migration of dental pulp stem cells (DPSCs), a population comprised of dental pulp cells (DPCs), are important processes for pulp tissue repair. Dental pulp is exposed to changes in extracellular pH under various conditions, such as acidosis and exposure to caries-associated bacteria or a pulp capping agent. The objective of this study was to investigate the effects of extracellular pH on DPC proliferation and migration in vitro. To evaluate the proliferation potency of DPCs in various extracellular pH conditions, 2 × 10(4) cells were seeded into 35-mm dishes. The following day, we changed to NaHCO3-free medium, which was adjusted to different extracellular pH levels. After 120 hours, DPCs cultured in media from a pH of 3.5 to 5.5 showed cell death, those cultured in conditions from a pH of 6.5 to 7.5 showed growth arrest or cell death, and those grown at a pH of 9.5 showed mild proliferation. The migratory activity of living DPCs was not affected by extracellular pH. For histologic analysis, human teeth possessing a small abscess in the coronal pulp chamber were sliced for histologic analysis. Proliferating cell nuclear antigen (PCNA) immunolocalization was used as an index of cell proliferation for the sections and cultured cells. Acidic extracellular pH conditions resulted in reduced numbers of PCNA-positive DPCs in the dishes. As for pulp tissue affected by a small abscess, a PCNA-negative pulp cell layer was observed in close proximity to the infectious lesion. Together, these results suggest that an acidic extracellular pH condition is associated with DPC growth arrest or cell death. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Preparation and characterization of 3,5-dinitro-1H-1,2,4-triazole.

PubMed

Haiges, R; Bélanger-Chabot, G; Kaplan, S M; Christe, K O

2015-04-28

Neat 3,5-dinitro-1H-1,2,4-triazole was obtained in quantitative yield from potassium 3,5-dinitro-1,2,4-triazolate and sulfuric acid. The compound was purified by sublimation in vacuo at 110 °C. Pure HDNT is a hygroscopic white solid that is impact and friction sensitive and decomposes explosively upon heating to 170 °C. However, the presence of impurities might lower the decomposition temperature and increase the sensitivity of the material. Potassium 3,5-dinitro-1,2,4-triazolate was prepared from commercially available 3,5-diamino-4H-1,2,4-triazole with sodium nitrite and sulfuric acid. The synthesis of HDNT from 2-cyanoguanidine and hydrazine hydrate without isolation and purification of the 3,5-diamino-4H-1,2,4-triazole intermediate can result in the formation of azidotriazole impurities. A triclinic and a monoclinic polymorph of 3,5-dinitro-1H-1,2,4-triazole were found by X-ray structure determination. In addition, the crystal structure of the hydrate (HDNT)3·4H2O, as well as those of several HDNT impurities and decomposition products were obtained.

Cyclic Mechanical Stretch Up-regulates Hepatoma-Derived Growth Factor Expression in Cultured Rat Aortic Smooth Muscle Cells.

PubMed

Kao, Ying-Hsien; Chen, Po-Han; Sun, Cheuk-Kwan; Chang, Yo-Chen; Lin, Yu-Chun; Tsai, Ming-Shian; Lee, Po-Huang; Cheng, Cheng-I

2018-02-21

Hepatoma-derived growth factor (HDGF) is a potent mitogen for vascular smooth muscle cells (SMCs) during embryogenesis and injury repair of vessel walls. Whether mechanical stimuli modulate HDGF expression remains unknown. This study aimed at investigating whether cyclic mechanical stretch plays a regulatory role in HDGF expression and regenerative cytokine production in aortic SMCs. A SMC cell line was grown on a silicone-based elastomer chamber with extracellular matrix coatings (either type I collagen or fibronectin) and received cyclic and uni-axial mechanical stretches with 10% deformation at frequency 1 Hz. Morphological observation showed that fibronectin coating provided better cell adhesion and spreading and that consecutive 6 hours of cyclic mechanical stretch remarkably induced reorientation and realignment of SMCs. Western blotting detection demonstrated that continuous mechanical stimuli elicited up-regulation of HDGF and PCNA, a cell proliferative marker. Signal kinetic profiling study indicated that cyclic mechanical stretch induced signaling activity in RhoA/ROCK and PI3K/Akt cascades. Kinase inhibition study further showed that blockade of PI3K activity suppressed the stretch-induced TNF-a, whereas RhoA/ROCK inhibition significantly blunted the IL-6 production and HDGF over-expression. Moreover, siRNA-mediated HDGF gene silencing significantly suppressed constitutive expression of IL-6, but not TNF-α, in SMCs. These findings support the role of HDGF in maintaining vascular expression of IL-6, which has been regarded a crucial regenerative factor for acute vascular injury. In conclusion, cyclic mechanical stretch may maintain constitutive expression of HDGF in vascular walls and be regarded an important biophysical regulator in vascular regeneration. ©2018 The Author(s).

Thyroid hormone enhanced human hepatoma cell motility involves brain-specific serine protease 4 activation via ERK signaling

PubMed Central

2014-01-01

Background The thyroid hormone, 3, 3′, 5-triiodo-L-thyronine (T3), has been shown to modulate cellular processes via interactions with thyroid hormone receptors (TRs), but the secretory proteins that are regulated to exert these effects remain to be characterized. Brain-specific serine protease 4 (BSSP4), a member of the human serine protease family, participates in extracellular matrix remodeling. However, the physiological role and underlying mechanism of T3-mediated regulation of BSSP4 in hepatocellular carcinogenesis are yet to be established. Methods The thyroid hormone response element was identified by reporter and chromatin immunoprecipitation assays. The cell motility was analyzed via transwell and SCID mice. The BSSP4 expression in clinical specimens was examined by Western blot and quantitative reverse transcription polymerase chain reaction. Results Upregulation of BSSP4 at mRNA and protein levels after T3 stimulation is a time- and dose-dependent manner in hepatoma cell lines. Additionally, the regulatory region of the BSSP4 promoter stimulated by T3 was identified at positions -609/-594. BSSP4 overexpression enhanced tumor cell migration and invasion, both in vitro and in vivo. Subsequently, BSSP4-induced migration occurs through the ERK 1/2-C/EBPβ-VEGF cascade, similar to that observed in HepG2-TRα1 and J7-TRα1 cells. BSSP4 was overexpressed in clinical hepatocellular carcinoma (HCC) patients, compared with normal subjects, and positively associated with TRα1 and VEGF to a significant extent. Importantly, a mild association between BSSP4 expression and distant metastasis was observed. Conclusions Our findings collectively support a potential role of T3 in cancer cell progression through regulation of the BSSP4 protease via the ERK 1/2-C/EBPβ-VEGF cascade. BSSP4 may thus be effectively utilized as a novel marker and anti-cancer therapeutic target in HCC. PMID:24980078

Thyroid hormone enhanced human hepatoma cell motility involves brain-specific serine protease 4 activation via ERK signaling.

PubMed

Chen, Cheng-Yi; Chung, I-Hsiao; Tsai, Ming-Ming; Tseng, Yi-Hsin; Chi, Hsiang-Cheng; Tsai, Chung-Ying; Lin, Yang-Hsiang; Wang, You-Ching; Chen, Chie-Pein; Wu, Tzu-I; Yeh, Chau-Ting; Tai, Dar-In; Lin, Kwang-Huei

2014-07-01

The thyroid hormone, 3, 3', 5-triiodo-L-thyronine (T3), has been shown to modulate cellular processes via interactions with thyroid hormone receptors (TRs), but the secretory proteins that are regulated to exert these effects remain to be characterized. Brain-specific serine protease 4 (BSSP4), a member of the human serine protease family, participates in extracellular matrix remodeling. However, the physiological role and underlying mechanism of T3-mediated regulation of BSSP4 in hepatocellular carcinogenesis are yet to be established. The thyroid hormone response element was identified by reporter and chromatin immunoprecipitation assays. The cell motility was analyzed via transwell and SCID mice. The BSSP4 expression in clinical specimens was examined by Western blot and quantitative reverse transcription polymerase chain reaction. Upregulation of BSSP4 at mRNA and protein levels after T3 stimulation is a time- and dose-dependent manner in hepatoma cell lines. Additionally, the regulatory region of the BSSP4 promoter stimulated by T3 was identified at positions -609/-594. BSSP4 overexpression enhanced tumor cell migration and invasion, both in vitro and in vivo. Subsequently, BSSP4-induced migration occurs through the ERK 1/2-C/EBPβ-VEGF cascade, similar to that observed in HepG2-TRα1 and J7-TRα1 cells. BSSP4 was overexpressed in clinical hepatocellular carcinoma (HCC) patients, compared with normal subjects, and positively associated with TRα1 and VEGF to a significant extent. Importantly, a mild association between BSSP4 expression and distant metastasis was observed. Our findings collectively support a potential role of T3 in cancer cell progression through regulation of the BSSP4 protease via the ERK 1/2-C/EBPβ-VEGF cascade. BSSP4 may thus be effectively utilized as a novel marker and anti-cancer therapeutic target in HCC.

The synergistic radiosensitizing effect of tirapazamine-conjugated gold nanoparticles on human hepatoma HepG2 cells under X-ray irradiation

PubMed Central

Liu, Xi; Liu, Yan; Zhang, Pengcheng; Jin, Xiaodong; Zheng, Xiaogang; Ye, Fei; Chen, Weiqiang; Li, Qiang

2016-01-01

Reductive drug-functionalized gold nanoparticles (AuNPs) have been proposed to enhance the damage of X-rays to cells through improving hydroxyl radical production by secondary electrons. In this work, polyethylene glycol-capped AuNPs were conjugated with tirapazamine (TPZ) moiety, and then thioctyl TPZ (TPZs)-modified AuNPs (TPZs-AuNPs) were synthesized. The TPZs-AuNPs were characterized by transmission electron microscopy, ultraviolet-visible spectra, dynamic light scattering, and inductively coupled plasma mass spectrometry to have a size of 16.6±2.1 nm in diameter and a TPZs/AuNPs ratio of ~700:1. In contrast with PEGylated AuNPs, the as-synthesized TPZs-AuNPs exhibited 20% increment in hydroxyl radical production in water at 2.0 Gy, and 19% increase in sensitizer enhancement ratio at 10% survival fraction for human hepatoma HepG2 cells under X-ray irradiation. The production of reactive oxygen species in HepG2 cells exposed to X-rays in vitro demonstrated a synergistic radiosensitizing effect of AuNPs and TPZ moiety. Thus, the reductive drug-conjugated TPZs-AuNPs as a kind of AuNP radiosensitizer with low gold loading provide a new strategy for enhancing the efficacy of radiation therapy. PMID:27555772

An Effect of Dexamethasone on Adenosine 3′,5′ -Monophosphate Content and Adenosine 3′,5′ -Monophosphate Phosphodiesterase Activity of Cultured Hepatoma Cells

PubMed Central

Manganiello, Vincent; Vaughan, Martha

1972-01-01

The effect of dexamethasone on adenosine 3′,5′-monophosphate (cAMP) phosphodiesterase activity in cultured HTC hepatoma cells was investigated. Homogenates of these cells contain phosphodiesterase activity with two apparent Michaelis constants for cAMP (2-5 μm and 50 μm). At all substrate concentrations tested, phosphodiesterase activity was decreased 25-40% in cells incubated for 36 hr or more with 1 μm dexamethasone. Acid phosphatase activity in the same cells was not decreased. α-Methyl testosterone, 1 μm, was without effect on phosphodiesterase activity. Incubation for 10 min with epinephrine plus theophylline increased the cAMP content of the HTC cells 3- to 6-fold. In cells incubated for 72 hr with dexamethasone, the basal concentration of cAMP was slightly increased and the increment produced by epinephrine plus theophylline was markedly increased. We believe that in many cells the so-called permissive effects of steroid hormones on cAMP mediated processes may be due to an effect of these hormones on cAMP phosphodiesterase activity similar to that observed in HTC cells incubated with dexamethasone. PMID:4341439

[Knockdown of STAT3 inhibits proliferation and migration of HepG2 hepatoma cells induced by IFN1].

PubMed

Li, Xiaofang; Wang, Yuqi; Yan, Ben; Fang, Peipei; Ma, Chao; Xu, Ning; Fu, Xiaoyan; Liang, Shujuan

2018-02-01

Objective To prepare lentiviruses expressing shRNA sequences targeting human signal transducer and activator of transcription 3 (STAT3) and detect the effect of STAT3 knockdown on type I interferon (IFN1)-induced proliferation and migration in HepG2 cells. Methods Four STAT3-targeting shRNA sequences (shRNA1-shRNA4) and one control sequence (Ctrl shRNA) were selected and cloned respectively into pLKO.1-sp6-pgk-GFP to construct shRNA-expressing vectors. Along with backbone psPAX2 and pMD2.G vectors, they were separately transfected into HEK293T cells to prepare lentiviruses. HepG2 cells were infected with the lentiviruses. Cytoplastic STAT3 level was detected by Western blotting to screen effective shRNA sequence(s) targeting STAT3. Proliferation and migration of HepG2 cells were analyzed by CCK-8 assay and Transwell TM migration and scratching assay, respectively. To detect the effect of IFN1 on cell proliferation and migration of HepG2 cells, the cells were treated with 2000 U/mL IFNα2b for indicated time and the activation of IFN-triggered STAT1 signal transduction was assayed by Western blotting. Results Two most effective STAT3-targeting shRNA sequences shRNA1 and shRNA2 were selected, and the expression of both STAT3 shRNA significantly decreased proliferation and migration of HepG2 cells. When treated with IFNα2b, 2000 U/mL of IFN1 showed more competent in attenuating growth and migration of HepG2 cells. Our data further proved that knockdown of STAT3 increased the phosphorylation of STAT1, and IFNα2b further enhanced the activation of STAT1 signaling in HepG2 cells. Conclusion Knockdown of STAT3 inhibits cell migration and growth, and rescues IFN response through up-regulating STAT1 signal transduction in HepG2 hepatoma cells.

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.

Hydrogen peroxide impairs autophagic flux in a cell model of nonalcoholic fatty liver disease

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

Jiang, Pengtao; University of Chinese Academy of Sciences, 19 Yuquan Road, Shijingshan District, Beijing 100049; Huang, Zhen

2013-04-19

Highlights: •Free fatty acids exposure induces elevated autophagy. •H{sub 2}O{sub 2} inhibits autophagic flux through impairing the fusion between autophagosomes and lysosomes. •Inhibition of autophagy potentiates H{sub 2}O{sub 2}-induced cell death. -- Abstract: Nonalcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease, but the pathogenesis of NAFLD is not fully clear. The aim of this study was to determine whether autophagy plays a role in the pathogenesis of NAFLD. We found that the levels of autophagy were elevated in hepatoma cells upon exposure to free fatty acids, as confirmed by the increase in the numbermore » of autophagosomes. However, exposure of hepatoma cells to H{sub 2}O{sub 2} and TNF-α, two typical “second hit” factors, increased the initiation of autophagy but inhibited the autophagic flux. The inhibition of autophagy sensitized cells to pro-apoptotic stimuli. Taken together, our results suggest that autophagy acts as a protective mechanism in the pathogenesis of NAFLD and that impairment of autophagy might induce more severe lesions of the liver. These findings will be a benefit to the understanding of the pathogenesis of NAFLD and might suggest a strategy for the prevention and cure of NAFLD.« less

Camel milk triggers apoptotic signaling pathways in human hepatoma HepG2 and breast cancer MCF7 cell lines through transcriptional mechanism.

PubMed

Korashy, Hesham M; Maayah, Zaid H; Abd-Allah, Adel R; El-Kadi, Ayman O S; Alhaider, Abdulqader A

2012-01-01

Few published studies have reported the use of crude camel milk in the treatment of stomach infections, tuberculosis and cancer. Yet, little research was conducted on the effect of camel milk on the apoptosis and oxidative stress associated with human cancer. The present study investigated the effect and the underlying mechanisms of camel milk on the proliferation of human cancer cells using an in vitro model of human hepatoma (HepG2) and human breast (MCF7) cancer cells. Our results showed that camel milk, but not bovine milk, significantly inhibited HepG2 and MCF7 cells proliferation through the activation of caspase-3 mRNA and activity levels, and the induction of death receptors in both cell lines. In addition, Camel milk enhanced the expression of oxidative stress markers, heme oxygenase-1 and reactive oxygen species production in both cells. Mechanistically, the increase in caspase-3 mRNA levels by camel milk was completely blocked by the transcriptional inhibitor, actinomycin D; implying that camel milk increased de novo RNA synthesis. Furthermore, Inhibition of the mitogen activated protein kinases differentially modulated the camel milk-induced caspase-3 mRNA levels. Taken together, camel milk inhibited HepG2 and MCF7 cells survival and proliferation through the activation of both the extrinsic and intrinsic apoptotic pathways.

Sphingoid bases from sea cucumber induce apoptosis in human hepatoma HepG2 cells through p-AKT and DR5.

PubMed

Hossain, Zakir; Sugawara, Tatsuya; Hirata, Takashi

2013-03-01

Biofunctional marine compounds have recently received substantial attention for their nutraceutical characteristics. In this study, we investigated the apoptosis-inducing effects of sphingoid bases prepared from sea cucumber using human hepatoma HepG2 cells. Apoptotic effects were determined by cell viability assay, DNA fragmentation assay, caspase-3 and caspase-8 activities. The expression levels of apoptosis-inducing death receptor-5 (DR5) and p-AKT were assayed by western blot analysis, and mRNA expression of bax, GADD45 and PPARγ was assayed by quantitative RT-PCR analysis. Sphingoid bases from sea cucumber markedly reduced the cell viability of HepG2 cells. DNA fragmentation indicative of apoptosis was observed in a dose-dependent manner. The expression levels of the apoptosis inducer protein Bax were increased by the sphingoid bases from sea cucumber. GADD45, which plays an important role in apoptosis-inducing pathways, was markedly upregulated by sphingoid bases from sea cucumber. Upregulation of PPARγ mRNA was also observed during apoptosis induced by the sphingoid bases. The expression levels of DR5 and p-AKT proteins were increased and decreased, respectively, as a result of the effects of sphingoid bases from sea cucumber. The results indicate that sphingoid bases from sea cucumber induce apoptosis in HepG2 cells through upregulation of DR5, Bax, GADD45 and PPARγ and downregulation of p-AKT. Our results show for the first time the functional properties of marine sphingoid bases as inducers of apoptosis in HepG2 cells.

Modulation of the DNA repair system and ATR-p53 mediated apoptosis is relevant for tributyltin-induced genotoxic effects in human hepatoma G2 cells.

PubMed

Li, Bowen; Sun, Lingbin; Cai, Jiali; Wang, Chonggang; Wang, Mengmeng; Qiu, Huiling; Zuo, Zhenghong

2015-01-01

The toxic effects of tributyltin (TBT) have been extensively documented in several types of cells, but the molecular mechanisms related to the genotoxic effects of TBT have still not been fully elucidated. Our study showed that exposure of human hepatoma G2 cells to 1-4 μmol/L TBT for 3 hr caused severe DNA damage in a concentration-dependent manner. Moreover, the expression levels of key DNA damage sensor genes such as the replication factor C, proliferating cell nuclear antigen and poly (ADP-ribose) polymerase-1 were inhabited in a concentration-dependent manner. We further demonstrated that TBT induced cell apoptosis via the p53-mediated pathway, which was most likely activated by the ataxia telangiectasia mutated and rad-3 related (ATR) protein kinase. The results also showed that cytochrome c, caspase-3, caspase-8, caspase-9, and the B-cell lymphoma 2 were involved in this process. Taken together, we demonstrated for the first time that the inhibition of the DNA repair system might be more responsible for TBT-induced genotoxic effects in cells. Then the generated DNA damage induced by TBT initiated ATR-p53-mediated apoptosis. Copyright © 2014. Published by Elsevier B.V.

A 21-35 kDa Mixed Protein Component from Helicobacter pylori Activates Mast Cells Effectively in Chronic Spontaneous Urticaria.

PubMed

Tan, Ran-Jing; Sun, He-Qiang; Zhang, Wei; Yuan, Han-Mei; Li, Bin; Yan, Hong-Tao; Lan, Chun-Hui; Yang, Jun; Zhao, Zhuo; Wu, Jin-Jin; Wu, Chao

2016-12-01

Helicobacter pylori (H. pylori) seem to involve in the etiology of chronic spontaneous urticaria (CSU). But studies of the pathogenic mechanism are very little. In this study, we detected the serum-specific anti-H. pylori IgG and IgE antibodies in 211 CSU and 137 normal subjects by enzyme-linked immunosorbent assay (ELISA), evaluated the direct activation effects of H. pylori preparations and its protein components on human LAD 2 mast cell line in vitro, and analyzed the specific protein ingredients and functions of the most effective H. pylori mixed protein component using liquid chromatography-mass spectrometry and ELISA assay. In CSU patients, the positive rate of anti-H. pylori IgG positive rate was significantly higher than that in normal controls, and the anti-H. pylori IgE levels had no statistical difference between H. pylori-infected patients with and without CSU. Further studies suggested that H. pylori preparations can directly activate human LAD 2 mast cell line in a dose-dependent manner and its most powerful protein component was a mixture of 21-35 kDa proteins. Moreover, the 21-35 kDa mixed protein component mainly contained 23 kinds of proteins, which can stimulate the release of histamine, TNF-a, IL-3, IFN-γ, and LTB4 by LAD 2 cells in a dose-dependent or time-dependent manner. A 21-35 kDa mixed protein component should be regarded as the most promising pathogenic factor contributing to the CSU associated with H. pylori infection. © 2016 John Wiley & Sons Ltd.

Progression of conventional hepatic cell culture models to bioengineered HepG2 cells for evaluation of herbal bioactivities.

PubMed

Kaur, Pardeep; Robin; Mehta, Rajendra G; Arora, Saroj; Singh, Balbir

2018-06-01

Cancer cell lines of human tissue origin have been extensively used to investigate antiproliferative activity and toxicity of herbal extracts, isolated compounds, and anticancer drugs. These cell lines are genetically and/or epigenetically well characterized to determine the altered expression of proteins within given cellular pathways and critical genes in cancer. Human derived hepatoma (HepG2) cell line has been extensively exploited to examine cytoprotective, antioxidative, hepatoprotective, anti-hepatoma, hypocholesterolemic, anti-steatosis, bioenergetic homeostatic and anti-insulin resistant properties. Moreover, mechanism of action of various botanicals and bioactive constituents has been reported using these cells. HepG2 cells have significant differences as compared to primary hepatocytes with respect to expression of cytochrome P450 enzymes and xenobiotic receptors in conventional in vitro culture conditions. Therefore, strategies have been employed to overcome limitations of two dimensional (2D) in vitro HepG2 cell culture in order to recognize functional biomarkers more accurately and to boost its predictive value in clinical research. In consequence, three dimensional (3D) human hepatoma cell culture models are being developed as a resource to achieve these goals of simulating the in vivo tumor microenvironment. It is assumed that bioengineered 3D hepatoma cell culture models can provide significant assistance in scrutinizing the molecular response of herbal natural products to recognize novel prognostic targets and crucial biomarkers in treatment strategies for cancer patients in near future.

Design, synthesis and anticonvulsant activity of some new 6,8-halo-substituted-2h-[1,2,4]triazino[5,6-b]indole-3(5h)-one/-thione and 6,8-halo-substituted 5-methyl-2h-[1,2,4]triazino[5,6-b]indol-3(5h)-one/-thione

PubMed Central

Kumar, Rajeev; Singh, Tejendra; Singh, Hariram; Jain, Sandeep; Roy, R. K.

2014-01-01

A new series of 6,8-halo-substituted-2H-[1,2,4]triazino[5,6-b]indole-3(5H)-one/-thione and 6,8-halo-substituted 5-methyl-2H-[1,2,4]triazino[5,6-b]indol-3(5H)-one/-thione (5a-5l) were designed and synthesized keeping in view of the structural requirement of pharmacophore. The above compounds were characterized by thin layer chromatography and spectral analysis. Anticonvulsant activity of the synthesized compounds was evaluated by the maximal electroshock (MES) test. Neurotoxicity and CNS depressant effects were evaluated by the rotarod motor impairment and Porsolt’s force swim tests, respectively. A computational study was carried out, for calculation of pharmacophore pattern, prediction of pharmacokinetic properties and toxicity properties. The above study revealed that the compounds 8-chloro-2H-[1,2,4]triazino[5,6-b]indol-3(5H)-one (5e), 6,8-dibromo-2H-[1,2,4]triazino[5,6-b]indol-3(5H)-one (5i) and 6,8-dibromo-5-methyl-2H-[1,2,4]triazino[5,6-b]indol-3(5H)-one (5k) possess excellent anticonvulsant activity in the series with little CNS depressant effect and no neurotoxicity as compared to standard drugs phenytoin and carbamazepine. PMID:26417257

Trout hepatoma--a preliminary report

USGS Publications Warehouse

Rucker, R.R.; Yasutake, W.T.; Wolf, H.

1961-01-01

Fish pathology and its role in fish culture were brought into prominence in the spring of 1960 by the disclosure of a high incidence of hepatomas in hatchery-reared rainbow trout. The current problem came to light as the result of a routine inspection of live trout shipments at a California border fish-disease checking station. This service is performed by personnel of the California Department of Fish and Game to preclude the introduction or further spread of communicable fish diseases into California watersheds. Collaborative studies which followed revealed the nationwide distribution of the disease. This unusual disease soon attracted the attention of the Bureau of Sport Fisheries and Wildlife, the Food and Drug Administration, Public Health Service, and several western State health and conservation agencies.

Recombinant interleukin-12 and interleukin-18 antitumor therapy in a guinea-pig hepatoma cell implant model.

PubMed

Shiratori, Ikuo; Suzuki, Yasuhiko; Oshiumi, Hiroyuki; Begum, Nasim A; Ebihara, Takashi; Matsumoto, Misako; Hazeki, Kaoru; Kodama, Ken; Kashiwazaki, Yasuo; Seya, Tsukasa

2007-12-01

Interleukin (IL)-12 and IL-18 are secreted by myeloid cells activated with adjuvants such as Bacillus Calmette-Guérin (BCG) cell wall. They induce T-helper 1 polarization in the host immune system and upregulate production of lymphocyte interferon-gamma, which leads to the induction of an antitumor gene program. It has been reported that humans have an immune system that more closely resembles that of the guinea pig in adjuvant-response features rather than the mouse system, which prevents the mouse results being extrapolated to human immunotherapy. Here we have constructed a tumor-implant system in guinea pigs to evaluate the antitumor potential of guinea pig IL-12 (gpIL-12) and guinea pig IL-18 (gpIL-18). Purified recombinant gpIL-12 and gpIL-18 were prepared and applied intraperitoneally to tumor-bearing (line 10 hepatoma) guinea pigs as the basis of the adjuvant immunotherapy. Intraperitoneal administration of gpIL-12 and gpIL-18 led to retardation of primary tumor growth and suppression of lymph-node metastasis in tumor-bearing guinea pigs. The permissible range of IL-12 appeared wider in guinea pigs than in mice. Even at an IL-12 dose higher than that in mice, there was no evidence of side-effects until day 26, when the guinea pigs were killed. gpIL-18 augmented the antitumor effect of gpIL-12 but exerted less ability to suppress lymph-node metastasis. The effects of gpIL-12 and gpIL-18 on the tumors implanted in guinea pigs will encourage us to use IL-12- and IL-18-inducible adjuvants for immunotherapy in human patients with solid cancer.

Clinical and biological significance of hepatoma-derived growth factor in Ewing's sarcoma.

PubMed

Yang, Yang; Li, Hui; Zhang, Fenfen; Shi, Huijuan; Zhen, Tiantian; Dai, Sujuan; Kang, Lili; Liang, Yingjie; Wang, Jin; Han, Anjia

2013-11-01

We sought to investigate the clinicopathological significance and biological function of hepatoma-derived growth factor (HDGF) in Ewing's sarcoma. Our results showed that HDGF expression is up-regulated in Ewing's sarcoma. Nuclear HDGF expression is significantly associated with tumour volume (p < 0.001), metastases at diagnosis (p < 0.001), low overall survival rate (p < 0.001) and low disease-free survival rate (p < 0.001). HDGF knock-down results in significant reduction of Ewing's sarcoma cell growth, proliferation and enhances tumourigenesis, both in vitro and in vivo. Meanwhile, HDGF knock-down causes cell cycle arrest and enhanced sensitization to serum starvation-induced apoptosis. Furthermore, recombinant HDGF promotes proliferation and colony formation of Ewing's sarcoma cells. Ninety-eight candidate HDGF downstream genes were identified in Ewing's sarcoma cells using cDNA microarray analysis. In addition, we found that HDGF knock-down inhibited FLI1 expression in Ewing's sarcoma cells at the mRNA and protein levels. Our findings suggest that HDGF exhibits oncogenic properties and may be a novel prognostic factor in Ewing's sarcoma. Targeting HDGF might be a potential therapeutic strategy for Ewing's sarcoma. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Anti-hepatoma activity and mechanism of corn silk polysaccharides in H22 tumor-bearing mice.

PubMed

Yang, Jingyue; Li, Xiao; Xue, Yan; Wang, Nan; Liu, Wenchao

2014-03-01

Corn silk is a well known traditional Chinese herbal medicine and corn silk polysaccharides (CSP) possess multiple pharmacological activities. However, the antitumor effect of CSP on hepatocarcinoma has not been studied. This study aimed to investigate the effects of CSP on tumor growth and immune functions in H22 hepatocarcinoma tumor-bearing mice. The results demonstrated that CSP could not only inhibit the tumor growth, but also extended the survival time of H22 tumor-bearing mice. Besides, CSP administration could increase the body weight, peripheral white blood cells (WBC) count, thymus index and spleen index of H22 tumor-bearing mice. Furthermore, the production of serum cytokines in H22 tumor-bearing mice, such as IL-2, IL-6 and TNF-α, was enhanced by CSP treatment. In addition, no toxicological effects were observed on hepatic function and renal function in CSP-treated mice transplanted H22 tumor cells. In summary, this experimental finding indicated that CSP could elevate the immune functions in H22 tumor-bearing mice to enhance its antitumor activity and CSP seems to be a safe and effective agent for the treatment of hepatocellular carcinoma. Copyright © 2013 Elsevier B.V. All rights reserved.

Synthesis of a novel adamantyl nitroxide derivative with potent anti-hepatoma activity in vitro and in vivo

PubMed Central

Sun, Jin; Wang, Shan; Bu, Wei; Wei, Meng-Ying; Li, Wei-Wei; Yao, Min-Na; Ma, Zhong-Ying; Lu, Cheng-Tao; Li, Hui-Hui; Hu, Na-Ping; Zhang, En-Hu; Yang, Guo-Dong; Wen, Ai-Dong; Zhu, Xiao-He

2016-01-01

In this study, a novel adamantyl nitroxide derivative was synthesized and its antitumor activities in vitro and in vivo were investigated. The adamantyl nitroxide derivative 4 displayed a potent anticancer activity against all the tested human hepatoma cells, especially with IC50 of 68.1 μM in Bel-7404 cells, compared to the positive control 5-FU (IC50=607.7 μM). The significant inhibition of cell growth was also observed in xenograft mouse model, with low toxicity. Compound 4 suppressed the cell migration and invasion, induced the G2/M phase arrest. Further mechanistic studies revealed that compound 4 induced cell death, which was accompanied with damaging mitochondria, increasing the generation of intracellular reactive oxygen species, cleavages of caspase-9 and caspase-3, as well as activations of Bax and Bcl-2. These results confirmed that adamantyl nitroxide derivative exhibited selective antitumor activities via mitochondrial apoptosis pathway in Bel-7404 cells, and would be a potential anticancer agent for liver cancer. PMID:27429843

Growth-inhibiting effects of taxol on human liver cancer in vitro and in nude mice

PubMed Central

Yuan, Jin Hui; Zhang, Ru Ping; Zhang, Ru Gang; Guo, Li Xia; Wang, Xing Wang; Luo, Dan; Xie, Yong; Xie, Hong

2000-01-01

AIM: To investigate the effects of taxol on SMMC-7721 human hepatoma and its mechanisms. METHODS: In vitro cell growth was assessed by trypan blue exclusion method. Experimental hepatoma model was established by seeding SMMC-7721 cells subcutaneously into Balb/c (nu/nu) nude mice. In vivo tumor growth was determined by measurement of tumor diameter with Vernier calipers. The syntheses of DNA, RNA and protein were analyzed by incorporation of 3H-thymidine, 3H-uridine and 3H-leucine respectively. Using light and electron microscopes to observe the morphological changes of cells including mitosis and apoptosis. RESULTS: Taxol was effective against SMMC-7721 human hepatoma cell growth in the ranges of 2.5 nmol/L-10 nmol/L- with mitotic arrest and apoptosis in vitro. DNA, RNA and protein syntheses in cells were also obviously suppressed by in vitro treatment of taxol for 72 h. Taxol at 2.5 nmol/L reduced 3H-thymidine uptake to about 34% of the control value (P ＜ 0.05). Increasing the dose of taxol to 20 nmol/L resulted in a greater decrease in 3H-thymidine incorporation to 60% of the control value (P ＜ 0.01). At a concentration of 20 nmol/L, the 3H-uridine and 3H-leucine uptakes were reduced to 52% (P ＜ 0.05) and 63% (P ＜ 0.01), respectively. In vivo, taxol significantly inhibited SMMC-7721 tumor growth at 10 mg/kg, i.p. once daily for 10 d. A more than 90% decrease in tumor volume was observed by day 11 (P ＜ 0.01) similarly with mitotic arrest and cell apoptosis. CONCLUSION: Taxol has a marked anticancer activity in SMMC-7721 human hepatoma both in vitro and in nude mice. Its mechanisms might be associated with mitotic arrest, subsequently, apoptosis of the hepatoma cells. No obvious toxicity was observed with in vivo administration of taxol. PMID:11819558

Possible reduction of hepatoma formation by Smmu 7721 cells in SCID mice and metastasis formation by B16F10 melanoma cells in C57BL/6 mice by Agaricus blazei murill extract.

PubMed

Wu, Ming-Fang; Lu, Hsu-Feng; Hsu, Yu-Ming; Tang, Ming-Chu; Chen, Hsueh-Chin; Lee, Ching-Sung; Yang, Yi-Yuan; Yeh, Ming-Yang; Chung, Hsiung-Kwang; Huang, Yi-Ping; Wu, Chih-Chung; Chung, Jing-Gung

2011-01-01

Agaricus blazei Murill extract (ABM) has been reported to possess antitumor effects. In this study, the role of ABM in tumor growth and metastasis in vivo was evaluated in experimental Smmu 7721 hepatoma cells in severe combined immunodeficiency (SCID) mice and B16F10 melanoma cells lung metastasis in C57BL/6 mice. For the tumor growth model, the size of the liver tumor mass was about 10 mm to 20 mm in the control group. In comparison with the control group, the tumor mass seem to grow slowly with ABM treatment, especially at the high dose. For the tumor metastasis model, after a six-week treatment, the survival rates of B6 mice were 0%, 30%, 10% and 50% for control group, low, median and high concentration ABM treatment groups, respectively. The survival rate showed that pretreatment of C57BL/6 (B6) mice with ABM lengthened their lifespan after tumor cell inoculation, which supports the notion that ABM successfully reduced lung metastasis formation by B16F10 melanoma cells. The treatment effect was dependent on the concentration of ABM for tumor growth and metastasis in these models.

Expression of the serine/threonine kinase hSGK1 in chronic viral hepatitis.

PubMed

Fillon, Sophie; Klingel, Karin; Wärntges, Simone; Sauter, Martina; Gabrysch, Sabine; Pestel, Sabine; Tanneur, Valerie; Waldegger, Siegfried; Zipfel, Annette; Viebahn, Richard; Häussinger, Dieter; Bröer, Stefan; Kandolf, Reinhard; Lang, Florian

2002-01-01

The human serine/threonine kinase hSGK1 is expressed ubiquitously with highest transcript levels in pancreas and liver. This study has been performed to determine the hSGK1 distribution in normal liver and its putative role in fibrosing liver disease. HSGK1-localization was determined by in situ hybridization, regulation of hSGK1-transcription by Northern blotting, fibronectin synthesis and hSGK1 phosphorylation by Western blotting. In normal liver hSGK1 was mainly transcribed by Kupffer cells. In liver tissue from patients with chronic viral hepatitis, hSGK1 transcript levels were excessively high in numerous activated Kupffer cells and inflammatory cells localized within fibrous septum formations. HSGK1 transcripts were also detected in activated hepatic stellate cells. Accordingly, Western blotting revealed that tissue from fibrotic liver expresses excessive hSGK1 protein as compared to normal liver. TGF-beta1 (2 ng/ml) increases hSGK1 transcription in both human U937 macro-phages and HepG2 hepatoma cells. H(2)O(2) (0.3 mM) activated hSGK1 and increased fibronectin formation in HepG2 cells overexpressing hSGK1 but not in HepG2 cells expressing the inactive mutant hSGK1(K127R). In conclusion hSGK1 is upregulated by TGF-beta1 during hepatitis and may contribute to enhanced matrix formation during fibrosing liver disease. Copyright 2002 S. Karger AG, Basel

Studies of formation and efflux of methotrexate polyglutamates with cultured hepatic cells

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

Galivan, J.; Balinska, M.

1983-01-01

Methotrexate polyglutamates are extensively synthesized when cultured hepatocytes and H35 hepatoma cells are exposed to micromolar concentrations of methotrexate. The predominant species found within the cell have from two to four additional gamma-linked glutamate residues. When either cell type containing a mixture of methotrexate and its polyglutamate derivatives is exposed to medium lacking methotrexate, there is a rapid release of methotrexate. This release has a T/sub 1/2/ of 2 to 4 min and is apparently complete within 30 to 60 min. Methotrexate polyglutamates leave the cells much more slowly and appear to do so by two mechanisms. Although cleavage tomore » methotrexate and subsequent efflux appears to be quantitatively the more important pathway, there is also a slow, finite loss of intact methotrexate polyglutamates from cells which exclude trypan blue. The T/sub 1/2/ for the loss of methotrexate polyglutamates by both cell types, when placed in medium lacking methotrexate, is approximately 6 to 8 hr. These results suggest that the polyglutamate derivatives are forms of methotrexate which are as cytotoxic as methotrexate but which offer a potentially greater capacity for cellular destruction because they are retained longer in the tissue.« less

GROWTH INHIBITORY ACTIONS OF PROTHROMBIN ON NORMAL HEPATOCYTES

PubMed Central

Carr, Brian I.; Kar, Siddhartha; Wang, Meifang; Wang, Ziqiu

2007-01-01

Most hepatomas have a defect in prothrombin carboxylation, and can secrete under-carboxylated prothrombin or des-γ-carboxy-prothrombin (DCP), the function of which is unknown. We considered that prothrombin-DCP axis might also be involved in growth control. Hepatocytes and hepatoma cells were treated with prothrombin, and DNA synthesis and cytoskeleton were studied. Prothrombin inhibited DNA synthesis in hepatocytes on fibronectin, but not collagen matrix. Hepatoma cell lines were not inhibited. We found that hepatoma cell matrix conferred resistance to hepatocytes. Prothrombin decreased fibronectin but not collagen amounts, but only in the presence of hepatocytes and not hepatoma cells, indicating that it has a differential action on matrix proteins. It also caused changes in cell shape and actin depolymerization. In vivo, there was a decrease in plasma prothrombin activity after a partial hepatectomy (PH) concomitant with a peak of DNA synthesis by the hepatocyte at 24 h after PH. Injection of warfarin at the time of PH, further inhibited PT activity and enhanced this 24 h peak of DNA synthesis. Furthermore, repeated injection of prothrombin lowered the peak DNA synthesis after PH. The data support the hypothesis that prothrombin can act as a hepatocyte growth inhibitor, likely at the level of fibronectin loss and result in cytoskeletal changes. Hepatomas resist this action, possibly due to their different matrix proteins. This represents a novel mechanism for growth regulation and provides a possible biological significance for the tumor marker DCP. PMID:17490900

pH regulation in anoxic rice coleoptiles at pH 3.5: biochemical pHstats and net H+ influx in the absence and presence of NO3−

PubMed Central

Greenway, Hank; Kulichikhin, Konstantin Y.; Cawthray, Gregory R.; Colmer, Timothy D.

2012-01-01

During anoxia, cytoplasmic pH regulation is crucial. Mechanisms of pH regulation were studied in the coleoptile of rice exposed to anoxia and pH 3.5, resulting in H+ influx. Germinating rice seedlings survived a combination of anoxia and exposure to pH 3.5 for at least 4 d, although development was retarded and net K+ efflux was continuous. Further experiments used excised coleoptile tips (7–10 mm) in anoxia at pH 6.5 or 3.5, either without or with 0.2 mM NO3−, which distinguished two processes involved in pH regulation. Net H+ influx (μmol g−1 fresh weight h−1) for coleoptiles with NO3− was ∼1.55 over the first 24 h, being about twice that in the absence of NO3−, but then decreased to 0.5–0.9 as net NO3− uptake declined from ∼1.3 to 0.5, indicating reduced uptake via H+–NO3− symports. NO3− reduction presumably functioned as a biochemical pHstat. A second biochemical pHstat consisted of malate and succinate, and their concentrations decreased substantially with time after exposure to pH 3.5. In anoxic coleoptiles, K+ balancing the organic anions was effluxed to the medium as organic anions declined, and this efflux rate was independent of NO3− supply. Thus, biochemical pHstats and reduced net H+ influx across the plasma membrane are important features contributing to pH regulation in anoxia-tolerant rice coleoptiles at pH 3.5. PMID:22174442

Iso-suillin from Suillus flavus Induces Apoptosis in Human Small Cell Lung Cancer H446 Cell Line

PubMed Central

Zhao, Jun-Xia; Zhang, Qing-Shuang; Chen, Ying; Yao, Sheng-Jie; Yan, Yong-Xin; Wang, Ying; Zhang, Jin-Xiu; Wang, Li-An

2016-01-01

Background: The suillin isoform iso-suillin is a natural substance isolated from a petroleum ether extract of the fruiting bodies of the mushroom Suillus flavus. Previous studies have found its inhibition effect on some cancer cells, and we aimed to study its effects on human small cell lung cancer H446 cell line. Methods: Cell viability was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Cellular morphological changes (apoptosis and necrosis) were evaluated using an electron microscope and Hoechst 33258 staining detected by the inverted microscope. Flow cytometry was used to detect cell apoptosis, cell cycle distribution, and mitochondrial membrane potential. Protein expression was determined by Western blotting analysis. Results: Here, we describe the ability of iso-suillin to inhibit the growth of H446 cells in time- and dose-dependent way. Iso-suillin had no obvious impact on normal human lymphocyte proliferation at low concentrations (9.09, 18.17, or 36.35 μmol/L) but promoted lymphocyte proliferation at a high concentration (72.70 μmol/L). After treatment of different concentrations of iso-suillin (6.82, 13.63, or 20.45 μmol/L), the apoptosis rate of H446 cells increased with increasing concentrations of iso-suillin (16.70%, 35.54%, and 49.20%, respectively, all P < 0.05 compared with the control), and the expression of related apoptotic proteins in the mitochondrial pathway including cytochrome c and caspase-9 were up-regulated compared with the control (all P < 0.05). On the contrary, Bcl-2/Bax ratio was down-regulated compared with the control. Besides, the expression of pro-apoptotic proteins in the death receptor apoptosis pathway, including Fas-associating protein with a novel death domain and caspase-8, and the expression of caspase-3, a downstream regulatory protein of apoptosis, were also increased compared with the control (all P < 0.05). Inhibitors of caspase-9 and caspase-8 reversed the apoptosis process in

Iso-suillin from Suillus flavus Induces Apoptosis in Human Small Cell Lung Cancer H446 Cell Line.

PubMed

Zhao, Jun-Xia; Zhang, Qing-Shuang; Chen, Ying; Yao, Sheng-Jie; Yan, Yong-Xin; Wang, Ying; Zhang, Jin-Xiu; Wang, Li-An

2016-05-20

The suillin isoform iso-suillin is a natural substance isolated from a petroleum ether extract of the fruiting bodies of the mushroom Suillus flavus. Previous studies have found its inhibition effect on some cancer cells, and we aimed to study its effects on human small cell lung cancer H446 cell line. Cell viability was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Cellular morphological changes (apoptosis and necrosis) were evaluated using an electron microscope and Hoechst 33258 staining detected by the inverted microscope. Flow cytometry was used to detect cell apoptosis, cell cycle distribution, and mitochondrial membrane potential. Protein expression was determined by Western blotting analysis. Here, we describe the ability of iso-suillin to inhibit the growth of H446 cells in time- and dose-dependent way. Iso-suillin had no obvious impact on normal human lymphocyte proliferation at low concentrations (9.09, 18.17, or 36.35 μmol/L) but promoted lymphocyte proliferation at a high concentration (72.70 μmol/L). After treatment of different concentrations of iso-suillin (6.82, 13.63, or 20.45 μmol/L), the apoptosis rate of H446 cells increased with increasing concentrations of iso-suillin (16.70%, 35.54%, and 49.20%, respectively, all P < 0.05 compared with the control), and the expression of related apoptotic proteins in the mitochondrial pathway including cytochrome c and caspase-9 were up-regulated compared with the control (all P < 0.05). On the contrary, Bcl-2/Bax ratio was down-regulated compared with the control. Besides, the expression of pro-apoptotic proteins in the death receptor apoptosis pathway, including Fas-associating protein with a novel death domain and caspase-8, and the expression of caspase-3, a downstream regulatory protein of apoptosis, were also increased compared with the control (all P < 0.05). Inhibitors of caspase-9 and caspase-8 reversed the apoptosis process in H446 cells to varying degrees

The effect of oleuropein from olive leaf (Olea europaea) extract on Ca²⁺ homeostasis, cytotoxicity, cell cycle distribution and ROS signaling in HepG2 human hepatoma cells.

PubMed

Cheng, Jin-Shiung; Chou, Chiang-Ting; Liu, Yuan-Yuarn; Sun, Wei-Chih; Shieh, Pochuen; Kuo, Daih-Huang; Kuo, Chun-Chi; Jan, Chung-Ren; Liang, Wei-Zhe

2016-05-01

Oleuropein, a phenolic compound found in the olive leaf (Olea europaea), has been shown to have biological activities in different models. However, the effects of oleuropein on Ca(2+) homeostasis, cytotoxicity, cell cycle distribution and ROS signaling in liver cells have not been analyzed. Oleuropein induced [Ca(2+)]i rises only in HepG2 cells but not in AML12, HA22T or HA59T cells due to the different status of 3-hydroxy-3-methylglutaryl-CoA reductase expression. In HepG2 cells, this Ca(2+) signaling response was reduced by removing extracellular Ca(2+), and was inhibited by the store-operated Ca(2+) channel blockers 2-APB and SKF96365. In Ca(2+)-free medium, pretreatment with the ER Ca(2+) pump inhibitor thapsigargin abolished oleuropein-induced [Ca(2+)]i rises. Oleuropein induced cell cycle arrest which was associated with the regulation of p53, p21, CDK1 and cyclin B1 levels. Furthermore, oleuropein elevated intracellular ROS levels but reduced GSH levels. Treatment with the intracellular Ca(2+) chelator BAPTA-AM or the antioxidant NAC partially reversed oleuropein-induced cytotoxicity. Together, in HepG2 cells, oleuropein induced [Ca(2+)]i rises by releasing Ca(2+) from the ER and causing Ca(2+) influx through store-operated Ca(2+) channels. Moreover, oleuropein induced Ca(2+)-associated cytotoxicity that involved ROS signaling and cell cycle arrest. This compound may offer a potential therapy for treatment of human hepatoma. Copyright © 2016 Elsevier Ltd. All rights reserved.

Suppression of Cytochrome P450 Reductase (POR) Expression in Hepatoma Cells Replicates the Hepatic Lipidosis Observed in Hepatic POR-Null Mice

PubMed Central

Banerjee, Subhashis; Stolarczyk, Elzbieta I.; Zou, Ling

2011-01-01

Cytochrome P450 reductase (POR) is a microsomal electron transport protein essential to cytochrome P450-mediated drug metabolism and sterol and bile acid synthesis. The conditional deletion of hepatic POR gene expression in mice results in a marked decrease in plasma cholesterol levels counterbalanced by the accumulation of triglycerides in lipid droplets in hepatocytes. To evaluate the role of cholesterol and bile acid synthesis in this hepatic lipidosis, as well as the possible role of lipid transport from peripheral tissues, we developed a stable, small interfering RNA (siRNA)-mediated cell culture model for the suppression of POR. POR mRNA and protein expression were decreased by greater than 50% in McArdle-RH7777 rat hepatoma cells 10 days after transfection with a POR-siRNA expression plasmid, and POR expression was nearly completely extinguished by day 20. Immunofluorescent analysis revealed a marked accumulation of lipid droplets in cells by day 15, accompanied by a nearly 2-fold increase in cellular triglyceride content, replicating the lipidosis seen in hepatic POR-null mouse liver. In contrast, suppression of CYP51A1 (lanosterol demethylase) did not result in lipid accumulation, indicating that loss of cholesterol synthesis is not the basis for this lipidosis. Indeed, addition of cholesterol to the medium appeared to augment the lipidosis in POR-suppressed cells, whereas removal of lipids from the medium reversed the lipidosis. Oxysterols did not accumulate in POR-suppressed cells, discounting a role for liver X receptor in stimulating triglyceride synthesis, but addition of chenodeoxycholate significantly repressed lipid accumulation, suggesting that the absence of bile acids and loss of farnesoid X receptor stimulation lead to excessive triglyceride synthesis. PMID:21368239

Suppression of cytochrome P450 reductase (POR) expression in hepatoma cells replicates the hepatic lipidosis observed in hepatic POR-null mice.

PubMed

Porter, Todd D; Banerjee, Subhashis; Stolarczyk, Elzbieta I; Zou, Ling

2011-06-01

Cytochrome P450 reductase (POR) is a microsomal electron transport protein essential to cytochrome P450-mediated drug metabolism and sterol and bile acid synthesis. The conditional deletion of hepatic POR gene expression in mice results in a marked decrease in plasma cholesterol levels counterbalanced by the accumulation of triglycerides in lipid droplets in hepatocytes. To evaluate the role of cholesterol and bile acid synthesis in this hepatic lipidosis, as well as the possible role of lipid transport from peripheral tissues, we developed a stable, small interfering RNA (siRNA)-mediated cell culture model for the suppression of POR. POR mRNA and protein expression were decreased by greater than 50% in McArdle-RH7777 rat hepatoma cells 10 days after transfection with a POR-siRNA expression plasmid, and POR expression was nearly completely extinguished by day 20. Immunofluorescent analysis revealed a marked accumulation of lipid droplets in cells by day 15, accompanied by a nearly 2-fold increase in cellular triglyceride content, replicating the lipidosis seen in hepatic POR-null mouse liver. In contrast, suppression of CYP51A1 (lanosterol demethylase) did not result in lipid accumulation, indicating that loss of cholesterol synthesis is not the basis for this lipidosis. Indeed, addition of cholesterol to the medium appeared to augment the lipidosis in POR-suppressed cells, whereas removal of lipids from the medium reversed the lipidosis. Oxysterols did not accumulate in POR-suppressed cells, discounting a role for liver X receptor in stimulating triglyceride synthesis, but addition of chenodeoxycholate significantly repressed lipid accumulation, suggesting that the absence of bile acids and loss of farnesoid X receptor stimulation lead to excessive triglyceride synthesis.

Protective Effect of Edaravone Against Aβ25-35-Induced Mitochondrial Oxidative Damage in SH-SY5Y Cells.

PubMed

Zhang, G-L; Zhang, L; Guo, Y-Y; Ma, Z-L; Wang, H-Y; Li, T; Liu, J; Du, Y; Yao, L; Li, T-T; Du, J-M

2017-05-20

Amyloid-β (Aβ)-induced oxidative stress plays an important role in the pathogenesis of Alzheimer's disease (AD). Recent studies show that Aβ accumulation may lead to mitochondrial oxidative damage. In the present study, we investigated the protective effect of edaravone on mitochondrial damage in SH-SY5Y cells treated with Aβ25-35. SH-SY5Y cells were pre-treated with 20, 40 or 80 μM edaravone before treatment with 25 μM Aβ25-35. After 24h cell culture, cellular apoptosis, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), ATP levels and mitochondrial morphology were evaluated. SH-SY5Y cells exposed to Aβ25-35 had high levels of apoptosis and ROS; loss of ΔΨm, decreased ATP levels and presence of mitochondrial swelling. However, these effects were significantly inhibited by edaravone pre-treatment. These results indicate that edaravone prevents mitochondria oxidative damage caused by Aβ in SH-SY5Y cells, which suggests that it may have potential clinical application in AD therapy.

Apolipoproteins A-I, A-II and E are independently distributed among intracellular and newly secreted HDL of human hepatoma cells

PubMed Central

Gillard, Baiba K.; Lin, Hu-Yu Alice; Massey, John B.; Pownall, Henry J.

2009-01-01

Whereas hepatocytes secrete the major human plasma high density lipoproteins (HDL)-protein, apo A-I, as lipid-free and lipidated species, the biogenic itineraries of apo A-II and apo E are unknown. Human plasma and HepG2 cell-derived apo A-II and apo E occur as monomers, homodimers and heterodimers. Dimerization of apo A-II, which is more lipophilic than apo A-I, is catalyzed by lipid surfaces. Thus, we hypothesized that lipidation of intracellular and secreted apo A-II exceeds that of apo A-I, and once lipidated, apo A-II dimerizes. Fractionation of HepG2 cell lysate and media by size exclusion chromatography showed that intracellular apo A-II and apo E are fully lipidated and occur on nascent HDL and VLDL respectively, while only 45% of intracellular apo A-I is lipidated. Secreted apo A-II and apo E occur on small HDL and on LDL and large HDL respectively. HDL particles containing both apo A-II and apo A-I form only after secretion from both HepG2 and Huh7 hepatoma cells. Apo A-II dimerizes intracellularly while intracellular apo E is monomeric but after secretion associates with HDL and subsequently dimerizes. Thus, HDL apolipoproteins A-I, A-II and E have distinct intracellular and post-secretory pathways of hepatic lipidation and dimerization in the process of HDL formation. These early forms of HDL are expected to follow different apolipoprotein-specific pathways through plasma remodeling and reverse cholesterol transport. PMID:19635584

CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

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

Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji

2009-05-15

Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1{sub C}YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carryingmore » humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+){sub s}evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.« less

TiF(4) and NaF at pH 1.2 but not at pH 3.5 are able to reduce dentin erosion.

PubMed

Wiegand, Annette; Magalhães, Ana Carolina; Sener, Beatrice; Waldheim, Elena; Attin, Thomas

2009-08-01

This study aimed to analyse and compare the protective effect of buffered (pH 3.5) and native (pH 1.2) TiF(4) in comparison to NaF solutions of same pH on dentin erosion. Bovine samples were pretreated with 1.50% TiF(4) or 2.02% NaF (both 0.48M F) solutions, each with a pH of 1.2 and 3.5. The control group received no fluoride pretreatment. Ten samples in each group were eroded with HCl (pH 2.6) for 10x60s. Erosion was analysed by determination of calcium release into the acid. Additionally, the surface and the elemental surface composition were examined by scanning electron microscopy (two samples in each group) and X-ray energy-dispersive spectroscopy in fluoridated but not eroded samples (six samples in each group). Cumulative calcium release (nmol/mm(2)) was statistically analysed by repeated measures ANOVA and one-way ANOVA at t=10min. TiF(4) and NaF at pH 1.2 decreased calcium release significantly, while TiF(4) and NaF at pH 3.5 were not effective. Samples treated with TiF(4) at pH 1.2 showed a significant increase of Ti, while NaF pretreatment increased F concentration significantly. TiF(4) at pH 1.2 led to the formation of globular precipitates occluding dentinal tubules, which could not be observed on samples treated with TiF(4) at pH 3.5. NaF at pH 1.2 but not at pH 3.5 induced the formation of surface precipitates covering dentinal tubules. Dentin erosion can be significantly reduced by TiF(4) and NaF at pH 1.2, but not at pH 3.5.

Efficacy and safety of cell-associated vaccines against Marek's disease virus grown in QT35 cells or JBJ-1 cells.

PubMed

Geerligs, Harm; Spijkers, Ine; Rodenberg, Jeff

2013-06-01

The Marek's disease virus (MDV) vaccine strain CVI 988 usually is grown in primary chicken embryo fibroblasts (CEFs). We found that the strains could be grown also in the QT35 and JBJ-1 cell lines to titers in the same range as in the CEFs. Both cell lines are fibroblast-like cell lines, which can be grown in flat-bottomed tissue-culture flasks, roller bottles, and on microcarriers. For growth in QT35 cells it was necessary to adapt the virus to the cell line; for growth in JBJ-1 cells this was not necessary. We investigated the efficacy of experimental CVI 988 vaccines grown in QT35 cells and JBJ-1 cells. The efficacy studies were performed in accordance with European Pharmacopoeia (EP) monograph for live MDV disease vaccines. Groups of 1-day-old specific-pathogen-free chicks were vaccinated. Nonvaccinated control groups were included in the studies. Five to 7 days after vaccination all chickens were challenged with the very virulent MDV strain RB1B. After challenge the chickens were observed for a period of 70 days for signs of MD. The protection induced by CVI 988 grown in QT35 cells as well as JBJ-1 cells complied with the requirements of the EP that prescribe that the protection index should be at least 80%. The safety of the vaccines grown in QT35 cells and JBJ-1 cells was tested in a field study in commercial layer chickens. The vaccine virus was not safe after passaging in QT35 cells. This can be explained by the presence of fragments of the genome of MDV strains in the QT35 cell line. No signs of MD were noticed in the study in which CVI988 grown in JBJ-1 cells was tested. It is concluded that the JBJ-1 cell line is a suitable substrate for the current vaccines against MD.

Effect of GAPDH-derived antimicrobial peptides on sensitive yeasts cells: membrane permeability, intracellular pH and H+-influx/-efflux rates.

PubMed

Branco, Patrícia; Albergaria, Helena; Arneborg, Nils; Prista, Catarina

2018-05-01

Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which induce the death of several non-Saccharomyces yeasts. Previously, we demonstrated that the naturally secreted GAPDH-derived AMPs (i.e. saccharomycin) caused a loss of culturability and decreased the intracellular pH (pHi) of Hanseniaspora guilliermondii cells. In this study, we show that chemically synthesised analogues of saccharomycin also induce a pHi drop and loss of culturability in H. guilliermondii, although to a lesser extent than saccharomycin. To assess the underlying causes of the pHi drop, we evaluated the membrane permeability to H+ cations of H. guilliermondii cells, after being exposed to saccharomycin or its synthetic analogues. Results showed that the H+-efflux decreased by 75.6% and the H+-influx increased by 66.5% in cells exposed to saccharomycin at pH 3.5. Since H+-efflux via H+-ATPase is energy dependent, reduced glucose consumption would decrease ATP production and consequently H+-ATPase activity. However, glucose uptake rates were not affected, suggesting that the AMPs rather than affecting glucose transporters may affect directly the plasma membrane H+-ATPase or increase ATP leakage due to cell membrane disturbance. Thus, our study revealed that both saccharomycin and its synthetic analogues induced cell death of H. guilliermondii by increasing the proton influx and inhibiting the proton efflux.

Comparison of DNA damage in human-derived hepatoma line (HepG2) exposed to the fifteen drinking water disinfection byproducts using the single cell gel electrophoresis assay.

PubMed

Zhang, Li; Xu, Liang; Zeng, Qiang; Zhang, Shao-Hui; Xie, Hong; Liu, Ai-Lin; Lu, Wen-Qing

2012-01-24

Disinfection of drinking water reduces pathogenic infection, but generates disinfection by-products (DBPs) in drinking water. In this study, the effect of fifteen DBPs on DNA damage in human-derived hepatoma line (HepG2) was investigated by the single cell gel electrophoresis (SCGE) assay. These fifteen DBPs are: four trihalomethanes (THMs), six haloacetic acides (HAAs), three haloacetonitriles (HANs), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), and chloral hydrate (CH). Based on the minimal effective concentration (MEC) at which DBPs induced significant increase in olive tail moment (OTM), the rank order of DNA-damaging potency is: bromodichloromethane (BDCM)>dibromochloromethane (DBCM)>tribromomethane (TBM)>trichloromethane (TCM) of the four THMs; iodoacetic acid (IA)>bromoacetic acid (BA)>dibromoacetic acid (DBA)>dichloracetic acid (DCA)>trichloroacetic acid (TCA) of the five HAAs; dibromoacetonitrile (DBN)approximately dichloroacetonitrile (DCN)>trichloroacetonitrile (TCN) of the three HANs. The DNA damaging potency of MX and CH is similar to TCA and DCA, respectively. IA is the most genotoxic DBP in the fifteen DBPs, followed by BA. Chloroacetic acid (CA) is not genotoxic in this assay. Our findings indicated that HepG2/SCGE is a sensitive tool to evaluate the genotoxicity of DBPs and iodinated DBPs are more genotoxic than brominated DBPs, but chlorinated DBPs are less genotoxic than brominated DBPs. © 2011 Elsevier B.V. All rights reserved.

The coordinated effects of Apatinib and Tripterine on the proliferation, invasiveness and apoptosis of human hepatoma Hep3B cells.

PubMed

Li, Huihui; Fan, Yichang; Yang, Fan; Zhao, Lei; Cao, Bangwei

2018-07-01

As a novel vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitor, Apatinib has exhibited antitumor effects in a variety of solid tumors. Extracts of Chinese herbal medicines have emerged as a promising alternative option to increase the sensitivity of patients to chemotherapeutics while alleviating side effects. The present study aimed to investigate the effects of Apatinib and the traditional Chinese herb Tripterine on the proliferation, invasion and apoptosis of human hepatoma Hep3B cells. The expression of VEGFR-2 in Hep3B cells was detected by western blotting and immunofluorescence assays. Hep3B cells were then divided into four different groups: Control group, Apatinib group, Tripterine group and Apatinib plus Tripterine group. The proliferation, invasion and apoptosis of these four groups of Hep3B cells were assessed by MTS, wound healing and Transwell assays, and flow cytometry, respectively. Finally, the levels of the proliferation-associated proteins phosphorylated protein kinase B (p-Akt) and phosphorylated extracellular signal-regulated kinase (p-ERK) and the apoptosis-associated proteins cleaved Caspase-3 and B-cell lymphoma-associated X protein (Bax) were detected by western blotting. The proliferation, migration and invasion of Hep3B cells were significantly inhibited by Apatinib and Tripterine, compared with the control group (P<0.01). The inhibitory effect of the combination group was markedly stronger than that of the Apatinib and Tripterine groups. The downregulation of p-Akt and p-ERK induced by Apatinib and Tripterine was further inhibited in the combination group (P<0.05), and the expression levels of Caspase-3 and Bax were also significantly increased in the combination group (P<0.05). The combination of Apatinib and Tripterine significantly inhibited the proliferation, migration and invasion ability and promoted the apoptosis of Hep3B cells by downregulating the expression of p-Akt and p-ERK, and upregulating the

Ilexgenin A exerts anti-inflammation and anti-angiogenesis effects through inhibition of STAT3 and PI3K pathways and exhibits synergistic effects with Sorafenib on hepatoma growth

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

Yang, Hao

Recently, we reported that Ilexgenin A exhibits anti-cancer activities and induces cell arrest. Here, we investigated the effect of Ilexgenin A on the inflammation, angiogenesis and tumor growth of hepatocellular carcinoma (HCC). Our current study revealed that Ilexgenin A significantly inhibited the inflammatory cytokines TNF-α and IL-6 levels and downregulated pro-angiogenic factor VEGF production and transcription in HepG2 cells. The underlying mechanism for Ilexgenin A effects appears to be through inhibiting STAT3 and PI3K pathways. Furthermore, we found that not only Ilexgenin A inhibited STAT3 and PI3K pathways in HepG2 cells but also blocked these signaling pathways in HUVECs. Mostmore » importantly, by employing two HCC xenografts models - HepG2 and H22, we showed that Ilexgenin A reduced tumor growth and exhibited synergy effect with Sorafenib. ELISA assay, histological analysis and immunohistochemistry examination revealed that the expression of VEGF and MVD was significantly decreased after the treatment with Ilexgenin A and the combination. Moreover, Ilexgenin A could enhance caspase-3/7 activity in vitro and transmission electron microscope indicated that the combination induced evident apoptosis of tumor cells and caused the structural changes of mitochondria in vivo. Although no apparent adverse effects occurred during the treatment period, Sorafenib monotherapy elicited hepatotoxicity for specific expression in the increased level of AST and the ratio of AST/ALT. However, the combination could remedy this adverse effect. In conclusion, the results described in the present study identifies Ilexgenin A as a promising therapeutic candidate that modulates inflammation, angiogenesis, and HCC growth. - Highlights: • Ilexgenin A exerts anti-inflammatory and anti-angiogenesis effects in hepatoma. • Ilexgenin A may exert these effects through inhibition of STAT3 and PI3K pathways. • Ilexgenin A exhibits synergistic effects with Sorafenib on

Measuring and modeling of binary mixture effects of pharmaceuticals and nickel on cell viability/cytotoxicity in the human hepatoma derived cell line HepG2

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

Rudzok, S., E-mail: susanne.rudzok@ufz.d; Schlink, U., E-mail: uwe.schlink@ufz.d; Herbarth, O., E-mail: olf.herbarth@medizin.uni-leipzig.d

2010-05-01

The interaction of drugs and non-therapeutic xenobiotics constitutes a central role in human health risk assessment. Still, available data are rare. Two different models have been established to predict mixture toxicity from single dose data, namely, the concentration addition (CA) and independent action (IA) model. However, chemicals can also act synergistic or antagonistic or in dose level deviation, or in a dose ratio dependent deviation. In the present study we used the MIXTOX model (EU project ENV4-CT97-0507), which incorporates these algorithms, to assess effects of the binary mixtures in the human hepatoma cell line HepG2. These cells possess a liver-likemore » enzyme pattern and a variety of xenobiotic-metabolizing enzymes (phases I and II). We tested binary mixtures of the metal nickel, the anti-inflammatory drug diclofenac, and the antibiotic agent irgasan and compared the experimental data to the mathematical models. Cell viability was determined by three different methods the MTT-, AlamarBlue (registered) and NRU assay. The compounds were tested separately and in combinations. We could show that the metal nickel is the dominant component in the mixture, affecting an antagonism at low-dose levels and a synergism at high-dose levels in combination with diclofenac or irgasan, when using the NRU and the AlamarBlue assay. The dose-response surface of irgasan and diclofenac indicated a concentration addition. The experimental data could be described by the algorithms with a regression of up to 90%, revealing the HepG2 cell line and the MIXTOX model as valuable tool for risk assessment of binary mixtures for cytotoxic endpoints. However the model failed to predict a specific mode of action, the CYP1A1 enzyme activity.« less

HuH-7 reference genome profile: complex karyotype composed of massive loss of heterozygosity.

PubMed

Kasai, Fumio; Hirayama, Noriko; Ozawa, Midori; Satoh, Motonobu; Kohara, Arihiro

2018-05-17

Human cell lines represent a valuable resource as in vitro experimental models. A hepatoma cell line, HuH-7 (JCRB0403), has been used extensively in various research fields and a number of studies using this line have been published continuously since it was established in 1982. However, an accurate genome profile, which can be served as a reliable reference, has not been available. In this study, we performed M-FISH, SNP microarray and amplicon sequencing to characterize the cell line. Single cell analysis of metaphases revealed a high level of heterogeneity with a mode of 60 chromosomes. Cytogenetic results demonstrated chromosome abnormalities involving every chromosome in addition to a massive loss of heterozygosity, which accounts for 55.3% of the genome, consistent with the homozygous variants seen in the sequence analysis. We provide empirical data that the HuH-7 cell line is composed of highly heterogeneous cell populations, suggesting that besides cell line authentication, the quality of cell lines needs to be taken into consideration in the future use of tumor cell lines.

Angiogenesis for tumor vascular normalization of Endostar on hepatoma 22 tumor-bearing mice is involved in the immune response.

PubMed

Xu, Qingyu; Gu, Junfei; Lv, You; Yuan, Jiarui; Yang, Nan; Chen, Juan; Wang, Chunfei; Hou, Xuefeng; Jia, Xiaobin; Feng, Liang; Yin, Guowen

2018-03-01

Tumor vascular normalization involved in immune response is beneficial to the chemotherapy of tumors. Recombinant human endostatin (Endostar), an angiogenesis inhibitor, has been demonstrated to be effective in hepatocellular cancer (HCC). However, its vascular normalization in HCC and the role of the immune response in angiogenesis were unclear. In the present study, effects of Endostar on tumor vascular normalization were evaluated in hepatoma 22 (H22) tumor-bearing mice. Endostar was able to inhibit the proliferation and infiltration of tumor cells and improve α-fetoprotein, tumor necrosis factor-α and cyclic adenosine 5'-phosphate levels in the serum of H22-bearing mice, as well as the protein expression levels of the immune factors interferon-γ and cluster of differentiation (CD)86 in liver tissue. Endostar also exhibited more marked downregulation of the levels of vascular endothelial growth factor, CD31, matrix metalloproteinase (MMP)-2, MMP-9 and interleukin-17 during day 3-9 treatment, resulting in short-term normalization of tumor blood vessels. The period of vascular normalization was 3-9 days. The results of the present study demonstrated that Endostar was able to induce the period of vascular normalization, contributing to a more efficacious means of HCC treatment combined with other chemotherapy, and this effect was associated with the immune response. It may be concluded that Endostar inhibited immunity-associated angiogenesis behaviors of vascular endothelial cells in response to HCC. The results of the present study provided more reasonable possibility for the combination therapy of Endostar for the treatment of HCC.

Synoviocytes-derived Interleukin 35 Potentiates B Cell Response in Patients with Osteoarthritis and Rheumatoid Arthritis.

PubMed

Kam, Ngar-Woon; Liu, Dehua; Cai, Zhe; Mak, Wah-Yan; Wong, Chun-Kwok; Chiu, Kwok-Hing; Wong, Kam-Yiu; Tsang, Wai-Leuk; Tam, Lai-Shan

2018-04-01

Elevated expression of interleukin 35 (IL-35) is associated with autoimmune disease, including rheumatoid arthritis (RA). The present study was undertaken to determine the functional interaction among IL-35, B cells, and stromal cells residing in the synovium of patients with RA and osteoarthritis (OA). IL-35 (EBI-3/p35) expression was investigated in RA and OA synovium using quantitative real-time PCR (qRT-PCR) and immunohistochemistry. IL-35 receptor (IL-35R) expression on B cells dissociated from synovium and periphery of patients with RA, OA, and healthy donor controls (HC) was determined by flow cytometry. The degree of B cells activation after IL-4 and/or IL-35 stimulation was measured by flow cytometry and qRT-PCR. Synovial fibroblasts (SF) purified from RA and OA synovium were cocultured with peripheral HC B cells in the presence/absence of tumor necrosis factor-α (TNF-α) and with/without anti-IL-35-blocking antibodies. EBI-3/p35 transcripts were expressed in close proximity to B cells residing in RA and OA synovium. IL-35R subunits, gp130 and IL-27Rα, but not IL-12Rβ2, were expressed in B cells extracted from the synovium and periphery of patients with RA/OA. Notably, RA synovium expressed the highest level of IL-27Rα on their cell surface. IL-35 induced proliferation and IgG production in HC B cells. Cocultures of HC B cells with RASF, but not OASF, exhibited significantly elevated B cells activation. TNF-α-induced, RASF-dependent secretion of IgG in B cells is partly IL-35-dependent. To our knowledge, for the first time we demonstrated that synovial/peripheral B cells expressed IL-35R and were responsive to IL-35 stimulation. SF residing in RA synovium can be linked to B cell activation and maintenance in RA synovium through IL-35.

CYP1A1 and CYP1A2 expression: Comparing ‘humanized’ mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

PubMed Central

Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

2009-01-01

Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how “human-like” can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1_CYP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+)_severe-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs. PMID:19285097

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

Liu, Fabao; Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071; You, Xiaona

Highlights: • Relative to wild type HBx, HBX mutant HBxΔ127 strongly enhances cell proliferation. • Relative to wild type HBx, HBxΔ127 remarkably up-regulates miR-215 in hepatoma cells. • HBxΔ127-elevated miR-215 promotes cell proliferation via targeting PTPRT mRNA. - Abstract: The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migrationmore » in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3′UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.« less

Different Temporal Effects of Ebola Virus VP35 and VP24 Proteins on Global Gene Expression in Human Dendritic Cells.

PubMed

Ilinykh, Philipp A; Lubaki, Ndongala M; Widen, Steven G; Renn, Lynnsey A; Theisen, Terence C; Rabin, Ronald L; Wood, Thomas G; Bukreyev, Alexander

2015-08-01

Ebola virus (EBOV) causes a severe hemorrhagic fever with a deficient immune response, lymphopenia, and lymphocyte apoptosis. Dendritic cells (DC), which trigger the adaptive response, do not mature despite EBOV infection. We recently demonstrated that DC maturation is unblocked by disabling the innate response antagonizing domains (IRADs) in EBOV VP35 and VP24 by the mutations R312A and K142A, respectively. Here we analyzed the effects of VP35 and VP24 with the IRADs disabled on global gene expression in human DC. Human monocyte-derived DC were infected by wild-type (wt) EBOV or EBOVs carrying the mutation in VP35 (EBOV/VP35m), VP24 (EBOV/VP24m), or both (EBOV/VP35m/VP24m). Global gene expression at 8 and 24 h was analyzed by deep sequencing, and the expression of interferon (IFN) subtypes up to 5 days postinfection was analyzed by quantitative reverse transcription-PCR (qRT-PCR). wt EBOV induced a weak global gene expression response, including markers of DC maturation, cytokines, chemokines, chemokine receptors, and multiple IFNs. The VP35 mutation unblocked the expression, resulting in a dramatic increase in expression of these transcripts at 8 and 24 h. Surprisingly, DC infected with EBOV/VP24m expressed lower levels of many of these transcripts at 8 h after infection, compared to wt EBOV. In contrast, at 24 h, expression of the transcripts increased in DC infected with any of the three mutants, compared to wt EBOV. Moreover, sets of genes affected by the two mutations only partially overlapped. Pathway analysis demonstrated that the VP35 mutation unblocked pathways involved in antigen processing and presentation and IFN signaling. These data suggest that EBOV IRADs have profound effects on the host adaptive immune response through massive transcriptional downregulation of DC. This study shows that infection of DC with EBOV, but not its mutant forms with the VP35 IRAD and/or VP24 IRAD disabled, causes a global block in expression of host genes. The temporal

Depletion of hepatoma-derived growth factor-related protein-3 induces apoptotic sensitization of radioresistant A549 cells via reactive oxygen species-dependent p53 activation

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

Yun, Hong Shik; Hong, Eun-Hee; Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791

2013-09-27

Highlights: •HRP-3 is a radiation- and anticancer drug-responsive protein in A549 cells. •Depletion of HRP-3 induces apoptosis of radio- and chemoresistant A549 cells. •Depletion of HRP-3 promotes ROS generation via inhibition of the Nrf2/HO-1 pathway. •Depletion of HRP-3 enhances ROS-dependent p53 activation and PUMA expression. -- Abstract: Biomarkers based on functional signaling have the potential to provide greater insight into the pathogenesis of cancer and may offer additional targets for anticancer therapeutics. Here, we identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistance-related gene and characterized the molecular mechanism by which its encoded protein regulates the radio- and chemoresistant phenotypemore » of lung cancer-derived A549 cells. Knockdown of HRP-3 promoted apoptosis of A549 cells and potentiated the apoptosis-inducing action of radio- and chemotherapy. This increase in apoptosis was associated with a substantial generation of reactive oxygen species (ROS) that was attributable to inhibition of the Nrf2/HO-1 antioxidant pathway and resulted in enhanced ROS-dependent p53 activation and p53-dependent expression of PUMA (p53 upregulated modulator of apoptosis). Therefore, the HRP-3/Nrf2/HO-1/ROS/p53/PUMA cascade is an essential feature of the A549 cell phenotype and a potential radiotherapy target, extending the range of targets in multimodal therapies against lung cancer.« less

Chemopreventive effect of 18β-glycyrrhetinic acid via modulation of inflammatory markers and induction of apoptosis in human hepatoma cell line (HepG2).

PubMed

Hasan, Syed Kazim; Siddiqi, Aisha; Nafees, Sana; Ali, Nemat; Rashid, Summya; Ali, Rashid; Shahid, Ayaz; Sultana, Sarwat

2016-05-01

Hepatocellular carcinoma is one of the most common lethal diseases worldwide and there is no effective treatment till date. Natural products derived from the plants play an important role in chemoprevention and act as therapeutic antitumor agents. Licorice is a plant that has been used in food and medicine for the treatment of various diseases. 18β-Glycyrrhetinic acid (18β-GA), a pentacyclic triterpenoid obtained from the roots of licorice plant, is reported to possess various pharmacological properties such as antitumor and antiinflammatory activities. The present study was designed to elucidate the chemopreventive effect of 18β-GA through antiinflammation, antiproliferation, and induction of apoptosis in human hepatoma cell line HepG2. 18β-GA significantly inhibits the proliferation of HepG2 cell without affecting the normal liver cell line (Chang's). In the present study, 18β-GA increased the formation of reactive oxygen species, nitric oxide production, and loss of mitochondrial membrane potential, suggesting the involvement of 18β-GA in apoptosis which was also confirmed by assessing the markers involved in apoptosis like caspase-3, caspase-9, Bax:Bcl-2 ratio, and cleaved PARP. 18β-GA also downregulated the expression of inflammatory proteins such as NF-κB, iNOS, and COX-2. Keeping these data into consideration, our results suggest that 18β-GA may be used as a chemopreventive agent in liver cancer.

Osteoactivin expressed during cirrhosis development in rats fed a choline-deficient, L-amino acid-defined diet, accelerates motility of hepatoma cells.

PubMed

Onaga, Masaaki; Ido, Akio; Hasuike, Satoru; Uto, Hirofumi; Moriuchi, Akihiro; Nagata, Kenji; Hori, Takeshi; Hayash, Katsuhiro; Tsubouchi, Hirohito

2003-11-01

Hepatocellular carcinoma (HCC) is closely associated with chronic liver diseases, particularly cirrhosis. However, the genes involved in hepatocarcinogenesis in the context of developing cirrhosis remain unknown. This study aims to identify genes associated with early cirrhosis-associated hepatocarcinogenesis. We examined genes differentially expressed between the livers of normal rats and rats fed a choline-deficient, L-amino acid-defined (CDAA) diet using suppression subtractive hybridization. We examined both the expression in the liver and HCC tissues of osteoactivin (OA), isolated in this screen, and its effect on invasiveness and metastasis. OA mRNA was strongly expressed in the livers of rats fed the CDAA diet for 1-3 months. Moderate expression was sustained for 18 months. OA overexpression increased the invasiveness and metastasis of rat hepatoma cells in vitro and in vivo. In humans, OA expression was not detectable in normal liver tissues. While OA transcripts were detectable in cirrhotic nontumorous liver tissues surrounding HCCs, the majority of HCC tissue samples exhibited higher levels of OA expression than the surrounding normal tissue. These results indicate that OA is a novel factor involved in the progression of HCC via stimulation of tumor invasiveness and metastatic potential.

Genotoxicity of 2,6- and 3,5-Dimethylaniline in Cultured Mammalian Cells: The Role of Reactive Oxygen Species

PubMed Central

Chao, Ming-Wei; Kim, Min Young; Wogan, Gerald N.

2012-01-01

Several alkylanilines with structures more complex than toluidines have been associated epidemiologically with human cancer. Their mechanism of action remains largely undetermined, and there is no reported evidence that it replicates that of multicyclic aromatic amines even though the principal metabolic pathways of P450-mediated hydroxylation and phase II conjugation are very similar. As a means to elucidate their mechanisms of action, lethality and mutagenicity in the adenine phosphoribosyltransferase (aprt +/−) gene induced in several Chinese hamster ovary cell types by 2,6- and 3,5-dimethylaniline (2,6-DMA, 3,5-DMA) and their N- and ring-hydroxyl derivatives (N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, 3,5-DMAP) were assessed. Dose-response relationships were determined in the parental AA8 cell line, its repair-deficient UV5 subclone and other repair-deficient 5P3NAT2 or -proficient 5P3NAT2R9 subclones engineered to express mouse cytochrome P4501A2 (CYP1A2) and human N-acetyltransferase (NAT2), and also in AS52 cells harboring the bacterial guanine-hypoxanthine phosphoribosyltransferase (gpt) gene. Mutations in the gpt gene of AS52 cells were characterized and found to be dominated by G:C to A:T and A:T to G:C transitions. Separately, treatment of AS52 cells with N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, 3,5-DMAP, and 3,5-DMAP led to intracellular production of reactive oxygen species (ROS) for at least 24h after removal of the mutagens in every case. Using the comet assay, DNA strand breaks were observed in a dose-dependent manner in AS52 cells when treated with each of the four N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, and 3,5-DMAP derivatives. Comparative evaluation of the results indicates that the principal mechanism of mutagenic action is likely to be through redox cycling of intracellularly bound aminophenol/quinone imine structures to generate ROS rather than through formation of covalent DNA adducts. PMID:22831970

Detection and functional characterization of Pgp1 (ABCB1) and MRP3 (ABCC3) efflux transporters in the PLHC-1 fish hepatoma cell line.

PubMed

Zaja, Roko; Klobucar, Roberta Sauerborn; Smital, Tvrtko

2007-03-30

The PLHC-1 hepatoma cell line derived from topminnow (Poeciliopsis lucida) is one of the most frequently used fish cell lines in aquatic ecotoxicology. These cells have been well characterized regarding the presence of phase I and phase II enzymes involved in the metabolism of xenobiotics. However, the presence of the ABC transport proteins possibly involved in the MultiXenobiotic Resistance (MXR) mechanism as phase III of cellular detoxification has never been described in the PLHC-1 cells. The main goal of this study was the detection and functional characterization of toxicologically relevant xenobiotic efflux transporters from ABCB and ABCC subfamily in the PLHC-1 cells. Using specific primer pairs two PCR products 1769 and 1023bp in length were successfully cloned and sequenced. Subsequent multiple alignment and phylogenetic analysis showed that these sequences share a high degree of homology with the P-glycoprotein (Pgp1; ABCB1) and the MRP3 (ABCC3). Functional experiments with fluorescent model substrates and specific inhibitors were used to verify that transport activities of Pgp- and MRP-related proteins are indeed present in PLHC-1 cells. Accumulation or efflux/retention rates of rhodamine 123, calcein-AM or monochlorbimane were time- and concentration-dependent. Cyclosporine A, MK571, verapamil, reversine 205, indomethacine and probenecid were used as specific inhibitors of Pgp1 and/or MRPs transport activities, resulting in a dose dependent inhibition of related transport activities in PLHC-1 cells. Similar to mammalian systems, the obtained IC(50) values were in the lower micromolar range. Taken together these data demonstrate that: (1) the PLHC-1 cells do express a functional MXR mechanism mediated by toxicologically relevant ABC efflux transporters; and (2) the presence of all three critical phases of cellular detoxification additionally affirms the PLHC-1 cells as a reliable in vitro model in aquatic toxicology.

Recombinant Newcastle disease virus expressing human TRAIL as a potential candidate for hepatoma therapy

USDA-ARS?s Scientific Manuscript database

Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical studies and are currently proved for clinical trials. We have previously reported, for the first time, NDV Anhinga strain has an efficient cancer therapeutic efficacy in hepatoma. Tumor necrosis factor-related apo...

Double strand breaks and cell-cycle arrest induced by the cyanobacterial toxin cylindrospermopsin in HepG2 cells.

PubMed

Alja, Štraser; Filipič, Metka; Novak, Matjaž; Žegura, Bojana

2013-08-21

The newly emerging cyanobacterial cytotoxin cylindrospermopsin (CYN) is increasingly found in surface freshwaters, worldwide. It poses a potential threat to humans after chronic exposure as it was shown to be genotoxic in a range of test systems and is potentially carcinogenic. However, the mechanisms of CYN toxicity and genotoxicity are not well understood. In the present study CYN induced formation of DNA double strand breaks (DSBs), after prolonged exposure (72 h), in human hepatoma cells, HepG2. CYN (0.1-0.5 µg/mL, 24-96 h) induced morphological changes and reduced cell viability in a dose and time dependent manner. No significant increase in lactate dehydrogenase (LDH) leakage could be observed after CYN exposure, indicating that the reduction in cell number was due to decreased cell proliferation and not due to cytotoxicity. This was confirmed by imunocytochemical analysis of the cell-proliferation marker Ki67. Analysis of the cell-cycle using flow-cytometry showed that CYN has an impact on the cell cycle, indicating G0/G1 arrest after 24 h and S-phase arrest after longer exposure (72 and 96 h). Our results provide new evidence that CYN is a direct acting genotoxin, causing DSBs, and these facts need to be considered in the human health risk assessment.

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.

Interleukin-35 administration counteracts established murine type 1 diabetes--possible involvement of regulatory T cells.

PubMed

Singh, Kailash; Kadesjö, Erik; Lindroos, Julia; Hjort, Marcus; Lundberg, Marcus; Espes, Daniel; Carlsson, Per-Ola; Sandler, Stellan; Thorvaldson, Lina

2015-07-30

The anti-inflammatory cytokine IL-35 is produced by regulatory T (Treg) cells to suppress autoimmune and inflammatory responses. The role of IL-35 in type 1 diabetes (T1D) remains to be answered. To elucidate this, we investigated the kinetics of Treg cell response in the multiple low dose streptozotocin induced (MLDSTZ) T1D model and measured the levels of IL-35 in human T1D patients. We found that Treg cells were increased in MLDSTZ mice. However, the Treg cells showed a decreased production of anti-inflammatory (IL-10, IL-35, TGF-β) and increased pro-inflammatory (IFN-γ, IL-2, IL-17) cytokines, indicating a phenotypic shift of Treg cells under T1D condition. IL-35 administration effectively both prevented development of, and counteracted established MLDSTZ T1D, seemingly by induction of Eos expression and IL-35 production in Treg cells, thus reversing the phenotypic shift of the Treg cells. IL-35 administration reversed established hyperglycemia in NOD mouse model of T1D. Moreover, circulating IL-35 levels were decreased in human T1D patients compared to healthy controls. These findings suggest that insufficient IL-35 levels play a pivotal role in the development of T1D and that treatment with IL-35 should be investigated in treatment of T1D and other autoimmune diseases.

Molecular basis for ebolavirus VP35 suppression of human dendritic cell maturation.

PubMed

Yen, Benjamin; Mulder, Lubbertus C F; Martinez, Osvaldo; Basler, Christopher F

2014-11-01

Zaire ebolavirus (EBOV) VP35 is a double-stranded RNA (dsRNA)-binding protein that inhibits RIG-I signaling and alpha/beta interferon (IFN-α/β) responses by both dsRNA-binding-dependent and -independent mechanisms. VP35 also suppresses dendritic cell (DC) maturation. Here, we define the pathways and mechanisms through which VP35 impairs DC maturation. Wild-type VP35 (VP35-WT) and two well-characterized VP35 mutants (F239A and R322A) that independently ablate dsRNA binding and RIG-I inhibition were delivered to primary human monocyte-derived DCs (MDDCs) using a lentivirus-based expression system. VP35-WT suppressed not only IFN-α/β but also proinflammatory responses following stimulation of MDDCs with activators of RIG-I-like receptor (RLR) signaling, including RIG-I activators such as Sendai virus (SeV) or 5'-triphosphate RNA, or MDA5 activators such as encephalomyocarditis virus (EMCV) or poly(I · C). The F239A and R322A mutants exhibited greatly reduced suppression of IFN-α/β and proinflammatory cytokine production following treatment of DCs with RLR agonists. VP35-WT also blocked the upregulation of DC maturation markers and the stimulation of allogeneic T cell responses upon SeV infection, whereas the mutants did not. In contrast to the RLR activators, VP35-WT and the VP35 mutants impaired IFN-β production induced by Toll-like receptor 3 (TLR3) or TLR4 agonists but failed to inhibit proinflammatory cytokine production induced by TLR2, TLR3, or TLR4 agonists. Furthermore, VP35 did not prevent lipopolysaccharide (LPS)-induced upregulation of surface markers of MDDC maturation and did not prevent LPS-triggered allogeneic T cell stimulation. Therefore, VP35 is a general antagonist of DC responses to RLR activation. However, TLR agonists can circumvent many of the inhibitory effects of VP35. Therefore, it may be possible to counteract EBOV immune evasion by using treatments that bypass the VP35-imposed block to DC maturation. The VP35 protein, which is an

Synthesis of 3,5-Isoxazolidinediones and 1H-2,3-Benzoxazine-1,4(3H)-diones from Aliphatic Oximes and Dicarboxylic Acid Chlorides

PubMed Central

2015-01-01

The synthesis of the title compounds was carried out by reacting dicarboxylic acid chlorides with oximes in the presence of excess triethylamine. Disubstituted malonyl chlorides gave 2-alkenyl-4,4-dialkyl-3,5-isoxazolidinediones (8a–f) and 2,2′-ethylidene-bis[4,4-dialkyl-3,5-isoxazolidinedione]s (9a–f). Compounds 9 were formed from 8 and its N-unsubstituted 3,5-isoxazolidinedione decomposition product. Phthaloyl chlorides reacted with acetone oxime to yield 3-(1-methylethenyl)-1H-2,3-benzoxazine-1,4(3H)-diones (16a–e). Products 16 spontaneously decomposed to give N-unsubstituted 1H-2,3-benzoxazine-1,4(3H)-diones (17a–e) at rates that were dependent on temperature and solvent. Kinetic studies showed that two of the compounds decomposed by zero-order kinetics under neutral conditions. Butanedioyl chloride did not produce a cyclic product. PMID:24620711

Synthesis of 3,5-isoxazolidinediones and 1H-2,3-benzoxazine-1,4(3H)-diones from aliphatic oximes and dicarboxylic acid chlorides.

PubMed

Izydore, Robert A; Jones, Joseph T; Mogesa, Benjamin; Swain, Ira N; Davis-Ward, Ronda G; Daniels, Dwayne L; Kpakima, Felicia Frazier; Spaulding-Phifer, Sharnelle T

2014-04-04

The synthesis of the title compounds was carried out by reacting dicarboxylic acid chlorides with oximes in the presence of excess triethylamine. Disubstituted malonyl chlorides gave 2-alkenyl-4,4-dialkyl-3,5-isoxazolidinediones (8a-f) and 2,2'-ethylidene-bis[4,4-dialkyl-3,5-isoxazolidinedione]s (9a-f). Compounds 9 were formed from 8 and its N-unsubstituted 3,5-isoxazolidinedione decomposition product. Phthaloyl chlorides reacted with acetone oxime to yield 3-(1-methylethenyl)-1H-2,3-benzoxazine-1,4(3H)-diones (16a-e). Products 16 spontaneously decomposed to give N-unsubstituted 1H-2,3-benzoxazine-1,4(3H)-diones (17a-e) at rates that were dependent on temperature and solvent. Kinetic studies showed that two of the compounds decomposed by zero-order kinetics under neutral conditions. Butanedioyl chloride did not produce a cyclic product.

Effects of the augmenter of liver regeneration on the biological behavior of hepatocellular carcinoma.

PubMed

Tang, Lin; Sun, Hang; Zhang, Lin; Deng, Jian C; Guo, Hui; Zhang, Ling; Liu, Qi

2009-08-01

To take advantage of the small interfering ribonucleic acid (siRNA) targeting the human augmenter of liver regeneration (hALR) and anti-hALR monoclonal antibody (McAb) to inhibit the function of hALR, and to demonstrate whether the growth of hepatoma is influenced by siRNA targeting hALR and anti-hALR McAb through inhibiting expression of hALR. This study was conducted in the Key Laboratory of Molecular Biology for Infectious Diseases, Ministry of Education, Institute for Viral Hepatitis, Chongqing Medical University, China, between January 2005 and May 2007. We transfected siRNA plasmid pSIALR-A, which targeted the complementary deoxyribonucleic acid (cDNA) of hALR and the unrelated control plasmid pSIALR-B into human hepatocellular liver carcinoma cell line (HepG2) cells. Then, the proliferation of HepG2 cells, after being treated with pSIALR-A and anti-hALR McAb was detected. The growth of the xenograft tumor was observed after being treated with pSIALR-A and anti-hALR McAb in nude mice. We successfully constructed expressing plasmid pSIALR-A and pSIALR-B. The pSIALR-A inhibited the expression of hALR in HepG2 cells significantly. The siRNA targeting hALR and anti-hALR McAb inhibited obviously the growth of HepG2 cells in vitro. siRNA targeting hALR and anti-hALR McAb significantly inhibited the growth of xenograft tumor in 5 nude mice. Anti-hALR McAb inhibited apparently the autonomous growth of HepG2 cells. Our results demonstrated that anti-hALR McAb inhibited the autonomous growth of hepatoma cells obviously, moreover, hALR maintained the autonomous growth of hepatoma cells in vitro through an autocrine mechanism.

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.

Hyperfine-resolved transition frequency list of fundamental vibration bands of H35Cl and H37Cl

NASA Astrophysics Data System (ADS)

Iwakuni, Kana; Sera, Hideyuki; Abe, Masashi; Sasada, Hiroyuki

2014-12-01

Sub-Doppler resolution spectroscopy of the fundamental vibration bands of H35Cl and H37Cl has been carried out from 87.1 to 89.9 THz. We have determined the absolute transition frequencies of the hyperfine-resolved R(0) to R(4) transitions with a typical uncertainty of 10 kHz. We have also yielded six molecular constants for each isotopomer in the vibrational excited state, which reproduce the determined frequencies with a standard deviation of about 10 kHz.

Body distribution of nanoparticle-containing adriamycin injected into the hepatic artery of hepatoma-bearing rats.

PubMed

Chen, Jiang-Hao; Wang, Ling; Ling, Rui; Li, Yu; Wang, Zhe; Yao, Qing; Ma, Zhong

2004-08-01

The aim of the study was to investigate the body distribution of nanoparticle-containing adriamycin (NADR) injected into the hepatic artery of hepatoma-bearing rats. Thirty Walker-256 hepatoma-bearing rats were divided into two groups at random, with 15 rats in each. NADR and free adriamycin (FADR) were injected into the hepatic artery of animals on the seventh day after tumor implantation. At 1, 5, and 15 hr, after administration, five animals in each group were sacrificed and the ADR concentrations in the plasma, liver, heart, spleen, lungs, kidneys, and tumor were determined. The results showed that NADR substantially increased the ADR concentrations in liver, spleen, and tumor of rats compared to FADR, whereas the concentrations in plasma, heart, and lungs were significantly decreased. In conclusion, the body distribution of ADR can be modified by its encapsulation into nanoparticles and administration via the hepatic artery.

Method for Salmonella concentration from water at pH 3.5, using micro-fiber glass filters.

PubMed Central

Block, J C; Rolland, D

1979-01-01

A method is described for the concentration of Salmonella from water. As is done with enterovirus, Salmonella bacteria were concentrated from water in two steps: by pH 3.5 adsorption on and pH 9.5 elution from 8-micron porosity micro-fiber glass filter tubes. This method worked in less than 30 min, and Salmonella typhimurium was inactivated only slightly in spite of rapid pH variations (pH 3.5 to 9.5). It was demonstrated that the retention by the filters stems from two phenomena: a low retention in the micro-fiber glass labyrinth for small filtered volumes, and a high retention by adsorption at pH 3.5 for any filtered volume (experiments done with 15- and 80-liter samples). Addition in tap water of trivalent ions like Al3+ did not increase Salmonella adsorption. In most of the trials, Salmonella recovery varied from 42 to 93%. Preliminary field investigations indicate that enterovirus and Salmonella may both be concentrated from the same water sample by this procedure. PMID:39501

Biological activity of the azlactone derivative EPA-35 against Trypanosoma cruzi.

PubMed

de Azeredo, Camila Maria Oliveira; Ávila, Eloah Pereira; Pinheiro, Danielle Lobo Justo; Amarante, Giovanni Wilson; Soares, Maurilio José

2017-02-01

Chagas disease, caused by Trypanosoma cruzi, affects six to seven million people worldwide. Treatment is based on benznidazole, producing several side effects and debatable efficacy, highlighting the need for new alternative drugs. We investigated the activity of four C-4 functionalized azlactone derivatives (EPA-27, EPA-35, EPA-63 and EPA-91) as potential T. cruzi inhibitors. Screening with epimastigotes indicated EPA-35 as the best compound (IC50/24 h: 33 μM). This compound was 14.1 times more potent against intracellular amastigotes (IC50/24 h: 2.34 μM). Treatment of infected Vero cells for 72 h (up to 30 μM EPA-35) resulted in a dose-dependent decrease in number of trypomastigotes and amastigotes released in the supernatant, but the amastigote/trypomastigote ratio remained constant, indicating that amastigote growth was disturbed, but cell differentiation was unaffected. Analysis of treated epimastigotes by flow cytometry indicated that the plasma membrane remained intact, but there was a significant decrease in mitochondrial membrane potential. The pattern of cell distribution in the cell cycle stages (G1, G2, M) was unaltered in treated epimastigotes, indicating a trypanocidal rather than a trypanostatic activity. Scanning electron microscopy and flow cytometry showed epimastigotes with a round shape and decrease in cell size. Taken together, our data indicate that the EPA-35 is effective against T. cruzi. Synthetic transformation of EPA-35 into other derivatives may provide promising compounds for further evaluation against this parasite. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Predictive performance of the human Cell Line Activation Test (h-CLAT) for lipophilic chemicals with high octanol-water partition coefficients.

PubMed

Takenouchi, Osamu; Miyazawa, Masaaki; Saito, Kazutoshi; Ashikaga, Takao; Sakaguchi, Hitoshi

2013-01-01

To meet the urgent need for a reliable alternative test for predicting skin sensitizing potential of many chemicals, we have developed a cell-based in vitro test, human Cell Line Activation Test (h-CLAT). However, the predictive performance for lipophilic chemicals in the h-CLAT still remains relatively unknown. Moreover, it's suggested that low water solubility of chemicals might induce false negative outcomes. Thus, in this study, we tested relatively low water soluble 37 chemicals with log Kow values above and below 3.5 in the h-CLAT. The small-scale assessment resulted in nine false negative outcomes for chemicals with log Kow values greater than 3.5. We then created a dataset of 143 chemicals by combining the existing dataset of 106 chemicals and examined the predictive performance of the h-CLAT for chemicals with a log Kow of less than 3.5; a total of 112 chemicals from the 143 chemicals in the dataset. The sensitivity and overall accuracy for the 143 chemicals were 83% and 80%, respectively. In contrast, sensitivity and overall accuracy for the 112 chemicals with log Kow values below 3.5 improved to 94% and 88%, respectively. These data suggested that the h-CLAT could successfully detect sensitizers with log Kow values up to 3.5. When chemicals with log Kow values greater than 3.5 that were deemed positive by h-CLAT were included with the 112 chemicals, the sensitivity and accuracy in terms of the resulting applicable 128 chemicals out of the 143 chemicals became 95% and 88%, respectively. The use of log Kow values gave the h-CLAT a higher predictive performance. Our results demonstrated that the h-CLAT could predict sensitizing potential of various chemicals, which contain lipophilic chemicals using a large-scale chemical dataset.

Combined effect of synthetic enterocin CRL35 with cell wall, membrane-acting antibiotics and muranolytic enzymes against Listeria cells.

PubMed

Salvucci, E; Hebert, E M; Sesma, F; Saavedra, L

2010-08-01

To evaluate the inhibition effectiveness of enterocin CRL35 in combination with cell wall, membrane-acting antibiotics and muranolytic enzymes against the foodborne pathogen Listeria. Synthetic enterocin CRL35 alone and in combination with monensin, bacitracin, gramicidin, mutanolysin and lysozyme were used in this study. Minimal inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) index assays were performed using Listeria innocua 7 and Listeria monocytogenes FBUNT as sensitive strains. Antibiotics showed positive interactions with the bacteriocin in both strains tested. On the other hand, when mutanolysin and enterocin CRL35 were added to resting cells in a buffer system, the lytic effect of mutanolysin was enhanced. However, the addition of mutanolysin showed no effect on the growth of L. innocua 7 cells in a culture medium. Moreover, mutanolysin allowed the overgrowth of L. innocua 7 cells to an OD similar to control cells in the presence of inhibitory concentration of enterocin CRL35. In contrast, the combination of lysozyme and enterocin CRL35 resulted in a 50% inhibition of the L. innocua 7 growth. Based on our results, we conclude that the combination of synthetic enterocin CRL35 with some antibiotics is effective against L. innocua 7 and L. monocytogenes FBUNT cells, and more importantly the amount of these agents to be used was considerably reduced. The effectiveness of the combination of synthetic enterocin CRL35 with muramidases seems to depend on complex environments, and more detailed studies need to be performed to elucidate this issue. Enterocin CRL35 represents a promising agent that not only can ensure the quality and safety of food but it can also be combined with several antimicrobial agents important in the medical field.

Effect of pH on whitening efficacy of 35% hydrogen peroxide and enamel microhardness.

PubMed

Jurema, Ana Luiza Barbosa; de Souza, Mauricio Yugo; Torres, Carlos Rocha Gomes; Borges, Alessandra Bühler; Caneppele, Taciana Marco Ferraz

2018-03-01

This study aimed to evaluate the effect of 35% hydrogen peroxide at different pH values and the degree of tooth staining on whitening efficacy and enamel microhardness. 90 enamel-dentin specimens were obtained from bovine incisors. They were randomly divided into 2 groups (n = 45), 1 group was immersed in a staining broth for 14 days, and another group was not stained and kept in distilled water at 37°C. Twenty-four hours after the staining procedure, each group was distributed into 3 subgroups that were whitened by 35% hydrogen peroxide with different pH values (5, 7, and 8.4) for 30 minutes. The color was measured at baseline and 7 days after whitening. Microhardness was measured at baseline, immediate, 24 hours, and 1 month after the whitening procedure. Data were submitted to 2-way analysis of variance (ANOVA) and the Tukey test for multiple comparisons for color analysis. Repeated measures ANOVA and the Tukey test were used to analyze microhardness data. The color change of the stained groups (ΔE 00  = 4.6) was significantly higher than that of the nonstained groups (ΔE 00  = 3.7). Microhardness value decreased significantly immediately after whitening for all subgroups and did not return to initial values. For each measurement time, microhardness was not significantly different among subgroups with different pH values. Despite the effectiveness of 35% hydrogen peroxide, changes on gel pH did not affect the whitening efficacy, and the enamel was superficially demineralized, regardless of pH values. Independently of the pH value of whitening gel, enamel undergoes superficial demineralization and with a reduction in superficial microhardness that does not return to the initial values. However, using hydrogen peroxide with different pH values does not alter the whitening effect. © 2018 Wiley Periodicals, Inc.

Tanshinone IIA protects PC12 cells from β-amyloid(25-35)-induced apoptosis via PI3K/Akt signaling pathway.

PubMed

Dong, Huimin; Mao, Shanping; Mao, Shanpin; Wei, Jiajun; Liu, Baohui; Zhang, Zhaohui; Zhang, Qian; Yan, Mingmin

2012-06-01

For the aging populations of any nation, Dementia is becoming a primary problem and Alzheimer’s dementia (AD) is the most common type. However, until now, there is no effective treatment for AD. Tanshinone IIA (Tan IIA) has been reported for neuroprotective potential to against amyloid β peptides (Aβ)-induced cytotoxicity in the rat pheochromocytoma cell line PC-12, which is widely used as AD research model, but the mechanism still remains unclear. To investigate the effect of Tan IIA and the possible molecular mechanism in the apoptosis of PC12 cells, we induced apoptosis in PC12 cells with β-amyloid(25-35), and treated cells with Tan IIA. After 24 h treatment, we found that Tan IIA increased the cell viability and reduced the number of apoptotic cells induced by Aβ(25-35). However, neuroprotection of Tan IIA was abolished by PI3K inhibitor LY294002. Meanwhile, Treatment with lithium chloride, a phosphorylation inhibitor of GSK3β, which is a downstream target of PI3K/Akt, can block Aβ(25-35)-induced cell apoptosis in a Tan IIA-like manner. Our findings suggest that Tan IIA is an effective neuroprotective agent and a viable candidate in AD therapy and PI3K/Akt activation and GSK3β phosphorylation are involved in the neuroprotection of Tan IIA.

Sonodynamic action of pyropheophorbide-a methyl ester induces mitochondrial damage in liver cancer cells.

PubMed

Xu, Jing; Xia, Xinshu; Leung, Albert Wingnang; Xiang, Junyan; Jiang, Yuan; Yu, Heping; Bai, Dingqun; Li, Xiaohong; Xu, Chuanshan

2011-05-01

Sonodynamic therapy with pyropheophorbide-a methyl ester (MPPa) presents a promising aspect in treating liver cancer. The present study aims to investigate the mitochondrial damage of liver cancer cells induced by MPPa-mediated sonodynamic action. Mouse hepatoma cell line H(22) cells were incubated with MPPa (2 μM) for 20 h and then exposed to ultrasound with an intensity of 0.97 W/cm(2) for 8 s. Cytotoxicity was investigated 24h after sonodynamic action using MTT assay and light microscopy. Mitochondrial membrane potential (ΔΨm) was analyzed using flow cytometry with rhodamine 123 staining and ultrastructural changes were observed using transmission electron microscopy (TEM). The cytotoxicity of MPPa-mediated SDT on H(22) cell line was 73.00±3.42%, greater than ultrasound treatment alone (28.12±5.19%) significantly while MPPa treatment alone had no significant effect on H(22) cells. Moreover, after MPPa-mediated SDT cancer cells showed swollen mitochondria under TEM and a significant collapse of mitochondrial membrane potential. Our findings demonstrated that MPPa-mediated SDT could remarkably induce cell death of H(22) cells, and highlighted that mitochondrial damage might be an important cause of cell death induced by MPPa-mediated SDT. Copyright © 2010 Elsevier B.V. All rights reserved.

Genotoxic activity and induction of biotransformation enzymes in two human cell lines after treatment by Erika fuel extract.

PubMed

Amat-Bronnert, Agnès; Castegnaro, Marcel; Pfohl-Leszkowicz, Annie

2007-01-01

On 12 December 1999, the tanker Erika broke in two parts at about 60km from the Brittany French coasts (Point of Penmarc'h, Sud Finistère, France). About 10,000tonnes of heavy oil fuel were released in the sea. DNA adduct have been detected in fish liver and mussels digestive gland exposed to the Erika oil spill. In order to investigate the mechanism by which Erika fuel extract exhibits genotoxic effects the induction of DNA adducts by an Erika fuel extract have been analysed on two cell lines, human epithelial bronchial cells (WI) and human hepatoma cells. DNA adducts, reflected by a diagonal radioactive zone and individual adducts are detected only in hepatoma cells indicating biotransformation via CYP 1A2 and CYP 1B1. In addition, Erika fuel extract induces some metabolizing enzymes such CYP 1A2, COX2 and 5-LOX, the two later are involved in cancer processes. Formation of leucotrienes B4 (LTB(4)), a mediator playing a role in inflammation, is induced in epithelial bronchial cells. Since inhalation is one of the ways of contamination for human, the above results are important for human health and prevention. Copyright © 2006 Elsevier B.V. All rights reserved.

[Inhibitory effect of migration-inducing gene-7-shRNA recombinant retrovirus combined with endostatin on growth and metastasis of hepatoma xenograft].

PubMed

Qu, B; Chen, G N; Sheng, G N; Yu, F; Lyu, Q; Gu, Y J; Guo, L; Lyu, Y

2016-09-20

Objective: To investigate the inhibitory effect of migration-inducing gene-7(Mig-7)interfered with retrovirus-mediated RNA(shRNA)combined with recombinant human endostatin(ES)on the growth and metastasis of subcutaneous xenograft of human hepatoma cells in nude mice. Methods: Two Mig-7-mRNA oligonucleotide sequences(Mig-7-shRNA-1 and Mig-7-shRNA-2)and one sequence as a negative control(Mig-7-shRNA-N)were designed. The specific Mig-7-shRNA recombinant retrovirus expression vector plasmid was constructed and used for the transfection of human hepatoma MHCC-97H cells with high expression of Mig-7. The subcutaneous xenograft tumor model of human hepatocellular carcinoma(HCC)in nude mice was established, and according to the condition of transfection and administration, the nude mice were divided into pSIREN-M1 group, pSIREN-MN group, ES group, and pSIREN-M1+ES group. The xenograft tumor volume, mass, and metastasis were compared between groups. Immunohistochemistry was used to observe the formation of vasculogenic mimicry(VM)in xenograft tumor and the difference in tumor microvascular density(MVD), and Western blot was used to measure the expression of Mig-7 and vascular endothelial growth factor(VEGF)in each group. A one-way analysis of variance was used for comparison between groups, and the Fisher's exact test was used for comparison of continuous data between groups. Results: Compared with the pSIREN-MN group, the pSIREN-M1 group had significantly lower xenograft tumor volume, mass, and metastasis rate, Mig-7 expression, and formation of VM( P < 0.05), as well as significantly higher VEGF expression and MVD( P < 0.05). Compared with the pSIREN-MN group, the ES group had significantly lower xenograft tumor volume, mass, and metastasis rate, VEGF expression, and MVD( P < 0.05), as well as significantly higher Mig-7 expression and formation of VM( P < 0.05). Compared with the pSIREN-M1 group and the ES group, the pSIREN-M1+ES group had significantly lower xenograft

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.

Interleukin 35 (IL-35) and IL-37: Intestinal and peripheral expression by T and B regulatory cells in patients with Inflammatory Bowel Disease.

PubMed

Fonseca-Camarillo, Gabriela; Furuzawa-Carballeda, Janette; Yamamoto-Furusho, Jesús K

2015-10-01

The aim of the study was to characterize and to quantify peripheral and tissue. IL-35- and IL-37-producing cells in Inflammatory Bowel Disease (IBD) patients. We studied a total of 38 active UC, 31 inactive UC, 17 active CD, and 13 inactive CD and 50 non-inflamed tissues as control group. Gene expression was measured by real time polymerase chain reaction (RT-PCR) and protein expression was evaluated in tissue by immunohistochemistry and in peripheral blood mononuclear cells by flow cytometry. Higher levels of IL-35 was produced by intestinal regulatory B cells and circulating regulatory CD4(+) and CD8(+) T cells in active vs. inactive disease or healthy donors (P<0.05). The IL-37 was conspicuously synthesized by circulating B cells, active natural killer cells and monocytes. These results suggest that down-regulation of inflammation in active IBD patients might be based on the increased expression of IL-35 and IL-37. Copyright © 2015 Elsevier Ltd. All rights reserved.

Connective tissue growth factor is overexpressed in human hepatocellular carcinoma and promotes cell invasion and growth.

PubMed

Xiu, Ming; Liu, Ya-Hui; Brigstock, David R; He, Fang-Hui; Zhang, Rui-Juan; Gao, Run-Ping

2012-12-21

To determine the expression characteristics of connective tissue growth factor (CTGF/CCN2) in human hepatocellular carcinoma (HCC) in histology and to elucidate the roles of CCN2 on hepatoma cell cycle progression and metastasis in vitro. Liver samples from 36 patients (who underwent hepatic resection for the first HCC between 2006 and 2011) and 6 normal individuals were examined for transforming growth factor β1 (TGF-β1) or CCN2 mRNA by in situ hybridization. Computer image analysis was performed to measure integrated optimal density of CCN2 mRNA-positive cells in carcinoma foci and the surrounding stroma. Fibroblast-specific protein-1 (FSP-1) and E-cadherin were examined to evaluate the process of epithelial to mesenchymal transition, α-smooth muscle actin and FSP-1 were detected to identify hepatic stellate cells, and CD34 was measured to evaluate the extent of vascularization in liver tissues by immunohistochemical staining. CCN2 was assessed for its stimulation of HepG2 cell migration and invasion using commercial kits while flow cytometry was used to determine CCN2 effects on HepG2 cell-cycle. In situ hybridization analysis showed that TGF-β1 mRNA was mainly detected in connective tissues and vasculature around carcinoma foci. In comparison to normal controls, CCN2 mRNA was enhanced 1.9-fold in carcinoma foci (12.36 ± 6.08 vs 6.42 ± 2.35) or 9.4-fold in the surrounding stroma (60.27 ± 28.71 vs 6.42 ± 2.35), with concomitant expression of CCN2 and TGF-β1 mRNA in those areas. Epithelial-mesenchymal transition phenotype related with CCN2 was detected in 12/36 (33.3%) of HCC liver samples at the edges between carcinoma foci and vasculature. Incubation of HepG2 cells with CCN2 (100 ng/mL) resulted in more of the cells transitioning into S phase (23.85 ± 2.35 vs 10.94 ± 0.23), and induced a significant migratory (4.0-fold) and invasive (5.7-fold) effect. TGF-β1-induced cell invasion was abrogated by a neutralizing CCN2 antibody showing that CCN2 is a

A Role of hIPI3 in DNA Replication Licensing in Human Cells.

PubMed

Huang, Yining; Amin, Aftab; Qin, Yan; Wang, Ziyi; Jiang, Huadong; Liang, Lu; Shi, Linjing; Liang, Chun

2016-01-01

The yeast Ipi3p is required for DNA replication and cell viability in Sacharomyces cerevisiae. It is an essential component of the Rix1 complex (Rix1p/Ipi2p-Ipi1p-Ipi3p) that is required for the processing of 35S pre-rRNA in pre-60S ribosomal particles and for the initiation of DNA replication. The human IPI3 homolog is WDR18 (WD repeat domain 18), which shares significant homology with yIpi3p. Here we report that knockdown of hIPI3 resulted in substantial defects in the chromatin association of the MCM complex, DNA replication, cell cycle progression and cell proliferation. Importantly, hIPI3 silencing did not result in a reduction of the protein level of hCDC6, hMCM7, or the ectopically expressed GFP protein, indicating that protein synthesis was not defective in the same time frame of the DNA replication and cell cycle defects. Furthermore, the mRNA and protein levels of hIPI3 fluctuate in the cell cycle, with the highest levels from M phase to early G1 phase, similar to other pre-replicative (pre-RC) proteins. Moreover, hIPI3 interacts with other replication-initiation proteins, co-localizes with hMCM7 in the nucleus, and is important for the nuclear localization of hMCM7. We also found that hIPI3 preferentially binds to the origins of DNA replication including those at the c-Myc, Lamin-B2 and β-Globin loci. These results indicate that hIPI3 is involved in human DNA replication licensing independent of its role in ribosome biogenesis.

Selective imaging of cancer cells with a pH-activatable lysosome-targeting fluorescent probe.

PubMed

Shi, Rongguang; Huang, Lu; Duan, Xiaoxue; Sun, Guohao; Yin, Gui; Wang, Ruiyong; Zhu, Jun-Jie

2017-10-02

Fluorescence imaging with tumor-specific fluorescent probe has emerged as a tool to aid surgeons in the identification and removal of tumor tissue. We report here a new lysosome-targeting fluorescent probe (NBOH) with BODIPY fluorephore to distinguish tumor tissue out of normal tissue based on different pH environment. The probe exhibited remarkable pH-dependent fluorescence behavior in a wide pH range from 3.0 to 11.0, especially a sensitive pH-dependent fluorescence change at pH range between 3.5 and 5.5, corresponding well to the acidic microenvironment of tumor cells, in aqueous solution. The response time of NBOH was extremely short and the photostability was proved to be good. Toxicity test and fluorescence cell imaging together with a sub-cellular localization study were carried out revealing its low biotoxicity and good cell membrane permeability. And NBOH was successfully applied to the imaging of tumor tissue in tumor-bearing mice suggesting potential application to surgery as a tumor-specific probe. Copyright © 2017 Elsevier B.V. All rights reserved.

pH- and redox-responsive nanoparticles composed of charge-reversible pullulan-based shells and disulfide-containing poly(ß-amino ester) cores for co-delivery of a gene and chemotherapeutic agent.

PubMed

Zhang, Sipei; Wang, Dan; Li, Yating; Li, Ling; Chen, Hongli; Xiong, Qingqing; Liu, Yuanyuan; Wang, Yinsong

2018-08-10

A novel pH- and redox-responsive nanoparticle system was designed based on a charge-reversible pullulan derivative (CAPL) and disulfide-containing poly(β-amino ester) (ssPBAE) for the co-delivery of a gene and chemotherapeutic agent targeting hepatoma. The end-alkene groups of ssPBAE were reacted with diethylenetriamine to form amino-modified ssPBAE (NH-ssPBAE). Methotrexate (MTX), a chemotherapy agent, was then conjugated to NH-ssPBAE via an amide bond to obtain the polymeric prodrug ssPBAE-MTX. ssPBAE-MTX exhibited a good capability for condensing genes, including plasmid DNA (pDNA) and tetramethyl rhodamine-labeled DNA (TAMRA-DNA), and almost completely condensed pDNA at the weight ratio of 5/1 to form spherical nanocomplexes with a uniform size. In a D,L-dithiothreitol solution, the ssPBAE-MTX/pDNA nanocomplexes showed rapid release of pDNA and MTX, indicating their redox-responsive capability. CAPL, a pullulan derivative containing β-carboxylic amide bond, was efficiently coated on the surfaces of ssPBAE-MTX/pDNA nanocomplexes to form polysaccharide shells, thus realizing co-loading of the gene and chemotherapeutic agent. CAPL/ssPBAE-MTX/pDNA nanoparticles displayed an obvious pH-responsive charge reversal ability due to the rupture of the β-carboxylic amide bond under the weakly acidic condition. In human hepatoma HepG2 cells, CAPL/ssPBAE-MTX/TAMRA-DNA nanoparticles were efficiently internalized via endocytosis and successfully escaped from the endo/lysosomes into the cytoplasm, and CAPL/ssPBAE-MTX/pDNA nanoparticles remarkably inhibited the cell growth. In summary, this nanoparticle system based on CAPL and ssPBAE showed great potential for combined gene/chemotherapy on hepatomas.

Guava Leaf Extract Inhibits Quorum-Sensing and Chromobacterium violaceum Induced Lysis of Human Hepatoma Cells: Whole Transcriptome Analysis Reveals Differential Gene Expression

PubMed Central

Tiwary, Bipransh Kumar; Kumar, Anoop

2014-01-01

Quorum sensing (QS) is a process mediated via small molecules termed autoinducers (AI) that allow bacteria to respond and adjust according to the cell population density by altering the expression of multitudinous genes. Since QS governs numerous bioprocesses in bacteria, including virulence, its inhibition promises to be an ideal target for the development of novel therapeutics. We found that the aqueous leaf extract of Psidium guajava (GLE) exhibited anti-QS properties as evidenced by inhibition of violacein production in Chromobacterium violaceum and swarming motility of Pseudomonas aeruginosa. The gram-negative bacterium, C. violaceum is a rare pathogen with high mortality rate. In this study, perhaps for the first time, we identified the target genes of GLE in C. violaceum MTCC 2656 by whole transcriptome analysis on Ion Torrent. Our data revealed that GLE significantly down-regulated 816 genes at least three fold, with p value≤0.01, which comprises 19% of the C. violaceum MTCC 2656 genome. These genes were distributed throughout the genome and were associated with virulence, motility and other cellular processes, many of which have been described as quorum regulated in C. violaceum and other gram negative bacteria. Interestingly, GLE did not affect the growth of the bacteria. However, consistent with the gene expression pattern, GLE treated C. violaceum cells were restrained from causing lysis of human hepatoma cell line, HepG2, indicating a positive relationship between the QS-regulated genes and pathogenicity. Overall, our study proposes GLE as a QS inhibitor (QSI) with the ability to attenuate virulence without affecting growth. To the best of our knowledge, this is the first report which provides with a plausible set of candidate genes regulated by the QS system in the neglected pathogen C. violaceum. PMID:25229331

Guava leaf extract inhibits quorum-sensing and Chromobacterium violaceum induced lysis of human hepatoma cells: whole transcriptome analysis reveals differential gene expression.

PubMed

Ghosh, Runu; Tiwary, Bipransh Kumar; Kumar, Anoop; Chakraborty, Ranadhir

2014-01-01

Quorum sensing (QS) is a process mediated via small molecules termed autoinducers (AI) that allow bacteria to respond and adjust according to the cell population density by altering the expression of multitudinous genes. Since QS governs numerous bioprocesses in bacteria, including virulence, its inhibition promises to be an ideal target for the development of novel therapeutics. We found that the aqueous leaf extract of Psidium guajava (GLE) exhibited anti-QS properties as evidenced by inhibition of violacein production in Chromobacterium violaceum and swarming motility of Pseudomonas aeruginosa. The gram-negative bacterium, C. violaceum is a rare pathogen with high mortality rate. In this study, perhaps for the first time, we identified the target genes of GLE in C. violaceum MTCC 2656 by whole transcriptome analysis on Ion Torrent. Our data revealed that GLE significantly down-regulated 816 genes at least three fold, with p value ≤ 0.01, which comprises 19% of the C. violaceum MTCC 2656 genome. These genes were distributed throughout the genome and were associated with virulence, motility and other cellular processes, many of which have been described as quorum regulated in C. violaceum and other gram negative bacteria. Interestingly, GLE did not affect the growth of the bacteria. However, consistent with the gene expression pattern, GLE treated C. violaceum cells were restrained from causing lysis of human hepatoma cell line, HepG2, indicating a positive relationship between the QS-regulated genes and pathogenicity. Overall, our study proposes GLE as a QS inhibitor (QSI) with the ability to attenuate virulence without affecting growth. To the best of our knowledge, this is the first report which provides with a plausible set of candidate genes regulated by the QS system in the neglected pathogen C. violaceum.

Suppressive effects of caraway (Carum carvi) extracts on 2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin-dependent gene expression of cytochrome P450 1A1 in the rat H4IIE cells.

PubMed

Naderi-Kalali, B; Allameh, A; Rasaee, M J; Bach, H-J; Behechti, A; Doods, K; Kettrup, A; Schramm, K-W

2005-04-01

Cytochrome P450 1A1 (CYP1A1) is among the cytochrome P450 classes known to convert xenobiotics and endogenous compounds to toxic and/or carcinogenic metabolites. Suppression of CYP1A1 over expression by certain compounds is implicated in prevention of cancer caused by chemical carcinogens. Chemopreventive agents containing high levels of flavonoids and steroids-like compounds are known to suppress CYP1A1. This study was carried out for assessment of the genomic and proteomic effects of caraway (Carum carvi) extracts containing high levels of both flavonoids and steroid-like substances on ethoxy resorufin dealkylation (EROD) activity and CYP1A1 at mRNA levels. Rat hepatoma cells co-treated with a CYP1A1 inducer i.e. TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin) and different preparations of caraway extracts at concentrations of 0, 0.13, 1.3, and 13 microM in culture medium. After incubation (37 degrees C and 7% CO2 for 20 h), changes in EROD specific activity recorded and compared in cells under different treatments. The results show that caraway seed extract prepared in three different organic solvents suppressed the enzyme activity in hepatoma cells in a dose-dependent manner. The extracts added above 0.13 microM could significantly inhibit EROD activity and higher levels of each extract (1.3 and 13 microM) caused approximately 10-fold suppression in the enzyme activity. Accordingly, data obtained from the RT-PCR (TaqMan) clearly showed the suppressive effects of plant extract on CYP1A1-related mRNA expression. These data clearly show that substances in caraway seeds extractable in organic solvents can potentially reverse the TCDD-dependent induction in cytochrome P450 1A1.

Influence of adhesive restorations on diffusion of H2O2 released from a bleaching agent and its toxic effects on pulp cells.

PubMed

Soares, Diana Gabriela; Pastana, Júlia Vieira; de Oliveira Duque, Carla Caroline; Dias Ribeiro, Ana Paula; Basso, Fernanda Gonçalves; Hebling, Josimeri; de Souza Costa, Carlos Alberto

2014-04-01

To assess the influence of adhesive restorations on hydrogen peroxide (H2O2) diffusion through enamel and dentin and its cytotoxicity to pulp (MDPC-23) cells. Sound and resin-restored enamel/dentin disks were stored in water for 24 h or 6 months and adapted to artificial pulp chambers. Bleaching gels with 20% or 35% H2O2 were applied to the enamel surface for 45 min, and a culture medium in direct contact with the dentin surface (extract) was applied for 1 h to the MDPC-23 cells. Cell metabolism (MTT assay) and cell morphology (SEM) were assessed. The amount of H2O2 in the extracts was also quantified (peroxidase/leuco-crystal violet reaction). A significant reduction in cell metabolism was observed between the group bleached with the 35% gel and the control group (sound, nonbleached) (p < 0.05). The H2O2 diffusion was directly related to its concentration in the bleaching gel. The variables "presence of restoration" and "time of water storage" did not significantly influence H2O2 diffusion or cell metabolism for either of the bleaching gels (p > 0.05). All bleached groups presented alterations in cell morphology related to the concentration of H2O2 in the bleaching gel. The reduction in cell metabolism and the changes in cell morphology were H2O2-concentration dependent, having no relationship with the presence of either new or aged adhesive restorations on teeth subjected to bleaching therapies.

Application of an octa-anionic 5,10,15,20-tetra[3,5-(nido-carboranylmethyl)phenyl]porphyrin (H2OCP) as dual sensitizer for BNCT and PDT

USDA-ARS?s Scientific Manuscript database

The applications of the octa-anionic 5,10,15,20-tetra[3,5-(nidocarboranylmethyl) phenyl]porphyrin (H2OCP) as a boron delivery agent in boron neutron capture therapy (BNCT) and a photosensitizer in photodynamic therapy (PDT) have been investigated. Using F98 Rat glioma cells, we evaluated the cytotox...

A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

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

Toki, Yasumichi; Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp; Tanaka, Hiroki

2016-08-05

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncatedmore » peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.« less

Construction of C35 gene bait recombinants and T47D cell cDNA library.

PubMed

Yin, Kun; Xu, Chao; Zhao, Gui-Hua; Liu, Ye; Xiao, Ting; Zhu, Song; Yan, Ge

2017-11-20

C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C35 1-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation. The three kinds of C35 genes were successfully amplified and inserted into pGBKT7 to construct bait recombinant plasmids pGBKT7-C351-348bp, pGBKT7-C351-153bp and pGBKT7-C35154-348bp, then transformed into Y187 yeast cells by the lithium acetate method. Auto-activation and toxicity of C35 baits were detected using nutritional deficient medium and X-α-Gal assays. The T47D cell ds cDNA was generated by SMART TM technology and the library was constructed using in vivo recombination-mediated cloning in the AH109 yeast strain using a pGADT7-Rec plasmid. The transformed Y187/pGBKT7-C351-348bp line was intensively inhibited while the truncated Y187/pGBKT7-C35 lines had no auto-activation and toxicity in yeast cells. The titer of established cDNA library was 2 × 10 7 pfu/mL with high transformation efficiency of 1.4 × 10 6 , and the insert size of ds cDNA was distributed homogeneously between 0.5-2.0 kb. Our research generated a T47D cell cDNA library with high titer, and the constructed two C35 "baits" contained a respective functional immunoreceptor tyrosine based activation motif (ITAM) and the conserved last four amino acids Cys-Ile-Leu-Val (CILV) motif, and therefore laid a foundation for screening the C35 interaction factors in a BC cell line.

Structural study of dehydration mechanisms of NH4Th(NO3)5·9H2O

NASA Astrophysics Data System (ADS)

Knyazev, A. V.; Komshina, M. E.; Baranov, E. V.; Savushkin, I. A.; Nipruk, O. V.; Lukoyanov, A. Yu.

2017-12-01

The new pentanitrate thorium compounds NH4Th(NO3)5·nH2O were synthesized and their crystal structures were determined by X-ray diffraction analysis: space group P21/n, a = 10.5476(5), b = 14.0444(7), c = 15.5287(8) Å, β = 109.4999(7)°, Z = 4; R = 0.0246 (NH4Th(NO3)5·9H2O); space group P212121, a = 8.7039(4), b = 11.9985(6), c = 16.3531(8) Å, Z = 4; R = 0.0259 (NH4Th(NO3)5·5H2O). Features of structural changes in the dehydration were revealed. Conditions of thermal decomposition of the thorium compound were established using differential scanning calorimetry. The compound was investigated by IR spectroscopy and its bands are assigned.

Potentiating effect of graphene nanomaterials on aromatic environmental pollutant-induced cytochrome P450 1A expression in the topminnow fish hepatoma cell line PLHC-1.

PubMed

Lammel, Tobias; Boisseaux, Paul; Navas, José M

2015-09-01

Graphene and its derivatives are an emerging class of carbon nanomaterial with great potential for a broad range of industrial and consumer applications. However, their increasing production and use is expected to result in release of nano-sized graphene platelets into the environment, where they may interact with chemical pollutants modifying their fate and toxic potential. The objective of this study was to assess whether graphene nanoplatelets can act as vector for aromatic environmental pollutants increasing their cellular uptake and associated hazardous effects in vitro. For this purpose, cell cultures of the topminnow fish (Poeciliopsis lucida) hepatoma cell line PLHC-1 were simultaneously (and successively) exposed to graphene nanoplatelets (graphene oxide (GO) or carboxyl graphene (CXYG)) and an aryl hydrocarbon receptor (AhR) agonist (β-naphthoflavone (β-NF), benzo(k)fluoranthene (BkF) or 3,3',4,4',5,5'-hexachlorobiphenyl (PCB169)). Following exposure cytochrome P450 1A (Cyp1A) induction was assessed by measuring cyp1A mRNA expression levels using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Cyp1A-dependent ethoxyresorufin-O-deethylase (EROD) activity. It was observed that pre- and co-exposure of cells to GO and CXYG nanoplatelets had a potentiating effect on β-NF, BkF, and PCB169-dependent Cyp1A induction suggesting that graphene nanoplatelets increase the effective concentration of AhR agonists by facilitating their passive diffusion into the cells by damaging the cells' plasma membrane and/or by transporting them over the plasma membrane via a Trojan horse-like mechanism. The results demonstrate the existence of combination effects between nanomaterials and environmental pollutants and stress the importance of considering these effects when evaluating their respective hazard. © 2014 Wiley Periodicals, Inc.

Fluorescent pH-Sensing Probe Based on Biorefinery Wood Lignosulfonate and Its Application in Human Cancer Cell Bioimaging.

PubMed

Xue, Yuyuan; Liang, Wanshan; Li, Yuan; Wu, Ying; Peng, Xinwen; Qiu, Xueqing; Liu, Jinbin; Sun, Runcang

2016-12-28

A water-soluble, ratiometric fluorescent pH probe, L-SRhB, was synthesized via grafting spirolactam Rhodamine B (SRhB) to lignosulfonate (LS). As the ring-opening product of L-SRhB, FL-SRhB was also prepared. The pH-response experiment indicated that L-SRhB showed a rapid response to pH changes from 4.60 to 6.20 with a pK a of 5.35, which indicated that L-SRhB has the potential for pH detection of acidic organelle. In addition, the two probes were internalized successfully by living cells through the endocytosis pathway and could distinguish normal cells from cancer cells by different cell staining rates. In addition, L-SRhB showed obvious cytotoxicity to cancer cells, whereas it was nontoxic to normal cells in the same condition. L-SRhB might have potential in cancer therapy. L-SRhB might be a promising ratiometric fluorescent pH sensor and bioimaging dye for the recognition of cancer cells. The results also provided a new perspective to the high-value utilization of lignin.

Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas.

PubMed

Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

2016-02-28

Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas

NASA Astrophysics Data System (ADS)

Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

2016-02-01

Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

Single-cell intracellular nano-pH probes†

PubMed Central

Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

2016-01-01

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution. PMID:27708772

Single-cell intracellular nano-pH probes.

PubMed

Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

2015-01-01

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

Decomposition of 3,5-dinitrobenzamide in aqueous solution during UV/H2O2 and UV/TiO2 oxidation processes.

PubMed

Yan, Yingjie; Liao, Qi-Nan; Ji, Feng; Wang, Wei; Yuan, Shoujun; Hu, Zhen-Hu

2017-02-01

3,5-Dinitrobenzamide has been widely used as a feed additive to control coccidiosis in poultry, and part of the added 3,5-dinitrobenzamide is excreted into wastewater and surface water. The removal of 3,5-dinitrobenzamide from wastewater and surface water has not been reported in previous studies. Highly reactive hydroxyl radicals from UV/hydrogen peroxide (H 2 O 2 ) and UV/titanium dioxide (TiO 2 ) advanced oxidation processes (AOPs) can decompose organic contaminants efficiently. In this study, the decomposition of 3,5-dinitrobenzamide in aqueous solution during UV/H 2 O 2 and UV/TiO 2 oxidation processes was investigated. The decomposition of 3,5-dinitrobenzamide fits well with a fluence-based pseudo-first-order kinetics model. The decomposition in both two oxidation processes was affected by solution pH, and was inhibited under alkaline conditions. Inorganic anions such as NO 3 - , Cl - , SO 4 2- , HCO 3 - , and CO 3 2- inhibited the degradation of 3,5-dinitrobenzamide during the UV/H 2 O 2 and UV/TiO 2 oxidation processes. After complete decomposition in both oxidation processes, approximately 50% of 3,5-dinitrobenzamide was decomposed into organic intermediates, and the rest was mineralized to CO 2 , H 2 O, and other inorganic anions. Ions such as NH 4 + , NO 3 - , and NO 2 - were released into aqueous solution during the degradation. The primary decomposition products of 3,5-dinitrobenzamide were identified using time-of-flight mass spectrometry (LCMS-IT-TOF). Based on these products and ions release, a possible decomposition pathway of 3,5-dinitrobenzamide in both UV/H 2 O 2 and UV/TiO 2 processes was proposed.

Mitochondrial free fatty acid β-oxidation supports oxidative phosphorylation and proliferation in cancer cells.

PubMed

Rodríguez-Enríquez, Sara; Hernández-Esquivel, Luz; Marín-Hernández, Alvaro; El Hafidi, Mohammed; Gallardo-Pérez, Juan Carlos; Hernández-Reséndiz, Ileana; Rodríguez-Zavala, José S; Pacheco-Velázquez, Silvia C; Moreno-Sánchez, Rafael

2015-08-01

Oxidative phosphorylation (OxPhos) is functional and sustains tumor proliferation in several cancer cell types. To establish whether mitochondrial β-oxidation of free fatty acids (FFAs) contributes to cancer OxPhos functioning, its protein contents and enzyme activities, as well as respiratory rates and electrical membrane potential (ΔΨm) driven by FFA oxidation were assessed in rat AS-30D hepatoma and liver (RLM) mitochondria. Higher protein contents (1.4-3 times) of β-oxidation (CPT1, SCAD) as well as proteins and enzyme activities (1.7-13-times) of Krebs cycle (KC: ICD, 2OGDH, PDH, ME, GA), and respiratory chain (RC: COX) were determined in hepatoma mitochondria vs. RLM. Although increased cholesterol content (9-times vs. RLM) was determined in the hepatoma mitochondrial membranes, FFAs and other NAD-linked substrates were oxidized faster (1.6-6.6 times) by hepatoma mitochondria than RLM, maintaining similar ΔΨm values. The contents of β-oxidation, KC and RC enzymes were also assessed in cells. The mitochondrial enzyme levels in human cervix cancer HeLa and AS-30D cells were higher than those observed in rat hepatocytes whereas in human breast cancer biopsies, CPT1 and SCAD contents were lower than in human breast normal tissue. The presence of CPT1 and SCAD in AS-30D mitochondria and HeLa cells correlated with an active FFA utilization in HeLa cells. Furthermore, the β-oxidation inhibitor perhexiline blocked FFA utilization, OxPhos and proliferation in HeLa and other cancer cells. In conclusion, functional mitochondria supported by FFA β-oxidation are essential for the accelerated cancer cell proliferation and hence anti-β-oxidation therapeutics appears as an alternative promising approach to deter malignant tumor growth. Copyright © 2015 Elsevier Ltd. All rights reserved.

Decreased interleukin 35 and CD4+EBI3+ T cells in patients with active systemic lupus erythematosus.

PubMed

Ouyang, Han; Shi, Yong-Bing; Liu, Zhi-Chun; Wang, Zhi; Feng, Sheng; Kong, Shu-Min; Lu, Ying

2014-08-01

Interleukin 35 (IL-35) is likely to contribute to the development of autoimmune diseases, as the Epstein-Barr virus-induced gene protein 3 (EBI3) is the specificity subunit of IL-35. Nevertheless, until recently, no studies have evaluated its role in systemic lupus erythematosus (SLE) in humans. The objective of this study was to investigate the serum IL-35 level and the percentage of CD4EBI3 T cells in the peripheral blood of patients with SLE and explore the roles of double-positive T cells and IL-35 in the pathogenesis of SLE and the effects of glucocorticoid on these roles. Fifty-five hospitalized patients with SLE were recruited, and 20 volunteers were enrolled as healthy controls. Serum IL-35 levels were measured by enzyme-linked immunosorbent assay, and the percentage of CD4EBI3 T cells was analyzed by flow cytometry. The serum IL-35 level and the percentage of CD4EBI3 T cells were significantly decreased in patients with active SLE compared with healthy controls and patients with inactive SLE. The serum IL-35 level and the percentage of CD4EBI3 T cells were negatively correlated with the SLE disease activity index. The percentages of CD4EBI3 T cells and serum IL-35 levels in 10 untreated patients with active SLE were increased at days l, 3, and 7 after the treatment with methylprednisolone (0.8 mg·kg·d) compared with the percentages before the treatment. These results demonstrate that abnormalities in IL-35 and CD4EBI3 T cells may play important roles in the pathogenesis of SLE; the percentage of double-positive T cells and the level of IL-35 are parameters for the evaluation of SLE activity and severity.

Crotalaria-induced pulmonary hypertension. Uptake of 3H-thymidine by the cells of the pulmonary circulation and alveolar walls.

PubMed Central

Meyrick, B. O.; Reid, L. M.

1982-01-01

Feeding with Crotalaria spectabilis seeds induces structural changes in the pulmonary arterial circulation characteristic of pulmonary hypertension: increased medial and adventitial thickness, the appearance of muscle in smaller arteries than normal, and reduction in the number of peripheral arteries. By autoradiographic techniques, after injection of 3H-thymidine into rats fed Crotalaria for 3, 7, 14, 21, 28, or 35 days, the contribution of hyperplasia to these changes has been assessed at two levels of the pulmonary artery--the hilum and the periphery. In the hilar pulmonary artery, a biphasic increase in labeling index (LI) is seen in each cell type. After 3 days of feeding, the medial smooth muscle cells show a slight but significant increase (1.5 times the control value), and, after 7 days, so do the adventitial fibroblasts (3 x) and the endothelial cells (EC) (2 x). After 14 days LI for all three cell types is again at control values, but after 21 days (wall thickness is no increased) each cell type shows at least a fivefold increase; by 35 days all are again near control levels. In the intra-acinar region, by 14 days, "newly" muscularized arteries are identified and increase in number and proportion up to 35 days; 3H-thymidine uptake is not evident in this cell type until 35 days have passed. The ECs of these arteries, however, show a striking increase in LI after 14 days as do those of the alveolar capillaries. The ECs of the intra-acinar veins show a biphasic response being increased after 7, 28, and 35 days. The present study has shown that Crotalaria ingestion induces hyperplasia and hypertrophy of pulmonary arterial cells at pre- and intra-acinar levels. The early increase in LI probably represents a response to the original cell injury, the later changes, a response to continuing damage or, in part, adaptation to the pulmonary hypertension now present. Images Figure 3 Figure 7 PMID:7055214

Intracellular pH gradients in migrating cells.

PubMed

Martin, Christine; Pedersen, Stine F; Schwab, Albrecht; Stock, Christian

2011-03-01

Cell polarization along the axis of movement is required for migration. The localization of proteins and regulators of the migratory machinery to either the cell front or its rear results in a spatial asymmetry enabling cells to simultaneously coordinate cell protrusion and retraction. Protons might function as such unevenly distributed regulators as they modulate the interaction of focal adhesion proteins and components of the cytoskeleton in vitro. However, an intracellular pH (pH(i)) gradient reflecting a spatial asymmetry of protons has not been shown so far. One major regulator of pH(i), the Na(+)/H(+) exchanger NHE1, is essential for cell migration and accumulates at the cell front. Here, we test the hypothesis that the uneven distribution of NHE1 activity creates a pH(i) gradient in migrating cells. Using the pH-sensitive fluorescent dye BCECF, pH(i) was measured in five cell lines (MV3, B16V, NIH3T3, MDCK-F1, EA.hy926) along the axis of movement. Differences in pH(i) between the front and the rear end (ΔpH(i) front-rear) were present in all cell lines, and inhibition of NHE1 either with HOE642 or by absence of extracellular Na(+) caused the pH(i) gradient to flatten or disappear. In conclusion, pH(i) gradients established by NHE1 activity exist along the axis of movement.

Antitumoral Activity of (20R)- and (20S)-Ginsenoside Rh2 on Transplanted Hepatocellular Carcinoma in Mice.

PubMed

Lv, Qun; Rong, Na; Liu, Li-Jia; Xu, Xiao-Lin; Liu, Jian-Ting; Jin, Feng-Xie; Wang, Chun-Mei

2016-05-01

Hepatocellular carcinoma is one of the leading causes of malignancy-related death in China. Its therapy in clinics is a big challenge. Ginsenoside Rh2 is one of the most notable cancer-preventing components from red ginseng and it has been reported that ginsenoside Rh2 exhibited potent cytotoxicity against human hepatoma cells. Rh2 exists as two different stereoisomeric forms, (20S)-ginsenoside Rh2 and (20R)-ginsenoside Rh2. Previous reports showed that the Rh2 epimers demonstrated different pharmacological activities and only (20S)-ginsenoside Rh2 showed potent proliferation inhibition on cancer cells in vitro. However, the in vivo anti-hepatoma activity of (20R)-ginsenoside Rh2 and (20S)-ginsenoside Rh2 has not been reported yet. This work assessed and compared the anti-hepatoma activities of (20S)-ginsenoside Rh2 and (20R)-ginsenoside Rh2 using H22 a hepatoma-bearing mouse model in vivo. In addition, hematoxylin and eosin staining, the deoxynucleotidyl transferase dUTP nick-end labeling assay, and the semiquantitative reverse transcriptase polymerase chain reaction method were used to further study the apoptosis of the tumors. The results showed that both (20S)-ginsenoside Rh2 and (20R)-ginsenoside Rh2 suppressed the growth of H22 transplanted tumors in vivo, and the highest inhibition rate could be up to 42.2 and 46.8 %, respectively (p < 0.05). Further, hematoxylin/eosin staining and the deoxynucleotidyl transferase dUTP nick-end labeling assay indicated that both (20R)-ginsenoside Rh2 and (20S)-ginsenoside Rh2 could induce H22 hepatoma tumor cell apoptosis, with apoptosis indexes of 3.87 %, and 3.80 %, respectively (p < 0.05). Moreover, this effect was accompanied by downregulating the level of Bcl-2 mRNA. In conclusion, both (20S)-ginsenoside Rh2 and (20R)-ginsenoside Rh2 can suppress the growth of H22 hepatomas without causing severe side effects, and this effect is associated with the induction of apoptosis. Georg Thieme Verlag KG Stuttgart �

Interleukin-35 Inhibits Endothelial Cell Activation by Suppressing MAPK-AP-1 Pathway.

PubMed

Sha, Xiaojin; Meng, Shu; Li, Xinyuan; Xi, Hang; Maddaloni, Massimo; Pascual, David W; Shan, Huimin; Jiang, Xiaohua; Wang, Hong; Yang, Xiao-feng

2015-07-31

Vascular response is an essential pathological mechanism underlying various inflammatory diseases. This study determines whether IL-35, a novel responsive anti-inflammatory cytokine, inhibits vascular response in acute inflammation. Using a mouse model of LPS-induced acute inflammation and plasma samples from sepsis patients, we found that IL-35 was induced in the plasma of mice after LPS injection as well as in the plasma of sepsis patients. In addition, IL-35 decreased LPS-induced proinflammatory cytokines and chemokines in the plasma of mice. Furthermore, IL-35 inhibited leukocyte adhesion to the endothelium in the vessels of lung and cremaster muscle and decreased the numbers of inflammatory cells in bronchoalveolar lavage fluid. Mechanistically, IL-35 inhibited the LPS-induced up-regulation of endothelial cell (EC) adhesion molecule VCAM-1 through IL-35 receptors gp130 and IL-12Rβ2 via inhibition of the MAPK-activator protein-1 (AP-1) signaling pathway. We also found that IL-27, which shares the EBI3 subunit with IL-35, promoted LPS-induced VCAM-1 in human aortic ECs and that EBI3-deficient mice had similar vascular response to LPS when compared with that of WT mice. These results demonstrated for the first time that inflammation-induced IL-35 inhibits LPS-induced EC activation by suppressing MAPK-AP1-mediated VCAM-1 expression and attenuates LPS-induced secretion of proinflammatory cytokines/chemokines. Our results provide insight into the control of vascular inflammation by IL-35 and suggest that IL-35 is an attractive novel therapeutic reagent for sepsis and cardiovascular diseases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Antitumor effects of a tumor cell vaccine expressing a membrane-bound form of the IL-12 p35 subunit.

PubMed

Lim, Ho Yong; Ju, Hee Young; Chung, Hee-Yong; Kim, Young Sang

2010-08-15

We investigated whether expression of the IL-12 p35 subunit in membrane-bound form in tumor cells enhanced their immunogenicity. Since p35 is only secreted when associated with the IL-12 p40 subunit, we generated tumor cells expressing membrane-bound forms of p35 and p40 as chimeras with the transmembrane/cytoplasmic region of TNFα (mbIL-12p35 and mbIL-12p40). The relevant vectors were transfected into MethA fibrosarcoma cells, and mbIL-12p35 or mbIL-12p40-expressing tumor clones were isolated and their ability to induce antitumor immunity studied. Cells of the mbIL-12p35 tumor clone induced CD69 expression and IFNγ production in purified CD8(+) T cells in vitro, and their in vivo tumorigenicity was reduced. Cells of the mbIL-12p40 tumor clone failed to show either of these activities. Mice that had rejected cells of the mbIL-12p35 tumor clone possessed systemic antitumor immunity to wild type tumor cells. The growth rate of mbIL-12p35 tumor cells was greater in CD8(+) T cell-depleted mice than in CD4(+) T-cell- and NK cell-depleted mice or normal mice, suggesting that CD8(+) T cells were mainly responsible for the antitumor immunity. These results indicate that expression of mbIL-12p35 on tumor cells enhances their immunogenicity by increasing their ability to activate CD8(+) T cells, possibly by direct priming.

Effect of ethanol on hydrogen peroxide-induced AMPK phosphorylation.

PubMed

Liangpunsakul, Suthat; Wou, Sung-Eun; Zeng, Yan; Ross, Ruth A; Jayaram, Hiremagalur N; Crabb, David W

2008-12-01

AMP-activated protein kinase (AMPK) responds to oxidative stress. Previous work has shown that ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMPK and reduced the amount of AMPK protein. Ethanol generates oxidative stress in the liver. Since AMPK is activated by reactive oxygen species, it seems paradoxical that ethanol would inhibit AMPK in the hepatoma cells. In an attempt to understand the mechanism whereby ethanol inhibits AMPK, we studied the effect of ethanol on AMPK activation by exogenous hydrogen peroxide. The effects of ethanol, hydrogen peroxide, and inhibitors of protein phosphatase 2A (PP2A) [either okadaic acid or PP2A small interference RNA (siRNA)] on AMPK phosphorylation and activity were examined in rat hepatoma cells (H4IIEC3) and HeLa cells. In H4IIEC3 cells, hydrogen peroxide (H(2)O(2), 1 mM) transiently increased the level of phospho-AMPK to 1.5-fold over control (P < 0.05). Similar findings were observed in HeLa cells, which do not express the upstream AMPK kinase, LKB1. H(2)O(2) markedly increased the phosphorylation of LKB1 in H4IIEC3 cells. Ethanol significantly inhibited the phosphorylation of PKC-zeta, LKB1, and AMPK caused by exposure to H(2)O(2). This inhibitory effect of ethanol required its metabolism. More importantly, the inhibitory effects of ethanol on H(2)O(2)-induced AMPK phosphorylation were attenuated by the presence of the PP2A inhibitor, okadaic acid, or PP2A siRNA. The inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of PKC-zeta and LKB1 phosphorylation and the activation of PP2A.

Exogenous regucalcin suppresses the growth of human liver cancer HepG2 cells in vitro.

PubMed

Yamaguchi, Masayoshi; Murata, Tomiyasu

2018-04-05

Regucalcin, which its gene is localized on the X chromosome, plays a pivotal role as a suppressor protein in signal transduction in various types of cells and tissues. Regucalcin gene expression has been demonstrated to be suppressed in various tumor tissues of animal and human subjects, suggesting a potential role of regucalcin in carcinogenesis. Regucalcin, which is produced from the tissues including liver, is found to be present in the serum of human subjects and animals. This study was undertaken to determine the effects of exogenous regucalcin on the proliferation in cloned human hepatoma HepG2 cells in vitro. Proliferation of HepG2 cells was suppressed after culture with addition of regucalcin (0.01 – 10 nM) into culture medium. Exogenous regucalcin did not reveal apoptotic cell death in HepG2 cells in vitro. Suppressive effects of regucalcin on cell proliferation were not enhanced in the presence of various signaling inhibitors including tumor necrosis factor-α (TNF-α), Bay K 8644, PD98059, staurosporine, worthomannin, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) or gemcitabine, which were found to suppress the proliferation. In addition, exogenous regucalcin suppressed the formation of colonies of cultured hepatoma cells in vitro. These findings demonstrated that exogenous regucalcin exhibits a suppressive effect on the growth of human hepatoma HepG2 cells, proposing a strategy with the gene therapy for cancer treatment.

Genotoxic and antigenotoxic effects of catechin and tannins from the bark of Hamamelis virginiana L. in metabolically competent, human hepatoma cells (Hep G2) using single cell gel electrophoresis.

PubMed

Dauer, Andreas; Hensel, Andreas; Lhoste, Evelyne; Knasmüller, Siegfried; Mersch-Sundermann, Volker

2003-05-01

The genotoxic and antigenotoxic activities of catechin, hamamelitannin and two proanthocyanidin fractions prepared from the bark of Hamamelis virginiana L. were investigated in a human derived, metabolically competent hepatoma cell line (Hep G2) using single cell gel electrophoresis (SCGE) for the detection of DNA-damage. DNA-migration was calculated as Olive tail moment (OTM). Catechin and a low-molecular weight proanthocyandin fraction (W(M)) caused only slight increases of OTM up to concentrations of 166 microg/ml whereas hamamelitannin and the proanthocyandin fraction with higher molecular weight (W(A)) led to a two-fold enhancement of OTM at the same concentrations. These effects were dose-independent. Treatment of the cells with the test compounds in a dose-range of 2-166 microg/ml prior to the exposure to benzo(a)pyrene (B(a)P, 10 microM, 2.5 microg/ml) led to a significant reduction of induced DNA damage which was dose-dependent for all test compounds, except for hamamelitannin. The inhibitory effects of proanthocyanidins were stronger than those of catechin and hamamelitannin; the lowest effective concentrations were about 2 microg/ml. In order to clarify the mechanisms of protection, possible effects of the test compounds on enzymes involved in toxification and detoxification of B(a)P were investigated. While B(a)P toxification by cytochrome P450 was not inhibited by the test compounds, detoxification by glutathion-S-transferase (GST) was induced by catechin and W(M). Combination experiments with the ultimate metabolite of B(a)P, (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE; 5 microM, 1.5 microg/ml), revealed strong inhibitory effects, indicating that the observed protective effects were caused by scavenging of the ultimate mutagen by the test compounds. Exposure of Hep G2 cells to the test compounds after B(a)P treatment did not influence B(a)P induced DNA damage, demonstrating that repair mechanisms were not affected.

Protective effects of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone to PC12 cells against cytotoxicity induced by hydrogen peroxide.

PubMed

Su, Ming-Yuan; Huang, Hai-Ya; Li, Lin; Lu, Yan-Hua

2011-01-26

Oxidative stress has been considered as a major cause of cellular injuries in various clinical abnormalities. One of the possible ways to prevent reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical therapies to augment the endogenous antioxidant defense capacity. The present study found that 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), a chalcone isolated from the buds of Cleistocalyx operculatus, possessed cytoprotective activity in PC12 cells treated with H(2)O(2). The results showed that DMC could effectively increase cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) reduction], decrease the cell apoptotic percentage [annexin V/propidium iodide (AV/PI) assay], prevent the membrane from damage [lactate dehydrogenase (LDH) release], scavenge ROS formation, reduce caspase-3 activity, and attenuate the decrease of mitochondrial membrane potential (MMP) in PC12 cells treated with H(2)O(2). Meanwhile, DMC increased the catalytic activity of superoxide dismutase (SOD) and the cellular amount of glutathione (GSH), decreased the cellular amount of malondialdehyde (MDA), and decreased the production of lipid peroxidation in PC12 cells treated with H(2)O(2).

Modulation of HepG2 cell net apolipoprotein B secretion by the citrus polymethoxyflavone, tangeretin.

PubMed

Kurowska, Elzbieta M; Manthey, John A; Casaschi, Adele; Theriault, Andre G

2004-02-01

The purpose of the present study was to examine the role of tangeretin, a polymethoxylated flavone from citrus fruits, on the regulation of apolipoprotein B (apoB) and lipid metabolism in the human hepatoma cell-line HepG2. The marked reduction in apoB secretion observed in cells incubated with 72.8 microM tangeretin was rapid, apoB-specific, and partly reversible. The reduction also was observed under lipid-rich conditions and found to be insensitive to proteasomal degradation of nascent apoB. We followed our study by examining lipid synthesis and mass. A 24-h exposure of cells to 72.8 microM tangeretin decreased intracellular synthesis of cholesteryl esters, free cholesterol, and TAG by 82, 45, and 64%, respectively; tangeretin also reduced the mass of cellular TAG by 37%. The tangeretin-induced suppression of TAG synthesis and mass were associated with decreased activities of DAG acyltransferase (up to -39.0 +/- 3.0% vs. control) and microsomal triglyceride transfer protein (up to -35.5 +/- 2.5% vs. control). Tangeretin was also found to activate the peroxisome proliferator-activated receptor, a transcription factor with a positive regulatory impact on FA oxidation and TAG availability (up to 36% increase vs. control). The data suggest that tangeretin modulates apoB-containing lipoprotein metabolism through multiple mechanisms.

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

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.

Polychromatic Light (480-3400 nm) Upregulates Sensitivity of Tumor Cells to Lysis by Natural Killers.

PubMed

Knyazev, Nickolay A; Samoilova, Kira A; Abrahamse, Heidi; Filatova, Natalia A

2016-09-01

This study evaluates the participation of immunological mechanisms of downregulation of murine hepatoma cells MH22a after direct exposure to polychromatic polarized light. Previous studies have shown that exposure to a combination of visible (VIS) and infrared (IR) light leads to decreased tumorigenicity of the murine hepatoma cells MH22a, which correlated with an increase in the amount of cells with reorganized cytoskeleton in the submembrane region. The mechanism of tumor inhibition and elimination has not been determined. Polychromatic light (480-3400 nm) has been used at doses of 4.8 and 9.6 J/cm(2) to determine the sensitivity of murine MH22a cells and human erythroleukemia cells K562 exposed to this light, to lysis by effector cells of innate immunity (NK cells), and enhancement of the glycocalyx of the studied tumor cells. This was determined using flow cytometry, the H(3)-uridine cytotoxic test followed by spectrophotometry. VIS-IR light increases the sensitivity of MH-22a cells at a dose 4.8 J/cm(2) and K562 cells at 9.6 J/cm(2). The enhancement of sensitivity of tumor cells to NK lysis changed their ability to absorb alcian blue, reflecting a change in the expression of the glycocalyx. Increasing the sensitivity of the murine tumor cells MH22a and human K562 irradiated VIS-IR light correlated with a change in the expression of their glycocalyx. The results of the present study demonstrate that the reduction of tumorigenicity of irradiated tumor cells is due to their sensitivity to lysis by NK cells of the immune system.

Bioelectricity production from food waste leachate using microbial fuel cells: effect of NaCl and pH.

PubMed

Li, Xiao Min; Cheng, Ka Yu; Wong, Jonathan W C

2013-12-01

Microbial fuel cells are a promising technology for simultaneous treatment and energy recovery from food waste leachate. This study evaluates the effects of NaCl (0-150 mM) and pH on the treatment of food waste leachate using microbial fuel cells. The food waste leachate amended with 100mM NaCl enabled the highest maximum power density (1000 mW/m(3)) and lowest internal resistance (371Ω). Increasing the anodic pH gradually from acidic to alkaline conditions (pH 4-9) resulted in a gradual increase in maximum power density to 9956 mW/m(3) and decrease in internal cell resistance to 35.3Ω. The coulombic efficiency obtained under acidic conditions was only 17.8%, but increased significantly to 60.0% and 63.4% in the neutral and alkaline pH's MFCs, respectively. Maintaining a narrow pH window (6.3-7.6) was essential for efficient bioelectricity production and COD removal using microbial fuel cells for the treatment of food waste leachate. Copyright © 2013 Elsevier Ltd. All rights reserved.

Attenuation of Aβ{sub 25–35}-induced parallel autophagic and apoptotic cell death by gypenoside XVII through the estrogen receptor-dependent activation of Nrf2/ARE pathways

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

Meng, Xiangbao; Wang, Min; Sun, Guibo, E-mail: sunguibo@126.com

Amyloid-beta (Aβ) has a pivotal function in the pathogenesis of Alzheimer's disease. To investigate Aβ neurotoxicity, we used an in vitro model that involves Aβ{sub 25–35}-induced cell death in the nerve growth factor-induced differentiation of PC12 cells. Aβ{sub 25–35} (20 μM) treatment for 24 h caused apoptotic cell death, as evidenced by significant cell viability reduction, LDH release, phosphatidylserine externalization, mitochondrial membrane potential disruption, cytochrome c release, caspase-3 activation, PARP cleavage, and DNA fragmentation in PC12 cells. Aβ{sub 25–35} treatment led to autophagic cell death, as evidenced by augmented GFP-LC3 puncta, conversion of LC3-I to LC3-II, and increased LC3-II/LC3-I ratio.more » Aβ{sub 25–35} treatment induced oxidative stress, as evidenced by intracellular ROS accumulation and increased production of mitochondrial superoxide, malondialdehyde, protein carbonyl, and 8-OHdG. Phytoestrogens have been proved to be protective against Aβ-induced neurotoxicity and regarded as relatively safe targets for AD drug development. Gypenoside XVII (GP-17) is a novel phytoestrogen isolated from Gynostemma pentaphyllum or Panax notoginseng. Pretreatment with GP-17 (10 μM) for 12 h increased estrogen response element reporter activity, activated PI3K/Akt pathways, inhibited GSK-3β, induced Nrf2 nuclear translocation, augmented antioxidant responsive element enhancer activity, upregulated heme oxygenase 1 (HO-1) expression and activity, and provided protective effects against Aβ{sub 25–35}-induced neurotoxicity, including oxidative stress, apoptosis, and autophagic cell death. In conclusion, GP-17 conferred protection against Aβ{sub 25–35}-induced neurotoxicity through estrogen receptor-dependent activation of PI3K/Akt pathways, inactivation of GSK-3β and activation of Nrf2/ARE/HO-1 pathways. This finding might provide novel insights into understanding the mechanism for neuroprotective effects of

The ML1Nx2 Phosphatidylinositol 3,5-Bisphosphate Probe Shows Poor Selectivity in Cells.

PubMed

Hammond, Gerald R V; Takasuga, Shunsuke; Sasaki, Takehiko; Balla, Tamas

2015-01-01

Phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P2) is a quantitatively minor phospholipid in eukaryotic cells that plays a fundamental role in regulating endocytic membrane traffic. Despite its clear importance for cellular function and organism physiology, mechanistic details of its biology have so far not been fully elucidated. In part, this is due to a lack of experimental tools that specifically probe for PtdIns(3,5)P2 in cells to unambiguously identify its dynamics and site(s) of action. In this study, we have evaluated a recently reported PtdIns(3,5)P2 biosensor, GFP-ML1Nx2, for its veracity as such a probe. We report that, in live cells, the localization of this biosensor to sub-cellular compartments is largely independent of PtdIns(3,5)P2, as assessed after pharmacological, chemical genetic or genomic interventions that block the lipid's synthesis. We therefore conclude that it is unwise to interpret the localization of ML1Nx2 as a true and unbiased biosensor for PtdIns(3,5)P2.

The Simultaneous Determination of Muscle Cell pH Using a Weak Acid and Weak Base

PubMed Central

Adler, Sheldon

1972-01-01

Should significant pH heterogeneity exist within cells then the simultaneous calculation of intracellular pH from the distribution of a weak acid will give a value closest to the highest pH in the system, whereas calculation from the distribution of a weak base will give a value closer to the lowest pH. These two values should then differ significantly. Intact rat diaphragms were exposed in vitro to varying bicarbonate concentrations (pure metabolic) and CO2 tensions (pure respiratory), and steady-state cell pH was measured simultaneously either by distribution of the weak acid 5,5-dimethyloxazolidine-2,4-dione-14C (pH DMO) or by distribution of the weak base nicotine-14C (pH nicotine). The latter compound was found suitable to measure cell pH since it was neither metabolized nor bound by rat diaphragms. At an external pH of 7.40, pH DMO was 7.17 while pH nicotine was 6.69—a pH difference of 0.48 pH units (P < 0.001). In either respiratory or metabolic alkalosis both DMO and pH nicotine rose so that differences between them remained essentially constant. Metabolic acidosis induced a decrease in both values though they fell more slowly than did extracellular pH. In contradistinction, in respiratory acidosis, decreasing extracellular pH from 7.40 to 6.80 resulted in 0.35 pH unit drop in pH DMO while pH nicotine remained constant. In every experiment, under all external conditions, pH DMO exceeded pH nicotine. These results indicate that there is significant pH heterogeneity within diaphragm muscle, but the degree of heterogeneity may vary under different external conditions. The metabolic implications of these findings are discussed. In addition, the data show that true overall cell pH is between 6.69 and 7.17—a full pH higher than would be expected from thermodynamic considerations alone. This implies the presence of active processes to maintain cell pH. PMID:5009113

Tumour-derived Interleukin 35 promotes pancreatic ductal adenocarcinoma cell extravasation and metastasis by inducing ICAM1 expression

PubMed Central

Huang, Chongbiao; Li, Na; Li, Zengxun; Chang, Antao; Chen, Yanan; Zhao, Tiansuo; Li, Yang; Wang, Xiuchao; Zhang, Wei; Wang, Zhimin; Luo, Lin; Shi, Jingjing; Yang, Shengyu; Ren, He; Hao, Jihui

2017-01-01

Interleukin 35 (IL-35) is a novel member of the IL-12 family, consisting of an EBV-induced gene 3 (EBI3) subunit and a P35 subunit. IL-35 is an immune-suppressive cytokine mainly produced by regulatory T cells. However, the role of IL-35 in cancer metastasis and progression is not well understood. Here we demonstrate that IL-35 is overexpressed in human pancreatic ductal adenocarcinoma (PDAC) tissues, and that IL-35 overexpression is associated with poor prognosis in PDAC patients. IL-35 has critical roles in PDAC cell extravasation and metastasis by facilitating the adhesion to endothelial cells and transendothelial extravasation. Mechanistically, IL-35 promotes ICAM1 overexpression through a GP130-STAT1 signalling pathway, which facilitates endothelial adhesion and transendothelial migration via an ICAM1–fibrinogen–ICAM1 bridge. In an orthotopic xenograft model, IL-35 promotes spontaneous pancreatic cancer metastasis in an ICAM1-dependent manner. Together, our results indicate additional functions of IL-35 in promoting PDAC metastasis through mediating ICAM1 expression. PMID:28102193

Reduced H3K27me3 expression in Merkel cell polyoma virus-positive tumors.

PubMed

Busam, Klaus J; Pulitzer, Melissa P; Coit, Daniel C; Arcila, Maria; Leng, Danielle; Jungbluth, Achim A; Wiesner, Thomas

2017-06-01

Merkel cell carcinoma is a primary cutaneous neuroendocrine carcinoma, which once metastatic is difficult to treat. Recent mutation analyses of Merkel cell carcinoma revealed a low number of mutations in Merkel cell polyomavirus-associated tumors, and a high number of mutations in virus-negative combined squamous cell and neuroendocrine carcinomas of chronically sun-damaged skin. We speculated that the paucity of mutations in virus-positive Merkel cell carcinoma may reflect a pathomechanism that depends on derangements of chromatin without alterations in the DNA sequence (epigenetic dysregulation). One central epigenetic regulator is the Polycomb repressive complex 2 (PRC2), which silences genomic regions by trimethylating (me3) lysine (K) 27 of histone H3, and thereby establishes the histone mark H3K27me3. Recent experimental research data demonstrated that PRC2 loss in mice skin results in the formation of Merkel cells. Prompted by these findings, we explored a possible contribution of PRC2 loss in human Merkel cell carcinoma. We examined the immunohistochemical expression of H3K27me3 in 35 Merkel cell carcinomas with pure histological features (22 primary and 13 metastatic lesions) and in 5 combined squamous and neuroendocrine carcinomas of the skin. We found a strong reduction of H3K27me3 staining in tumors with pure histologic features and virus-positive Merkel cell carcinomas. Combined neuroendocrine carcinomas had no or only minimal loss of H3K27me3 labeling. Our findings suggest that a PRC2-mediated epigenetic deregulation may play a role in the pathogenesis of virus-positive Merkel cell carcinomas and in tumors with pure histologic features.

Hepatoma-derived growth factor: A survival-related protein in prostate oncogenesis and a potential target for vitamin K2.

PubMed

Shetty, Aditya; Dasari, Subramanyam; Banerjee, Souresh; Gheewala, Taher; Zheng, Guoxing; Chen, Aoshuang; Kajdacsy-Balla, Andre; Bosland, Maarten C; Munirathinam, Gnanasekar

2016-11-01

Hepatoma-derived growth factor (HDGF) is a heparin-binding growth factor, which has previously been shown to be expressed in a variety of cancers. HDGF overexpression has also previously been correlated with a poor prognosis in several cancers. The significance of HDGF in prostate cancer, however, has not been investigated. Here, we show that HDGF is overexpressed in both androgen-sensitive LNCaP cells and androgen-insensitive DU145, 22RV1, and PC-3 cells. Forced overexpression enhanced cell viability of RWPE-1 cells, whereas HDGF knockdown reduced cell proliferation in human prostate cancer cells. We also show that HDGF may serve as a survival-related protein as ectopic overexpression of HDGF in RWPE cells up-regulated the expression of antiapoptosis proteins cyclin E and BCL-2, whereas simultaneously down-regulating proapoptotic protein BAX. Western blot analysis also showed that HDGF overexpression modulated the activity of phospho-AKT as well as NF-kB, and these results correlated with in vitro migration and invasion assays. We next assessed the therapeutic potential of HDGF inhibition with a HDGF monoclonal antibody and vitamin k 2 , showing reduced cell proliferation as well as inhibition of NF-kB expression in HDGF overexpressed RWPE cells treated with a HDGF monoclonal antibody and vitamin K 2 . Collectively, our results suggest that HDGF is a relevant protein in prostate oncogenesis and may serve as a potential therapeutic target in prostate cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Quantitative studies of sulphate conjugation by isolated rat liver cells using [35S]sulphate.

PubMed

Dawson, J; Knowles, R G; Pogson, C I

1991-06-21

We have developed a simple, rapid and sensitive method for the study of sulphate conjugation in isolated liver cells based on the incorporation of 35S from [35S]sulphate. Excess [35S]sulphate is removed by a barium precipitation procedure, leaving [35S]sulphate conjugates in solution. We have used this method to examine the kinetics of sulphation of N-acetyl-p-aminophenol (acetaminophen), 4-nitrophenol and 1-naphthol in isolated rat liver cells. The efficiency of recovery of the sulphate conjugates was greater than 86%. The method is applicable to the quantitative study of sulphate conjugation of any substrate which forms a sulphate conjugate that is soluble in the presence of barium, without the need for standards or radiolabelled sulphate acceptors.

Nqrs Data for C26H35Br2CuP2 (Subst. No. 1603)

NASA Astrophysics Data System (ADS)

Chihara, H.; Nakamura, N.

This document is part of Subvolume B 'Substances Containing C10H16 … Zn' of Volume 48 'Nuclear Quadrupole Resonance Spectroscopy Data' of Landolt-Börnstein - Group III 'Condensed Matter'. It contains an extract of Section '3.2 Data tables' of the Chapter '3 Nuclear quadrupole resonance data' providing the NQRS data for C26H35Br2CuP2 (Subst. No. 1603)

Microwave-assisted solid-phase synthesis of highly fluorescent carbon nanoparticles and its application in intracellular pH sensing.

PubMed

Yang, Shenghong; Chen, Xiao; Liu, Shuqin; Wang, Fuxin; Ouyang, Gangfeng

2018-08-15

Fluorescent carbon nanoparticles (FCNPs) have been deeply researched and widely applied in recent years due to their good optics performance, chemical stability and biocompatibility. Herein, a green and rapid microwave-assisted solid-phase synthesis (solvent-free) approach was proposed for the fabrication of highly FCNPs in a very short period of time, 4 min. The as-prepared FCNPs can emit a blue emission with quantum yield of up to 63.2% in water solution and show yellow fluorescence in the solid state. The FCNPs also exhibit special solvent effect that the fluorescence emission can be adjusted by controlling the solvent ratio of ethanol and water. Most importantly, the FCNPs possess a narrow-range pH response. The probe responds linearly and rapidly to minor pH fluctuations within the range of 3.47-5.10 and the correlation coefficient is above 0.99. The proposed FCNPs also exhibit high photostability and reusability. As expected, the cell imaging and intracellular pH monitoring was achieved successfully in living SMMC 7721 hepatoma cells by this probe. The FCNPs is promising as a convenient and general fluorescent pH sensor for bioimaging applications. Copyright © 2018. Published by Elsevier B.V.

Anti-invasion effects of 6-shogaol and 6-gingerol, two active components in ginger, on human hepatocarcinoma cells.

PubMed

Weng, Chia-Jui; Wu, Cheng-Feng; Huang, Hsiao-Wen; Ho, Chi-Tang; Yen, Gow-Chin

2010-11-01

Hepatocellular carcinoma is the most common type of liver cancer and is highly metastatic. Metastasis is considered to be the major cause of death in cancer patients. Ginger is a natural dietary rhizome with anti-oxidative, anti-inflammatory, and anti-carcinogenic activities. The aims of this study were to evaluate the anti-invasion activity of 6-shogaol and 6-gingerol, two compounds found in ginger, on hepatoma cells. The migratory and invasive abilities of phorbol 12-myristate 13-acetate (PMA)-treated HepG2 and PMA-untreated Hep3B cells were both reduced in a dose-dependent manner by treatment with 6-shogaol and 6-gingerol. Upon incubation of PMA-treated HepG2 cells and PMA-untreated Hep3B cells with 6-shogaol and 6-gingerol, matrix metalloproteinase (MMP)-9 activity decreased, whereas the expression of tissue inhibitor metalloproteinase protein (TIMP)-1 increased in both cell types. Additionally, urokinase-type plasminogen activator activity was dose-dependently decreased in Hep3B cells after incubation with 6-shogaol for 24 h. Analysis with semi-quantitative reverse transcription-PCR showed that the regulation of MMP-9 by 6-shogaol and 6-gingerol and the regulation of TIMP-1 by 6-shogaol in Hep3B cells may on the transcriptional level. These results suggest that 6-shogaol and 6-gingerol might both exert anti-invasive activity against hepatoma cells through regulation of MMP-9 and TIMP-1 and that 6-shogaol could further regulate urokinase-type plasminogen activity.

Vildagliptin and its metabolite M20.7 induce the expression of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells

PubMed Central

Asakura, Mitsutoshi; Karaki, Fumika; Fujii, Hideaki; Atsuda, Koichiro; Itoh, Tomoo; Fujiwara, Ryoichi

2016-01-01

Vildagliptin is a potent, orally active inhibitor of dipeptidyl peptidase-4 (DPP-4) for the treatment of type 2 diabetes mellitus. It has been reported that vildagliptin can cause hepatic dysfunction in patients. However, the molecular-mechanism of vildagliptin-induced liver dysfunction has not been elucidated. In this study, we employed an expression microarray to determine hepatic genes that were highly regulated by vildagliptin in mice. We found that pro-inflammatory S100 calcium-binding protein (S100) a8 and S100a9 were induced more than 5-fold by vildagliptin in the mouse liver. We further examined the effects of vildagliptin and its major metabolite M20.7 on the mRNA expression levels of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells. In HepG2 cells, vildagliptin, M20.7, and sitagliptin – another DPP-4 inhibitor – induced S100A9 mRNA. In HL-60 cells, in contrast, S100A8 and S100A9 mRNAs were significantly induced by vildagliptin and M20.7, but not by sitagliptin. The release of S100A8/A9 complex in the cell culturing medium was observed in the HL-60 cells treated with vildagliptin and M20.7. Therefore, the parental vildagliptin- and M20.7-induced release of S100A8/A9 complex from immune cells, such as neutrophils, might be a contributing factor of vildagliptin-associated liver dysfunction in humans. PMID:27759084

Vildagliptin and its metabolite M20.7 induce the expression of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells.

PubMed

Asakura, Mitsutoshi; Karaki, Fumika; Fujii, Hideaki; Atsuda, Koichiro; Itoh, Tomoo; Fujiwara, Ryoichi

2016-10-19

Vildagliptin is a potent, orally active inhibitor of dipeptidyl peptidase-4 (DPP-4) for the treatment of type 2 diabetes mellitus. It has been reported that vildagliptin can cause hepatic dysfunction in patients. However, the molecular-mechanism of vildagliptin-induced liver dysfunction has not been elucidated. In this study, we employed an expression microarray to determine hepatic genes that were highly regulated by vildagliptin in mice. We found that pro-inflammatory S100 calcium-binding protein (S100) a8 and S100a9 were induced more than 5-fold by vildagliptin in the mouse liver. We further examined the effects of vildagliptin and its major metabolite M20.7 on the mRNA expression levels of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells. In HepG2 cells, vildagliptin, M20.7, and sitagliptin - another DPP-4 inhibitor - induced S100A9 mRNA. In HL-60 cells, in contrast, S100A8 and S100A9 mRNAs were significantly induced by vildagliptin and M20.7, but not by sitagliptin. The release of S100A8/A9 complex in the cell culturing medium was observed in the HL-60 cells treated with vildagliptin and M20.7. Therefore, the parental vildagliptin- and M20.7-induced release of S100A8/A9 complex from immune cells, such as neutrophils, might be a contributing factor of vildagliptin-associated liver dysfunction in humans.

Differential expression and signaling of the human histamine H3 receptor isoforms of 445 and 365 amino acids expressed in human neuroblastoma SH-SY5Y cells.

PubMed

Nieto-Alamilla, Gustavo; Escamilla-Sánchez, Juan; López-Méndez, María-Cristina; Molina-Hernández, Anayansi; Guerrero-Hernández, Agustín; Arias-Montaño, José-Antonio

2018-04-01

In stably-transfected human neuroblastoma SH-SY5Y cells, we have compared the effect of activating two isoforms of 445 and 365 amino acids of the human histamine H 3 receptor (hH 3 R 445 and hH 3 R 365 ) on [ 35 S]-GTPγS binding, forskolin-induced cAMP formation, depolarization-induced increase in the intracellular concentration of Ca 2+ ions ([Ca 2+ ]i) and depolarization-evoked [ 3  H]-dopamine release. Maximal specific binding (B max ) of [ 3  H]-N-methyl-histamine to cell membranes was 953 ± 204 and 555 ± 140 fmol/mg protein for SH-SY5Y-hH 3 R 445 and SH-SY5Y-hH 3 R 365 cells, respectively, with similar dissociation constants (K d , 0.86 nM and 0.81 nM). The mRNA of the hH 3 R 365 isoform was 40.9 ± 7.9% of the hH 3 R 445 isoform. No differences in receptor affinity were found for the H 3 R ligands histamine, immepip, (R)(-)-α-methylhistamine (RAMH), A-331440, clobenpropit and ciproxifan. Both the stimulation of [ 35 S]-GTPγS binding and the inhibition of forskolin-stimulated cAMP accumulation by the agonist RAMH were significantly larger in SH-SY5Y-hH 3 R 445 cells ([ 35 S]-GTPγS binding, 158.1 ± 7.5% versus 136.5 ± 3.6% for SH-SY5Y-hH 3 R 365 cells; cAMP accumulation, -74.0 ± 4.9% versus -43.5 ± 5.3%), with no significant effect on agonist potency. In contrast, there were no differences in the efficacy and potency of RAMH to inhibit [ 3  H]-dopamine release evoked by 100 mM K + (-18.9 ± 3.0% and -20.5 ± 3.3%, for SH-SY5Y-hH 3 R 445 and SH-SY5Y-hH 3 R 365 cells), or the inhibition of depolarization-induced increase in [Ca 2+ ]i (S2/S1 ratios: parental cells 0.967 ± 0.069, SH-SY5Y-hH 3 R 445 cells 0.639 ± 0.049, SH-SY5Y-hH 3 R 365 cells 0.737 ± 0.045). These results indicate that in SH-SY5Y cells, hH 3 R 445 and hH 3 R 365 isoforms regulate in a differential manner the signaling pathways triggered by receptor activation.

Inhibition of growth of Morris hepatomas 7777 and 7800 by corn oil.

PubMed

Gilbertson, J R; Gelman, R A; Ove, P; Coetzee, M L

1977-01-01

Intraperitoneal injection of trace amounts of corn oil prior to and following the injection of 40-50 mg of tissue from hepatoma 7777 or 7800 into the thigh of adult male Buffalo rats resulted in a marked decrease in the growth rate of both tumors. Exhaustive extraction of the corn oil with water indicated that the active component was not water soluble. Similar injections of safflower oil or isotonic saline had no effect on tumor growth rate. Analysis of the tissue phospholipid fatty acids revealed that the injected corn oil caused no change in the esterified fatty acids in this lipid fraction.

Effect of Tumor Microenvironment on Selective Uptake of Boric Acid in HepG2 Human Hepatoma Cells.

PubMed

Bai, Yu-Chi; Hsia, Yu-Chun; Lin, Yu-Ting; Chen, Kuan-Hao; Chou, Fong-In; Yang, Chia-Min; Chuang, Yung-Jen

2017-11-01

Feasibility and efficacy of boric acid (BA)-mediated boron neutron capture therapy (BNCT) was first demonstrated by eliminating hepatocellular carcinoma (HCC) in a rat model. Furthermore, selective uptake of BA by liver tumor cells was shown in a rabbit model. To gain further insight, this study aimed to investigate the mechanisms of transportation and selective uptake of BA in HepG2 liver tumor cells. Transportation of BA in HepG2 cells was analyzed by time-course assays and by analyzing the rate of diffusion versus the concentration of BA. The effect of different tumor conditions on BA uptake was studied by treating HepG2 cells with 25 μg 10 B/ml BA under different concentrations of glucose, at different pH and in the presence of water-soluble cholesterol. HepG2 cells mainly uptake BA by simple diffusion. Cell membrane permeability may also contribute to tumor-specific uptake of BA. The selective uptake of BA was achieved primarily by diffusion, while other factors, such as low pH and increased membrane fluidity, which are hallmarks of HCC, might further enhance BA uptake. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Establishment of MicroRNA delivery system by PP7 bacteriophage-like particles carrying cell-penetrating peptide.

PubMed

Sun, Yanli; Sun, Yanhua; Zhao, Ronglan

2017-08-01

MicroRNAs have great therapeutic potential in cancer and other diseases. However, their instability and low in vivo delivery efficiency limits their application. Recombinant PP7 bacteriophage-based virus-like particles (VLPs) could protect microRNAs against rapid degradation by RNase by packaging specific exogenous pre-microRNAs using the pac site. Insertion of a cell-penetrating peptide (CPP) into the AB-loop of VLPs could significantly improve the delivery efficiency of microRNAs into mammalian cells. Unlike other microRNA delivery methods (viral or non-viral vectors), recombinant PP7 VLPs carrying a CPP and microRNA could be efficiently expressed in Escherichia coli using the one-plasmid double expression system. Here we showed that PP7 VLPs carrying a CPP penetrated hepatoma SK-HEP-1 cells and delivered the pre-microRNA-23b, which was processed into a mature product within 24 h; a concentration of 10 nM was sufficient for the inhibition of hepatoma cell migration via the downregulation of liver-intestine cadherin expression. Furthermore, PP7 VLPs carrying a CPP and a pre-microRNA were not infectious, replicative, or cytotoxic. Therefore, recombinant PP7 VLPs can be used for simultaneous and targeted delivery of both microRNAs and peptides because of their ability to package specific exogenous RNA using the pac site and to display peptides. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Endoplasmic reticulum factor ERLIN2 regulates cytosolic lipid content in cancer cells

PubMed Central

Wang, Guohui; Zhang, Xuebao; Lee, Jin-Sook; Wang, Xiaogang; Yang, Zeng-Quan; Zhang, Kezhong

2013-01-01

Increased de novo lipogenesis is a hallmark of aggressive cancers. Lipid droplets, the major form of cytosolic lipid storage, have been implicated in cancer cell proliferation and tumorigenesis. Recently, we identified the ERLIN2 [ER (endoplasmic reticulum) lipid raft-associated 2) gene that is amplified and overexpressed in aggressive human breast cancer. Previous studies demonstrated that ERLIN2 plays a supporting oncogenic role by facilitating the transformation of human breast cancer cells. In the present study, we found that ERLIN2 supports cancer cell growth by regulating cytosolic lipid droplet production. ERLIN2 is preferably expressed in human breast cancer cells or hepatoma cells and is inducible by insulin signalling or when cells are cultured in lipoprotein-deficient medium. Increased expression of ERLIN2 promotes the accumulation of cytosolic lipid droplets in breast cancer cells or hepatoma cells in response to insulin or overload of unsaturated fatty acids. ERLIN2 regulates activation of SREBP (sterol regulatory element-binding protein) 1c, the key regulator of de novo lipogenesis, in cancer cells. ERLIN2 was found to bind to INSIG1 (insulin-induced gene 1), a key ER membrane protein that blocks SREBP activation. Consistent with the role of ERLIN2 in regulating cytosolic lipid content, down-regulation of ERLIN2 in breast cancer or hepatoma cells led to lower cell proliferation rates. The present study revealed a novel role for ERLIN2 in supporting cancer cell growth by promoting the activation of the key lipogenic regulator SREBP1c and the production of cytosolic lipid droplets. The identification of ERLIN2 as a regulator of cytosolic lipid content in cancer cells has important implications for understanding the molecular basis of tumorigenesis and the treatment of cancer. PMID:22690709

The VP35 protein of Ebola virus impairs dendritic cell maturation induced by virus and lipopolysaccharide.

PubMed

Jin, Huali; Yan, Zhipeng; Prabhakar, Bellur S; Feng, Zongdi; Ma, Yijie; Verpooten, Dustin; Ganesh, Balaji; He, Bin

2010-02-01

Ebola virus causes rapidly progressive haemorrhagic fever, which is associated with severe immuosuppression. In infected dendritic cells (DCs), Ebola virus replicates efficiently and inhibits DC maturation without inducing cytokine expression, leading to impaired T-cell proliferation. However, the underlying mechanism remains unclear. In this study, we report that Ebola virus VP35 impairs the maturation of mouse DCs. When expressed in mouse immature DCs, Ebola virus VP35 prevents virus-stimulated expression of CD40, CD80, CD86 and major histocompatibility complex class II. Further, it suppresses the induction of cytokines such as interleukin (IL)-6, IL-12, tumour necrosis factor alpha and alpha/beta interferon (IFN-alpha/beta). Notably, Ebola VP35 attenuates the ability of DCs to stimulate the activation of CD4(+) T cells. Addition of type I IFN to mouse DCs only partially reverses the inhibitory effects of VP35. Moreover, VP35 perturbs mouse DC functions induced by lipopolysaccharide, an agonist of Toll-like receptor 4. Deletion of the amino terminus abolishes its activity, whereas a mutation in the RNA binding motif has no effect. Our work highlights a critical role of VP35 in viral interference in DC function with resultant deficiency in T-cell function, which may contribute to the profound virulence of Ebola virus infection.

Comprehensive benchmarking reveals H2BK20 acetylation as a distinctive signature of cell-state-specific enhancers and promoters.

PubMed

Kumar, Vibhor; Rayan, Nirmala Arul; Muratani, Masafumi; Lim, Stefan; Elanggovan, Bavani; Xin, Lixia; Lu, Tess; Makhija, Harshyaa; Poschmann, Jeremie; Lufkin, Thomas; Ng, Huck Hui; Prabhakar, Shyam

2016-05-01

Although over 35 different histone acetylation marks have been described, the overwhelming majority of regulatory genomics studies focus exclusively on H3K27ac and H3K9ac. In order to identify novel epigenomic traits of regulatory elements, we constructed a benchmark set of validated enhancers by performing 140 enhancer assays in human T cells. We tested 40 chromatin signatures on this unbiased enhancer set and identified H2BK20ac, a little-studied histone modification, as the most predictive mark of active enhancers. Notably, we detected a novel class of functionally distinct enhancers enriched in H2BK20ac but lacking H3K27ac, which was present in all examined cell lines and also in embryonic forebrain tissue. H2BK20ac was also unique in highlighting cell-type-specific promoters. In contrast, other acetylation marks were present in all active promoters, regardless of cell-type specificity. In stimulated microglial cells, H2BK20ac was more correlated with cell-state-specific expression changes than H3K27ac, with TGF-beta signaling decoupling the two acetylation marks at a subset of regulatory elements. In summary, our study reveals a previously unknown connection between histone acetylation and cell-type-specific gene regulation and indicates that H2BK20ac profiling can be used to uncover new dimensions of gene regulation. © 2016 Kumar et al.; Published by Cold Spring Harbor Laboratory Press.

Comprehensive benchmarking reveals H2BK20 acetylation as a distinctive signature of cell-state-specific enhancers and promoters

PubMed Central

Kumar, Vibhor; Rayan, Nirmala Arul; Muratani, Masafumi; Lim, Stefan; Elanggovan, Bavani; Xin, Lixia; Lu, Tess; Makhija, Harshyaa; Poschmann, Jeremie; Lufkin, Thomas; Ng, Huck Hui; Prabhakar, Shyam

2016-01-01

Although over 35 different histone acetylation marks have been described, the overwhelming majority of regulatory genomics studies focus exclusively on H3K27ac and H3K9ac. In order to identify novel epigenomic traits of regulatory elements, we constructed a benchmark set of validated enhancers by performing 140 enhancer assays in human T cells. We tested 40 chromatin signatures on this unbiased enhancer set and identified H2BK20ac, a little-studied histone modification, as the most predictive mark of active enhancers. Notably, we detected a novel class of functionally distinct enhancers enriched in H2BK20ac but lacking H3K27ac, which was present in all examined cell lines and also in embryonic forebrain tissue. H2BK20ac was also unique in highlighting cell-type-specific promoters. In contrast, other acetylation marks were present in all active promoters, regardless of cell-type specificity. In stimulated microglial cells, H2BK20ac was more correlated with cell-state-specific expression changes than H3K27ac, with TGF-beta signaling decoupling the two acetylation marks at a subset of regulatory elements. In summary, our study reveals a previously unknown connection between histone acetylation and cell-type-specific gene regulation and indicates that H2BK20ac profiling can be used to uncover new dimensions of gene regulation. PMID:26957309

Analysis of Chameleonic Change of Red Cabbage Depending on Broad pH Range for Dye-Sensitized Solar Cells.

PubMed

Park, Kyung Hee; Kim, Tae Young; Ko, Hyun Seok; Han, Eun Mi; Lee, Suk-Ho; Kim, Jung-Hun; Lee, Jae Wook

2015-08-01

Dye-sensitized solar cells (DSSCs) were assembled using natural dyes extracted from red cabbage as a sensitizer. In this work, we investigated the adsorption characteristics and the electrochemical behavior for harvesting sunlight and electron transfer in red cabbage DSSCs under different solvents and pH. For the red cabbage dye-sensitized electrode adsorbed at pH 3.5, the solar cell yields a short-circuit current density (Jsc) of 1.60 mA/cm2, a photovoltage (Vcc) of 0.46 V, and a fill factor of 0.55, corresponding to an energy conversion efficiency (η) of 0.41%.

CLEFMA- An Anti-Proliferative Curcuminoid from Structure Activity Relationship Studies on 3,5-bis(benzylidene)-4-piperidones

PubMed Central

Lagisetty, Pallavi; Vilekar, Prachi; Sahoo, Kaustuv; Anant, Shrikant; Awasthi, Vibhudutta

2010-01-01

3,5-bis(benzylidene)-4-piperidones are being advanced as synthetic analogs of curcumin for anticancer and anti-inflammatory properties. We performed structure-activity relationship studies, by testing several synthesized 3,5-bis(benzylidene)-4-piperidones for anti-proliferative activity in lung adenocarcinoma H441 cells. Compared to the lead compound 1, or 3,5-bis(2-fluorobenzylidene)-4-piperidone, five compounds were found to be more potent (IC50 < 30 μM), and sixteen compounds possessed reduced cell-killing efficacy (IC50 > 50 μM). Based on the observations, we synthesized 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] (29 or CLEFMA) as a novel analog of 1. CLEFMA was evaluated for anti-proliferative activity in H441 cells, and was found to be several folds more potent than compound 1. We did not find apoptotic cell population in flow cytometry, and the absence of apoptosis was confirmed by the lack of caspase cleavage. The electron microscopy of H441cells indicated that CLEFMA and compound 1 induce autophagic cell death that was inhibited by specific autophagy inhibitor 3-methyladenine. The results suggest that the potent and novel curcuminoid, CLEFMA, offers an alternative mode of cell death in apoptosis-resistant cancers. PMID:20638855

Proton induces apoptosis of hypoxic tumor cells by the p53-dependent and p38/JNK MAPK signaling pathways.

PubMed

Lee, Kheun Byeol; Kim, Kye-Ryung; Huh, Tae-Lin; Lee, You Mie

2008-12-01

Tumor hypoxia is a main obstacle for radiation therapy. To investigate whether exposure to a proton beam can overcome radioresistance in hypoxic tumor cells, three kinds of cancer cells, Lewis lung carcinoma (LLC) cells, hepatoma HepG2 and Molt-4 leukemia cells, were treated with a proton beam (35 MeV, 1, 2, 5, 10 Gy) in the presence or absence of hypoxia. Cell death rates were determined 72 h after irradiation. Hypoxic cells exposed to the proton beam underwent a typical apoptotic program, showing condensed nuclei, fragmented DNA ladders, and poly-ADP-ribose polymerase (PARP) cleavage. Fluorescence-activated cell sorter analysis revealed a significant increase in Annexin-V-positive cells. Cells treated with the proton beam and hypoxia displayed increased expression of p53, p21 and Bax, but decreased levels of phospho-Rb, Bcl-2 and XIAP, as well as activated caspase-9 and -3. The proton beam with hypoxia induced cell death in wild-type HCT116 cells, but not in a p53 knockout cell line, demonstrating a requirement for p53. As reactive oxygen species (ROS) were also significantly increased, apoptosis could also be abolished by treatment with the anti-oxidant N-acetyl cysteine (NAC). P38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) were activated by the treatment, and their respective DN mutants restored the cell death induced by either proton therapy alone or with hypoxia. In conclusion, proton beam treatment did not differently regulate cancer cell apoptosis either in normoxic or hypoxic conditions via a p53-dependent mechanism and by the activation of p38/JNK MAPK pathways through ROS.

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

Zhao, Yu; Wang, Wenhui; Wang, Qi

Highlights: Black-Right-Pointing-Pointer 5-LOX is able to upregulate expression of NF-{kappa}B p65. Black-Right-Pointing-Pointer 5-LOX enhances nuclear translocation of NF-{kappa}B p65 via increasing p-I{kappa}B-{alpha} level. Black-Right-Pointing-Pointer 5-LOX stimulates transcriptional activity of NF-{kappa}B in hepatoma cells. Black-Right-Pointing-Pointer LTB4 activates transcriptional activity of NF-{kappa}B in hepatoma cells. -- Abstract: The issue that lipid metabolism enzyme and its metabolites regulate transcription factors in cancer cell is not fully understood. In this study, we first report that the lipid metabolism enzyme 5-Lipoxygenase (5-LOX) and its metabolite leukotriene B4 (LTB4) are capable of activating nuclear factor-{kappa}B (NF-{kappa}B) in hepatoma cells. We found that the treatment of MK886more » (an inhibitor of 5-LOX) or knockdown of 5-LOX was able to downregulate the expression of NF-{kappa}B p65 at the mRNA level and decreased the phosphorylation level of inhibitor {kappa}B{alpha} (I{kappa}B{alpha}) in the cytoplasm of hepatoma HepG2 or H7402 cells, which resulted in the decrease of the level of nuclear NF-{kappa}B p65. These were confirmed by immunofluorescence staining in HepG2 cell. Moreover, the above treatments were able to decrease the transcriptional activity of NF-{kappa}B in the cells. The LTB4, one of metabolites of 5-LOX, is responsible for 5-LOX-activated NF-{kappa}B in a dose-dependent manner. Thus, we conclude that the lipid metabolism enzyme 5-LOX and its metabolite LTB4 are capable of activating transcription factor NF-{kappa}B in hepatoma cells. Our finding provides new insight into the significance of lipid metabolism in activation of transcription factors in cancer.« less

Long life 80Ah standard IPV NiH2 battery cell

NASA Technical Reports Server (NTRS)

Armantrout, Jon D.; Waller, J. S.

1995-01-01

A standard Nickel-Hydrogen (NiH2) Individual Pressure Vessel (IPV) battery cell is needed to meet future low cost, high performance mission requirements for NASA, military, and civil space programs. A common or standard cell design has evolved from the heritage of HST, Milstar, and other Air Force Mantech cell designs with substantial flight experience, while incorporating some of the historical COMSAT cell design features described in a previous NASA publication. Key features include slurry process nickel electrodes having high strength, long life and high yield (lower cost), and dual layer zircar separators for improved KOH retention, uniformality, and longer life. The cell design will have a zirconium oxide wall wick inside the pressure vessel to redistribute electrolyte and extend life. The slurry electrode will be 35 mils thick to take advantage of qualified cell mechanical configurations and proven assembly and activation techniques developed by Eagle Picher Industries (EPI) for the Hubble Space Telescope (HST) RNH-90-3 and 'Generic HST' RNH-90-5 cell designs with back-to-back nickel electrodes produced by the dry sinter process. The 80Ah common cell design can be scaled to meet capacity requirements from 60Ah to 100Ah. Producibility, commonality, and long life performance will be enhanced with the robust cell design described herein.

Regulation of intracellular pH in LLC-PK1 cells by Na+/H+ exchange.

PubMed

Montrose, M H; Murer, H

1986-01-01

Suspensions of LLC-PK1 cells (a continuous epitheliod cell line with renal characteristics) are examined for mechanisms of intracellular pH regulation using the fluorescent probe BCECF. Initial experiments determine suitable calibration procedures for use of the BCECF fluorescent signal. They also determine that the cell suspension contains cells which (after 4 hr in suspension) have Na+ and K+ gradients comparable to those of cells in monolayer culture. The steady-state intracellular pH (7.05 +/- 0.01, n = 5) of cells which have recovered in (pH 7.4) Na+-containing medium is not affected over several minutes by addition of 100 microM amiloride or removal of extracellular Na+ (Na+o less than 1 mM). In contrast, when the cells recover from an acid load (caused by NH4 preincubation and removal), the recovery is largely Na+ dependent and is sensitive to 100 microM amiloride. These results suggest that with resting pH near neutrality, both Na+o/H+i and Na+i/H+o exchange reactions are functionally inactive (compared to cellular buffering capacity). In contrast, Na+o/H+i exchange is activated by an increased cellular acid load. This activation may be observed directly either as a stimulation of net H+ efflux or net Na+ influx with decreasing intracellular pH. The extrapolation of this latter data suggests a "set point" of Na+/H+ exchange of approximately pH 7.0, consistent with the observed resting intracellular pH of approximately 7.05.

A combination hepatoma-targeted therapy based on nanotechnology: pHRE-Egr1-HSV-TK/131I-antiAFPMcAb-GCV/MFH

NASA Astrophysics Data System (ADS)

Lin, Mei; Huang, Junxing; Jiang, Xingmao; Zhang, Jia; Yu, Hong; Ye, Jun; Zhang, Dongsheng

2016-09-01

Combination targeted therapy is a promising cancer therapeutic strategy. Here, using PEI-Mn0.5Zn0.5Fe2O4 nanoparticles (PEI-MZF-NPs) as magnetic media for MFH (magnetic fluid hyperthermia) and gene transfer vector for gene-therapy, a combined therapy, pHRE-Egr1-HSV-TK/131I-antiAFPMcAb-GCV/MFH, for hepatoma is developed. AntiAFPMcAb (Monoclonal antibody AFP) is exploited for targeting. The plasmids pHRE-Egr1-HSV-TK are achieved by incorporation of pEgr1-HSV-TK and pHRE-Egr1-EGFP. Restriction enzyme digestion and PCR confirm the recombinant plasmids pHRE-Egr1-HSV-TK are successfully constructed. After exposure to the magnetic field, PEI-MZF-NPs/pHRE-Egr1-EGFP fluid is warmed rapidly and then the temperature is maintained at 43 °C or so, which is quite appropriate for cancer treatment. The gene expression reaches the peak when treated with 200 μCi 131I for 24 hours, indicating that the dose of 200 μCi might be the optimal dose for irradiation and 24 h irradiation later is the best time to initiate MFH. The in vitro and in vivo experiments demonstrate that pHRE-Egr1-HSV-TK/131I-antiAFPMcAb-GCV/MFH can greatly suppress hepatic tumor cell proliferation and induce cell apoptosis and necrosis and effectively inhibit the tumor growth, much better than any monotherapy does alone. Furthermore, the combination therapy has few or no adverse effects. It might be applicable as a strategy to treat hepatic cancer.

Improved specific energy Ni-H2 cell

NASA Astrophysics Data System (ADS)

Miller, L.

1985-07-01

Design optimization activities which have evolved and validated the necessary technology to produce Ni-H2 battery cells exhibiting a specific energy of 75-80 Whr/Kg (energy density approximately 73 Whr/L are summarized. Final design validation is currently underway with the production of battery cells for qualification and life testing. The INTELSAT type Ni-H2 battery cell design has been chosen for expository purposes. However, it should be recognized portions of the improved technology could be applied to the Air Force type Ni-H2 battery cell design with equal benefit.

Improved Specific Energy Ni-h2 Cell

NASA Technical Reports Server (NTRS)

Miller, L.

1985-01-01

Design optimization activities which have evolved and validated the necessary technology to produce Ni-H2 battery cells exhibiting a specific energy of 75-80 Whr/Kg (energy density approximately 73 Whr/L are summarized. Final design validation is currently underway with the production of battery cells for qualification and life testing. The INTELSAT type Ni-H2 battery cell design has been chosen for expository purposes. However, it should be recognized portions of the improved technology could be applied to the Air Force type Ni-H2 battery cell design with equal benefit.

Dynamic regulation of EZH2 from HPSc to hepatocyte-like cell fate

PubMed Central

Helsen, Nicky; Vanhove, Jolien; Boon, Ruben; Xu, Zhuofei; Ordovas, Laura; Verfaillie, Catherine M.

2017-01-01

Currently, drug metabolization and toxicity studies rely on the use of primary human hepatocytes and hepatoma cell lines, which both have conceivable limitations. Human pluripotent stem cell (hPSC)—derived hepatocyte-like cells (HLCs) are an alternative and valuable source of hepatocytes that can overcome these limitations. EZH2 (enhancer of zeste homolog 2), a transcriptional repressor of the polycomb repressive complex 2 (PRC2), may play an important role in hepatocyte development, but its role during in vitro hPSC-HLC differentiation has not yet been assessed. We here demonstrate dynamic regulation of EZH2 during hepatic differentiation of hPSC. To enhance EZH2 expression, we inducibly overexpressed EZH2 between d0 and d8, demonstrating a significant improvement in definitive endoderm formation, and improved generation of HLCs. Despite induction of EZH2 overexpression until d8, EZH2 transcript and protein levels decreased from d4 onwards, which might be caused by expression of microRNAs predicted to inhibit EZH2 expression. In conclusion, our studies demonstrate that EZH2 plays a role in endoderm formation and hepatocyte differentiation, but its expression is tightly post-transcriptionally regulated during this process. PMID:29091973

Cyanidin attenuates Aβ25-35-induced neuroinflammation by suppressing NF-κB activity downstream of TLR4/NOX4 in human neuroblastoma cells.

PubMed

Thummayot, Sarinthorn; Tocharus, Chainarong; Jumnongprakhon, Pichaya; Suksamrarn, Apichart; Tocharus, Jiraporn

2018-04-19

Cyanidin is polyphenolic pigment found in plants. We have previously demonstrated that cyanidin protects nerve cells against Aβ 25-35 -induced toxicity by decreasing oxidative stress and attenuating apoptosis mediated by both the mitochondrial apoptotic pathway and the ER stress pathway. To further elucidate the molecular mechanisms underlying the neuroprotective effects of cyanidin, we investigated the effects of cyanidin on neuroinflammation mediated by the TLR4/NOX4 pathway in Aβ 25-35 -treated human neuroblastoma cell line (SK-N-SH). SK-N-SH cells were exposed to Aβ 25-35 (10 μmol/L) for 24 h. Pretreatment with cyanidin (20 μmol/L) or NAC (20 μmol/L) strongly inhibited the NF-κB signaling pathway in the cells evidenced by suppressing the degradation of IκBα, translocation of the p65 subunit of NF-κB from the cytoplasm to the nucleus, and thereby reducing the expression of iNOS protein and the production of NO. Furthermore, pretreatment with cyanidin greatly promoted the translocation of the Nrf2 protein from the cytoplasm to the nucleus; upregulating cytoprotective enzymes, including HO-1, NQO-1 and GCLC; and increased the activity of SOD enzymes. Pretreatment with cyanidin also decreased the expression of TLR4, directly improved intracellular ROS levels and regulated the activity of inflammation-related downstream pathways including NO production and SOD activity through TLR4/NOX4 signaling. These results demonstrate that TLR4 is a primary receptor in SK-N-SH cells, by which Aβ 25-35 triggers neuroinflammation, and cyanidin attenuates Aβ-induced inflammation and ROS production mediated by the TLR4/NOX4 pathway, suggesting that inhibition of TLR4 by cyanidin could be beneficial in preventing neuronal cell death in the process of Alzheimer's disease.

DEVELOPMENT OF AN ENVIRONMENTAL ESTROGEN SCREEN USING TRANSIENTLY TRANSFECTED RAINBOW TROUT CELL LINES

EPA Science Inventory

Rainbow troutp hepatoma (RTH-149) and gonad cells (RTG-2) were used to develop a screening protocol for estrogen disrupting chemicals. Transfection of an estrogen-responsive luciferase reporter plasmid into...

Persian shallot, Allium hirtifolium Boiss, induced apoptosis in human hepatocellular carcinoma cells.

PubMed

Hosseini, Farzaneh Sadat; Falahati-Pour, Soudeh Khanamani; Hajizadeh, Mohammad Reza; Khoshdel, Alireza; Mirzaei, Mohammad Reza; Ahmadirad, Hadis; Behroozi, Reza; Jafari, Nesa; Mahmoodi, Mehdi

2017-08-01

This study investigated the potential of Persian shallot extract as an anticancer agent in HepG2 tumor cell line, an in vitro human hepatoma cancer model system. The inhibitory effect of Persian shallot on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of Persian shallot, the activity of Persian shallot in inducing apoptosis was investigated through the detection of annexin V signal by flow cytometry and expression of some apoptosis related genes such p21, p53, puma, caspase-8 family-Bcl-2 proteins like bid, bim, bcl-2 and bax were measured by real-time PCR in HepG2 cells. Persian shallot extract inhibited the growth of HepG2 cells in a dose-dependent manner. The IC 50 value (inhibiting cell growth by 50%) was 149 μg/ml. The results of real-time PCR revealed a significant up-regulation of bid, bim, caspase-8, puma, p53, p21 and bax genes and a significant downregulation of bcl-2 gene in HepG2 cells treated with Persian shallot extract significantly. Therefore, this is the first report on an increased expression of bid, bim, caspase-8, puma, p53, p21 and bax genes and down regulation of bcl-2 gene indicating that the Persian shallot extract possibly induced the process of cell death through the intrinsic and extrinsic apoptosis pathways and triggers the programmed cell death in HepG2 tumor cell lines by modulating the expression of pro-/anti-apoptotic genes. Furthermore, we showed that Persian shallot extract increased annexin V signal and expression, resulting in apoptotic cell death of HepG2 cells after 24 h treatment. Therefore, according to the results of this study, the Persian shallot extract could be considered as a potential candidate for production of drug for the prevention or treatment of human hepatoma.

Comparative proteomics analysis of apoptotic Spodoptera frugiperda cells during p35 knockout Autographa californica multiple nucleopolyhedrovirus infection.

PubMed

Yu, Qian; Xiong, Youhua; Liu, Jianliang; Wang, Qin; Qiu, Yuanxin; Wen, Dongling

2016-06-01

Infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutants lacking a functional p35 gene can induce host cell apoptosis, which provides the possibility to use the potential of these viruses in the biological control of pest insects. Nonetheless, the proteomics or the protein changes of Spodoptera frugiperda (Sf9) cells infected with p35 knockout AcMNPV have not yet been studied. To further improve the use of AcMNPV, we set out to analyze the protein composition and protein changes of Sf9 cells of different infection stages by isobaric tag for relative and absolute quantification (iTRAQ) techniques. A total of 4004 sf9 proteins were identified by iTRAQ. After comparation of the significantly expressed 483 proteins from p35koAcMNPV-infected Sf9 cells and the significantly expressed 413 proteins from wtAcMNPV-infected Sf9 cells, we found that 226 proteins were specific to p35koAcMNPV-infected Sf9 cells. The 226 proteins were categorized according to GO classification for insects and were categorized into: biological processes, molecular functions and cellular components. Of interest, the most up-regulated proteins related to Epstein-Barr virus infection, RNA transport, Calcium signaling pathway, cGMP-PKG signaling pathway, oxidative phosphorylation and N-Glycan biosynthesis. Determination of the protein changes in p35 knockout AcMNPV-infected Sf9 cells would facilitate the better use of this virus-host cell interaction in pest insect control and other related fields. Copyright © 2016 Elsevier Inc. All rights reserved.

The Role of Biotransformation and Oxidative Stress in 3,5-Dichloroaniline (3,5-DCA) Induced Nephrotoxicity in Isolated Renal Cortical Cells from Male Fischer 344 Rats

PubMed Central

Racine, Christopher R.; Ferguson, Travis; Preston, Debbie; Ward, Dakota; Ball, John; Anestis, Dianne; Valentovic, Monica; Rankin, Gary O.

2016-01-01

Among the mono- and dichloroanilines, 3,5-Dichloroaniline (3,5-DCA) is the most potent nephrotoxicant in vivo and in vitro. However, the role of renal biotransformation in 3,5-DCA induced nephrotoxicity is unknown. The current study was designed to determine the in vitro nephrotoxic potential of 3,5-DCA in isolated renal cortical cells (IRCC) obtained from male Fischer 344 rats, and the role of renal bioactivation and oxidative stress in 3,5-DCA nephrotoxicity. IRCC (~4 million cells/ml) from male rats were exposed to 3,5-DCA (0-1.0 mM) for up to 120 min. In IRCC, 3,5-DCA was cytotoxic at 1.0 mM by 60 min as evidenced by the increased release of lactate dehydrogenase (LDH), but 120 min was required for 3,5-DCA 0.5 mM to increase LDH release. In subsequent studies, IRCC were exposed to a pretreatment (antioxidant or enzyme inhibitor) prior to exposure to 3,5-DCA (1.0 mM) for 90 min. Cytotoxicity induced by 3,5-DCA was attenuated by pretreatment with inhibitors of flavin-containing monooxygenase (FMO; methimazole, N-octylamine), cytochrome P450 (CYP; piperonyl butoxide, metyrapone), or peroxidase (indomethacin, mercaptosuccinate) enzymes. Use of more selective CYP inhibitors suggested that the CYP 2C family contributed to 3,5-DCA bioactivation. Antioxidants (glutathione, N-acetyl-L-cysteine, α-tocopherol, ascorbate, pyruvate) also attenuated 3,5-DCA nephrotoxicity, but oxidized glutathione levels and the oxidized/reduced glutathione ratios were not increased. These results indicate that 3,5-DCA may be activated via several renal enzyme systems to toxic metabolites, and that free radicals, but not oxidative stress, contribute to 3,5-DCA induced nephrotoxicity in vitro. PMID:26808022

Sustained induction of cytochrome P4501A1 in human hepatoma cells by co-exposure to benzo[a]pyrene and 7H-dibenzo[c,g]carbazole underlies the synergistic effects on DNA adduct formation

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

Gábelová, Alena, E-mail: alena.gabelova@savba.sk; Poláková, Veronika; Prochazka, Gabriela

To gain a deeper insight into the potential interactions between individual aromatic hydrocarbons in a mixture, several benzo[a]pyrene (B[a]P) and 7H-dibenzo[c,g]carbazole (DBC) binary mixtures were studied. The biological activity of the binary mixtures was investigated in the HepG2 and WB-F344 liver cell lines and the Chinese hamster V79 cell line that stably expresses the human cytochrome P4501A1 (hCYP1A1). In the V79 cells, binary mixtures, in contrast to individual carcinogens, caused a significant decrease in the levels of micronuclei, DNA adducts and gene mutations, but not in cell survival. Similarly, a lower frequency of micronuclei and levels of DNA adducts weremore » found in rat liver WB-F344 cells treated with a binary mixture, regardless of the exposure time. The observed antagonism between B[a]P and DBC may be due to an inhibition of Cyp1a1 expression because cells exposed to B[a]P:DBC showed a decrease in Cyp1a1 mRNA levels. In human liver HepG2 cells exposed to binary mixtures for 2 h, a reduction in micronuclei frequency was also found. However, after a 24 h treatment, synergism between B[a]P and DBC was determined based on DNA adduct formation. Accordingly, the up-regulation of CYP1A1 expression was detected in HepG2 cells exposed to B[a]P:DBC. Our results show significant differences in the response of human and rat cells to B[a]P:DBC mixtures and stress the need to use multiple experimental systems when evaluating the potential risk of environmental pollutants. Our data also indicate that an increased expression of CYP1A1 results in a synergistic effect of B[a]P and DBC in human cells. As humans are exposed to a plethora of noxious chemicals, our results have important implications for human carcinogenesis. - Highlights: • B[a]P:DBC mixtures were less genotoxic in V79MZh1A1 cells than B[a]P and DBC alone. • An antagonism between B[a]P and DBC was determined in rat liver WB-F344 cells. • The inhibition of CYP1a1 expression by B

Pharmacological characterization of the human histamine H2 receptor stably expressed in Chinese hamster ovary cells.

PubMed Central

Leurs, R.; Smit, M. J.; Menge, W. M.; Timmerman, H.

1994-01-01

1. The gene for the human histamine H2 receptor was stably expressed in Chinese hamster ovary (CHO) cells and characterized by [125I]-iodoaminopotentidine binding studies. In addition, the coupling of the expressed receptor protein to a variety of signal transduction pathways was investigated. 2. After cotransfection of CHO cells with pCMVhumH2 and pUT626, a phleomycine-resistant clonal cell line (CHOhumH2) was isolated that expressed 565 +/- 35 fmol kg-1 protein binding sites with high affinity (0.21 +/- 0.02 nM) for the H2 antagonist, [125I]-iodoaminopotentidine. 3. Displacement studies with a variety of H2 antagonists indicated that the encoded protein was indistinguishable from the H2 receptor identified in human brain membranes and guinea-pig right atrium. The Ki-values observed in the various preparations correlated very well (r2 = 0.996-0.920). 4. Displacement studies with histamine showed that a limited fraction (32 +/- 6%) of the binding sites showed a high affinity for histamine (2 +/- 1.2 microM); the shallow displacement curves were reflected by a Hill-coefficient significantly different from unity (nH = 0.58 +/- 0.09). The addition of 100 microM Gpp(NH)p resulted in a steepening of the displacement curve (nH = 0.79 +/- 0.02) and a loss of high affinity sites for histamine. 5. Displacement studies with other agonists indicated that the recently developed specific H2 agonists, amthamine and amselamine, showed an approximately 4-5 fold higher affinity for the human H2 receptor than histamine. 6. Stimulation of CHOhumH2 cells with histamine resulted in a rapid rise of the intracellular cyclic AMP levels. After 10 min an approximately 10 fold increase in cyclic AMP could be measured.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 4 PMID:7921611

75 FR 55327 - Tetrahydro-3,5-dimethyl-2H-1,3,5-thiadiazine-2-thione (Dazomet); Notice of Receipt of Request to...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-10

... is granted, any sale, distribution, or use of products listed in this notice will be permitted after...-3,5-dimethyl-2H-1,3,5-thiadiazine-2-thione product registrations to terminate or delete one or more... would not terminate the last tetrahydro-3,5-dimethyl-2H-1,3,5-thiadiazine-2- thione products registered...

MicroRNA-1 promotes apoptosis of hepatocarcinoma cells by targeting apoptosis inhibitor-5 (API-5).

PubMed

Li, Dong; Liu, Yu; Li, Hua; Peng, Jing-Jing; Tan, Yan; Zou, Qiang; Song, Xiao-Feng; Du, Min; Yang, Zheng-Hui; Tan, Yong; Zhou, Jin-Jun; Xu, Tao; Fu, Zeng-Qiang; Feng, Jian-Qiong; Cheng, Peng; chen, Tao; Wei, Dong; Su, Xiao-Mei; Liu, Huan-Yi; Qi, Zhong-Chun; Tang, Li-Jun; Wang, Tao; Guo, Xin; Hu, Yong-He; Zhang, Tao

2015-01-02

Although microRNA-1 (miR-1) is a known liver cancer suppressor, the role of miR-1 in apoptosis of hepatoma cells has remained largely unknown. Our study shows that ectopic miR-1 overexpression induced apoptosis of liver hepatocellular carcinoma (HepG2) cells. Apoptosis inhibitor 5 (API-5) was found to be a potential regulator of miR-1 induced apoptosis, using a bioinformatics approach. Furthermore, an inverse relationship between miR-1 and API-5 expression was observed in human liver cancer tissues and adjacent normal liver tissues. Negative regulation of API-5 expression by miR-1 was demonstrated to promote apoptosis of HepG2 cells. Our study provides a novel regulatory mechanism of miR-1 in the apoptosis of hepatoma cells. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Syringin from stem bark of Fraxinus rhynchophylla protects Abeta(25-35)-induced toxicity in neuronal cells.

PubMed

Yang, Eun-Ju; Kim, Sang-In; Ku, Hyun-Yeong; Lee, Dong-Seok; Lee, Jong-Won; Kim, Yae-Sil; Seong, Yeon-Hee; Song, Kyung-Sik

2010-04-01

The medicinal herb Jinpi, derived from the dried stem barks of Fraxinus rhynchophylla belonging to Oleaceae is widely used as a variety of Korean folk remedies for anti-inflammatory, febricide, antidiarrhea, and antileukorrhea diseases. In the course of screening antidementia agents from natural products, F. rhynchophylla showed significant inhibitory activity toward Abeta(25-35)-induced neuronal cell death. An active principle was isolated and identified as syringin. When the neuroblastoma cells were exposed to 50 microM Abeta(25-35), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction rate (survival rate) decreased to 60.21 +/- 2.16% over control while syringin treated ones recovered cell viability up to 79.12 +/- 1.39% at 20 microM. In addition, 20 microM syringin almost completely removed Abeta(25-35)-induced reactive oxygen species. The neuroprotective effect of syringin seemed to be originated from the reduction of apoptosis since decrease in caspase-3 activity and expression, reduction in cleaved PARP, and DNA fragmentation were observed. These results suggest that F. rhynchophylla and syringin are expected to be useful for preventing Abeta(25-35)-induced neuronal cell damage.

21 CFR 176.230 - 3,5-Dimethyl-1,3,5,2H-tetrahydrothiadiazine-2-thione.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true 3,5-Dimethyl-1,3,5,2H-tetrahydrothiadiazine-2-thione. 176.230 Section 176.230 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) INDIRECT FOOD ADDITIVES: PAPER AND...

Cell cycle-dependent changes in H3K56ac in human cells

PubMed Central

Stejskal, Stanislav; Stepka, Karel; Tesarova, Lenka; Stejskal, Karel; Matejkova, Martina; Simara, Pavel; Zdrahal, Zbynek; Koutna, Irena

2015-01-01

The incorporation of histone H3 with an acetylated lysine 56 (H3K56ac) into the nucleosome is important for chromatin remodeling and serves as a marker of new nucleosomes during DNA replication and repair in yeast. However, in human cells, the level of H3K56ac is greatly reduced, and its role during the cell cycle is controversial. Our aim was to determine the potential of H3K56ac to regulate cell cycle progression in different human cell lines. A significant increase in the number of H3K56ac foci, but not in H3K56ac protein levels, was observed during the S and G2 phases in cancer cell lines, but was not observed in embryonic stem cell lines. Despite this increase, the H3K56ac signal was not present in late replication chromatin, and H3K56ac protein levels did not decrease after the inhibition of DNA replication. H3K56ac was not tightly associated with the chromatin and was primarily localized to active chromatin regions. Our results support the role of H3K56ac in transcriptionally active chromatin areas but do not confirm H3K56ac as a marker of newly synthetized nucleosomes in DNA replication. PMID:26645646

Hexachlorobenzene induces cell proliferation, and aryl hydrocarbon receptor expression (AhR) in rat liver preneoplastic foci, and in the human hepatoma cell line HepG2. AhR is a mediator of ERK1/2 signaling, and cell cycle regulation in HCB-treated HepG2 cells.

PubMed

de Tomaso Portaz, Ana Clara; Caimi, Giselle Romero; Sánchez, Marcela; Chiappini, Florencia; Randi, Andrea S; Kleiman de Pisarev, Diana L; Alvarez, Laura

2015-10-02

Hexachlorobenzene (HCB) is a widespread environmental pollutant, and a liver tumor promoter in rodents. Depending on the particular cell lines studied, exposure to these compounds may lead to cell proliferation, terminal differentiation, or apoptosis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is involved in drug and xenobiotic metabolism. AhR can also modulate a variety of cellular and physiological processes that can affect cell proliferation and cell fate determination. The mechanisms by which AhR ligands, both exogenous and endogenous, affect these processes involve multiple interactions between AhR and other signaling pathways. In the present study, we examined the effect of HCB on cell proliferation and AhR expression, using an initiation-promotion hepatocarcinogenesis protocol in rat liver and in the human-derived hepatoma cell line, HepG2. Female Wistar rats were initiated with a single dose of 100 mg/kg of diethylnitrosamine (DEN) at the start of the experiment. Two weeks later, daily dosing of 100 mg/kg HCB was maintained for 10 weeks. Partial hepatectomy was performed 3 weeks after initiation. The number and area of glutathione S-transferase-P (GST-P)-positive foci, in the rat liver were used as biomarkers of liver precancerous lesions. Immunohistochemical staining showed an increase in proliferating cell nuclear antigen (PCNA)-positive cells, along with enhanced AhR protein expression in hepatocytes within GST-P-positive foci of (DEN HCB) group, when compared to DEN. In a similar manner, Western blot analysis demonstrated that HCB induced PCNA and AhR protein expression in HepG2 cells. Flow cytometry assay indicated that the cells were accumulated at S and G2/M phases of the cell cycle. HCB increased cyclin D1 protein levels and ERK1/2 phosphorylation in a dose-dependent manner. Treatment of cells with a selective MEK1 inhibitor, prevented HCB-stimulatory effect on PCNA and cyclinD1, indicating that these effects

Silibinin down-regulates expression of secreted phospholipase A2 enzymes in cancer cells.

PubMed

Hagelgans, Albert; Nacke, Brit; Zamaraeva, Maria; Siegert, Gabriele; Menschikowski, Mario

2014-04-01

Silibinin, a naturally-occurring flavonoid produced by milk thistle, possesses antioxidant, anti-inflammatory and cancer-preventive activities. In the current study, we examined the effects of silibinin on the expression of secreted phospholipase A2 (sPLA2) enzymes, especially those of group IIA (hGIIA), which play a crucial role in inflammation and carcinogenesis. The effects of silibinin on sPLA2 expressions in human HepG2 hepatoma and PC-3 prostate cancer cells were analyzed using quantitative reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay technique. Silibinin inhibited the expression of hGIIA in unstimulated and cytokine-primed HepG2 and PC-3 cells. The mRNA levels of sPLA2 of groups IB, III and V were also significantly decreased by silibinin. Analyses of transcription factor activation suggest that nuclear factor-κB, but not specificity protein 1 (SP1) is implicated in the silibinin-mediated down-regulation of hGIIA. Silibinin exhibits inhibitory effects on basal and cytokine-induced expression of sPLA2s in cancer cells and therefore, may have the potential to protect against up-regulation of hGIIA and other sPLA2 isoforms during inflammation and cancer.

Cloning, expression and characterization of a novel human CAP10-like gene hCLP46 from CD34(+) stem/progenitor cells.

PubMed

Teng, Yun; Liu, Qiaohong; Ma, Jie; Liu, Feng; Han, Zeguang; Wang, Youxin; Wang, Wei

2006-04-12

A novel human gene, named as human CAP10-like protein 46 kDa (hCLP46), was isolated and identified from human acute myeloid leukemia transformed from myelodysplastic syndrome (MDS-AML) CD34(+) cells. hCLP46 (3q13.33) contains 11 exons encoding a putative protein of 392 amino acids, with a highly conserved CAP10 domain, a hydrophobic signal peptide at its N-terminus, and an endoplasmic reticulum (ER) retention signal motif KTEL at the C-terminus. The homologs of hCLP46 exist in different organisms from plants to animal kingdoms. Subcellular localization analysis showed that hCLP46 is an ER-resident protein. hCLP46 expressed in most human adult tissues at different intensities, with lengths of 3.5 kb and 1.9 kb. Transcript of hCLP46 was not detectable in colon, thymus, and small intestine, but was abundant in liver, indicating that hCLP46 may be involved in important physiological functions in the liver. hCLP46 over-expressed U937 cells had higher growth rate than the cells without exogenic hCLP46 protein expression, suggesting that hCLP46 protein possess the ability of promoting cell proliferation.

HLA-B35 Upregulates Endothelin-1 and Downregulates Endothelial Nitric Oxide Synthase via Endoplasmic Reticulum Stress Response in Endothelial Cells

PubMed Central

Lenna, Stefania; Townsend, Danyelle M.; Tan, Filemon K.; Kapanadze, Bagrat; Markiewicz, Malgorzata; Trojanowska, Maria; Scorza, Raffaella

2013-01-01

The presence of the HLA-B35 allele has emerged as an important risk factor for the development of isolated pulmonary hypertension in patients with scleroderma, however the mechanisms underlying this association have not been fully elucidated. The goal of our study was to determine the molecular mechanisms that mediate the biological effects of HLA-B35 in endothelial cells (ECs). Our data demonstrate that HLA-B35 expression at physiological levels via adenoviral vector resulted in significantly increased endothelin-1 (ET-1) and a significantly decreased endothelial NO synthase (eNOS), mRNA, and protein levels. Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the endoplasmic reticulum (ER) stress and unfolded protein response in ECs. Examination of selected mediators of the unfolded protein response, including H chain binding protein (BiP; GRP78), C/Ebp homologous protein (CHOP; GADD153), endoplasmic reticulum oxidase, and protein disulfide isomerase has revealed a consistent increase of BiP expression levels. Accordingly, thapsigargin, a known ER stress inducer, stimulated ET-1mRNAand protein levels in ECs. This study suggests that HLA-B35 could contribute to EC dysfunction via ER stress-mediated induction of ET-1 in patients with pulmonary hypertension. PMID:20335527

HLA-B35 upregulates endothelin-1 and downregulates endothelial nitric oxide synthase via endoplasmic reticulum stress response in endothelial cells.

PubMed

Lenna, Stefania; Townsend, Danyelle M; Tan, Filemon K; Kapanadze, Bagrat; Markiewicz, Malgorzata; Trojanowska, Maria; Scorza, Raffaella

2010-05-01

The presence of the HLA-B35 allele has emerged as an important risk factor for the development of isolated pulmonary hypertension in patients with scleroderma, however the mechanisms underlying this association have not been fully elucidated. The goal of our study was to determine the molecular mechanisms that mediate the biological effects of HLA-B35 in endothelial cells (ECs). Our data demonstrate that HLA-B35 expression at physiological levels via adenoviral vector resulted in significantly increased endothelin-1 (ET-1) and a significantly decreased endothelial NO synthase (eNOS), mRNA, and protein levels. Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the endoplasmic reticulum (ER) stress and unfolded protein response in ECs. Examination of selected mediators of the unfolded protein response, including H chain binding protein (BiP; GRP78), C/Ebp homologous protein (CHOP; GADD153), endoplasmic reticulum oxidase, and protein disulfide isomerase has revealed a consistent increase of BiP expression levels. Accordingly, thapsigargin, a known ER stress inducer, stimulated ET-1 mRNA and protein levels in ECs. This study suggests that HLA-B35 could contribute to EC dysfunction via ER stress-mediated induction of ET-1 in patients with pulmonary hypertension.

Histone h1 depletion impairs embryonic stem cell differentiation.

PubMed

Zhang, Yunzhe; Cooke, Marissa; Panjwani, Shiraj; Cao, Kaixiang; Krauth, Beth; Ho, Po-Yi; Medrzycki, Magdalena; Berhe, Dawit T; Pan, Chenyi; McDevitt, Todd C; Fan, Yuhong

2012-01-01

Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes.

Dietary Factors and Hepatoma in Rainbow Trout (Salmo gairdneri). I. Aflatoxins in Vegetable Protein Feedstuffs

USGS Publications Warehouse

Sinnhuber, R.O.; Wales, J.H.; Ayers, J.L.; Engebrecht, R.H.; Amend, D.F.

1968-01-01

Aflatoxins (toxic metabolites of the mold Aspergillus flavus) were present in a commercial trout ration causing hepatoma in rainbow trout. Cottonseed meal and solvent extracts of cottonseed meal and of rations containing cottonseed meal and peanut meal were found by chemical assay and confirmed by duckling assay to contain aflatoxins. Diets containing these materials and a purified test diet to which aflatoxins had been added produced microscopic tumors in 6 months and gross lesions of hepatocarcinoma in 9 months. Similar diets without aflatoxin were negative.

Thyroid epithelial cell hyperplasia in IFN-gamma deficient NOD.H-2h4 mice.

PubMed

Yu, Shiguang; Sharp, Gordon C; Braley-Mullen, Helen

2006-01-01

The role of inflammatory cells in thyroid epithelial cell (thyrocyte) hyperplasia is unknown. Here, we demonstrate that thyrocyte hyperplasia in IFN-gamma-/- NOD.H-2h4 mice has an autoimmune basis. After chronic exposure to increased dietary iodine, 60% of IFN-gamma-/- mice had severe thyrocyte hyperplasia with minimal or moderate lymphocyte infiltration, and thyroid dysfunction with reduced serum T4. All mice produced anti-thyroglobulin autoantibody. Some wild-type NOD.H-2h4 mice had isolated areas of thyrocyte hyperplasia with predominantly lymphocytic infiltration, whereas IL-4-/- and 50% of wild-type NOD.H-2h4 mice developed lymphocytic thyroiditis but no thyrocyte hyperplasia. Both thyroid infiltrating inflammatory cells and environmental factors (iodine) were required to induce thyrocyte hyperplasia. Splenocytes from IFN-gamma-/- mice with thyrocyte hyperplasia, but not splenocytes from naïve IFN-gamma-/- mice, induced hyperplasia in IFN-gamma-/- NOD.H-2h4.SCID mice. These results may provide clues for understanding the mechanisms underlying development of epithelial cell hyperplasia not only in thyroids but also in other tissues and organs.

21 CFR 176.230 - 3,5-Dimethyl-1,3,5,2H-tetrahydrothiadiazine-2-thione.

Code of Federal Regulations, 2014 CFR

2014-04-01

... manufacture and coating of paper and paperboard intended for use in contact with food in accordance with the... 21 Food and Drugs 3 2014-04-01 2014-04-01 false 3,5-Dimethyl-1,3,5,2H-tetrahydrothiadiazine-2-thione. 176.230 Section 176.230 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

Mitochondrial DNA maintenance is regulated in human hepatoma cells by glycogen synthase kinase 3β and p53 in response to tumor necrosis factor α.

PubMed

Vadrot, Nathalie; Ghanem, Sarita; Braut, Françoise; Gavrilescu, Laura; Pilard, Nathalie; Mansouri, Abdellah; Moreau, Richard; Reyl-Desmars, Florence

2012-01-01

During chronic liver inflammation, up-regulated Tumor Necrosis Factor alpha (TNF-α) targets hepatocytes and induces abnormal reactive oxygen species (ROS) production responsible for mitochondrial DNA (mtDNA) alterations. The serine/threonine Glycogen Synthase Kinase 3 beta (GSK3β) plays a pivotal role during inflammation but its involvement in the maintenance of mtDNA remains unknown. The aim of this study was to investigate its involvement in TNF-α induced mtDNA depletion and its interrelationship with p53 a protein known to maintain mtDNA copy numbers. Using quantitative polymerase chain reaction (qPCR) we found that at 30 min in human hepatoma HepG2 cells TNF-α induced 0.55±0.10 mtDNA lesions per 10 Kb and a 52.4±2.8% decrease in mtDNA content dependent on TNF-R1 receptor and ROS production. Both lesions and depletion returned to baseline from 1 to 6 h after TNF-α exposure. Luminol-amplified chemiluminescence (LAC) was used to measure the rapid (10 min) and transient TNF-α induced increase in ROS production (168±15%). A transient 8-oxo-dG level of 1.4±0.3 ng/mg DNA and repair of abasic sites were also measured by ELISA assays. Translocation of p53 to mitochondria was observed by Western Blot and co-immunoprecipitations showed that TNF-α induced p53 binding to GSK3β and mitochondrial transcription factor A (TFAM). In addition, mitochondrial D-loop immunoprecipitation (mtDIP) revealed that TNF-α induced p53 binding to the regulatory D-loop region of mtDNA. The knockdown of p53 by siRNAs, inhibition by the phosphoSer(15)p53 antibody or transfection of human mutant active GSK3βS9A pcDNA3 plasmid inhibited recovery of mtDNA content while blockade of GSK3β activity by SB216763 inhibitor or knockdown by siRNAs suppressed mtDNA depletion. This study is the first to report the involvement of GSK3β in TNF-α induced mtDNA depletion. We suggest that p53 binding to GSK3β, TFAM and D-loop could induce recovery of mtDNA content through mtDNA repair.

1H nuclear magnetic resonance studies of sarcoplasmic oxygenation in the red cell-perfused rat heart.

PubMed

Jelicks, L A; Wittenberg, B A

1995-05-01

The proximal histidine N delta H proton of deoxymyoglobin experiences a large hyperfine shift resulting in its 1H nuclear magnetic resonance (NMR) signal appearing at approximately 76 ppm (at 35 degrees C), downfield of the diamagnetic spectral region. 1H NMR of this proton is used to monitor sarcoplasmic oxygen pressure in isolated perfused rat heart. This method monitors intracellular oxygenation in the whole heart and does not reflect oxygenation in a limited region. The deoxymyoglobin resonance intensity is reduced upon conversion of myoglobin to the ferric form by sodium nitrite. 1H resonances of the N delta H protons of the alpha and beta subunits of bovine deoxyhemoglobin do not interfere with the measurement of myoglobin deoxygenation in blood-perfused rat heart. We find that steady-state myoglobin deoxygenation is increased progressively (and reversibly) as oxygenation of the perfusing medium is decreased in both saline and red blood cell-perfused hearts at constant work output. An eightfold increase in the heart rate of the blood-perfused heart resulted in no change in the deoxymyoglobin signal intensity. Intracellular PO2 of myoglobin-containing cells is maintained remarkably constant in changing work states.

Aryl hydrocarbon receptor-mediated toxic potency of dissolved lipophilic organic contaminants collected from Lincoln Creek, Milwaukee, Wisconsin, USA, to PLHC-1 (Poeciliopsis lucida) fish hepatoma cells

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

Villeneuve, D.L.; Crunkilton, R.L.; DeVita, W.M.

1997-05-01

Lincoln Creek is a severely degraded urban stream located in Milwaukee County, Wisconsin, USA. As part of a comprehensive study on effects of urban storm water runoff on the stream biota, an in vitro bioassay with PLHC-1 (Poeciliopsis lucida) fish hepatoma cells was used to assess potential toxic potency of aryl hydrocarbon receptor (AhR)-active compounds, collected by semipermeable membrane devices (SPMDs) exposed to Lincoln Creek water. Dialysates from SPMDs exposed to Lincoln Creek water caused marked cytochrome P4501A induction in PLHC-1. Toxic potency of dialysates, expressed as bioassay-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQ) ranged from 1,300 to 6,600 pg TCDD-EQ/g SPMD formore » 14-d exposures. Dialysates from SPMDs exposed to stream water at base flow had potencies consistently lower than those exposed to storm-flow (high-flow) events that occurred during the same 14-d period. Polychlorinated biphenyls were not detectable in the dialysates. Gas chromatography-mass spectrometry analysis identified polycyclic aromatic hydrocarbons (PAHs) as major contaminants in the dialysates. A log-log correlation of total PAHs and TCDD-EQ yielded an r{sup 2} of 0.802. Empirical evidence suggests that AhR-active PAHs can account for about 20 to 50% of the potency observed.« less

Genotoxic potential of montmorillonite clay mineral and alteration in the expression of genes involved in toxicity mechanisms in the human hepatoma cell line HepG2.

PubMed

Maisanaba, Sara; Hercog, Klara; Filipic, Metka; Jos, Ángeles; Zegura, Bojana

2016-03-05

Montmorillonite, also known as Cloisite(®)Na(+) (CNa(+)), is a natural clay with a wide range of well-documented and novel applications, such as pharmaceutical products or food packaging. Although considered a low toxic product, the expected increased exposure to CNa(+) arises concern on the potential consequences on human and environmental health especially as its genotoxicity has scarcely been investigated so far. Thus, we investigated, for the first time, the influence of non-cytotoxic concentrations of CNa(+) (15.65, 31.25 and 62.5 μg/mL) on genomic instability of human hepatoma cell line (HepG2) by determining the formation of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) with the Cytokinesis block micronucleus cytome assay. Further on we studied the influence of CNa(+) on the expression of several genes involved in toxicity mechanisms using the real-time quantitative PCR. The results showed that CNa(+) increased the number of MNi, while the numbers of NBUDs and NPBs were not affected. In addition it deregulated genes in all the groups studied, mainly after longer time of exposure. These findings provide the evidence that CNa(+) is potentially genotoxic. Therefore further studies that will elucidate the molecular mechanisms involved in toxic activity of CNa(+) are needed for hazard identification and human safety assessment. Copyright © 2015 Elsevier B.V. All rights reserved.

The RING-Type E3 Ligase XBAT35.2 Is Involved in Cell Death Induction and Pathogen Response1[OPEN

PubMed Central

Liu, Hongxia; Ravichandran, Sridhar; McVey, Sarah; Lilley, Carly; Teresinski, Howard J.; Gonzalez-Ferrer, Carmen; Prithiviraj, Balakrishnan

2017-01-01

XBAT35 belongs to a subfamily of Arabidopsis (Arabidopsis thaliana) RING-type E3s that are similar in domain architecture to the rice (Oryza sativa) XA21 Binding Protein3, a defense protein. The XBAT35 transcript undergoes alternative splicing to produce two protein isoforms, XBAT35.1 and XBAT35.2. Here, we demonstrate that XBAT35.2 localizes predominantly to the Golgi and is involved in cell death induction and pathogen response. XBAT35.2, but not XBAT35.1, was found to trigger cell death when overexpressed in tobacco (Nicotiana benthamiana) leaves and does so in a manner that requires its RING domain. Loss of XBAT35 gene function disrupts the plant’s ability to defend against pathogen attack, whereas overexpression of XBAT35.2 enhances resistance to pathogens. XBAT35.2 was found to be unstable and promotes its own degradation, suggesting self-regulation. Inoculation with virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv tomato DC3000 results in a drastic reduction in the levels of ubiquitinated XBAT35.2 and an increase in the abundance of the E3. This implies that pathogen infection prohibits XBAT35.2 self-regulation and stabilizes the E3. In agreement with a role in defending against pathogens, XBAT35.2 interacts with defense-related Accelerated Cell Death11 (ACD11) in planta and promotes the proteasome-dependent turnover of ACD11 in cell-free degradation assays. In accordance with regulation by a stabilized XBAT35.2, the levels of ubiquitinated ACD11 increased considerably, and the abundance of ACD11 was reduced following pathogen infection. In addition, treatment of transgenic seedlings with a proteasome inhibitor results in the accumulation of ACD11, confirming proteasome-dependent degradation. Collectively, these results highlight a novel role for XBAT35.2 in cell death induction and defense against pathogens. PMID:28951488

Anti-tumor effect of in vivo IL-2 and GM-CSF electrogene therapy in murine hepatoma model.

PubMed

Chi, Chau-Hwa; Wang, Yu-Shan; Lai, Yen-Shuae; Chi, Kwan-Hwa

2003-01-01

We evaluated the effect of in vivo electrogene therapy (EGT), a newly-developed gene transfer method using electroporation on the induction of anti-cancer immunity. The in vivo EGT was carried out by direct injection of plasmid DNAs encoding mouse interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in a subcutaneous murine hepatoma model of 1MEA.7R.1 cells. Six electric pulses were generated in situ from a square-wave electroporator fitted with a circular, six-needle electrode array. 1MEA.7R1 cells in vitro were modified to secret IL-2 (1MEA.7R.1/IL-2 cells). The 1MEA.7R.1/IL-2 cells had a similar cell doubling-time as their parent cells but showed a much slower growth rate on Balb/C mice. One, or 3 rounds of single gene EGT with IL-2 gene showed a dose-responsive effect of growth retardation. Co-administration of 3 rounds of IL-2/GM-CSF double genes EGT had a stronger growth inhibition effect than 3 rounds of IL-2 single gene EGT. Three rounds of IL-2/GM-CSF EGT rendered the tumor to a growth rate of stably transfected 1MEA.7R.1/IL-2 cells. Seven rounds of IL-2/GM-CSF EGT markedly inhibited the tumor growth. Reverse transciptase-polymerase chain reaction confirmed the expression of IL-2, GM-CSF and interferon-gamma within treated tumors. Systemic inhibitory effects can be demonstrated from tumor-re-challenged experiments on mice which received 3 rounds of double-gene EGT. The T cell proliferation assay revealed an increased T cell proliferation in double-gene EGT-treated mice. This experiment showed that partial systemic immunity can be provoked by IL-2/GM-CSF double-gene EGT. These findings suggest that our immuno-gene therapy protocol has the potential for future clinical applications.

Analysis of microRNA and gene expression profiling in triazole fungicide-treated HepG2 cell line.

PubMed

An, Yu Ri; Kim, Seung Jun; Oh, Moon-Ju; Kim, Hyun-Mi; Shim, Il-Seob; Kim, Pil-Je; Choi, Kyunghee; Hwang, Seung Yong

2013-01-07

MicroRNA (miRNA) plays an important role in various diseases and in cellular and molecular responses to toxicants. In the present study, we investigated differential expression of miRNAs in response to three triazole fungicides (myclobutanil, propiconazole, and triadimefon). The human hepatoma cell line (HepG2) was treated with the above triazoles for 3 h or 48 h. miRNA-based microarray experiments were carried out using the Agilent human miRNA v13 array. At early exposure (3h), six miRNAs were differentially expressed and at late exposure (48 h), three miRNAs were significantly expressed. Overall, this study provides an array of potential biomarkers for the above triazole fungicides. Furthermore, these miRNAs induced by triazoles could be the foundation for the development of a miRNA-based toxic biomarker library that can predict environmental toxicity. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Alterations in microRNA expression profile in HCV-infected hepatoma cells: Involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway

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

Ishida, Hisashi; Tatsumi, Tomohide; Hosui, Atsushi

2011-08-19

Highlights: {yields} HCV infection upregulated miR-192, -194, -215, downregulated miR-320, -491. {yields} Transfection of miR-192, -215, and -491 enhanced HCV replication. {yields} Transfection of miR-491 inhibited Akt phosphorylation. {yields} Akt inhibition could be responsible for augmentation of HCV replication by miR-491. -- Abstract: The aim of this study was to investigate the role of microRNA (miRNA) on hepatitis C virus (HCV) replication in hepatoma cells. Using miRNA array analysis, miR-192/miR-215, miR-194, miR-320, and miR-491 were identified as miRNAs whose expression levels were altered by HCV infection. Among them, miR-192/miR-215 and miR-491 were capable of enhancing replication of the HCV repliconmore » as well as HCV itself. HCV IRES activity or cell proliferation was not increased by forced expression of miR-192/miR-215 or miR-491. Investigation of signaling pathways revealed that miR-491 specifically suppressed the phosphoinositol-3 (PI3) kinase/Akt pathway. Under inhibition of PI3 kinase by LY294002, the suppressive effect of miR-491 on HCV replication was abolished, indicating that suppression of HCV replication by miR-491 was dependent on the PI3 kinase/Akt pathway. miRNAs altered by HCV infection would then affect HCV replication, which implies a complicated mechanism for regulating HCV replication. HCV-induced miRNA may be involved in changes in cellular properties including hepatocarcinogenesis.« less

Novel polymerizable surfactants with pH-sensitive amphiphilicity and cell membrane disruption for efficient siRNA delivery.

PubMed

Wang, Xu-Li; Ramusovic, Sergej; Nguyen, Thanh; Lu, Zheng-Rong

2007-01-01

Small interfering RNA (siRNA) is a promising new therapeutic modality that can specifically silence disease-related genes. The main challenge for successful clinical development of therapeutic siRNA is the lack of efficient delivery systems. In this study, we have designed and synthesized a small library of novel multifunctional siRNA carriers, polymerizable surfactants with pH-sensitive amphiphilicity based on the hypothesis that pH-sensitive amphiphilicity and environmentally sensitive siRNA release can result in efficient siRNA delivery. The polymerizable surfactants comprise a protonatable amino head group, two cysteine residues, and two lipophilic tails. The surfactants demonstrated pH-sensitive amphiphilic hemolytic activity or cell membrane disruption with rat red blood cells. Most of the surfactants resulted in low hemolysis at pH 7.4 and high hemolysis at reduced pH (6.5 and 5.4). The pH-sensitive cell membrane disruption can facilitate endosomal-lysosomal escape of siRNA delivery systems at the endosomal-lysosomal pH. The surfactants formed compact nanoparticles (160-260 nm) with siRNA at N/P ratios of 8 and 10 via charge complexation with the amino head group, lipophilic condensation, and autoxidative polymerization of dithiols. The siRNA complexes with the surfactants demonstrated low cytotoxicity. The cellular siRNA delivery efficiency and RNAi activity of the surfactants correlated well with their pH-sensitive amphiphilic cell membrane disruption. The surfactants mediated 40-88% silencing of luciferase expression with 100 nM siRNA and 35-75% with 20 nM siRNA in U87-luc cells. Some of the surfactants resulted in similar or higher gene silencing efficiency than TransFast. EHCO with no hemolytic activity at pH 7.4 and 6.5 and high hemolytic activity at pH 5.4 resulted in the best siRNA delivery efficiency. The polymerizable surfactants with pH-sensitive amphiphilicity are promising for efficient siRNA delivery.

Effects of short-chain chlorinated paraffins exposure on the viability and metabolism of human hepatoma HepG2 cells.

PubMed

Geng, Ningbo; Zhang, Haijun; Zhang, Baoqin; Wu, Ping; Wang, Feidi; Yu, Zhengkun; Chen, Jiping

2015-03-03

Short-chain chlorinated paraffins (SCCPs) have attracted considerable attention for their characteristic of persistent organic pollutants. However, very limited information is available for their toxic effects at environmentally relevant doses, limiting the evaluation of their health risks. In this study, cell viability assay and targeted metabolomic approach was used to evaluate the environmental dose (<100 μg/L) effect of SCCPs on HepG2 cells. Cell viability was found to be decreased with increases in exposure dose of SCCPs. Exposure for 48 h to C10-CPs resulted in a significant reduction in cell viability compared with 24 h, even at 1 μg/L. SCCPs exposure altered the intracellular redox status and caused significant metabolic disruptions. As a kind of peroxisome proliferator, SCCPs specifically stimulated the β-oxidation of unsaturated fatty acids and long-chain fatty acids. Meanwhile, SCCPs exposure disturbed glycolysis and amino acid metabolism, and led to the up-regulation of glutamate metabolism and urea cycle. The toxic effects of SCCPs might mainly involve the perturbation of energy production, protein biosynthesis, fatty acid metabolism, and ammonia recycling.

¹H-MAS-NMR chemical shifts in hydrogen-bonded complexes of chlorophenols (pentachlorophenol, 2,4,6-trichlorophenol, 2,6-dichlorophenol, 3,5-dichlorophenol, and p-chlorophenol) and amine, and H/D isotope effects on ¹H-MAS-NMR spectra.

PubMed

Honda, Hisashi

2013-04-22

Chemical shifts (CS) of the ¹H nucleus in N···H···O type hydrogen bonds (H-bond) were observed in some complexes between chlorophenols [pentachlorophenol (PCP), 2,4,6-tricholorophenol (TCP), 2,6-dichlorophenol (26DCP), 3,5-dichlorophenol (35DCP), and p-chlorophenol (pCP)] and nitrogen-base (N-Base) by solid-state high-resolution ¹H-NMR with the magic-angle-spinning (MAS) method. Employing N-Bases with a wide range of pKa values (0.65-10.75), ¹H-MAS-NMR CS values of bridging H atoms in H-bonds were obtained as a function of the N-Base's pKa. The result showed that the CS values were increased with increasing pKa values in a range of DpKa < 0 [DpKa = pKa(N-Base)-pKa(chlorophenols)] and decreased when DpKa > 2: The maximum CS values was recorded in the PCP (pKa = 5.26)-4-methylpyridine (6.03), TCP (6.59)-imidazole (6.99), 26DCP (7.02)-2-amino-4-methylpyridine (7.38), 35DCP (8.04)-4-dimethylaminopyridine (9.61), and pCP (9.47)-4-dimethylaminopyridine (9.61) complexes. The largest CS value of 18.6 ppm was recorded in TCP-imidazole crystals. In addition, H/D isotope effects on ¹H-MAS-NMR spectra were observed in PCP-2-amino-3-methylpyridine. Based on the results of CS simulation using a B3LYP/6-311+G** function, it can be explained that a little changes of the N-H length in H-bond contribute to the H/D isotope shift of the ¹H-MAS-NMR peaks.

Using high haze (> 90%) light-trapping film to enhance the efficiency of a-Si:H solar cells

NASA Astrophysics Data System (ADS)

Chu, Wei-Ping; Lin, Jian-Shian; Lin, Tien-Chai; Tsai, Yu-Sheng; Kuo, Chen-Wei; Chung, Ming-Hua; Hsieh, Tsung-Eong; Liu, Lung-Chang; Juang, Fuh-Shyang; Chen, Nien-Po

2012-07-01

The high haze light-trapping (LT) film offers enhanced scattering of light and is applied to a-Si:H solar cells. UV glue was spin coated on glass, and then the LT pattern was imprinted. Finally, a UV lamp was used to cure the UV glue on the glass. The LT film effectively increased the Haze ratio of glass and decreased the reflectance of a-Si:H solar cells. Therefore, the photon path length was increased to obtain maximum absorption by the absorber layer. High Haze LT film is able to enhance short circuit current density and efficiency of the device, as partial composite film generates broader scattering light, thereby causing shorter wave length light to be absorbed by the P layer so that the short circuit current density decreases. In case of lab-made a-Si:H thin film solar cells with v-shaped LT films, superior optoelectronic performances have been found (Voc = 0.74 V, Jsc = 15.62 mA/cm2, F.F. = 70%, and η = 8.09%). We observed ~ 35% enhancement of the short-circuit current density and ~ 31% enhancement of the conversion efficiency.

RESPONSES OF CELLS TO pH CHANGES IN THE MEDIUM

PubMed Central

Taylor, A. Cecil

1962-01-01

Studies were made with time-lapse motion pictures of the reactions of cells in culture to changes in their environment. The concentrations of H+, HCO3 -and CO2 in the medium were altered in such a way that each, in turn, could be maintained constant while the others were varied. Observations were made on the shape of the cells, their activity, and their relation to the substratum. Characteristic reversible changes in the cells were observed whenever environmental pH was altered. Elevation of the pH accelerated cell movements and caused contraction of the cytoplasm, while lowering of the pH retarded and eventually stopped all cell activity, causing apparent gelation of the protoplasm. These responses did not occur when HCO3 - and CO2 were varied without changing the pH. It is suggested that local pH changes in the micro-environment of a cell's surface may be a significant factor in controlling cell behavior in culture and in vivo. PMID:13993539

Mesenchymal stem cell expression of interleukin-35 protects against ulcerative colitis by suppressing mucosal immune responses.

PubMed

Yan, Yongjia; Zhao, Na; He, Xianghui; Guo, Hao; Zhang, Zhixiang; Liu, Tong

2018-06-12

Interleukin-35 (IL-35) has recently been identified as an immunosuppressive cytokine that has been used as a potential therapy for chronic inflammatory and autoimmune diseases. However, there remains a paucity of data regarding its potential benefits after integration into mesenchymal stem cells (MSCs). We used a dextran sulfate sodium (DSS)-induced colitis mice model and treated them with IL-35-MSCs, MSCs or saline. The body weight was recorded daily and inflammatory processes were determined. Cytokine secretion by lamina propria lymphocytes (LPLs) and percentage of regulatory T cells (Tregs) were also measured. The data showed that mice in the two treated groups recovered their body weight more rapidly than mice treated with saline in the later stage of colitis. The colon lengths of IL-35-MSC-treated mice were markedly longer than those in the other two groups and the inflammation reduced significantly. Furthermore, the percentage of Foxp3 + Tregs increased significantly and the level of proinflammatory cytokines produced by LPLs decreased significantly in the IL-35-MSC-treated group. The results demonstrate that IL-35-MSCs could ameliorate ulcerative colitis by down-regulating the expression of pro-inflammatory cytokines. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Computational approach for predicting the conserved B-cell epitopes of hemagglutinin H7 subtype influenza virus.

PubMed

Wang, Xiangyu; Sun, Qi; Ye, Zhonghua; Hua, Ying; Shao, Na; Du, Yanli; Zhang, Qiwei; Wan, Chengsong

2016-10-01

An avian-origin influenza H7N9 virus epidemic occurred in China in 2013-2014, in which >422 infected people suffered from pneumonia, respiratory distress syndrome and septic shock. H7N9 viruses belong to the H7 subtype of avian-origin influenza viruses (AIV-H7). Hemagglutinin (HA) is a vital membrane protein of AIV that has an important role in host recognition and infection. The epitopes of HA are significant determinants of the regularity of epidemic and viral mutation and recombination mechanisms. The present study aimed to predict the conserved B-cell epitopes of AIV-H7 HA using a bioinformatics approach, including the three most effective epitope prediction softwares available online: Artificial Neural Network based B-cell Epitope Prediction (ABCpred), B-cell Epitope Prediction (BepiPred) and Linear B-cell Epitope Prediction (LBtope). A total of 24 strains of Euro-Asiatic AIV-H7 that had been associated with a serious poultry pandemic or had infected humans in the past 30 years were selected to identify the conserved regions of HA. Sequences were obtained from the National Center for Biotechnology Information and Global Initiative on Sharing Avian Influenza Data databases. Using a combination of software prediction and sequence comparisons, the conserved epitopes of AIV-H7 were predicted and clarified. A total of five conserved epitopes [amino acids (aa) 37-52, 131-142, 215-234, 465-484 and 487-505] with a suitable length, high antigenicity and minimal variation were predicted and confirmed. Each obtained a score of >0.80 in ABCpred, 60% in LBtope and a level of 0.35 in Bepipred. In addition, a representative amino acid change (glutamine 235 -to-leucine 235 ) in the HA protein of the 2013 AIV-H7N9 was discovered. The strategy adopted in the present study may have profound implications on the rapid diagnosis and control of infectious disease caused by H7N9 viruses, as well as by other virulent viruses, such as the Ebola virus.