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Sample records for hepatoma

  1. Solamargine triggers hepatoma cell death through apoptosis

    PubMed Central

    XIE, XIAODONG; ZHU, HAITAO; YANG, HUIJIAN; HUANG, WENSI; WU, YINGYING; WANG, YING; LUO, YANLING; WANG, DONGQING; SHAO, GENBAO

    2015-01-01

    Solamargine (SM), a steroidal alkaloid glycoside extracted from the traditional Chinese herb Solanum incanum, has been evidenced to inhibit the growth and induce apoptosis in a number of human cancer cell lines. In the present study, the anticancer effect of SM and underlying molecular mechanism of SM-induced apoptosis were investigated on the human hepatocellular carcinoma cells, SMMC7721 and HepG2. The proliferation effects of SM on the SMMC7721 and HepG2 cell lines were evaluated using MTT and colony formation assays. In addition, the percentage of apoptosis was measured using an Annexin V/propidium iodide staining method and the cell cycle distribution mediated by SM was analyzed using flow cytometry. The expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3, caspase-9, proliferating cell nuclear antigen (pcna) and Ki67 proteins were examined to further demonstrate the proliferate and apoptosis effects of SM on the hepatoma cells. The results indicated that SM effectively inhibited hepatoma cell proliferation and promoted apoptosis. SM resulted in cell cycle arrest at the G2/M phase in the two cell lines. In addition, SM downregulated the levels of proliferation-associated (Ki67 and pcna) and anti-apoptotic (Bcl-2) proteins, and promoted the activity of apoptosis-associated proteins (Bax, caspase-3 and caspase-9). Therefore, the activation of the Bcl-2/Bax and caspase signaling pathways may be involved in the SM-induced apoptosis of hepatoma cells. PMID:26170994

  2. Liver epithelial cells inhibit proliferation and invasiveness of hepatoma cells.

    PubMed

    Jeng, Kuo-Shyang; Jeng, Chi-Juei; Jeng, Wen-Juei; Sheen, I-Shyan; Li, Shih-Yun; Hung, Zih-Hang; Hsiau, Hsin-I; Yu, Ming-Che; Chang, Chiung-Fang

    2016-03-01

    Hepatocellular carcinoma (HCC) is a worldwide malignancy with poor prognosis. Liver progenitors or stem cells could be a potential therapy for HCC treatment since they migrate toward tumors. Rat liver epithelial (RLE) cells have both progenitor and stem cell-like properties. Therefore, our study elucidated the therapeutic effect of RLE cells in rat hepatoma cells. RLE cells were isolated from 10-day old rats and characterized for stem cell marker expression. RLE cells and rat hepatoma cells (H4-IIE-C3 cells) were co-cultured and divided into four groups with different ratios of RLE and hepatoma cells. Group A had only rat hepatoma cells as a control group. The ratios of rat hepatoma and RLE cells in group B, C and D were 5:1, 1:1 and 1:5, respectively. Effective inhibition of cell proliferation and migration was found in group D when compared to group A. There was a significant decrease in Bcl2 expression and increase in late apoptosis of rat hepatoma cells when adding more RLE cells. RLE cells reduced cell proliferation and migration of rat hepatoma cells. These results suggested that RLE cells could be used as a potential cell therapy. PMID:26647726

  3. The selective reduction in PTPdelta expression in hepatomas.

    PubMed

    Urushibara, N; Karasaki, H; Nakamura, K; Mizuno, Y; Ogawa, K; Kikuchi, K

    1998-03-01

    The mRNA levels for receptor-like protein tyrosine phosphatases (PTPases), PTPalpha, PTPdelta, PTPgamma and LAR, were evaluated by Northern blot analysis in two types of chemically-induced rat primary hepatomas. In the four PTPases the PTPdelta mRNA was selectively reduced in these hepatoma tissues. It was also diminished in HepG2 hepatoblastoma cell line and in all of the poorly differentiated ascites hepatoma cells examined. PTPalpha, PTPgamma and LAR did not show such a characteristic decrease. This selective reduction in PTPdelta expression strongly suggests PTPdelta plays an important role in hepatocarcinogenesis, possibly as a tumor suppressor gene. PMID:9472099

  4. Comparative Study of Light Scattering from Hepatoma Cells and Hepatocytes

    NASA Astrophysics Data System (ADS)

    Lin, Xiaogang; Wang, Rongrong; Guo, Yongcai; Gao, Chao; Guo, Xiaoen

    2012-11-01

    Primary liver cancer is one of the highest mortality malignant tumors in the world. China is a high occurrence area of primary liver cancer. Diagnosis of liver cancer, especially early diagnosis, is essential for improving patients' survival. Light scattering and measuring method is an emerging technology developed in recent decades, which has attracted a large number of biomedical researchers due to its advantages, such as fast, simple, high accuracy, good repeatability, and non-destructive. The hypothesis of this project is that there may be some different light scattering information between hepatoma cells and hepatocyte. Combined with the advantages of the dynamic light scattering method and the biological cytology, an experimental scheme to measure the light scattering information of cells was formulated. Hepatoma cells and hepatic cells were irradiated by a semiconductor laser (532 nm). And the Brookhaven BI-200SM wide-angle light scattering device and temperature control apparatus were adopted. The light scattering information of hepatoma cells and hepatic cells in vitro within the 15°C to 30°C temperature range was processed by a BI-9000AT digital autocorrelator. The following points were found: (a) the scattering intensities of human hepatic cells and hepatoma cells are nearly not affected by the temperature factor, and the former is always greater than the latter and (b) the relaxation time of hepatoma cells is longer than that of hepatic cells, and both the relaxation time are shortened with increasing temperature from 15°C to 25°C. It can be concluded that hepatoma cells could absorb more incident light than hepatic cells. The reason may be that there exists more protein and nucleic acid in cancerous cells than normal cells. Furthermore, based on the length relaxation time, a conclusion can be inferred that the Brownian movement of cancer cells is greater.

  5. Trichloroethylene toxicity in a human hepatoma cell line

    SciTech Connect

    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.

  6. Energy substrate utilization in a poorly differentiated rat hepatoma

    SciTech Connect

    Mares-Perlman, J.A.

    1987-01-01

    Metabolism of energy substrates in a transplantable, poorly differentiated rat hepatoma and the effect of high fat total parenteral nutrition (TPN) on growth of this neoplasms and host were studied. Although high-fat TPN better maintained host weight and nitrogen balance than oral feeding and did not increase tumor growth, adverse consequences of high-fat TPN were found. These included liver lipid infiltration and indications of the possible development of insulin resistance. A method for isolating fresh hepatoma cells was designed to study the metabolism of energy substrates by this neoplasms. The metabolic viability of cells obtained by this procedure in sustained incubations was demonstrated by observations of linear rates of leucine and uridine incorporation into acid-insoluble material, retention of cellular ATP and ADP content and stable rates of oxygen consumption. Cells isolated by this procedure were used to determine whether this hepatoma was capable of oxidizing fatty acids and ketones and to estimate the contribution oxidation of these substrates made to ATP production relative to glucose and glutamine. Incorporation of radiolabel from both palmitate and ..beta..-hydroxybutyrate carbon into CO/sub 2/ was observed.

  7. Repression of the albumin gene in Novikoff hepatoma cells.

    PubMed Central

    Capetanaki, Y G; Flytzanis, C N; Alonso, A

    1982-01-01

    Novikoff hepatoma cells have lost their capacity to synthesize albumin. As a first approach to study the mechanisms underlying this event, in vitro translation in a reticulocyte system was performed using total polyadenylated mRNA from rat liver and Novikoff hepatoma cells. Immunoprecipitation of the in vitro translation products with albumin-specific antibody revealed a total lack of albumin synthesis in Novikoff hepatoma, suggesting the absence of functional albumin mRNA in these cells. Titration experiments using as probe albumin cDNA cloned in pBR322 plasmid demonstrated the absence of albumin-specific sequences in both polysomal and nuclear polyadenylated and total RNA from Novikoff cells. This albumin recombinant plasmid was obtained by screening a rat liver cDNA library with albumin [32P]cDNA reverse transcribed from immuno-precipitated mRNA. The presence of an albumin-specific gene insert was documented with translation assays as well as by restriction mapping. Repression of the albumin gene at the transcriptional level was further demonstrated by RNA blotting experiments using the cloned albumin cDNA probe. Genomic DNA blots using the cloned albumin cDNA as probe did not reveal any large-scale deletions, insertions, or rearrangements in the albumin gene, suggesting that the processes involved in the suppression of albumin mRNA synthesis do not involve extensive genomic rearrangements. Images PMID:6180302

  8. Synergistic Inhibitory Effect of Hyperbaric Oxygen Combined with Sorafenib on Hepatoma Cells

    PubMed Central

    Peng, Hai-Shan; Liao, Ming-Bin; Zhang, Mei-Yin; Xie, Yin; Xu, Li; Zhang, Yao-Jun; Zheng, X. F. Steven; Wang, Hui-Yun; Chen, Yi-Fei

    2014-01-01

    Objectives Hypoxia is a common phenomenon in solid tumors, associated with chemotherapy and radiotherapy resistance, recurrence and metastasis. Hyperbaric oxygen (HBO) therapy can increase tissue oxygen pressure and content to prevent the resistance, recurrence and metastasis of cancer. Presently, Sorafenib is a first-line drug, targeted for hepatocellular carcinoma (HCC) but effective in only a small portion of patients and can induce hypoxia. The purpose of this study is to investigate the effect of HBO in combination with sorafenib on hepatoma cells. Methods Hepatoma cell lines (BEL-7402 and SK-Hep1) were treated with HBO at 2 atmosphere absolute pressure for 80 min per day or combined with sorafenib or cisplatin. At different time points, cells were tested for cell growth, colony formation, apoptosis, cell cycle and migration. Finally, miRNA from the hepatoma cells was detected by microRNA array and validated by qRT-PCR. Results Although HBO, sorafenib or cisplatin alone could inhibit growth of hepatoma cells, HBO combined with sorafenib or cisplatin resulted in much greater synergistic growth inhibition (cell proliferation and colony formation) in hepatoma cells. Similarly, the synergistic effect of HBO and sorafenib on induction of apoptosis was also observed in hepatoma cells. HBO induced G1 arrest in SK-Hep1 not in BEL-7402 cells, but enhanced cell cycle arrest induced by sorafenib in BEL-7402 treated cells. However, HBO had no obvious effect on the migration of hepatoma cells, and microRNA array analysis showed that hepatoma cells with HBO treatment had significantly different microRNA expression profiles from those with blank control. Conclusions We show for the first time that HBO combined with sorafenib results in synergistic growth inhibition and apoptosis in hepatoma cells, suggesting a potential application of HBO combined with sorafenib in HCC patients. Additionally, we also show that HBO significantly altered microRNA expression in hepatoma cells

  9. Synthesis and targeting of hexokinase to mitochondria in hepatoma cells

    SciTech Connect

    Kabir, F.; Nelson, B.D. )

    1989-10-01

    The synthesis and turnover of hexokinase has been measured in Zajdela hepatoma ascites cells labeled for short periods with ({sup 35}S)methionine. Digitonin fractionation of the labeled cells into a soluble and a membrane fraction showed that only a small part of the newly labeled hexokinase is transferred to mitochondrial binding sites. The soluble enzyme disappears, however, with a half-life of less than 2 h. Glucose had no effect on the stability of the soluble enzyme in intact cells. Our experiments suggest that Zajdela cell hexokinase is synthesized in excess of binding sites and that the excess enzyme is not stable.

  10. Effect of hepatoma H22 on lymphatic endothelium in vitro

    PubMed Central

    Yu, Hua; Zhou, Hong-Zhi; Wang, Chun-Mei; Gu, Xiao-Ming; Pan, Bo-Rong

    2004-01-01

    AIM: To determine the effect of metastatic hepatoma cells on lymphangioma-derived endothelium, and to establish in vitro model systems for assessing metastasis-related response of lymphatic endothelium. METHODS: Benign lymphangioma, induced by intraperitoneal injection of the incomplete Freund’s adjuvant in BALB/c mice, was embedded in fibrin gel or digested and then cultured in the conditioned medium derived from hepatoma H22. Light and electron microscopy, and the transwell migration assay were used to determine the effect of H22 on tissue or cell culture. Expressions of Flt-4, c-Fos, proliferating cell nuclear antigen (PCNA), and inducible nitric oxide synthase (iNOS) in cultured cells, and content of nitric oxide in culture medium were also examined. RESULTS: The embedded lymphangioma pieces gave rise to array of capillaries, while separated cells from lymphangioma grew to a cobblestone-like monolayer. H22 activated growth and migration of the capillaries and cells, induced expressions of Flt-4, c-Fos, PCNA and iNOS in cultured cells, and significantly increased the content of NO in the culture medium. CONCLUSION: Lymphangioma-derived cells keep the differentiated phenotypes of lymphatic endothelium, and the models established in this study are feasible for in vitro study of metastasis-related response of lymphatic endothelium. PMID:15526361

  11. 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

  12. Membrane Glycolipids Content Variety in Gastrointestinal Tumors and Transplantable Hepatomas in Mice

    PubMed Central

    Lv, Jun; Lv, Can Qun; Wang, Bo-Liang; Mei, Ping; Xu, Lei

    2016-01-01

    Background The aim of this study was to investigate the variety of plasma contents of membrane glycolipids in 65 gastrointestinal tumors and 31 transplant hepatomas in mice. Material/Methods The experimental model was a transplantable murine hepatoma. Experimental mice were divided into 3 groups. Results The LSA and TSA content in the 2 groups were significantly difference (p<0.01), and were significantly lower in the therapeutic group than in the control group (p<0.01). Conclusions These results indicate that membrane glycolipids index LSA and TSA are sensitive markers in gastrointestinal tumors. In the transplanted hepatomas in mice, they may be considered as ancillary indicators for judging the therapeutic effect of hepatoma. PMID:27554918

  13. Permissiveness of human hepatoma cell lines for HCV infection

    PubMed Central

    2012-01-01

    Background Although primary and established human hepatoma cell lines have been evaluated for hepatitis C virus (HCV) infection in vitro, thus far only Huh7 cells have been found to be highly permissive for infectious HCV. Since our understanding of the HCV lifecycle would benefit from the identification of additional permissive cell lines, we assembled a panel of hepatic and non-hepatic cell lines and assessed their ability to support HCV infection. Here we show infection of the human hepatoma cell lines PLC/PRF/5 and Hep3B with cell culture-derived HCV (HCVcc), albeit to lower levels than that achieved in Huh7 cells. To better understand the reduced permissiveness of PLC and Hep3B cells for HCVcc infection, we performed studies to evaluate the ability of each cell line to support specific steps of the viral lifecycle (i.e. entry, replication, egress and spread). Results We found that while the early events in HCV infection (i.e. entry plus replication initiation) are cumulatively equivalent or only marginally reduced in PLC and Hep3B cells, later steps of the viral life cycle such as steady-state replication, de novo virus production and/or spread are impaired to different degrees in PLC and Hep3B cultures compared to Huh7 cell cultures. Interestingly, we also observed that interferon stimulated gene (i.e. ISG56) expression was significantly and differentially up-regulated in PLC and Hep3B cells following viral infection. Conclusions We conclude that the restrictions observed later during HCV infection in these cell lines could in part be attributed to HCV-induced innate signaling. Nevertheless, the identification of two new cell lines capable of supporting authentic HCVcc infection, even at reduced levels, expands the current repertoire of cell lines amendable for the study of HCV in vitro and should aid in further elucidating HCV biology and the cellular determinants that modulate HCV infection. PMID:22273112

  14. Screening and identification of a specific peptide for targeting hypoxic hepatoma cells.

    PubMed

    Liu, Yiming; Xia, Xiangwen; Wang, Yong; Li, Xin; Zhou, Guofeng; Liang, Huiming; Feng, Gansheng; Zheng, Chuansheng

    2016-08-01

    The biological behaviors of residual hepatoma cells after transarterial embolization therapy, which exist in a hypoxic or even anaerobic tumor microenvironment, differ from the tumor cells under normoxic conditions. This study aimed to use a phage display peptide library for in vivo and in vitro screening to obtain a peptide which could specifically bind to hypoxic hepatoma cells, allowing further targeted diagnosis and treatment for liver cancer. In this study, hypoxic hepatoma cells HepG2 (targeted cells), and normal liver cells HL-7702 (control cells), were utilized to perform three rounds of in vitro screening using a phage-displayed 7-mer peptide library. In addition, hypoxic HepG2 were subcutaneously injected into nude mice to establish a hepatocarcinoma model, followed by performing three rounds of in vivo screening on the phages identified from the in vitro screening. The products from the screening were further identified using ELISA and immunofluorescence staining on cells and tissues. The results indicated that the P11 positive clone had the highest binding effect with hypoxic hepatoma cells. The sequence of the exogenous insert fragment of P11 positive clone was obtained by sequencing: GSTSFSK. The binding assay indicated that GSTSFSK could specifically bind to hypoxic hepatoma cells and hepatocarcinoma tissues. This 7-mer peptide has the potential to be developed as an useful molecular to the targeting diagnosis and treatment of residual hepatoma cells after transarterial chemoembolization. PMID:27381416

  15. Regulated expression of erythropoietin by two human hepatoma cell lines

    SciTech Connect

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  16. 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. PMID:23830978

  17. Coordinate secretion of mouse alphafetoprotein, mouse albumin and rat albumin by mouse hepatoma-rat hepatoma hybrid cells.

    PubMed

    Cassio, D; Hassoux, R; Dupiers, M; Uriel, J; Weiss, M C

    1980-09-01

    Mouse heptoma cells that secrete large amounts of alpha-fetoprotein (AFP) and albumin have been crossed with rat hepatoma cells that secret only albumin, and in relatively small amounts, to investigate the influence of each parental genome upon the expression of serum proteins. All of the ten independent hybrid clones examined produce mouse AFP and both mouse and rat albumin; none produces rat AFP. The absence of production of rat AFP by the hybrids suggests that different mechanisms are involved in the initiation and in the maintenance of expression of this function. The secretion of the three proteins by the hybrid cells is coordinate: Whatever the growth phase (exponential or stationary) and irrespective of the amounts produced over a wide range, the ratio secreted of mouse AFP to mouse albumin is near to one, and that of mouse albumin to rat albumin is near to five. In addition, even though the pattern of protein secretion during the growth cycle of hybrid cells is different from those of both parents, the products of both parental genomes conform to the new hybrid pattern. Finally, some hybrids secrete less of the proteins with increasing numbers of cell generations, yet all three continue to be secreted in coordinate fashion. Since the rates of secretion of serum proteins probably reflect their rates of synthesis, we conclude that coordinate secretion indicates coordinate synthesis, and may reflect coordinate transcription of the relevant genes. PMID:6158520

  18. 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.

  19. Rapid internalization of the insulin receptor in rat hepatoma cells

    SciTech Connect

    Backer, J.M.; White, M.F.; Kahn, C.R.

    1987-05-01

    The authors have studied the internalization of the insulin receptor (IR) in rat hepatoma cells (Fao). The cells were surface-iodinated at 4C, stimulated with insulin at 37C, and then cooled rapidly, trypsinized at 4C and solubilized. The IR was immunoprecipitated with a specific antibody, and internalization of the IR was assessed by the appearance of trypsin-resistant bands on SDS-PAGE. Insulin induced the internalization of surface receptors with a t 1/2 of 9-10 mins; cells not exposed to insulin internalized less than 20% of the IR during 1 h at 37C. Further experiments demonstrated that the accumulation of trypsin-resistant IR paralleled a loss of receptor from the cell surface. Insulin-stimulated cells were chilled and iodinated at 4C, followed by solubilization, immunoprecipitation and SDS-PAGE; alternatively, insulin-stimulated cells were chilled, surface-bound ligand removed by washing the cells at pH 4.2, and specific ( SVI)insulin binding measured at 4C. Both techniques confirmed the disappearance of IR from the cell surface at rates comparable to the insulin-stimulated internalization described above. The total amount of phosphotyrosine-containing IR, as assessed by immunoprecipitation with an anti-phosphotyrosine antibody, remained constant during this time interval, suggesting that active kinase is translocated into the cell. In summary, the authors data indicate that insulin binding increases the rate of IR internalization of Fao cells. This relocation may facilitate the interaction of the activated tyrosine kinase in the IR with intracellular substrates, thus transmitting the insulin signal to metabolic pathways.

  20. The triad of lichen planus, thymoma and liver cirrhosis-hepatoma. First reported case.

    PubMed

    Hassan, J A; Saadiah, S; Roslina, A M; Atan, M; Masir, N; Hussein, S; Ganesapillai, T

    2000-07-01

    We describe a patient with liver cirrhosis who presented with erosive oral and cutaneous lichen planus (LP) and incidentally was found simultaneously to have thymoma and hepatoma. We support the notion forwarded earlier that LP and chronic liver disease is more than a mere coincidence and that there is a non-coincidental association between LP and thymoma. We believe this is also the first reported case in the English Literature of coexistence of the three condition LP, thymoma and hepatoma complicating liver disease. PMID:22977389

  1. The Triad of Lichen Planus, Thymoma and Liver Cirrhosis-Hepatoma. First Reported Case

    PubMed Central

    Hassan, J. A.; Saadiah, S; Roslina, A M; Atan, M; Masir, Noraidah; Hussein, S; Ganesapillai, T

    2000-01-01

    We describe a patient with liver cirrhosis who presented with erosive oral and cutaneous lichen planus (LP) and incidentally was found simultaneously to have thymoma and hepatoma. We support the notion forwarded earlier that LP and chronic liver disease is more than a mere coincidence and that there is a non-coincidental association between LP and thymoma. We believe this is also the first reported case in the English Literature of coexistence of the three condition LP, thymoma and hepatoma complicating liver disease. PMID:22977389

  2. End products of glutamine oxidation in MC-29 virus-induced chicken hepatoma mitochondria.

    PubMed

    Matsuno, T

    1989-10-01

    Products of glutamine metabolism were examined in the MC-29 virus-induced chicken hepatoma mitochondria incubated in vitro. Glutamine oxidation proceeded in the tumor mitochondria exclusively via a pathway involving glutamic-oxalacetic transaminase. Malate stimulated aspartate production from glutamine, while pyruvate exerted suppressive effect on aspartate production with little alanine formation. The mitochondria of this hepatoma are unique in that the metabolic pattern and response to malate and pyruvate are essentially inconsistent with those reported in normal cells as well as those proposed by Moreadith and Lehninger in various tumor cells. PMID:2571353

  3. Hepatoma-derived growth factor/nucleolin axis as a novel oncogenic pathway in liver carcinogenesis.

    PubMed

    Chen, San-Cher; Hu, Tsung-Hui; Huang, Chao-Cheng; Kung, Mei-Lang; Chu, Tian-Huei; Yi, Li-Na; Huang, Shih-Tsung; Chan, Hoi-Hung; Chuang, Jiin-Haur; Liu, Li-Feng; Wu, Han-Chung; Wu, Deng-Chyang; Chang, Min-Chi; Tai, Ming-Hong

    2015-06-30

    Hepatoma-derived growth factor (HDGF) overexpression is involved in liver fibrosis and carcinogenesis. However, the receptor(s) and signaling for HDGF remain unclear. By using affinity chromatography and proteomic techniques, nucleolin (NCL) was identified and validated as a HDGF-interacting membrane protein in hepatoma cells. Exogenous HDGF elicited the membrane NCL accumulation within 0.5 hour by protein stabilization and transcriptional NCL upregulation within 24 hours. Blockade of surface NCL by antibodies neutralization potently suppressed HDGF uptake and HDGF-stimulated phosphatidylinositol 3-kinase (PI3K)/Akt signaling in hepatoma cells. By using rescectd hepatocellular carcinoma (HCC) tissues, immunohistochemical analysis revealed NCL overexpression was correlated with tumour grades, vascular invasion, serum alpha-fetoprotein levels and the poor survival in HCC patients. Multivariate analysis showed NCL was an independent prognostic factor for survival outcome of HCC patients after surgery. To delineate the role of NCL in liver carcinogenesis, ectopic NCL overexpression promoted the oncogenic behaviours and induced PI3K/Akt activation in hepatoma cells. Conversely, NCL knockdown by RNA interference attenuated the oncogenic behaviours and PI3K/Akt signaling, which could be partially rescued by exogenous HDGF supply. In summary, this study provides the first evidence that surface NCL transmits the oncogenic signaling of HDGF and facilitates a novel diagnostic and therapeutic target for HCC. PMID:25938538

  4. Biodistribution of TAT-LHRH conjugated chitosan/DNA nanoparticles in the mice bearing hepatoma xenografts.

    PubMed

    Liu, Lanxia; Wang, Hai; Liu, Qi; Duan, Mingli; Dong, Xia; Zhu, Dunwan; Zhu, Yingjun; Leng, Xigang

    2016-10-01

    Hepatocellular carcinoma (HCC) is the fifth most prevalent malignancy and the third leading cause of cancer-related deaths worldwide. More effective cures for HCC patients are urgently needed, of which gene therapy is among those with the most potential. We previously developed a novel gene carrier by conjugating low molecular weight chitosan with TAT (transactivator of transcription) peptide and LHRH (luteinizing hormone-releasing hormone) analog, with the resultant TAT-LHRH-chitosan conjugate (TLC) demonstrating high selectivity for hepatoma cells in vitro. However, it remains unclear whether TLC can deliver the genes to the target organs and tissues in vivo, which is one of the critical features determining their medical application potential. The current study further investigated the in vivo distribution of TLC/DNA nanoparticles (TLCDNPs) in the nude mice with subcutaneous hepatoma xenografts. It was found that TLCDNPs delayed the renal clearance of DNA and prolonged its circulation time as compared with CS/DNA complexes (CDNPs) and naked DNA, but failed to demonstrate enhanced accumulation of DNA in the hepatoma xenografts. The mechanisms regarding the failure of TLCDNPs' tumor targeting in the mice bearing subcutaneous hepatoma xenografts remain unclear and need to be further addressed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2394-2400, 2016. PMID:27153405

  5. Hepatoma-derived growth factor/nucleolin axis as a novel oncogenic pathway in liver carcinogenesis

    PubMed Central

    Huang, Chao-Cheng; Kung, Mei-Lang; Chu, Tian-Huei; Yi, Li-Na; Huang, Shih-Tsung; Chan, Hoi-Hung; Chuang, Jiin-Haur; Liu, Li-Feng; Wu, Han-Chung; Wu, Deng-Chyang; Chang, Min-Chi; Tai, Ming-Hong

    2015-01-01

    Hepatoma-derived growth factor (HDGF) overexpression is involved in liver fibrosis and carcinogenesis. However, the receptor(s) and signaling for HDGF remain unclear. By using affinity chromatography and proteomic techniques, nucleolin (NCL) was identified and validated as a HDGF-interacting membrane protein in hepatoma cells. Exogenous HDGF elicited the membrane NCL accumulation within 0.5 hour by protein stabilization and transcriptional NCL upregulation within 24 hours. Blockade of surface NCL by antibodies neutralization potently suppressed HDGF uptake and HDGF-stimulated phosphatidylinositol 3-kinase (PI3K)/Akt signaling in hepatoma cells. By using rescectd hepatocellular carcinoma (HCC) tissues, immunohistochemical analysis revealed NCL overexpression was correlated with tumour grades, vascular invasion, serum alpha-fetoprotein levels and the poor survival in HCC patients. Multivariate analysis showed NCL was an independent prognostic factor for survival outcome of HCC patients after surgery. To delineate the role of NCL in liver carcinogenesis, ectopic NCL overexpression promoted the oncogenic behaviours and induced PI3K/Akt activation in hepatoma cells. Conversely, NCL knockdown by RNA interference attenuated the oncogenic behaviours and PI3K/Akt signaling, which could be partially rescued by exogenous HDGF supply. In summary, this study provides the first evidence that surface NCL transmits the oncogenic signaling of HDGF and facilitates a novel diagnostic and therapeutic target for HCC. PMID:25938538

  6. A Long Noncoding RNA Perturbs the Circadian Rhythm of Hepatoma Cells to Facilitate Hepatocarcinogenesis12

    PubMed Central

    Cui, Ming; Zheng, Minying; Sun, Baodi; Wang, Yue; Ye, Lihong; Zhang, Xiaodong

    2015-01-01

    Clock circadian regulator (CLOCK)/brain and muscle arnt-like protein-1 (BMAL1) complex governs the regulation of circadian rhythm through triggering periodic alterations of gene expression. However, the underlying mechanism of circadian clock disruption in hepatocellular carcinoma (HCC) remains unclear. Here, we report that a long noncoding RNA (lncRNA), highly upregulated in liver cancer (HULC), contributes to the perturbations in circadian rhythm of hepatoma cells. Our observations showed that HULC was able to heighten the expression levels of CLOCK and its downstream circadian oscillators, such as period circadian clock 1 and cryptochrome circadian clock 1, in hepatoma cells. Strikingly, HULC altered the expression pattern and prolonged the periodic expression of CLOCK in hepatoma cells. Mechanistically, the complementary base pairing between HULC and the 5' untranslated region of CLOCK mRNA underlay the HULC-modulated expression of CLOCK, and the mutants in the complementary region failed to achieve the event. Moreover, immunohistochemistry staining and quantitative real-time polymerase chain reaction validated that the levels of CLOCK were elevated in HCC tissues, and the expression levels of HULC were positively associated with those of CLOCK in clinical HCC samples. In functional experiments, our data exhibited that CLOCK was implicated in the HULC-accelerated proliferation of hepatoma cells in vitro and in vivo. Taken together, our data show that an lncRNA, HULC, is responsible for the perturbations in circadian rhythm through upregulating circadian oscillator CLOCK in hepatoma cells, resulting in the promotion of hepatocarcinogenesis. Thus, our finding provides new insights into the mechanism by which lncRNA accelerates hepatocarcinogenesis through disturbing circadian rhythm of HCC. PMID:25622901

  7. Baicalein inhibits the migration and invasive properties of human hepatoma cells

    SciTech Connect

    Chiu, Yung-Wei; Lin, Tseng-Hsi; Huang, Wen-Shih; Teng, Chun-Yuh; Liou, Yi-Sheng; Kuo, Wu-Hsien; Lin, Wea-Lung; Huang, Hai-I; Tung, Jai-Nien; Huang, Chih-Yang; Liu, Jer-Yuh; Wang, Wen-Hung; Hwang, Jin-Ming

    2011-09-15

    Flavonoids have been demonstrated to exert health benefits in humans. We investigated whether the flavonoid baicalein would inhibit the adhesion, migration, invasion, and growth of human hepatoma cell lines, and we also investigated its mechanism of action. The separate effects of baicalein and baicalin on the viability of HA22T/VGH and SK-Hep1 cells were investigated for 24 h. To evaluate their invasive properties, cells were incubated on matrigel-coated transwell membranes in the presence or absence of baicalein. We examined the effect of baicalein on the adhesion of cells, on the activation of matrix metalloproteinases (MMPs), protein kinase C (PKC), and p38 mitogen-activated protein kinase (MAPK), and on tumor growth in vivo. We observed that baicalein suppresses hepatoma cell growth by 55%, baicalein-treated cells showed lower levels of migration than untreated cells, and cell invasion was significantly reduced to 28%. Incubation of hepatoma cells with baicalein also significantly inhibited cell adhesion to matrigel, collagen I, and gelatin-coated substrate. Baicalein also decreased the gelatinolytic activities of the matrix metalloproteinases MMP-2, MMP-9, and uPA, decreased p50 and p65 nuclear translocation, and decreased phosphorylated I-kappa-B (IKB)-{beta}. In addition, baicalein reduced the phosphorylation levels of PKC{alpha} and p38 proteins, which regulate invasion in poorly differentiated hepatoma cells. Finally, when SK-Hep1 cells were grown as xenografts in nude mice, intraperitoneal (i.p.) injection of baicalein induced a significant dose-dependent decrease in tumor growth. These results demonstrate the anticancer properties of baicalein, which include the inhibition of adhesion, invasion, migration, and proliferation of human hepatoma cells in vivo. - Highlight: > Baicalein inhibits several essential steps in the onset of metastasis.

  8. A long noncoding RNA perturbs the circadian rhythm of hepatoma cells to facilitate hepatocarcinogenesis.

    PubMed

    Cui, Ming; Zheng, Minying; Sun, Baodi; Wang, Yue; Ye, Lihong; Zhang, Xiaodong

    2015-01-01

    Clock circadian regulator (CLOCK)/brain and muscle arnt-like protein-1 (BMAL1) complex governs the regulation of circadian rhythm through triggering periodic alterations of gene expression. However, the underlying mechanism of circadian clock disruption in hepatocellular carcinoma (HCC) remains unclear. Here, we report that a long noncoding RNA (lncRNA), highly upregulated in liver cancer (HULC), contributes to the perturbations in circadian rhythm of hepatoma cells. Our observations showed that HULC was able to heighten the expression levels of CLOCK and its downstream circadian oscillators, such as period circadian clock 1 and cryptochrome circadian clock 1, in hepatoma cells. Strikingly, HULC altered the expression pattern and prolonged the periodic expression of CLOCK in hepatoma cells. Mechanistically, the complementary base pairing between HULC and the 5' untranslated region of CLOCK mRNA underlay the HULC-modulated expression of CLOCK, and the mutants in the complementary region failed to achieve the event. Moreover, immunohistochemistry staining and quantitative real-time polymerase chain reaction validated that the levels of CLOCK were elevated in HCC tissues, and the expression levels of HULC were positively associated with those of CLOCK in clinical HCC samples. In functional experiments, our data exhibited that CLOCK was implicated in the HULC-accelerated proliferation of hepatoma cells in vitro and in vivo. Taken together, our data show that an lncRNA, HULC, is responsible for the perturbations in circadian rhythm through upregulating circadian oscillator CLOCK in hepatoma cells, resulting in the promotion of hepatocarcinogenesis. Thus, our finding provides new insights into the mechanism by which lncRNA accelerates hepatocarcinogenesis through disturbing circadian rhythm of HCC. PMID:25622901

  9. Merocyanine 540 and Photofrin II as photosensitizers for in vitro killing of duck hepatitis B virus and human hepatoma cells

    NASA Astrophysics Data System (ADS)

    Lin, Tsung-I.; Shien, Yong-Shau; Kao, Ming-Chien

    1994-03-01

    The feasibility of using merocyanine 540 (MC 540) and Photofrin II (PII) as effective photodynamic therapeutic (PDT) agents for killing hepatoma cells and duck hepatitis B virus (DHBV) in vitro was investigated. Cultured duck hepatocytes infected with DHBV and hepatoma cells, Hep 3B and HCC 36, were used as models. MC 540 and PII effectively inhibits the DHBV growth by 90 - 99% in a dose- and light-dependent manner. Photodynamic killing of MC 540 in the two hepatoma cell lines results in 94 - 99% growth inhibition. However, both photosensitizers exhibit dark cytotoxicity (37 - 56%). The present results suggest that MC 540 and PII could be promising and effective photodynamic agents for killing HBV and hepatoma cells.

  10. The characteristic gene expressions of MAPK phosphatases 1 and 2 in hepatocarcinogenesis, rat ascites hepatoma cells, and regenerating rat liver.

    PubMed

    Yokoyama, A; Karasaki, H; Urushibara, N; Nomoto, K; Imai, Y; Nakamura, K; Mizuno, Y; Ogawa, K; Kikuchi, K

    1997-10-29

    mRNA levels of mitogen-activated protein kinase phosphatases, MKP-1 and MKP-2, were determined during chemical hepatocarcinogenesis and during regeneration of rat liver. In chemical hepatocarcinogenesis, the mRNA levels of MKP-1 were increased in primary hepatomas but decreased in rat ascites hepatomas as compared with normal liver. MKP-2 was undetectable in normal liver but strongly expressed in hepatomas. The MKP-2 mRNA level was increased with expression of malignant phenotypes in hepatomas. In regenerating liver, the mRNA level of MKP-1 increased immediately but transiently after partial hepatectomy, and peaked again on day 10, the time when hepatocytes cease proliferation. The elevated expression of MKP-1 on day 10 suggests some roles of MKP-1 as a negative regulator in hepatocyte proliferation. PMID:9367840

  11. A Gelatinases-targeting scFv-based Fusion Protein Shows Enhanced Antitumour Activity with Endostar against Hepatoma.

    PubMed

    Gao, Ruijuan; Li, Liang; Shang, Boyang; Zhao, Chunyan; Sheng, Weijin; Li, Diandong

    2015-08-01

    Gelatinases play important roles in tumour invasion and metastasis and are thus considered promising targets for cancer therapy. In this study, a new single-chain variable fragment (scFv)-based fusion protein Fv-LDP, composed of the anti-gelatinases scFv and lidamycin apoprotein (LDP), was prepared, and its combination with angiogenesis inhibitor Endostar was then investigated. The fusion protein Fv-LDP specifically bound to various tumour cells, and its binding capability to human pulmonary giant cell carcinoma (PG) cells was higher than that of LDP. Fv-LDP inhibited the expression and secretion of gelatinases and could be internalized into tumour cells via endocytosis. Fv-LDP also suppressed the growth of human hepatoma cells and murine hepatoma 22 transplanted in Kunming mice in various degrees. In addition, Endostar could enhance the synergistic or additive inhibition of Fv-LDP on the growth, migration or invasion of human hepatoma cells shown by a colony formation assay and a transwell-based migration or invasion assay, respectively. In vivo, Fv-LDP/Endostar combination showed a significantly synergistic effect on the growth of a human hepatoma xenograft, with an inhibition rate of 80.8% compared with the Fv-LDP (44.1%) or Endostar (8.9%)-treated group. The above-mentioned results indicate that the fusion protein Fv-LDP is effective against transplantable hepatoma in mice and human hepatoma xenografts in athymic mice. Moreover, Endostar can potentiate the inhibition effect of Fv-LDP on the growth of human hepatoma cells and xenografts. These data will provide a new combined strategy for improving the therapeutic efficacy of treatments for hepatoma or other gelatinase-overexpressing tumours. PMID:25615234

  12. Astaxanthin Inhibits Proliferation and Induces Apoptosis and Cell Cycle Arrest of Mice H22 Hepatoma Cells.

    PubMed

    Shao, Yiye; Ni, Yanbo; Yang, Jing; Lin, Xutao; Li, Jun; Zhang, Lixia

    2016-01-01

    BACKGROUND It is widely recognized that astaxanthin (ASX), a member of the carotenoid family, has strong biological activities including antioxidant, anti-inflammation, and immune-modulation activities. Previous studies have confirmed that ASX can effectively inhibit hepatoma cells in vitro. MATERIAL AND METHODS MTT was used to assay proliferation of mice H22 cells, and flow cytometry was used to determine apoptosis and cell cycle arrest of H22 cells in vitro and in vivo. Moreover, anti-tumor activity of ASX was observed in mice. RESULTS ASX inhibited the proliferation of H22 cells, promoted cell necrosis, and induced cell cycle arrest in G2 phase in vitro and in vivo. CONCLUSIONS This study indicated that ASX can inhibit proliferation and induce apoptosis and cell cycle arrest in mice H22 hepatoma cells in vitro and in vivo. PMID:27333866

  13. Astaxanthin Inhibits Proliferation and Induces Apoptosis and Cell Cycle Arrest of Mice H22 Hepatoma Cells

    PubMed Central

    Shao, Yiye; Ni, Yanbo; Yang, Jing; Lin, Xutao; Li, Jun; Zhang, Lixia

    2016-01-01

    Background It is widely recognized that astaxanthin (ASX), a member of the carotenoid family, has strong biological activities including antioxidant, anti-inflammation, and immune-modulation activities. Previous studies have confirmed that ASX can effectively inhibit hepatoma cells in vitro. Material/Methods MTT was used to assay proliferation of mice H22 cells, and flow cytometry was used to determine apoptosis and cell cycle arrest of H22 cells in vitro and in vivo. Moreover, anti-tumor activity of ASX was observed in mice. Results ASX inhibited the proliferation of H22 cells, promoted cell necrosis, and induced cell cycle arrest in G2 phase in vitro and in vivo. Conclusions This study indicated that ASX can inhibit proliferation and induce apoptosis and cell cycle arrest in mice H22 hepatoma cells in vitro and in vivo. PMID:27333866

  14. Glucocorticoid-Dependent Complementation of a Hepatoma Cell Variant Defective in Viral Glycoprotein Sorting

    NASA Astrophysics Data System (ADS)

    John, Nancy J.; Bravo, Deborah A.; Haffar, Omar K.; Firestone, Gary L.

    1988-02-01

    We have utilized the rat hepatoma (HTC) cell sorting variant CR4 to examine the glucocorticoid-regulated pathways that localize mouse mammary tumor virus glycoproteins to the cell surface. The defective sorting of cell surface mouse mammary tumor virus glycoproteins in CR4 cells was complemented after fusion with either normal rat hepatocytes or uninfected HTC cells. Indirect immunofluorescence of transient heterokaryons revealed that the regulated localization of mouse mammary tumor virus glycoproteins was dependent upon glucocorticoid treatment and required de novo RNA and protein synthesis. Thus, a glucocorticoid-regulated trafficking activity, unrelated to mouse mammary tumor virus sequences, which is induced in both adult rat liver and cultured hepatoma cells, can act in trans to mediate an intracellular sorting pathway for membrane glycoproteins.

  15. Creation of a murine orthotopic hepatoma model with intra-abdominal metastasis

    PubMed Central

    Harris, Jamie; Kajdacsy-Balla, Andre; Chiu, Bill

    2016-01-01

    Aim: To create an orthotopic hepatoma model with local metastasis monitored with ultrasound could be created as a platform for testing new treatments. Background: Hepatoma accounts for 25% of liver tumors in children with poor overall survival. Intraabdominal metastasis are present in 35% of patients at time of diagnosis. We hypothesized that an orthotopic tumor model with local metastasis could be created as a platform for testing treatment modalities and could be monitored with ultrasound. Patients and methods: One million human hepatoma cells (Hep3B) were injected into the left lobe of the liver of immunocompromised mice. Tumor volume was monitored with high frequency-ultrasound until it reached 1,000mm3. At that time animals were sacrificed and examined for gross metastatic disease. Tumor sections were analyzed with hematoxylin and eosin (H&E) staining. Results: Tumor formed in 8/15 mice. The tumor was detected as small as 19.59mm3 on ultrasound. Of the forming tumors, tumor size was 145±177.93mm3 at 60 days post-injection, 665±650.39mm3 at 67 days, and reached >1000mm3 by 76.6±9.9 days. At necropsy, four mice (50%) had tumor only within the liver, four (50%) had additional tumors in omentum, pelvis and peritoneum. H&E showed tumor within the normal liver parenchyma, with multiple mitotic figures, small areas of necrosis, and hemorrhage within the tumor. Conclusion: We have successfully established an orthotopic hepatoma murine model, with a local metastatic rate of 50%. Non-invasive tumor monitoring is feasible via ultrasound. PMID:27458509

  16. Anti-hepatoma activity of a novel compound glaucocalyxin H in vivo and in vitro.

    PubMed

    Hai, Guangfan; Zhang, Chong; Jia, Yanlong; Bai, Suping; Han, Jinfen; Guo, Lanqing; Cui, Taizhen; Niu, Bingxuan; Huang, Feng; Song, Yu

    2015-06-01

    Glaucocalyxin H (GLH) is a new compound isolated from a traditional Chinese medical herb Isodon japonica var. glaucocalyx which has been used for folk medicine. This study was carried out for the first time to investigate the potential role of GLH in anti-hepatoma activity and underlying mechanisms in it. GLH could inhibit the growth of tumor in mice and induce HepG2 cells to death as assessed by the tumor reduction assay, toxic assay, morphological change, and survival rate assay. Many antitumor drugs originated from plants could inhibit the growth of tumor by inducing cells to apoptosis. The morphological changes of HepG2 cells treated with different concentrations of GLH under fluorescence and electron microscope and apoptotic rates were detected to verify its effect on apoptosis. As shown in the study, GLH could induce HepG2 cells to apoptosis in a dose-dependent manner. Bcl2 and Bax proteins played important roles in apoptosis and the disequilibrium between Bcl2 and Bax might result in apoptosis. The expression of Bax protein was upregulated and Bcl2 protein was downregulated in HepG2 cells treated with GLH assessed by Western blotting, and they were in a dose-dependent manner. Taken together, GLH can inhibit the growth of hepatoma cells in vivo and in vitro by inducing cell apoptosis due to the decreased Bcl2 and increased Bax proteins suggesting that GLH could be a potential candidate as an anti-hepatoma agent for the therapeutic treatment of hepatoma. PMID:25374342

  17. In vitro cultivation of the exoerythrocytic stage of Plasmodium berghei in irradiated hepatoma cells

    SciTech Connect

    Hollingdale, M.R.; Leland, P.; Sigler, C.I.

    1985-01-01

    Growth of cultures of human hepatoma cells was inhibited by exposure to doses of gamma irradiation as low as 1000 rad., and the monolayers remained viable for up to 35 days. Irradiated cells were at least as susceptible to Plasmodium berghei sporozoite invasion as non-irradiated cells, and supported the entire exoerythrocytic cycle producing more infectious merozoites. Irradiated cultures may have use for culture of human malarias, and drug studies requiring synchronous cultures.

  18. In vitro infectivity of irradiated Plasmodium berghei sporozoites to cultured hepatoma cells

    SciTech Connect

    Sigler, C.I.; Leland, P.; Hollingdale, M.R.

    1984-07-01

    The invasion of gamma-irradiated Plasmodium berghei sporozoites into cultured hepatoma cells and their transformation into trophozoites was similar to invasion and transformation of non-irradiated sporozoites. However, trophozoites from irradiated sporozoites did not further develop into schizonts, but persisted within the cells for up to 3 days. Sporozoite surface protective antigen was present in trophozoites from irradiated and non-irradiated sporozoites, suggesting that hepatocyte antigen processing may contribute to the induction of anti-malarial immunity.

  19. Expression of activins C and E induces apoptosis in human and rat hepatoma cells.

    PubMed

    Vejda, Susanne; Erlach, Natascha; Peter, Barbara; Drucker, Claudia; Rossmanith, Walter; Pohl, Jens; Schulte-Hermann, Rolf; Grusch, Michael

    2003-11-01

    Activins C and E (homodimers of the betaC and betaE subunits), which are almost exclusively expressed in the liver, are members of the transforming growth factor beta (TGFbeta) superfamily of growth factors. We examined their expression in three different hepatoma cell lines and found that, compared with normal liver or primary hepatocytes, human hepatoblastoma (HepG2), human hepatocellular carcinoma (Hep3B) and rat hepatoma (H4IIEC3) cells have either completely lost or drastically reduced the expression of activins C and E. In order to elucidate the biological function of these proteins we transiently transfected HepG2, Hep3B and H4IIEC3 cell lines with rat activin betaC or betaE cDNA to study the consequences of restoring activin expression in hepatoma cells. Transfection with activin betaA, a known inhibitor of hepatic DNA synthesis and inducer of apoptosis, served as a positive control. We found that transfection of the three cell lines with activin betaC or betaE, as well as with activin betaA, reduced the increase in cell number by up to 40% compared with cells transfected with a control plasmid. Co-culture with a CHO cell clone secreting activin C also inhibited HepG2 cell multiplication. Furthermore, the three hepatoma cell lines studied showed an enhanced rate of apoptosis and elevated levels of active caspases in response to activin transfection. These results indicate that activins C and E share the potential to induce apoptosis in liver derived cell lines with activin A and TGFbeta1. PMID:12949049

  20. PLC/PRF/5 (Alexander) hepatoma cell line: further characterization and studies of infectivity.

    PubMed Central

    Daemer, R J; Feinstone, S M; Alexander, J J; Tully, J G; London, W T; Wong, D C; Purcell, R H

    1980-01-01

    The Alexander hepatoma cell line, PLC/PRF/5, was studied for evidence of hepatitis B virus markers and alpha-fetoprotein. Only hepatitis B surface antigen and alpha-fetoprotein were detected. Induction experiments with 5-iodo-2'-deoxyuridine and inoculation of chimpanzees with whole cells or tissue culture fluid did not reveal evidence of synthesis of additional hepatitis B virus markers or of production of infectious virus. Images Fig. 1 Fig. 2 Fig. 3 PMID:6160110

  1. Anti-hepatoma effect of safrole from Cinnamomum longepaniculatum leaf essential oil in vitro.

    PubMed

    Song, Xu; Yin, Zhongqiong; Ye, Kuichuan; Wei, Qin; Jia, Renrong; Zhou, Lijun; Du, Yonghua; Xu, Jiao; Liang, Xiaoxia; He, Changliang; Shu, Gang; Yin, Lizi; Lv, Cheng

    2014-01-01

    The aim of this study was to study the anti-hepatoma effect of safrole and elucidate its molecular mechanism, the human hepatoma BEL-7402 cells were incubated with various concentrations (40, 80, 160, 320 and 640 μg/ml) of safrole and the cell proliferation and apoptosis were evaluated. The results showed that both the cell proliferation determined by 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium brominde (MTT) assay and cell colony determined by soft agar assay were significantly suppressed by safrole in a dose-time-dependent manner. Characteristic morphological and biochemical changes associated with apoptosis, including cells shrinkage, deformation and vacuolization of mitochondria, nuclear chromatin condensation and fragmentation, formation of apoptotic bodies were observed when treated with safrole for 24 h and 48 h. Cell cycle changes evaluated by flow cytometry analysis showed that the safrole could induce accumulation of cells arrested at G1 and S phases of the cell cycle. These results demonstrated that safrole is potent anti-hepatoma agent and the underlying mechanism may be attributed to suppress tumor cell growth by inducing cell apoptosis. PMID:24966935

  2. Hepatoma-Targeted Radionuclide Immune Albumin Nanospheres: 131I-antiAFPMcAb-GCV-BSA-NPs

    PubMed Central

    Lin, Mei; Huang, Junxing; Zhang, Dongsheng; Jiang, Xingmao; Zhang, Jia; Yu, Hong; Xiao, Yanhong; Shi, Yujuan; Guo, Ting

    2016-01-01

    An effective strategy has been developed for synthesis of radionuclide immune albumin nanospheres (131I-antiAFPMcAb-GCV-BSA-NPs). In vitro as well as in vivo targeting of 131I-antiAFPMcAb-GCV-BSA-NPs to AFP-positive hepatoma was examined. In cultured HepG2 cells, the uptake and retention rates of 131I-antiAFPMcAb-GCV-BSA-NPs were remarkably higher than those of 131I alone. As well, the uptake rate and retention ratios of 131I-antiAFPMcAb-GCV-BSA-NPs in AFP-positive HepG2 cells were also significantly higher than those in AFP-negative HEK293 cells. Compared to 131I alone, 131I-antiAFPMcAb-GCV-BSA-NPs were much more easily taken in and retained by hepatoma tissue, with a much higher T/NT. Due to good drug-loading, high encapsulation ratio, and highly selective affinity for AFP-positive tumors, the 131I-antiAFPMcAb-GCV-BSA-NPs are promising for further effective radiation-gene therapy of hepatoma. PMID:26981334

  3. Polyunsaturated Branched-Chain Fatty Acid Geranylgeranoic Acid Induces Unfolded Protein Response in Human Hepatoma Cells

    PubMed Central

    Iwao, Chieko; Shidoji, Yoshihiro

    2015-01-01

    The acyclic diterpenoid acid geranylgeranoic acid (GGA) has been reported to induce autophagic cell death in several human hepatoma-derived cell lines; however, the molecular mechanism for this remains unknown. In the present study, several diterpenoids were examined for ability to induce XBP1 splicing and/or lipotoxicity for human hepatoma cell lines. Here we show that three groups of diterpenoids emerged: 1) GGA, 2,3-dihydro GGA and 9-cis retinoic acid induce cell death and XBP1 splicing; 2) all-trans retinoic acid induces XBP1 splicing but little cell death; and 3) phytanic acid, phytenic acid and geranylgeraniol induce neither cell death nor XBP1 splicing. GGA-induced ER stress/ unfolded protein response (UPR) and its lipotoxicity were both blocked by co-treatment with oleic acid. The blocking activity of oleic acid for GGA-induced XBP1 splicing was not attenuated by methylation of oleic acid. These findings strongly suggest that GGA at micromolar concentrations induces the so-called lipid-induced ER stress response/UPR, which is oleate-suppressive, and shows its lipotoxicity in human hepatoma cells. PMID:26186544

  4. R-ETODOLAC DECREASES BETA-CATENIN LEVELS ALONG WITH SURVIVAL AND PROLIFERATION OF HEPATOMA CELLS

    PubMed Central

    Behari, Jaideep; Zeng, Gang; Otruba, Wade; Thompson, Michael; Muller, Peggy; Micsenyi, Amanda; Sekhon, Sandeep S.; Leoni, Lorenzo; Monga, Satdarshan P. S.

    2007-01-01

    Background Inhibition of hepatoma cells by cyclooxygenase (COX)-2 dependent and independent mechanisms has been shown previously. Here, we examine the effect of Celecoxib, a COX-2-inhibitor and R-Etodolac, an enantiomer of the nonsteroidal anti-inflammatory drug Etodolac, which lacks COX-inhibitory activity, on the Wnt/β-catenin pathway and human hepatoma cells. Methods Hep3B and HepG2 cell lines were treated with Celecoxib or R-Etodolac, and examined for viability, DNA synthesis, Wnt/β-catenin pathway components, and downstream target gene expression. Results Celecoxib at high doses affected β-catenin protein by inducing its degradation via GSK3β and APC along with diminished tumor cell proliferation and survival. R-Etodolac at physiological doses caused decrease in total and activated β-catenin protein secondary to decrease in its gene expression and post-translationally through GSK3β activation. In addition, increased β-catenin-E-cadherin was also observed at the membrane. An associated inhibition of β-catenin-dependent Tcf reporter activity, decreased levels of downstream target gene products glutamine synthetase and cyclin-D1, and decreased proliferation and survival of hepatoma cells was evident. Conclusion The antitumor effects of Celecoxib (at high concentrations) and R-Etodolac (at physiological doses) on HCC cells were accompanied by the down-regulation of β-catenin demonstrating a useful therapeutic strategy in hepatocellular cancer. PMID:17275129

  5. [Effect of Conditioned Medium from Endothelial Cells on Cancer Stem Cell Phenotype of Hepatoma Cells].

    PubMed

    Feng, Chuan; Yang, Xianjiong; Sun, Jinghui; Luo, Qing; Song, Guanbin

    2015-10-01

    In this study, we aimed to investigate the influences of conditioned medium from human umbilical vein endothelial cells (HUVEC) on cancer stem cell phenotype of human hepatoma cells. HUVEC and human hepatoma cells (MHCC97H) were cultured, respectively, and then the MHCC97H cells were co-cultured with conditioned medium from HUVEC (EC-CM) with Transwell system. Anti-cancer drug sensitivity, colony-formation, migration/invasion ability, expression of cancer stem cell marker and sphere formation were performed to determine the cancer stem cell phenotype in MHCC97H cells. We found that MHCC97H cells co-cultured with EC-CM exhibited significantly higher colony-formation ability and lower sensitivity of anti-cancer drugs 5-FU and Cis. Transwell assay showed that treatment with EC-CM obviously increased migration and invasion of MHCC97H cells. Moreover, increased sphere forming capability and expression of CD133 in MHCC97H cells were observed after co-cultured with EC-CM. These results suggested that EC-CM could promote cancer stem cell phenotype of hepatoma cells. PMID:26964312

  6. 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

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

    PubMed

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

    2015-02-21

    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. PMID:25613320

  8. Aberrant hedgehog signaling is responsible for the highly invasive behavior of a subpopulation of hepatoma cells.

    PubMed

    Fan, Y-H; Ding, J; Nguyen, S; Liu, X-J; Xu, G; Zhou, H-Y; Duan, N-N; Yang, S-M; Zern, M A; Wu, J

    2016-01-01

    Hepatoma exhibits a series of heterogeneous subpopulations in its cell surface markers, tumorigenicity, invasion and metastatic capability. We previously demonstrated that the CD133(-)/EpCAM(-) hepatoma subpopulation was more metastatic than its counterpart; however, the controlling mechanisms are unexplored. The present study aimed to delineate the significance of aberrant hedgehog (Hh) signaling in the mediation of metastases. Fluorescence-activated cell sorting-enriched CD133(-)/EpCAM(-) (double negative, DN), Huh-7 cells underwent a transwell selection for metastatic cells (transwell-selected, TS). The TS cells displayed much greater metastatic activity as evidenced by an increased invasion rate, extremely upregulated expression of matrix metalloproteinase (MMP)-1/2/9 genes compared with DN and double-positive (DP) subpopulations. In contrast to DP cells, TS cells lost E-cadherin and were all vimentin-positive as shown by immunocytochemistry. There was a transitional increase in Gli-1/2 gene expression levels from DP, DN to TS subpopulations, which was consistent with elevated Gli-1/2 or Twist-1 protein levels in the nuclear fraction. Furthermore, truncated Gli-1 (tGli-1), which transactivates molecules involved in metastasis, was detected in the highly invasive Huh-7 cell subpopulation, but not in less metastatic hepatoma cells or normal hepatocytes. The enhanced metastatic features with increased expression of MMPs as well as the presence of twist and snail genes in TS Huh-7 cells were reversed by LDE225, a potent Smoothened antagonist. In conclusion, the highly metastatic capability of a unique TS subpopulation was highly attributed to significant epithelial-mesenchymal transition, enhanced Hh activity and aberrant occurrence of a tGli-1 variant, which appears to be responsible for the highly invasive behavior. PMID:25772244

  9. Role of apolipoprotein A-I in HDL binding to a rat hepatoma cell in culture

    SciTech Connect

    Gottlieb, B.A.

    1985-01-01

    The binding of HDL to rat Fu5AH hepatoma cells at 4/sup 0/, and uptake and degradation at 37/sup 0/, was investigated in monolayer cultures. HDL, free of apo E and apo A-IV, was obtained from the plasma of nephrotic rats (HDLne). /sup 125/I-labeled HDLne bound to the cells in a specific, saturable manner. By Scatchard analysis, two classes of binding sites were obtained: a high affinity binding site (Kd = 1.25 +/- 0.023 ..mu..g/ml, or 5 x 10/sup -9/ M), and a lower affinity site (Kd = 45 +/- 15 ..mu..g/ml, or 1.8 x 10/sup -7/ M). In competitive binding experiments, normal rat HDL was nearly as effective as HDLne, but rat VLDL and human lipoproteins were ineffective. Rat apo A-I/phospholipid complexes also did not complete effectively for HDLne binding, although they were capable of binding to the cells. However, LDL (1.02 < d < 1.063) from nephrotic rat plasma, containing 20% of apo A-I, was as effective as rat HDL in competing for HDLne binding when the competition was expressed as a function of apo A-I content. Control experiments indicated that labeled apo A-I from HDLne did not exchange appreciably with unlabeled apo A-I on the LDLne. When the hepatoma cells were allowed to internalize and degrade HDLne at 37/sup 0/, the acid-soluble products (iodotyrosine and iodide) were derived almost entirely from the breakdown of apo A-I. We conclude that the rat hepatoma cell (Fu5AH) has high affinity HDL binding sites which recognize apo A-I-lipid complexes in which apo A-I an appropriate conformation.

  10. Demonstration of extensive chromatin cleavage in transplanted Morris hepatoma 7777 tissue: apoptosis or necrosis?

    PubMed Central

    Fukuda, K.; Kojiro, M.; Chiu, J. F.

    1993-01-01

    Cell death may occur by either of two mechanisms: necrosis or apoptosis (programmed cell death). In this paper, we demonstrate extensive chromatin cleavage into oligonucleosome-length fragments (DNA ladder) in transplanted Morris hepatoma 7777 tissue, which is suggestive of the stimulation of an endogenous endonuclease activity previously found to be involved in the process of apoptosis. The existence of many apoptotic cells, which are morphologically characterized by condensed cytoplasm and basophilic nuclear fragments, were also seen in this tissue. In vivo and in vitro experiments were designed to further differentiate the morphological and biochemical features of necrosis and apoptosis in liver and hepatoma cells. Liver tissue undergoing ischemic necrosis showed a distinct DNA ladder pattern without demonstrating the morphology of apoptosis, indicating that chromatin cleavage into oligonucleosomal-length fragments is not confined to apoptotic cell death, at least in liver cells. In in vitro-cultured McA-RH7777 cells, however, DNA ladder pattern was detected only in cells showing characteristic morphology of apoptosis. From these two criteria (i.e., characteristic morphology and DNA ladder), it was strongly suggested that the apoptotic process is highly activated in the transplanted 7777 tissue. Based on the results obtained from in vitro experiments, it was suggested that tumor apoptosis may represent a residual attempt at autoregulation within the expanding tumor population and/or may result from mild cellular injuries such as hypoxia, nutrient deficiency, or other unknown noxious factor(s). We also showed evidence that apoptosis is inducible in hepatoma cells in vitro by a wide range of mild injuries or stimuli. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8384410

  11. Osmoregulated taurine transport in H4IIE hepatoma cells and perfused rat liver.

    PubMed Central

    Warskulat, U; Wettstein, M; Häussinger, D

    1997-01-01

    The effects of aniso-osmotic exposure on taurine transport were studied in H4IIE rat hepatoma cells. Hyperosmotic (405 mosmol/l) exposure of H4IIE cells stimulated Na+-dependent taurine uptake and led to an increase in taurine transporter (TAUT) mRNA levels, whereas hypo-osmotic (205 mosmol/l) exposure diminished both taurine uptake and TAUT mRNA levels when compared with normo-osmotic (305 mosmol/l) control incubations. Taurine uptake increased 30-40-fold upon raising the ambient osmolarity from 205 to 405 mosmol/l. When H4IIE cells and perfused livers were preloaded with taurine, hypo-osmotic cell swelling led to a rapid release of taurine from the cells. The taurine efflux, but not taurine uptake, was sensitive to 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS), suggestive of an involvement of DIDS-sensitive channels in mediating volume-regulatory taurine efflux. Whereas in both H4IIE rat hepatoma cells and primary hepatocytes TAUT mRNA levels were strongly dependent upon ambient osmolarity, mRNAs for other osmolyte transporters, i.e. the betaine transporter BGT-1 and the Na+/myo-inositol transporter SMIT, were not detectable. In line with this, myo-inositol uptake by H4IIE hepatoma cells was low and was not stimulated by hyperosmolarity. However, despite the absence of BGT-1 mRNA, a slight osmosensitive uptake of betaine was observed, but the rate was less than 10% of that of taurine transport. This study identifies a constitutively expressed and osmosensitive TAUT in H4IIE cells and the use of taurine as a main osmolyte, whereas betaine and myo-inositol play little or no role in the osmolyte strategy in these cells. This is in contrast with rat liver macrophages, in which betaine has been shown to be a major osmolyte. PMID:9032454

  12. Regulation of P53 stability in p53 mutated human and mouse hepatoma cells.

    PubMed

    Hailfinger, Stephan; Jaworski, Maike; Marx-Stoelting, Philip; Wanke, Ines; Schwarz, Michael

    2007-04-01

    The tumor suppressor p53 is frequently mutated in cancer. We have investigated the regulation of P53 in p53 wild type mouse hepatoma cells (line 55.1c), in p53 heterozygeously mutated cells (56.1b) and in p53 defective cells (lines 56.1d, 70.4 and HUH7) under various experimental settings. The basal levels of P53 were low in 55.1c cells, but nuclear accumulation occurred upon UV-irradiation. Similarly, UV-exposure induced stabilization of P53 in the heterozygeously p53 mutated 56.1b hepatoma cells. By contrast, the 3 hepatoma lines, which lack transcriptionally active P53, demonstrated high basal nuclear concentrations of P53 protein and, unexpectedly, showed loss of P53 upon UV-irradiation. Expression of p53 mRNA was also decreased in p53 defective cells after 24 hr post UV-irradiation, which may be linked to induction of apoptosis of the irradiated cells under these conditions. Other stressors like H2O2 also mediated a decrease in P53 concentration in p53 defective cells. This effect occurred at very low concentrations and was already detectable 1-2 hr after exposure of cells. There were no signs of apoptosis of H2O2-exposed cells at this time point and no significant changes in p53 mRNA or MDM2 level. These unexpected findings indicate a new aspect related to regulation of P53 stability in cells with a defect in the tumor suppressor protein. PMID:17205518

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

    SciTech Connect

    Singaravelu, Ragunath; Lyn, Rodney K.; Srinivasan, Prashanth; Delcorde, Julie; Steenbergen, Rineke H.; Tyrrell, D. Lorne; Pezacki, John P.

    2013-11-15

    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 limited 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.

  14. Irradiation-induced localization of IL-12-expressing mesenchymal stem cells to enhance the curative effect in murine metastatic hepatoma.

    PubMed

    Jeong, Keun-Yeong; Lee, Eun-Jung; Kim, Su Jin; Yang, Seung-Hyun; Sung, Young Chul; Seong, Jinsil

    2015-08-01

    Irradiation in conjunction with gene therapy is considered for efficient cancer treatment. Mesenchymal stem cells (MSCs), due to their irradiation-promotable tumor tropism, are ideal delivery vehicles for gene therapy. In this study, we investigated whether treatment with radiation and interleukin (IL)-12-expressing MSCs (MSCs/IL-12) exerts improved antitumor effects on murine metastatic hepatoma. HCa-I and Hepa 1-6 cells were utilized to generate heterotopic murine hepatoma models. Tumor-bearing mice were treated with irradiation or MSCs/IL-12 alone, or a combination. Monocyte chemoattractant protein-1 (MCP-1/CCL2) expression was assessed in irradiated hepatoma tissues to confirm a chemotactic effect. Combination treatment strategies were established and their therapeutic efficacies were evaluated by monitoring tumor growth, metastasis and survival rate. IL-12 expression was assessed and the apoptotic activity and immunological alterations in the tumor microenvironment were examined. MCP-1/CCL2 expression and localization of MSCs/IL-12 increased in the irradiated murine hepatoma cells. The antitumor effects, including suppression of pulmonary metastasis and survival rate improvements, were increased by the combination treatment with irradiation and MSCs/IL-12. IL-12 expression was increased in tumor cells, causing proliferation of cluster of differentiation 8(+) T-lymphocytes and natural killer cells. The apoptotic activity increased, indicating that the cytotoxicity of immune cells was involved in the antitumor effect of the combined treatment. Treatment with irradiation and MSCs/IL-12 showed effectiveness in treating murine metastatic hepatoma. IL-12-induced proliferation of immune cells played an important role in apoptosis of tumor cells. Our results suggest that treatment with irradiation and MSCs/IL-12 may be a useful strategy for enhancing antitumor activity in metastatic hepatoma. PMID:25639194

  15. Lysosomal accumulation of gallium-67 in Morris hepatoma-7316A and Shionogi mammary carcinoma-115.

    PubMed

    Takeda, S; Okuyama, S; Takusagawa, K; Matsuzawa, T

    1978-04-01

    Intracellular localization of gallium-67 was investigated in Morris hepatoma-7316A and Shionogi mammary carcinoma-115 cells by the cell fractionation method 48 hr after an intraperitoneal injection of the nuclide. When lysosomes were purified from both tumors by discontinuous sucrose density gradient centrifugation, they had a strikingly high relative specific activity of the nuclide. From these results it was confirmed that gallium-67 is concentrated most specifically in the lysosomes of both tumor cells, which consist chiefly of phagolysosomes and can engulf only limited amount of foreign materials such as Triton and gallium-67. PMID:210077

  16. 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.

  17. Phosphorylation of the insulin receptor in cultured hepatoma cells and a solubilized system

    SciTech Connect

    Kasuga, M.; White, M.F.; Kahn, C.R.

    1985-01-01

    Methods are described which have been used successfully to study insulin receptor autophosphorylation in cultured cells (hepatoma cell line Fao) and detergent solubilized receptor systems. Intact cultured cells were labelled with /sup 32/PO/sub 4//sup 3 -/. Details are given for the solubilization and purification of the insulin receptor and insulin dose-response curves for phosphorylation of the solubilized insulin receptor. Trypsin digestion of a phosphorylated subunit suggests that at least peptides containing sites of /sup 32/P incorporation exist in the receptor molecule.

  18. Metabolism and cytotoxic effects of phosphatidylcholine hydroperoxide in human hepatoma HepG2 cells.

    PubMed

    Suzuki, Yuuri; Nakagawa, Kiyotaka; Kato, Shunji; Tatewaki, Naoto; Mizuochi, Shunsuke; Ito, Junya; Eitsuka, Takahiro; Nishida, Hiroshi; Miyazawa, Teruo

    2015-03-20

    In this study, we investigated cellular uptake and metabolism of phosphatidylcholine hydroperoxide (PCOOH) in human hepatoma HepG2 cells by high performance liquid chromatography-tandem mass spectrometry, and then evaluated whether PCOOH or its metabolites cause pathophysiological effects such as cytotoxicity and apoptosis. Although we found that most PCOOH was reduced to PC hydroxide in HepG2 cells, the remaining PCOOH caused cytotoxic effects that may be mediated through an unusual apoptosis pathway. These results will enhance our fundamental understanding of how PCOOH, which is present in oxidized low density lipoproteins, is involved in the development of atherosclerosis. PMID:25704087

  19. Different sensitivity of Zajdela hepatoma mitochondrial ATPase activity to uncouplers in digitonin-treated cells and isolated mitochondria.

    PubMed

    Luciaková, K; Kuzela, S

    1983-01-01

    Digitonin-treated Zajdela hepatoma cells and rat hepatocytes devoid of almost all cytosol but retaining intact mitochondria were found to represent a suitable system for direct measurement of mitochondrial ATPase activity. The enzyme activity in digitonin-treated Zajdela hepatoma cells in contrast to that of isolated coupled mitochondria was stimulated by uncouplers. No difference in response of mitochondrial ATPase activity to uncouplers in digitonin-treated hepatocytes and isolated liver mitochondria was found. It is concluded that uncoupler-insensitive mitochondrial ATPase activity does not occur in intact in situ tumor mitochondria but is acquired during the isolation of the organelles. PMID:6310422

  20. Characterization of novel hepadnaviral RNA species accumulated in hepatoma cells treated with viral DNA polymerase inhibitors.

    PubMed

    Zhang, Pinghu; Liu, Fei; Guo, Fang; Zhao, Qiong; Chang, Jinhong; Guo, Ju-Tao

    2016-07-01

    Inhibitors of hepadnaviral DNA polymerases are predicted to inhibit both minus and plus strand of viral DNA synthesis and arrest viral DNA replication at the stage of pregenomic (pg) RNA-containing nucleocapsids. However, analyses of the RNA species of human and duck hepatitis B viruses (HBV and DHBV, respectively) in hepatoma cells treated with viral DNA polymerase inhibitors revealed the genesis of novel RNA species migrating slightly faster than the full-length pgRNA. The DNA polymerase inhibitor-induced accumulation of these RNA species were abolished in the presence of alpha-interferon or HBV nucleocapsid assembly inhibitors. Moreover, they were protected from microccocal nuclease digestion and devoid of a poly-A tail. These characteristics suggest that the novel RNA species are most likely generated from RNase H cleavage of encapsidated pgRNA, after primer translocation and synthesis of the 5' terminal portion of minus strand DNA. In support of this hypothesis, DNA polymerase inhibitor treatment of chicken hepatoma cells transfected with a DHBV genome encoding an RNase H inactive DNA polymerase (E696H) failed to produce such RNA species. Our results thus suggest that the currently available DNA polymerase inhibitors do not efficiently arrest minus strand DNA synthesis at the early stage in hepatocytes. Hence, development of novel antiviral agents that more potently suppress viral DNA synthesis or viral nucleocapsid assembly inhibitors that are mechanistically complementary to the currently available DNA polymerase inhibitors are warranted. PMID:27083116

  1. Regulatory aspects of the glutamylation of methotrexate in cultured hepatoma cells

    SciTech Connect

    Nimec, Z.; Galivan, J.

    1983-10-15

    The glutamylation of methotrexate has been evaluated in H35 hepatoma cells in vitro as a function of the conditions of culture. Glutamylation yields methotrexate polyglutamate with two to five additional glutamate residues and is a saturable process. The rate of glutamylation increases little above 10 microM extracellular methotrexate which corresponds to an intracellular concentration of approximately 4 microM. The rate of glutamylation measured over a 6-h period was stimulated by a reduction in cellular folates and prior incubation of the cells with insulin. Glutamylation was also more rapid in dividing cultures than in confluent cells. The combination of insulin inclusion and folate reduction, which was additive, caused approximately a fourfold increase in the rate of glutamylation over control cells under the conditions tested. The maximal rate of methotrexate glutamylation, which was 100 nmol/g/h, occurred in folate-depleted, insulin-supplemented cells. Supplementing folate-depleted cells with reduced folate coenzymes caused the glutamylation to be reduced by more than 90%. In addition to showing that folates can modify the rates of methotrexate polyglutamate formation, data are presented suggesting that methotrexate polyglutamates can regulate their own synthesis. The consequences of the formation of these retained forms of methotrexate in H35 hepatoma cells and the effects of potential regulators of this process are discussed in terms of the glutamylation of folates in the cells and the chemotherapeutic effects of antifolates.

  2. Effects of glycyl-histidyl-lysine on Morris hepatoma 7777 cells.

    PubMed

    Barra, R

    1987-01-01

    Glycyl-histidyl-lysine (GHL) has been shown to have growth stimulatory effects on a number of different cell types including hepatocytes and hepatoma cells. In this study, the effects of GHL on Morris hepatoma 7777 cells were investigated. The greatest stimulatory effects on 3H-thymidine and 3H-leucine incorporation were observed at a GHL concentration of 2 ng/ml. In randomly proliferating cells, the incorporation of 3H-thymidine into DNA increased by 50% and that of 3H-leucine into protein by 29%. In addition, synergistic effects were observed when insulin and glucagon were included with GHL in the incubation mixture. Experiments with cells rendered quiescent by serum starvation indicated that cells in the G1 phase of the cell cycle are more sensitive to GHL stimulation. In these experiments, 3H-thymidine incorporation increased earlier and peaked at a higher value than in the control cells. This finding suggests that GHL may play a role in stimulating quiescent cells to re-enter the cell cycle. PMID:3319436

  3. Hepatoma-Derived Growth Factor: Its Possible Involvement in the Progression of Hepatocellular Carcinoma

    PubMed Central

    Enomoto, Hirayuki; Nakamura, Hideji; Liu, Weidong; Nishiguchi, Shuhei

    2015-01-01

    The development of hepatocellular carcinoma (HCC) is an important complication of viral infection induced by hepatitis virus C, and our major research theme is to identify a new growth factor related to the progression of HCC. HDGF (hepatoma-derived growth factor) is a novel growth factor that belongs to a new gene family. HDGF was initially purified from the conditioned medium of a hepatoma cell line. HDGF promotes cellular proliferation as a DNA binding nuclear factor and a secreted protein acting via a receptor-mediated pathway. HDGF is a unique multi-functional protein that can function as a growth factor, angiogenic factor and anti-apoptotic factor and it participates in the development and progression of various malignant diseases. The expression level of HDGF may be an independent prognostic factor for predicting the disease-free and overall survival in patients with various malignancies, including HCC. Furthermore, the overexpression of HDGF promotes the proliferation of HCC cells, while a reduction in the HDGF expression inhibits the proliferation of HCC cells. This article provides an overview of the characteristics of HDGF and describes the potential role of HDGF as a growth-promoting factor for HCC. PMID:26101867

  4. Modified nucleotides in T1 RNase oligonucleotides of 18S ribosomal RNA of the Novikoff hepatoma.

    PubMed

    Choi, Y C; Busch, H

    1978-06-27

    The primary structure of 18S rRNA of the Novikoff hepatoma cells was investigated. Regardless of whether the primary sequence of 18S rRNA is finally determined by RNA sequencing methods or DNA sequencing methods, it is important to identify numbers and types of the modified nucleotides and accordingly the present study was designed to localize the modified regions in T1 RNase derived oligonucleotide. Modified nucleotides found in 66 different oligonucleotide sequences included 2 m62A, 1 m6A, 1 m7G, 1m1cap3psi, 7 Cm, 13 Am, 9 Gm, 11 Um, and 38 psi residues. A number of these modified nucleotides are now placed in defined sequences of T1 RNase oligonucleotides which are now being searched for in larger fragments derived from partial T1 RNase digests of 18S rRNA. Improved homochromatography fingerprinting (Choi et al. (1976) Cancer Res. 36, 4301) of T1 RNase derived oligonucleotides provided a distinctive pattern for 18S rRNA of Novikoff hepatoma ascites cells. The 116 spots obtained by homochromatography contain 176 oligonucleotide sequences. PMID:209819

  5. Anti-proliferative effect of pterostilbene on rat hepatoma cells in culture.

    PubMed

    Dewi, Novi Indriana; Yagasaki, Kazumi; Miura, Yutaka

    2015-08-01

    Pterostilbene, a methoxylated analogue of resveratrol, is a natural compound primarily found in blueberries and several types of grapes. However, little is known about the effect of pterostilbene on the proliferation of hepatoma cells and its modes of actions. This study was undertaken to characterize its ability to suppress the proliferation of hepatoma AH109A cells and the possible mechanism(s) involved. Pterostilbene showed a significant and dose-dependent effect on the anti-proliferative activity against AH109A cells. Pterostilbene exerted little or no effect on the proliferation of rat L6 myoblasts and rat skin fibroblasts. Pterostilbene-loaded rat sera could significantly inhibit the proliferation of AH109A cells, which suggests that pterostilbene could be absorbed through gastrointestinal tract and retain its anti-proliferative activity. Pterostilbene arrested the cell cycle of AH109A cells at G0/G1 phase and reduced the protein expression of cyclin-dependent kinase 4 and cyclin-dependent kinase 6 dose-dependently. We also found that pterostilbene could significantly increase the intracellular peroxide level of AH109A cells, which may be involved in its anti-proliferative activity. PMID:24985197

  6. 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.

  7. Kinetics and regulation of hepatoma mitochondrial NAD(P) malic enzyme.

    PubMed

    Teller, J K; Fahien, L A; Davis, J W

    1992-05-25

    Kinetic studies of Morris 7777 hepatoma mitochondrial NAD(P) malic enzyme were consistent with an ordered mechanism where NAD adds to the enzyme before malate and dissociation of NADH from the enzyme is rate-limiting. In addition to its active site, malate apparently also associates with a lower affinity with an activator site. The activator fumarate competes with malate at the activator site and facilitates dissociation of NADH from the enzyme. The ratio of NAD(P) malic enzyme to malate dehydrogenase activity in the hepatoma mitochondrial extract was found to be too low, even in the presence of known inhibitors of malate dehydrogenase, to account for the known ability of NAD(P) malic enzyme to intercept exogenous malate from malate dehydrogenase in intact tumor mitochondria (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221). However, NAD(P) malic enzyme may be able to intercept exogenous malate because according to the present results, it can associate with the pyruvate dehydrogenase complex, which could localize NAD(P) malic enzyme in the vicinity of the inner mitochondrial membrane. The activity levels of some key metabolic enzymes were found to be different in Morris 7777 mitochondria than in liver or mitochondria of other rapidly dividing tumors. These results are discussed in terms of differences among tumors in their ability to utilize malate, glutamate, and citrate as respiratory fuels. PMID:1587826

  8. Production of infectious duck hepatitis B virus in a human hepatoma cell line.

    PubMed Central

    Galle, P R; Schlicht, H J; Fischer, M; Schaller, H

    1988-01-01

    The differentiated human hepatoma cell line Hep-G2 was transfected with cloned duck hepatitis B virus (DHBV) DNA. Introduction of closed circular DNA into the human liver cells resulted in the production of viral proteins: core antigen was detected in the cytoplasm, and e antigen, a related product, was secreted into the medium. Moreover, viral particles were released into the tissue culture medium which were indistinguishable from authentic DHBV by density, antigenicity, DNA polymerase activity, and morphology. Intravenous injection of tissue culture-derived DHBV particles into Pekin ducks established DHBV infection. In conclusion, transfection of human hepatoma cells with cloned DHBV DNA results in the production of infectious virus, as occurs with cloned human hepatitis B virus DNA. Human liver cells are therefore competent to support production of the avian and mammalian hepadnaviruses, indicating that liver-specific viral gene expression is controlled by evolutionarily conserved mechanisms. This new DHBV transfection system offers the opportunity to rapidly produce mutated DHBV which then can be further investigated in Pekin ducks. Images PMID:2833623

  9. Biogenesis and cytotoxicity of APOL1 renal risk variant proteins in hepatocytes and hepatoma cells.

    PubMed

    Cheng, Dongmei; Weckerle, Allison; Yu, Yi; Ma, Lijun; Zhu, Xuewei; Murea, Mariana; Freedman, Barry I; Parks, John S; Shelness, Gregory S

    2015-08-01

    Two APOL1 gene variants, which likely evolved to protect individuals from African sleeping sickness, are strongly associated with nondiabetic kidney disease in individuals with recent African ancestry. Consistent with its role in trypanosome killing, the pro-death APOL1 protein is toxic to most cells, but its mechanism of cell death is poorly understood and little is known regarding its intracellular trafficking and secretion. Because the liver appears to be the main source of circulating APOL1, we examined its secretory behavior and mechanism of toxicity in hepatoma cells and primary human hepatocytes. APOL1 is poorly secreted in vitro, even in the presence of chemical chaper-ones; however, it is efficiently secreted in wild-type transgenic mice, suggesting that APOL1 secretion has specialized requirements that cultured cells fail to support. In hepatoma cells, inducible expression of APOL1 and its risk variants promoted cell death, with the G1 variant displaying the highest degree of toxicity. To explore the basis for APOL1-mediated cell toxicity, endoplasmic reticulum stress, pyroptosis, autophagy, and apoptosis were examined. Our results suggest that autophagy represents the predominant mechanism of APOL1-mediated cell death. Overall, these results increase our understanding of the basic biology and trafficking behavior of circulating APOL1 from the liver. PMID:26089538

  10. Targeted transfection and expression of hepatitis B viral DNA in human hepatoma cells.

    PubMed Central

    Liang, T J; Makdisi, W J; Sun, S; Hasegawa, K; Zhang, Y; Wands, J R; Wu, C H; Wu, G Y

    1993-01-01

    A soluble DNA carrier system consisting of an asialoglycoprotein covalently linked to poly-L-lysine was used to bind DNA and deliver hepatitis B virus (HBV) DNA constructs to asialoglycoprotein receptor-positive human hepatoma cells. 4 d after transfection with surface or core gene expression constructs, HBsAg and HBeAg in the media were measured to be 16 ng/ml and 32 U/ml per 10(7) cells, respectively. Antigen production was completely inhibited by the addition of an excess of asialoorosomucoid. On the other hand, asialoglycoprotein receptor-negative human hepatoma cells, SK-Hep1, did not produce any viral antigens under identical conditions after incubation with HBV DNA complexed to a conjugate composed of asialoorosomucoid and poly-L-lysine. Using a complete HBV genome construct, HBsAg and HBeAg levels reached 16 ng/ml and 16 U/ml per 10(7) cells, respectively. Northern blots revealed characteristic HBV RNA transcripts including 3.5-, 2.4-, and 2.1-kb fragments. Intracellular and extracellular HBV DNA sequences including relaxed circular, linear and single stranded forms were detected by Southern blot hybridization. Finally, 42-nm Dane particles purified from the spent cultures medium were visualized by electron microscopy. This study demonstrates that a targetable DNA carrier system can transfect HBV DNA in vitro resulting in the production of complete HBV virions. Images PMID:8383700

  11. Inhibition of water activated by far infrared functional ceramics on proliferation of hepatoma cells.

    PubMed

    Zhang, Dongmei; Liang, Jinsheng; Ding, Yan; Meng, Junping; Zhang, Guangchuan

    2014-05-01

    Rare earth (RE)/tourmaline composite materials prepared by the precipitation method are added to the ceramic raw materials at a certain percentage and sintered into RE functional ceramics with high far infrared emission features. Then the far infrared functional ceramics are used to interact with water. The influence of the ceramics on the physical parameters of water is investigated, and the effect of the activated water on the growth of Bel-7402 hepatoma cells cultured in vitro is further studied. The results indicate that, compared with the raw water, the water activated by the ceramics can inhibit the proliferation of hepatoma cells, with statistical probability P < 0.01, which means that the effect is significant. It can be explained that the water activated by the ceramics has a higher concentration of H+, which decreases the potential difference across the cell membrane to release the apoptosis inducing factor (AIF). After entering the cells, the activated water stimulates the mitochondria to produce immune substances that lead tumor cells to apoptosis. PMID:24734643

  12. alpha 1-Fetoprotein mRNA of rat yolk sac and hepatoma.

    PubMed Central

    Chiu, J F; Decha-Umphai, W; Commer, P

    1979-01-01

    Rat alpha 1-fetoprotein mRNA was isolated and purified to apparent homogeneity by means of immunoadsorption and oligo (dT) cellulose affinity chromatography. Purified AFP mRNA migrated as a 21S peak in 2.5% SDS-polyacrylamide gels. The translation product of this mRNA in micrococcal nuclease treated reticulocyte lysate was identified as AFP by specific immunoprecipitation, SDS-gel electrophoresis and tryptic digestion analysis. DNA complimentary to AFP mRNA was synthesized with avian meyloblastosis virus RNA-dependent DNA polymerase. This AFP cDNA was used as a probe to quantitate AFP mRNA in the developing rat liver and to compare the complexity and diversity of AFP mRNA derived from the normal rat liver and Morris hepatoma 7777. We found that the amount of functional AFP mRNA is decreasing during liver development. There is very little, if any, AFP mRNA in the adult rat liver. A high degree of homology between the AFP mRNA sequences of yolk sac and hepatoma was also found. PMID:91159

  13. Antitumor Efficacy and Mechanism in Hepatoma H22-Bearing Mice of Brucea javanica Oil

    PubMed Central

    Shi, Wen-Rong; Liu, Yan; Wang, Xiao-Ting; Huang, Qiong-Ying; Cai, Xue-Rong; Wu, Shao-Rong

    2015-01-01

    Brucea javanica is a traditional herbal medicine in China, and its antitumor activities are of research interest. Brucea javanica oil, extracted with ether and refined with 10% ethyl alcohol from Brucea javanica seed, was used to treat hepatoma H22-bearing mice in this study. The antitumor effect and probable mechanisms of the extracted Brucea javanica oil were studied in H22-bearing mice by WBC count, GOT, GPT levels, and western blotting. The H22 tumor inhibition ratio of 0.5, 1, and 1.5 g/kg bw Brucea javanica oil were 15.64%, 23.87%, and 38.27%. Brucea javanica oil could inhibit the involution of thymus induced by H22 tumor-bearing, but it could not inhibit the augmentation of spleen and liver. Brucea javanica oil could decrease the levels of WBC count and GOT and GPT in H22-bearing mice. The protein levels of GAPDH, Akt, TGF-β1, and α-SMA in tumor tissues decreased after being treated with Brucea javanica oil. Disturbing energy metabolism and neoplastic hyperplasia controlled by Akt and immunoregulation activity were its probable antitumor mechanisms in hepatoma H22-bearing mice. PMID:26508976

  14. Growth inhibition of BEL-7404 human hepatoma cells by expression of mutant telomerase reverse transcriptase.

    PubMed

    Zhang, Rugang; Wang, Xingwang; Guo, Lixia; Xie, Hong

    2002-01-10

    Human hepatocellular carcinoma (HCC) is one of the most common malignancies in Asia and Africa. Human telomerase reverse transcriptase (hTERT) is expressed in HCC but absent in normal human liver cells, which is consistent with the expression pattern of telomerase. In the present study, expression of a dominant-negative form of hTERT (DN-hTERT) resulted in inhibition of telomerase activity and decreased mean telomeric length of BEL-7404 human hepatoma cells, whereas expression of wild-type hTERT (WT-hTERT) and control vector had no such effects. Cell growth was inhibited by this mutant (DN-hTERT), which was consistent with the changes in telomerase level. Flattened large cells were found in late generations with the DN-hTERT treatment. When mean telomeric length of DN-hTERT-transfected cells reached a critical length (about 1.7 kb), apoptosis was induced. Tumorigenicity of DN-hTERT-expressing cells was eliminated in vivo. These data indicated that hTERT was essential for the growth of hepatoma cells. hTERT can also be used as an important target for anti-HCC drug screening. PMID:11774261

  15. 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.

  16. Novel fluorinated docetaxel analog for anti-hepatoma: Molecular docking and biological evaluation.

    PubMed

    Hao, Yun-Peng; Liu, Zheng-Yu; Xie, Cheng; Zhou, Lu; Sun, Xun

    2016-06-10

    N-De-tert-butoxycarbonyl-N-[2-(1,1,1-trifluoro-2-methyl)propyloxycarbonyl]-2-debenzoyl-2-(m-fluorobenzoyl)-docetaxel (4FDT), a novel fluorinated docetaxel analog, was evaluated for its anti-hepatoma effect and possible druggability. In molecular docking studies, 4FDT coincided with paclitaxel in a part of the nucleus. In in vitro studies, 4FDT demonstrated higher anti-hepatoma activity approximately 1.5 times greater than that of docetaxel. More interestingly, 4FDT had been determined to have better anticancer effects, even 90 times greater in patient-derived xenografts (PDX) liver cancer cell lines than sorafenib. In the in vivo studies, 4FDT could effectively reduce the growth rate of liver cancer H22 and HepG2 cells. Furthermore, in a preliminary study on the ex vivo distribution of 4FDT, 4FDT-IR783 was primarily concentrated in the liver 1h after injection, and most of it was metabolized from the liver in 24h. Finally, the acute toxicity test revealed fewer side effects for 4FDT (approximately 16% than docetaxel). The water solubility, which was 11 times greater than that of docetaxel, confirmed the good druggability of 4FDT. All of these results demonstrated 4FDT's great potential to be a candidate drug for liver cancer treatment. PMID:27058438

  17. Peroxisomal oxidation of very long chain fatty acids (VLCFA) by human hepatoma cells

    SciTech Connect

    Watkins, P.A.; Ferrell, E.V. Jr.

    1986-05-01

    Beta-oxidation of VLCFA was studied in a human hepatoma cell line (HEP-G2). These cells, disrupted by exposure to low concentrations of digitonin, oxidize (1-/sup 14/C)palmitate (C16:0) and (1-/sup 14/C)lignocerate (C24:0) to /sup 14/CO/sub 2/ and water-soluble products. It was recently reported that in rat liver the beta-oxidation of VLCFA takes place primarily in the peroxisome rather than the mitochondrion. The precise site of VLCFA oxidation in human tissues has not been clearly elucidated. The peroxisome has been implicated since there is impaired VLCFA oxidation in fibroblasts from Zellweger syndrome patients, in which this organelle is deficient. In order to define the subcellular localization of human VLCFA oxidation, homogenates of HEP-G2 cells were fractionated on a discontinuous sucrose gradient. Fractions enriched in the peroxisomal marker catalase oxidized C24:0 at significantly greater rates than fractions enriched in the mitochondrial marker succinate:cytochrome c reductase. C16:0 oxidation was catalyzed by both peroxisomal and mitochondrial fractions. These results suggest that the subcellular site of VLCFA oxidation in human hepatoma cells and rat liver is similar.

  18. Cytotoxicity of the MEIC reference chemicals in rat hepatoma-derived Fa32 cells.

    PubMed

    Dierickx, P J

    2000-09-01

    The cytotoxicity of the MEIC reference chemicals was investigated in rat hepatoma-derived Fa32 cells. The total protein content was measured as an endpoint after exposure times of 30 min and 24 h, both in normal and glutathione-depleted cells. The neutral red uptake inhibition and the MTT conversion were also measured after 30 min. On average, the cytotoxicity was higher in glutathione-depleted cells when compared to normal cells, and was lower after 30 min than after 24 h. Evidence was obtained for lysosomal attack (of five chemicals) or mitochondrial dysfunction (of six chemicals) as the primary intoxication mechanism. Malathion and mercuric chloride belong to both series of chemicals. Good to excellent correlations were observed when the 50% inhibitory concentrations of the six different in vitro assays were compared. When the six in vitro assays in Fa32 cells were compared with the human toxicity, the correlation coefficient was almost always identical to that obtained previously in human hepatoma-derived Hep G2 cells. The latter was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. Altogether the results integrate very well with the basal cytotoxicity concept (Ekwall, B., 1995. The basal cytotoxicity concept. In: Goldberg, A.M., Van Zutphen, L.F.M. (Eds.), The World Congress on Alternatives and Animal Use in the Life Sciences: Education, Research, Testing. Mary Ann Liebert Publishers, New York, pp. 721-725). PMID:10996672

  19. Selenoprotein Genes Exhibit Differential Expression Patterns Between Hepatoma HepG2 and Normal Hepatocytes LO2 Cell Lines.

    PubMed

    Zhao, Hua; Tang, Jiayong; Xu, Jingyang; Cao, Lei; Jia, Gang; Long, Dingbiao; Liu, Guangmang; Chen, Xiaoling; Wang, Kangning

    2015-10-01

    The purpose of this study was to compare messenger RNA (mRNA) expression of selenoprotein genes between hepatoma HepG2 and normal hepatocytes LO2 cell lines. Liver HepG2 and LO2 cells were cultured in 12-well plates under the same condition until cells grew to complete confluence, and then cells were harvested for total RNA and protein extraction. The qPCRs were performed to compare gene expression of 14 selenoprotein genes and 5 cancer signaling-related genes. Enzyme activities were also assayed. The results showed that human hepatoma HepG2 cells grew faster than normal hepatocytes LO2 cells. Among the genes investigated, 10 selenoprotein genes (Gpx1, Gpx3, Gpx4, Selx, Sepp, Sepw1, Sepn1, Selt, Seli, Selh) and 3 cancer signaling-related genes (Bcl-2A, caspase-3, and P38) were upregulated (P < 0.05), while Selo and Bcl-2B were downregulated (P < 0.05) in hepatoma HepG2 cells compared to LO2 cells. Significant correlations were found between selenoprotein genes and the cancer signaling-related genes Caspase3, P53, Bc1-2A, and Bc1-2B. Our results revealed that selenoprotein genes were aberrantly expressed in hepatoma HepG2 cells compared to normal liver LO2 cells, which indicated that those selenoprotein genes may play important roles in the occurrence and development of liver carcinogenesis. PMID:25846212

  20. Radiosensitivity of hepatoma cell lines and human normal liver cell lines exposed to 12C6+ ions

    NASA Astrophysics Data System (ADS)

    Jing, X.; Yang, J.; Li, W.; Guo, C.; Dang, B.; Wang, J.; Zhou, L.; Wei, W.; Gao, Q.

    AIM To investigate the radiosensitivity of hepatoma cell lines and human normal liver cell lines METHODS Accelerated carbon ions by heavy ion research facility in Lanzhou HIRFL have high LET We employed it to study the radiosensitivity of hepatoma cell lines SMMC-7721 and human normal liver cell lines L02 using premature chromosome condensation technique PCC Cell survive was documented by a colony assay Chromatid breaks were measured by counting the number of chromatid breaks and isochromatid breaks immediately after prematurely chromosome condensed by Calyculin-A RESULTS The survival curve of the two cell lines presented a good linear relationship and the survival fraction of L02 is higher than that of SMMC-7721 Additionally the two types of G 2 phase chromosome breaks chromatid breaks and isochromatid breaks of L02 are lower than that of SMMC-7721 CONCLUSION Human normal liver cell line have high radioresistance than that of hepatoma cell line It imply that it is less damage to normal organs when radiotherapy to hepatoma

  1. Immunological screening of a glycoprotein antigen expressed by Zajdela ascites hepatoma cells on normal rat tissues and tumour cells.

    PubMed

    Nato, F; Goulut, C; Mirshahi, M; Bourrillon, R

    1991-10-01

    Expression of the glycoprotein MII2 antigen originally identified in Zajdela ascites hepatoma cells was investigated in several normal rat tissues and in more or less differentiated tumours using biochemical and immunological approaches. SDS-polyacrylamide gel electrophoresis followed by fluorography or immunoblotting with an antiserum raised against the purified MII2 antigen revealed that this antigen was absent from normal liver cells. ELISA assays, indirect immunofluorescence and immunoprecipitation experiments using the same antiserum showed that this glycoprotein was not expressed in various normal tissues such as liver, spleen, lung, pancreas, intestine and stomach, but it was unexpectedly detected in kidney and thymic tissues. However, the molecular weight of the antigens immunoprecipitated from kidney and thymus was lower than the one of MII2 (Mr of 60,000 versus 110,000-160,000 for purified MII2). No staining was observed in embryonic rat liver at 10 and 20 days of development. Moreover, this antigen was present on the surface of Morris hepatoma 7777, another rapidly proliferating and poorly differentiated hepatocellular carcinoma. In contrast, this antigen was not detected on the surface of in vitro Zajdela hepatoma cells (ZHC) or of partially differentiated hepatomas (Faza) which have recovered some hepatic functions. In addition, the MII2 antigen was found on the human non-hepatic HT-29 tumour cell line, under its undifferentiated form (HT-29 G+ subline). The possible relationships between the expression of this antigen and both the malignant transformation process and the differentiation process are discussed. PMID:1656518

  2. Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

    PubMed Central

    Zhu, Juanjuan; Liu, Shanshan; Ye, Fuqiang; Shen, Yuan; Tie, Yi; Zhu, Jie; Wei, Lixin; Jin, Yinghua; Fu, Hanjiang; Wu, Yongge; Zheng, Xiaofei

    2015-01-01

    Maternally Expressed Gene 3 (MEG3) encodes a lncRNA which is suggested to function as a tumor suppressor. Previous studies suggested that MEG3 functioned through activation of p53, however, the functional properties of MEG3 remain obscure and their relevance to human diseases is under continuous investigation. Here, we try to illuminate the relationship of MEG3 and p53, and the consequence in hepatoma cells. We find that transfection of expression construct of MEG3 enhances stability and transcriptional activity of p53. Deletion analysis of MEG3 confirms that full length and intact structure of MEG3 are critical for it to activate p53-mediated transactivation. Interestingly, our results demonstrate for the first time that MEG3 can interact with p53 DNA binding domain and various p53 target genes are deregulated after overexpression of MEG3 in hepatoma cells. Furthermore, results of qRT-PCR have shown that MEG3 RNA is lost or reduced in the majority of HCC samples compared with adjacent non-tumorous samples. Ectopic expression of MEG3 in hepatoma cells significantly inhibits proliferation and induces apoptosis. In conclusion, our data demonstrates that MEG3 functions as a tumor suppressor in hepatoma cells through interacting with p53 protein to activate p53-mediated transcriptional activity and influence the expression of partial p53 target genes. PMID:26444285

  3. Uptake mechanism and endosomal fate of drug-phospholipid lipid nanoparticles in subcutaneous and in situ hepatoma.

    PubMed

    Wang, Qi; Liu, Peifeng; Gong, Tao; Sun, Xun; Duan, Yourong; Zhang, Zhirong

    2014-06-01

    Drug-phospholipid lipid nanoparticles (DPLNs) can effectively enhance the properties of traditional solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs), as previously demonstrated by our research group and others. To date, however, very few studies have focused on the cellular uptake mechanism and fate of DPLNs in hepatoma. Therefore, we systematically studied the cellular uptake mechanism and endosomal fate of DPLNs through in vitro and in vivo experiments. Confocal laser scanning microscopy (CLSM) and flow cytometry demonstrated that the Raw264.7 cell line (macrophage Raw264.7 cells), Chang cells (a human liver cell line) and HepG2 cells (a human hepatoma cell line) exhibited distinct uptake mechanisms. The Raw264.7 cells served as a model for examining liver-targeting ability. The results from mice with subcutaneous hepatomas and in situ hepatomas confirmed that the liver tumor-targeting property of the DPLNs was associated with the liver drug reservoir function. These findings further improve our understanding of DPLNs for clinical applications. PMID:24749394

  4. Human lung cancer-derived microparticles enhanced angiogenesis and growth of hepatoma cells in rodent lung parenchyma

    PubMed Central

    Ko, Sheung-Fat; Hsu, Shu-Yuan; Chen, Chih-Hung; Sung, Pei-Hsun; Zhen, Meng-Shen TongYen-Yi; Chen, Yi-Ling; Huang, Tien-Hung; Chen, Sheng-Yi; Kao, Gour-Shenq; Chen, Hong-Hwa; Chang, Chia-Lo; Sun, Cheuk-Kwan; Chang, Hsueh-Wen; Yip, Hon-Kan

    2016-01-01

    This study tested the hypothesis that human lung cancer-derived microparticles (LcD-MPs) played an important role in tumor angiogenesis and growth. Fischer 344 rats (F344, n=18) were equally categorized into group 1 [Sham Control (3.0 mL normal saline intravenous injection (IV))], group 2 [hepatoma cell line (2.0 x 106 cells, IV)], and group 3 [hepatoma cell line + LcD-MPs (3.0 x 106, IV)]. Animals were euthanized by day 28 after hepatoma cells transfusion. Our result showed that the gross pathology confirmed growth of hepatoma cell line in lung parenchyma. The size and weight of the lungs were significantly increased in group 2 and further elevated in group 3 than in group 1 (all p<0.001). Histopathological analysis demonstrated that the lung crowded score and number of small vessel exhibited an identical pattern, whereas the number of alveolar sacs showed an opposite pattern compared to that of total lung weight among the three groups (all p<0.0001). The cellular expressions of CD34+, CXCR4+, c-Kit+, CK19+, VEGF+ and vimentin+ cells in lung parenchyma exhibited an identical pattern compared to those of total lung weight among all groups (all p<0.001). The protein expressions of apoptotic (Bax, cleaved caspase-3 and c-PARP), fibrotic (Smad3, TGF-β), and tumor suppression (PTEN) biomarkers showed an identical pattern, whereas that of anti-apoptotic (Bcl-2) and anti-fibrotic (Smad1/5, BMP-2) biomarkers were displayed an opposite pattern compared to that of total lung weight among all groups (all p<0.001). The MPs could enhance angiogenesis and accelerated hepatoma cell growth in rodent lung parenchyma. PMID:27186261

  5. Telomerase antagonists GRN163 and GRN163L inhibit tumor growth and increase chemosensitivity of human hepatoma.

    PubMed

    Djojosubroto, Meta W; Chin, Allison C; Go, Ning; Schaetzlein, Sonja; Manns, Michael P; Gryaznov, Sergei; Harley, Calvin B; Rudolph, K Lenhard

    2005-11-01

    Most cancer cells have an immortal growth capacity as a consequence of telomerase reactivation. Inhibition of this enzyme leads to increased telomere dysfunction, which limits the proliferative capacity of tumor cells; thus, telomerase inhibition represents a potentially safe and universal target for cancer treatment. We evaluated the potential of two thio-phosphoramidate oligonucleotide inhibitors of telomerase, GRN163 and GRN163L, as drug candidates for the treatment of human hepatoma. GRN163 and GRN163L were tested in preclinical studies using systemic administration to treat flank xenografts of different human hepatoma cell lines (Hep3B and Huh7) in nude mice. The studies showed that both GRN163 and GRN163L inhibited telomerase activity and tumor cell growth in a dose-dependent manner in vitro and in vivo. The potency and efficacy of the lipid-conjugated antagonist, GRN163L, was superior to the nonlipidated parent compound, GRN163. Impaired tumor growth in vivo was associated with critical telomere shortening, induction of telomere dysfunction, reduced rate of cell proliferation, and increased apoptosis in the treatment groups. In vitro, GRN163L administration led to higher prevalence of chromosomal telomere-free ends and DNA damage foci in both hepatoma cell lines. In addition, in vitro chemosensitivity assay showed that pretreatment with GRN163L increased doxorubicin sensitivity of Hep3B. In conclusion, our data support the development of GRN163L, a novel lipidated conjugate of the telomerase inhibitor GRN163, for systemic treatment of human hepatoma. In addition to limiting the proliferative capacity of hepatoma, GRN163L might also increase the sensitivity of this tumor type to conventional chemotherapy. PMID:16114043

  6. Human lung cancer-derived microparticles enhanced angiogenesis and growth of hepatoma cells in rodent lung parenchyma.

    PubMed

    Ko, Sheung-Fat; Hsu, Shu-Yuan; Chen, Chih-Hung; Sung, Pei-Hsun; Zhen, Meng-Shen TongYen-Yi; Chen, Yi-Ling; Huang, Tien-Hung; Chen, Sheng-Yi; Kao, Gour-Shenq; Chen, Hong-Hwa; Chang, Chia-Lo; Sun, Cheuk-Kwan; Chang, Hsueh-Wen; Yip, Hon-Kan

    2016-01-01

    This study tested the hypothesis that human lung cancer-derived microparticles (LcD-MPs) played an important role in tumor angiogenesis and growth. Fischer 344 rats (F344, n=18) were equally categorized into group 1 [Sham Control (3.0 mL normal saline intravenous injection (IV))], group 2 [hepatoma cell line (2.0 x 10(6) cells, IV)], and group 3 [hepatoma cell line + LcD-MPs (3.0 x 10(6), IV)]. Animals were euthanized by day 28 after hepatoma cells transfusion. Our result showed that the gross pathology confirmed growth of hepatoma cell line in lung parenchyma. The size and weight of the lungs were significantly increased in group 2 and further elevated in group 3 than in group 1 (all p<0.001). Histopathological analysis demonstrated that the lung crowded score and number of small vessel exhibited an identical pattern, whereas the number of alveolar sacs showed an opposite pattern compared to that of total lung weight among the three groups (all p<0.0001). The cellular expressions of CD34(+), CXCR4(+), c-Kit(+), CK19(+), VEGF(+) and vimentin+ cells in lung parenchyma exhibited an identical pattern compared to those of total lung weight among all groups (all p<0.001). The protein expressions of apoptotic (Bax, cleaved caspase-3 and c-PARP), fibrotic (Smad3, TGF-β), and tumor suppression (PTEN) biomarkers showed an identical pattern, whereas that of anti-apoptotic (Bcl-2) and anti-fibrotic (Smad1/5, BMP-2) biomarkers were displayed an opposite pattern compared to that of total lung weight among all groups (all p<0.001). The MPs could enhance angiogenesis and accelerated hepatoma cell growth in rodent lung parenchyma. PMID:27186261

  7. Chronic exposure to agmatine results in the selection of agmatine-resistant hepatoma cells.

    PubMed

    Bandino, Andrea; Andrea, Bandino; Battaglia, Valentina; Valentina, Battaglia; Bravoco, Vittoria; Vittoria, Bravoco; Busletta, Chiara; Chiara, Busletta; Compagnone, Alessandra; Alessandra, Compagnone; Cravanzola, Carlo; Carlo, Cravanzola; Meli, Floriana; Floriana, Meli; Agostinelli, Enzo; Enzo, Agostinelli; Parola, Maurizio; Maurizio, Parola; Colombatto, Sebastiano; Sebastiano, Colombatto

    2012-02-01

    During our study of the cytostatic effect of agmatine, we were able to isolate an agmatine resistant clone from a parental hepatoma cell line, HTC. These cells, called Agres, had slower growth rate than the parental cells when cultured in normal medium. The modification in polyamine content induced by agmatine was much lower in these cells and ornithine decarboxylase, S-adenosylmethionine decarboxylase and spermidine/spermine acetyltransferase activities were much less affected. By investigating the mechanism responsible for these modifications, it was shown that agmatine and polyamines were not taken up by Agres cells. Their resistance to the antiproliferative effects of agmatine may thus arise from a lack of the polyamine transport system. Moreover, Agres cells were able to take up both glutamic acid and arginine at a rate significantly higher than that detected for HTC cells, most likely to provide components for compensatory increase of PA synthesis. These results emphasize the importance of polyamine transport for cell growth. PMID:21901471

  8. Kinetics of steroid induction and deinduction of tyrosine aminotransferase synthesis in cultured hepatoma cells.

    PubMed Central

    Steinberg, R A; Levinson, B B; Tomkins, G M

    1975-01-01

    The specific rate of synthesis of tyrosine aminotransferase (EC 2.6.1.5; L-tyrosine:2-oxoglutarate aminotransferase) is used as a measure of the level of functional, cytoplasmic, tyrosine aminotransferase-specific mRNA in cultured rat hepatoma cells. An analysis of the kinetics of change in this rate after the addition or withdrawal of glucocorticosteroids sets an upper limit on the half-life of the enzyme-specific mRNA of 1-1.5 hr, whether or not steroid is present. The inactivation rate of the enzyme mRNA is independent of the growth condition of the cells, occuring equally rapidly in the presence or absence of serum or insulin, both of which induce tyrosine aminotransferase in these cells. The implications of these results for the mechanism of steroid induction are discussed. PMID:237268

  9. Cu,Zn Superoxide Dismutase is a Peroxisomal Enzyme in Human Fibroblast and Hepatoma Cells

    NASA Astrophysics Data System (ADS)

    Keller, Gilbert-Andre; Warner, Thomas G.; Steimer, Kathelyn S.; Hallewell, Robert A.

    1991-08-01

    The intracellular localization of Cu,Zn superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) has been examined by immunofluorescence using four monoclonal anti-Cu,Zn superoxide dismutase antibodies raised against a recombinant human Cu,Zn superoxide dismutase derivative produced and purified from Escherichia coli. Colocalization with catalase, a peroxisomal matrix enzyme, was used to demonstrate the peroxisomal localization of Cu,Zn superoxide dismutase in human fibroblasts and hepatoma cells. In the fibroblasts of Zellweger syndrome patients, the enzyme is not transported to the peroxisomal ghosts but, like catalase, remains in the cytoplasm. In addition, immunocryoelectron microscopy of yeast cells expressing human Cu,Zn superoxide dismutase showed that the enzyme is translocated to the peroxisomes.

  10. Elemene injection induced autophagy protects human hepatoma cancer cells from starvation and undergoing apoptosis.

    PubMed

    Lin, Yan; Wang, Keming; Hu, Chunping; Lin, Lin; Qin, Shukui; Cai, Xueting

    2014-01-01

    Elemene, a compound found in an herb used in traditional Chinese medicine, has shown promising anticancer effects against a broad spectrum of tumors. In an in vivo experiment, we found that apatinib, a tyrosine kinase inhibitor that selectively inhibits VEGFR2, combined with elemene injection (Ele) for the treatment of H22 solid tumor in mice resulted in worse effectiveness than apatinib alone. Moreover, Ele could protect HepG2 cells from death induced by serum-free starvation. Further data on the mechanism study revealed that Ele induced protective autophagy and prevented human hepatoma cancer cells from undergoing apoptosis. Proapoptosis effect of Ele was enhanced when proautophagy effect was inhibited by hydroxychloroquine. Above all, Ele has the effect of protecting cancer cells from death either in apatinib induced nutrient deficient environment or in serum-free induced starvation. A combination of elemene injection with autophagy inhibitor might thus be a useful therapeutic option for hepatocellular carcinoma. PMID:25152762

  11. Cathepsin H regulated by the thyroid hormone receptors associate with tumor invasion in human hepatoma cells.

    PubMed

    Wu, S-M; Huang, Y-H; Yeh, C-T; Tsai, M-M; Liao, C-H; Cheng, W-L; Chen, W-J; Lin, K-H

    2011-04-28

    Thyroid hormone, 3, 3', 5-triiodo-L-thyronine (T(3)), mediates cell growth, development and differentiation by binding to its nuclear receptors (TRs). The role of TRs in cancer is still undefined. Notably, hyperthyroxinemia has been reported to influence the rate of colon cancer in an experimental model of carcinogenesis in rats. Previous microarray analysis revealed that cathepsin H (CTSH) is upregulated by T(3) in HepG2-TR cells. We verified that mRNA and protein expression of CTSH are induced by T(3) in HepG2-TR cells and in thyroidectomized rats following administration of T(3). The possible thyroid hormone-responsive elements of the CTSH promoter localized to the nucleotides -2038 to -1966 and -1565 to -1501 regions. An in vitro functional assay showed that CTSH can increase metastasis. J7 cells overexpressing CTSH were inoculated into severe combined immune-deficient mice and these J7-CTSH mice displayed a greater metastatic potential than did J7-control mice. The clinicopathologic significance of CTSH expression in hepatocellular carcinoma (HCC) was also investigated. The CTSH overexpressing in HCC was associated with the presence of microvascular invasion (P=0.037). The microvascular invasion characteristic is closely related to our in vitro characterization of CTSH function. Our results show that T(3)-mediated upregulation of CTSH led to matrix metallopeptidase or extracellular signal-regulated kinase activation and increased cell migration. This study demonstrated that CTSH overexpression in a subset hepatoma may be TR dependent and suggests that this overexpression has an important role in hepatoma progression. PMID:21217776

  12. Plasmid Transfer of Plasminogen K1-5 Reduces Subcutaneous Hepatoma Growth by Affecting Inflammatory Factors

    PubMed Central

    Koch, Lea A.; Strassburg, Christian P.; Raskopf, Esther

    2014-01-01

    There is evidence that plasminogen K1-5 (PlgK1-5) directly affects tumour cells and inflammation. Therefore, we analysed if PlgK1-5 has immediate effects on hepatoma cells and inflammatory factors in vitro and in vivo. In vitro, effects of plasmid encoding PlgK1-5 (pK1-5) on Hepa129, Hepa1-6, and HuH7 cell viability, apoptosis, and proliferation as well as VEGF and TNF-alpha expression and STAT3-phosphorylation were investigated. In vivo, tumour growth, proliferation, vessel density, and effects on vascular endothelial growth factor (VEGF) and tumour necrosis factor alpha (TNF-alpha) expression were examined following treatment with pK1-5. In vivo, pK1-5 halved cell viability; cell death was increased by up to 15% compared to the corresponding controls. Proliferation was not affected. VEGF, TNF-alpha, and STAT3-phosphorylation were affected following treatment with pK1-5. In vivo, ten days after treatment initiation, pK1-5 reduced subcutaneous tumour growth by 32% and mitosis by up to 77% compared to the controls. Vessel density was reduced by 50%. TNF-alpha levels in tumour and liver tissue were increased, whereas VEGF levels in tumours and livers were reduced after pK1-5 treatment. Taken together, plasmid gene transfer of PlgK1-5 inhibits hepatoma (cell) growth not only by reducing vessel density but also by inducing apoptosis, inhibiting proliferation, and triggering inflammation. PMID:24895598

  13. Anti-hepatoma human single-chain Fv antibody and adriamycin conjugates with potent antitumor activity.

    PubMed

    Chen, Lin; Liu, Yan-Hong; Li, Yue-Hui; Jiang, Yan; Xie, Ping-Li; Zhou, Guo-Hua; Li, Guan-Cheng

    2014-01-01

    To construct an improved biological missile, an immunoconjugate ADM-Dex-ScFv-SA3 was synthesized, which was composed of a hepatocellular carcinoma-specific, single-chain Fv antibody (ScFv-SA3) and a highly potent cytotoxic drug, adriamycin (ADM), as the warhead. Oxidized Dextran T10 (Dex-T10) was used as a linker to connect these two moieties. The 40 KD soluble anti-hepatoma human Trx-ScFv-SA3 protein was expressed in E. coli BL21 (DE3), using a prokaryotic expression vector, pET21a (+)-Trx-ScFv-SA3-His. It was purified using a His-Tag Ni-Agarose column and identified by western blot. The activity of Trx-ScFv-SA3 was verified by enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry to confirm that it specifically binds to the hepatocellular carcinoma cell line HepG2. To prepare ADM-Dex-ScFv-SA3, ADM was conjugated to the antibody at a molar ratio of 14.21:1. The antitumor effect of the conjugate was tested by MTT assay, plate colony formation assay and xenografts in a nude mice experimental model. In vitro experiments revealed that ADM-Dex-ScFv-SA3 could bind to tumor cells selectively and inhibit the proliferation and the colony formation ability of HepG2 cells. In vivo experiments showed that ADM-Dex-ScFv-SA3 suppressed the tumor growth and prolonged the median survival time in tumor-bearing mice. Tumor histology slides indicated a significantly slower tumor tissue proliferation in the ADM-Dex-ScFv-SA3 group. These data indicate that the targeted drug, ADM-Dex-ScFv-SA3, may be a highly potent and selective therapy for the treatment of hepatoma. PMID:24239629

  14. Differential thioredoxin reductase activity from human normal hepatic and hepatoma cell lines.

    PubMed

    Jung, Haeng-Im; Lim, Hye-Won; Kim, Byung-Chul; Park, Eun-Hee; Lim, Chang-Jin

    2004-04-30

    Thioredoxin reductase (TrxR), a component of the thioredoxin system, including thioredoxin (Trx) and NADPH, catalyzes the transfer of electrons from NADPH to Trx, acts as a reductant of disulfide-containing proteins and participates in the defense system against oxidative stresses. In this study, the regulation pattern of TrxR in the presence of various stressful reagents was compared between Chang (human normal hepatic cell) and HepG2 (human hepatoma cell) cell lines. Aluminum chloride (0.5 mM) and zinc chloride (0.5 mM) enhanced the TrxR activity in the Chang cell line to a higher degree than in the HepG2 cell line, but cupric chloride (0.2 mM) and cadmium chloride (0.1 mM) enhanced the TrxR activity in the HepG2 cell line to a greater degree. The TrxR activities in both Chang and HepG2 cell lines were similarly induced by treatment with sodium selenite (0.02 mM) and menadione (0.5 and 1.0 mM). Lipopolysaccharide (2 micro g/m1) increased the TrxR activity upto 4.02- and 2.2-fold in the Chang and HepG2 cell lines, respectively, in time-dependent manners. Hydrogen peroxide (5 mM) markedly enhanced the TrxR activity in the HepG2 cell line, but not in the Chang cell line. NO-generating sodium nitroprusside (3.0 and 6.0 mM) induced TrxR activities in both human liver cell lines. The TrxR activity was also induced in human liver cells under limited growth conditions by serum deprivation. These results imply that the TrxR activities in normal hepatic and hepatoma cell lines are subject to different regulatory responses to various stresses. PMID:15118998

  15. Policosanol inhibits cholesterol synthesis in hepatoma cells by activation of AMP-kinase.

    PubMed

    Singh, Dev K; Li, Li; Porter, Todd D

    2006-09-01

    Policosanol is a mixture of long-chain primary alcohols that has been shown to decrease serum cholesterol in animals and in humans. The hypocholesterolemic effect results from a decrease in cholesterol synthesis by suppression of HMG-CoA reductase activity, but the mechanism of this suppression and the active components of policosanol have not been established. In the present study, we investigated the ability of policosanol and its principal components to inhibit cholesterol synthesis in cultured rat hepatoma cells. Maximal inhibition by policosanol yielded a 30% decrease in [(14)C]acetate incorporation without evidence of cellular toxicity. Octacosanol (C28, the major constituent of policosanol), heptacosanol (C27), and hexacosanol (C26) yielded smaller and statistically insignificant decreases in cholesterol synthesis, whereas triacontanol (1-hydroxytriacontane; C30) replicated the inhibition obtained with policosanol. At pharmacological concentrations (<5 microg/ml), policosanol and triacontanol decreased [(14)C]acetate incorporation into cholesterol without affecting the incorporation of [(14)C]mevalonate, indicating that these compounds act at or above HMG-CoA reductase. Policosanol and triacontanol did not directly inhibit HMG-CoA reductase, and incubation of these compounds with hepatoma cells did not affect reductase enzyme levels. However, reductase activity was decreased by up to 55% in lysates prepared from these cells, suggesting that HMG-CoA reductase activity was down-regulated by policosanol treatment. Consistent with this hypothesis, a 3-fold increase in AMP-kinase phosphorylation was noted in policosanol-treated cells. Because AMP-kinase is activated by phosphorylation and is well established to suppress HMG-CoA reductase activity, these results suggest that policosanol or a metabolite decreases HMG-CoA reductase activity by activating AMP-kinase. PMID:16714400

  16. 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

  17. [The morphometric analysis of cytotoxical action of rats and mice splenocytes against confluent monolayer cell lines of hepatomas].

    PubMed

    Teriukova, N P; Pogodina, O N; Blinova, G I; Ivanov, V A

    2009-01-01

    The present study was aimed to examine the possibility to use of the morphometric analysis for estimation of the total natural cytotoxic activity of rat and mice C3HA splenocytes against the cultured target cells which formed confluent monolayers. By means of this method we have revealed that two rat monolayer hepatoma cell lines -- HTC and Zajdela -- were sensitive to cytolysis mediated by rat effector cells, but not splenocytes of C3HA mice. The mild pretreatment with 0.5% paraformaldehyde of the rat splenocytes produced a significant decrease only in the cytotoxic activity against the target cells line Zajdela and don't affect the lysis of the other target HTC cells. These results suggest that the natural cytotoxic rat cells may mediate their lytic functions toward tested target lines by different ways. In the case of HTC cells as the targets the effectors induce death receptor-mediated apoptosis, as to target cells Zajdela they deliver lethal hit by perforin-granzyme exocytosis mechanism. The cultured cell monolayers of mice hepatoma -- MH-22a and BWTG3 -- cells showed under conditions of our experiments the resistance to cytolysis by homological effector cells; however, the hepatoma MH-22a cells were susceptible to killing mediated by rat cytotoxic splenocytes. PMID:19505047

  18. Ethanol extracts of Cinnamomum kanehirai Hayata leaves induce apoptosis in human hepatoma cell through caspase-3 cascade

    PubMed Central

    Liu, Yu-Kuo; Chen, Kuan-Hsing; Leu, Yann-Lii; Way, Tzong-Der; Wang, Ling-Wei; Chen, Yu-Jen; Liu, Yu-Ming

    2015-01-01

    Inducing apoptosis to susceptible cells is the major mechanism of most cytotoxic anticancer drugs in current use. Cinnamomum kanehirai Hayata (Lauraceae), a unique and native tree of Taiwan, is the major host for the medicinal fungus Antrodia cinnamomea which exhibits anti-cancer activity. Because of the scarcity of A. cinnamomea, C. kanehirai Hayata instead, is used as fork medicine in liver cancer. Here we observed the C. kanehirai Hayata ethanol extract could inhibit the cellular viability of both HepG2 and HA22T/VGH human hepatoma cell lines in a dose- and time-dependent manner. We found the mode of cell death was apoptosis according to cell morphological changes by Liu’s stain, oligonucleosomal DNA fragmentation by gel electrophoresis, externalization of phosphotidyl serine by detecting Annexin V and hypoploid population by cell cycle analysis. Our results showed that the extracts caused cleavage of caspase-3 and increased enzyme activity of caspase-8 and caspase-9. Caspase 3 inhibitor partially reversed the viability inhibition by the extract. Furthermore, the up-regulation of Bax and down-regulation of Bcl-2 were also noted by the extract treatment. In conclusion, C. kanehirai Hayata ethanol extract induced intrinsic pathway of apoptosis through caspase-3 cascade in human hepatoma HA22T/VGH and HepG2 cells, which might shed new light on hepatoma therapy. PMID:25678797

  19. Calcium antagonists and low density lipoproteins metabolism by human fibroblasts and by human hepatoma cell line HEP G2.

    PubMed

    Corsini, A; Granata, A; Fumagalli, R; Paoletti, R

    1986-01-01

    The effect of Ca2+ antagonists (CA) on the receptor-mediated low density lipoprotein pathway has been investigated "in vitro" in human skin fibroblasts (HSF) and in human hepatoma cell line Hep G2. The specific binding and internalization of human 125I-labeled LDL are dose-dependently increased in HSF by CA of the verapamil series (verapamil, anipamil, gallopamil, ronipamil, and diltiazem), but neither by CA of the dihydropyridine series (nifedipine, nitrendipine) nor by flunarizine. BAY K 8644, a Ca2+ agonist, elicited an opposite effect. In the presence of the tested CA, LDL degradation is either unaffected (lower concentrations) or inhibited (higher concentrations). 125I-LDL uptake is stimulated also in fibroblasts from type IIa hypercholesterolemic patients, heterozygous for defective expression of LDL receptor. The enhanced cellular uptake of 125I-LDL was prevented by cycloheximide and by alpha-amanitin. CA of the verapamil series including diltiazem retained their effect in human hepatoma cell line Hep G2, a model proposed for hepatic metabolism of LDL. Our studies show that a) CA stimulate the high affinity binding and internalization of LDL in HSF and in human hepatoma cell line Hep G2; b) this stimulation involves DNA transcription and new protein synthesis; c) this effect is specific to one subgroup of Ca2+ antagonists (the verapamil class only). PMID:3006091

  20. Optimization modeling of single-chain antibody against hepatoma based on similarity algorithm.

    PubMed

    Zhao, Zhi-Jun; Chen, Jing-Tao; Yuan, Jia-Ying; Yin, Xiao-Xiang; Song, Hua-Yong; Wang, Xin-Chun

    2015-01-01

    The purposes was to establish optimal modeling of single-chain antibody molecules based on similarity algorithm and seek the connecting peptides that had the minimal effect on the structure and bioactivity of the variable region of heavy chain (VH) and that of light chain (VL) in a single-chain antibody against liver cancer. After the Linker with different lengths (n=0~7) had been added into single chain fragment variable (ScFv), modeling of the overall sequences of VH, VL and ScFv were conducted respectively. Meanwhile, the peptide chain structure of (Gly4Ser)n was adopted for the connecting peptide. Then the spatial spherical shell layer alignment algorithm based on spherical polar coordinates was utilized for comparing the structural similarity of VH and VL before and after adding connecting peptide. Equally, in order to determine the stability of VH and VL, MATLAB was applied for analysis of the fore and aft distances and the diffusion radius. Indirect ELISA method was used to detect single-chain antibody immunological activity of Linker with different lengths. The MTT assay was utilized for the examination of the inhibition rate of single-chain antibody with different lengths of Linker to liver cancer cell. When n=4, the structural similarity between VH together with VL and their original ones was the highest. When n=3, the influence of connecting peptide on the stability of VH and VL was minimum. When n>3, the fore and aft distances changed little due to the increase and fold of the length of peptide chain. The results of ELISA detection showed that when n=4, affinity of single chain antibody to liver cancer cells was much higher. The MTT test also indicated that when n=4, the inhibition rate of the connecting peptide on hepatoma carcinoma cell reached the highest, and that came second when n=3. When n=4, the structural stability and biological functions of anti-hepatoma single-chain antibody were both favorable. This study has provided a basis for the design

  1. Impairment of the mitochondrial respiratory chain activity in diethylnitrosamine-induced rat hepatomas: possible involvement of oxygen free radicals.

    PubMed

    Boitier, E; Merad-Boudia, M; Guguen-Guillouzo, C; Defer, N; Ceballos-Picot, I; Leroux, J P; Marsac, C

    1995-07-15

    Alterations in the energy metabolism of cancer cells have been reported for many years. However, the deleterious mechanisms involved in these deficiencies have not yet been clearly proved. The main goal of this study was to decipher the harmful mechanisms responsible for the respiratory chain deficiencies in the course of diethylnitrosamine (DENA)-induced rat hepatocarcinogenesis, where mitochondrial DNA abnormalities had been previously reported. The respiratory activity of freshly isolated hepatoma mitochondria, assessed by oxygen consumption experiments and enzymatic assays, presented a severe complex I deficiency 19 months after DENA treatment, and later on, in addition, a defective complex III activity. Since respiratory complex subunits are encoded by both nuclear and mitochondrial genes, we checked whether the respiratory chain defects were due to impaired synthesis processes. The specific immunodetection of complex I failed to show any alterations in the steady-state levels of both nuclear and mitochondrial encoded subunits in the hepatomas. Moreover, in vitro protein synthesis experiments carried out on freshly isolated hepatoma mitochondria did not bring to light any modifications in the synthesis of the mitochondrial subunits of the respiratory complexes, whatever the degree of tumor progression. Finally, Southern blot analysis of mitochondrial DNA did not show any major mitochondrial DNA rearrangements in DENA-induced hepatomas. Because the synthetic processes of respiratory complexes did not seem to be implicated in the respiratory chain impairment, these deficiencies could be partly ascribed to a direct toxic impact of highly reactive molecules on these complexes, thus impairing their function. The mitochondrial respiratory chain is an important generator of noxious, reactive oxygen free radicals such as superoxide and H2O2, which are normally catabolized by powerful antioxidant scavengers. Nineteen months after DENA treatment, a general collapse of

  2. Delta-aminolevulinic acid as a photosensitizer precursor for the treatment of hepatoma cells in vitro

    NASA Astrophysics Data System (ADS)

    Laukka, Mark A.; Wang, Kenneth K.

    1994-07-01

    Delta-aminolevulinic acid ((delta) -ALA) has been recently proposed as a tumor photosensitizer precursor with increased selectivity and decreased toxicity for the treatment of neoplasms. We investigated the conversion and cytotoxicity of (delta) -ALA in a human hepatoma cell line to determine its clinical potential. SK-HEP-1 (ATCC) cells were plated on 35 mm coverslips in media for use in a digital fluorescence microscopic imaging system. (delta) -ALA was added to achieve final concentrations between 0-5 mM. Cells were excited with 450-490 nm light while a 610 nm long pass filter was used to assess fluorescence from conversion to protoporphyrin IX, the putative photosensitizer. After maximal fluorescence was obtained at each initial concentration of (delta) -ALA, cells were radiated with 10 J/cm2 of light from a xenon lamp fitted with a 515 nm band pass filter. After photoradiation, cell death was assessed by flow cytometry using propidium iodide labeling. Protoporphyrin IX accumulation was constant at Ksequals0.001 until a plateau was achieved 2 hours after the addition of (delta -ALA. Photoradiation with 10 J/cm2 at a concentration of 1 mM (delta ALA resulted in a linear increase in cell death over time with 5% cell death at 2 hours and 12% at 5 hours compared to controls. Interestingly, controls with (delta) -ALA alone demonstrated a cytoprotective effect with a logarithmic relationship between increasing cell survival and increasing dose of drug.

  3. Sequence of hepatitis B virus DNA incorporated into the genome of a human hepatoma cell line.

    PubMed Central

    Ziemer, M; Garcia, P; Shaul, Y; Rutter, W J

    1985-01-01

    Seven copies of integrated hepatitis B virus (HBV) DNA and contiguous genomic DNA from a human hepatoma cell line (PLC/PRF/5) have been isolated by molecular cloning and have been partially sequenced. The HBV sequences are fragmented and rearranged. Thus, the surface antigen gene is the only intact HBV transcription unit present in these integrated sequences. The sites of integration-recombination are dispersed over the entire viral genome; there is some preference for integration within the double-stranded portion of the genome. There are no repeats at the ends of the integrated HBV DNA fragments. Thus, recombination does not take place in a manner resembling the integration of retroviruses. The sequence data suggest that each HBV fragment is of the adw subtype. However, the integrated DNAs show an unexpected degree of sequence divergence. Direct evidence for the duplication, transposition, and subsequent divergence of two sequences is presented. The data surprisingly suggest that infection-integration of four distinct adw strains occurred. PMID:2983098

  4. Pomegranate peel extract polyphenols induced apoptosis in human hepatoma cells by mitochondrial pathway.

    PubMed

    Song, Bingbing; Li, Jia; Li, Jianke

    2016-07-01

    This study was aimed to investigate the influence of pomegranate peel polyphenols (PPPs) on the proliferation and apoptosis of HepG2 cells (a kind of human hepatoma cells) and the related mechanism. The inverted fluorescence microscope and the flow cytometer (FCM) were used to test the changes of the cellular morphology, cell cycle, apoptosis, reactive oxygen species (ROS) and mitochondrial transmembrane potential (Δψm). The kit was used to measure the activities of caspase-3/9, and Western Blot was used to detect the expressions of apoptosis-associated proteins including p53, Bcl-2/Bax, Cyt-c and PARP. The results showed that the cells cycle of HepG2 arrested at the S-phase by PPPs and the amount of the early apoptotic cells and ROS level were increased obviously, the level of Cyt-c and the activity of Caspase-3/9 markedly were also increased by PPPs, as well as the ratio of Bax/Bcl-2 and the protein expressions of P53. It was concluded that PPPs could inhibit the growth of HepG2 cells by blocking the cell cycle and inducing the mitochondrial apoptotic pathway in a dose-dependent manner. PMID:27120393

  5. Dexamethasone blocks arachidonate biosynthesis in isolated hepatocytes and cultured hepatoma cells

    SciTech Connect

    Marra, C.A.; de Alaniz, M.J.; Brenner, R.R.

    1986-03-01

    The effect of dexamethasone on the incorporation and conversion of (1-14C)eicosa-8,11,14-trienoic acid to arachidonic acid in isolated hepatocytes and in hepatoma tissue culture (HTC) cells was studied. In both kinds of cells, no changes in the exogenous acid incorporation were found when the hormone was added to the incubation media at 0.1 or 0.2 mM concentration, while the biosynthesis of arachidonic acid was significantly depressed. The effect on the biosynthesis was faster in isolated normal liver cells (60 min) than in tumoral cells (120 min) and reached an inhibition of ca. 50% after 3 hr of treatment. The addition of cycloheximide (10(-6) M) also caused a marked decrease in the biosynthesis of this polyunsaturated fatty acid, but when dexamethasone was added to the media simultaneously with cycloheximide, a synergistic action was not observed. The results obtained show that protein synthesis would be involved in the modulation of the biosynthesis of arachidonic acid by glucocorticoids. The changes in the delta 5 desaturation of labeled 20:3 omega 6 to arachidonic acid correlated with changes in the fatty acid composition in isolated cells.

  6. Extinction of hepatitis C virus by ribavirin in hepatoma cells involves lethal mutagenesis.

    PubMed

    Ortega-Prieto, Ana M; Sheldon, Julie; Grande-Pérez, Ana; Tejero, Héctor; Gregori, Josep; Quer, Josep; Esteban, Juan I; Domingo, Esteban; Perales, Celia

    2013-01-01

    Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV. PMID:23976977

  7. Metabolic Flux Distribution during Defatting of Steatotic Human Hepatoma (HepG2) Cells.

    PubMed

    Yarmush, Gabriel; Santos, Lucas; Yarmush, Joshua; Koundinyan, Srivathsan; Saleem, Mubasher; Nativ, Nir I; Schloss, Rene S; Yarmush, Martin L; Maguire, Timothy J; Berthiaume, Francois

    2016-01-01

    Methods that rapidly decrease fat in steatotic hepatocytes may be helpful to recover severely fatty livers for transplantation. Defatting kinetics are highly dependent upon the extracellular medium composition; however, the pathways involved are poorly understood. Steatosis was induced in human hepatoma cells (HepG2) by exposure to high levels of free fatty acids, followed by defatting using plain medium containing no fatty acids, or medium supplemented with a cocktail of defatting agents previously described before. We measured the levels of 28 extracellular metabolites and intracellular triglyceride, and fed the data into a steady-state mass balance model to estimate strictly intracellular fluxes. We found that during defatting, triglyceride content decreased, while beta-oxidation, the tricarboxylic acid cycle, and the urea cycle increased. These fluxes were augmented by defatting agents, and even more so by hyperoxic conditions. In all defatting conditions, the rate of extracellular glucose uptake/release was very small compared to the internal supply from glycogenolysis, and glycolysis remained highly active. Thus, in steatotic HepG2 cells, glycolysis and fatty acid oxidation may co-exist. Together, these pathways generate reducing equivalents that are supplied to mitochondrial oxidative phosphorylation. PMID:26742084

  8. 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. PMID:22754333

  9. 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

  10. Hepatoma-derived growth factor stimulates smooth muscle cell growth and is expressed in vascular development

    PubMed Central

    Everett, Allen D.; Lobe, David R.; Matsumura, Martin E.; Nakamura, Hideji; McNamara, Coleen A.

    2000-01-01

    Hepatoma-derived growth factor (HDGF) is the first member identified of a new family of secreted heparin-binding growth factors highly expressed in the fetal aorta. The biologic role of HDGF in vascular growth is unknown. Here, we demonstrate that HDGF mRNA is expressed in smooth muscle cells (SMCs), most prominently in proliferating SMCs, 8–24 hours after serum stimulation. Exogenous HDGF and endogenous overexpression of HDGF stimulated a significant increase in SMC number and DNA synthesis. Rat aortic SMCs transfected with a hemagglutinin-epitope–tagged rat HDGF cDNA contain HA-HDGF in their nuclei during S-phase. We also detected native HDGF in nuclei of cultured SMCs, of SMCs and endothelial cells from 19-day fetal (but not in the adult) rat aorta, of SMCs proximal to abdominal aortic constriction in adult rats, and of SMCs in the neointima formed after endothelial denudation of the rat common carotid artery. Moreover, HDGF colocalizes with the proliferating cell nuclear antigen (PCNA) in SMCs in human atherosclerotic carotid arteries, suggesting that HDGF helps regulate SMC growth during development and in response to vascular injury. PMID:10712428

  11. Silver nanoparticles affect glucose metabolism in hepatoma cells through production of reactive oxygen species

    PubMed Central

    Lee, Mi Jin; Lee, Seung Jun; Yun, Su Jin; Jang, Ji-Young; Kang, Hangoo; Kim, Kyongmin; Choi, In-Hong; Park, Sun

    2016-01-01

    The silver nanoparticle (AgNP) is a candidate for anticancer therapy because of its effects on cell survival and signaling. Although numerous reports are available regarding their effect on cell death, the effect of AgNPs on metabolism is not well understood. In this study, we investigated the effect of AgNPs on glucose metabolism in hepatoma cell lines. Lactate release from both HepG2 and Huh7 cells was reduced with 5 nm AgNPs as early as 1 hour after treatment, when cell death did not occur. Treatment with 5 nm AgNPs decreased glucose consumption in HepG2 cells but not in Huh7 cells. Treatment with 5 nm AgNPs reduced nuclear factor erythroid 2-like 2 expression in both cell types without affecting its activation at the early time points after AgNPs’ treatment. Increased reactive oxygen species (ROS) production was detected 1 hour after 5 nm AgNPs’ treatment, and lactate release was restored in the presence of an ROS scavenger. Our results suggest that 5 nm AgNPs affect glucose metabolism by producing ROS. PMID:26730190

  12. In vitro anti-hepatoma activity of fifteen natural medicines from Canada.

    PubMed

    Lin, Liang-Tzung; Liu, Li-Teh; Chiang, Lien-Chai; Lin, Chun-Ching

    2002-08-01

    Fifteen crude drugs, Stellaria media Cyrill. (Caryophyllaceae), Calendula officinalis L. (Compositae), Achillea millefolium L. (Compositae), Verbascum thapsus L. (Scrophulariaceae), Plantago major L. (Plantaginaceae), Borago officinalis L. (Boraginaceae), Satureja hortensis L. (Labiatae), Coptis groenlandica Salisb. (Ranunculaceae), Cassia angustifolia Vahl. (Leguminosae), Origanum majorana L. (Labiatae), Centella asiatica L. (Umbelliferae), Caulophyllum thalictroides Mich. (Berberidaceae), Picea rubens Sargent. (Pinaceae), Rhamnus purshiana D.C. (Rhamnaceae) and Hibiscus sabdariffa L. (Malvaceae), which have been used as folk medicine in Canada, were evaluated for their anti-hepatoma activity on five human liver-cancer cell lines, i.e. HepG2/C3A, SK-HEP-1, HA22T/VGH, Hep3B and PLC/PRF/5. The samples were examined by in vitro evaluation for their cytotoxicity. The results showed that the effects of crude drugs on hepatitis B virus genome-containing cell lines were different from those against non hepatitis B virus genome-containing cell lines. C. groenlandica was observed to be the most effective against the growth of all five cell lines and its chemotherapeutic values will be of interest for further studies. PMID:12203264

  13. 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. PMID:25445350

  14. Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination.

    PubMed

    Baude, Annika; Aaes, Tania Løve; Zhai, Beibei; Al-Nakouzi, Nader; Oo, Htoo Zarni; Daugaard, Mads; Rohde, Mikkel; Jäättelä, Marja

    2016-03-18

    We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage. PMID:26721387

  15. DNA triplex-mediated inhibition of MET leads to cell death and tumor regression in hepatoma

    PubMed Central

    Singhal, G; Akhter, MZ; Stern, DF; Gupta, SD; Ahuja, A; Sharma, U; Jagannathan, NR; Rajeswari, MR

    2016-01-01

    Mesenchymal epithelial transition factor (MET) is one of the critical cell signaling molecules whose aberrant expression is reported in several human cancers. The aim of the study is to investigate the antigene and antiproliferative effect of short triplex forming oligonucleotides, TFO-1 (part of the positive regulatory element) and TFO-2 (away from the transcription start site) on MET expression. HepG2 cells transfected only with TFO-1 (but not with TFO-2 and non-specific TFO) significantly decreased MET levels, which is accompanied by decrease in antiapoptotic proteins and increase in pro-apoptotic proteins. Phosphoproteome-array analysis of 46 intracellular kinases revealed hypophosphorylation of about 15 kinases including ERK, AKT, Src and MEK, suggesting the growth inhibitory effect of TFO-1. Further, the efficacy of TFO-1 was tested on diethylnitrosamine-induced liver tumors in wistar rats. T2-weighted magnetic resonance imaging showed decrease in liver tumor volume up to 90% after treatment with TFO-1. Decreased MET expression and elevated apoptotic activity further indicate that TFO-1 targeted to c-met leads to cell death and tumor regression in hepatoma. Formation of stable DNA triplex between TFO-1 and targeted gene sequence was confirmed by circular dichroic spectroscopy and gel retardation assay. Therefore, it can be concluded that DNA triplex-based therapeutic approaches hold promise in the treatment of malignancies associated with MET overexpression. PMID:21660063

  16. Apoptotic and autophagic responses to photodynamic therapy in 1c1c7 murine hepatoma cells

    PubMed Central

    Andrzejak, Michelle; Price, Michael

    2011-01-01

    Photodynamic therapy (PDT) is a process that can induce apoptosis, autophagy or both depending on the cell phenotype. Apoptosis is a pathway to cell death while autophagy can protect from photokilling or act as a death pathway. In a previous study, we reported a cytoprotective effect of autophagy in murine leukemia cell lines where both autophagy and apoptosis occur within minutes after irradiation of photosensitized cells. In this study, we examined the effects of mitochondrial photodamage catalyzed by low (≤1 µM) concentrations of the photosensitizing agent termed benzoporphyrin derivative (BPD, Verteporfin) on murine hepatoma 1c1c7 cells. Apoptosis was not observed until several hours after irradiation of photosensitized cells. Autophagy was clearly cytoprotective since PDT efficacy was significantly enhanced in a knockdown sub-line (KD) in which the level of a critical autophagy protein (Atg7) was markedly reduced. This result indicates that autophagy can protect from phototoxicity even when apoptosis is substantially delayed. Much higher concentrations (≥10 µM) of BPD had previously been shown to inhibit autophagosome formation. Phototoxicity studies performed with 10 µM BPD and a proportionally reduced light dose were consistent with the absence of an autophagic process in wild-type (WT) cells under these conditions. PMID:21555918

  17. Cytotoxic effect of Eucalyptus citriodora resin on human hepatoma HepG2 cells.

    PubMed

    Shen, Kun-Hung; Chen, Zong-Tsi; Duh, Pin-Der

    2012-01-01

    The aim of this study was to evaluate the antiproliferative effect of Eucalyptus citriodora resin (ECR) on human hepatoma HepG2 cells. The results from MTT assay and LDH leakage analysis showed that water extracts of ECR (WEECR) in the dose range of 0-500 μg/ml displayed stronger cytotoxic effects on HepG2 cells than other organic solvent extracts of ECR. By flow cytometry analysis, WEECR slowed down the cell cycle at the G0/G1 phase after 24 h of incubation. Moreover, WEECR treatment induced an apoptotic response in HepG2 cells. WEECR-induced apoptosis was in association with the attenuation of mitochondrial transmembrane potentials (ΔΨ(m)), increased Bax/Bcl-2 ratio and activation of caspase-3. In addition, WEECR contained high concentration of phenolics and flavonoids, which may be responsible for the potent cytotoxicity of WEECR on HepG2 cells. Taken together, WEECR may be a potent antihepatoma agent due to apoptosis in HepG2 cells. PMID:22419432

  18. Insulin regulation of Na/K pump activity in rat hepatoma cells

    SciTech Connect

    Gelehrter, T.D.; Shreve, P.D.; Dilworth, V.M.

    1984-05-01

    Insulin rapidly increases Na/K pump activity in HTC rat hepatoma cells in tissue culture, as measured by the ouabain-sensitive influx of the potassium analogue 86Rb+. Increased influx is observed within minutes and is maximal (70% above control) within 1-2 h. The effect appears to be mediated by the insulin receptors, as: the concentration dependence on insulin is identical to that for insulin induction of tyrosine aminotransferase and stimulation of 2-aminoisobutyric acid transport, proinsulin is 6% as potent as insulin, and the effect is blocked by anti-receptor antibodies. The early stimulation of potassium influx is not blocked by cycloheximide and is not associated with an increased number of pump sites as measured by /sup 3/H-ouabain binding. The insulin effect is blocked by amiloride, which blocks sodium influx, and is mimicked by the sodium ionophore monensin, which increases sodium influx and intracellular accumulation. Insulin also rapidly increases the initial rate of /sup 22/Na+ influx, suggesting that insulin may enhance Na/K pump activity, in part, by increasing intracellular sodium concentration. Incubation of HTC cells with insulin for 24 h causes complete unresponsiveness to the insulin induction of transaminase and stimulation of amino acid transport, a phenomenon mediated by postbinding mechanisms. In contrast, similar incubation with insulin does not cause unresponsiveness to the insulin stimulation of Na/K pump activity. Therefore, the site of regulation of responsiveness to insulin must be distal to, or separate from, those events causing stimulation of ion fluxes.

  19. Solution structure of the PWWP domain of the hepatoma-derived growth factor family

    PubMed Central

    Nameki, Nobukazu; Tochio, Naoya; Koshiba, Seizo; Inoue, Makoto; Yabuki, Takashi; Aoki, Masaaki; Seki, Eiko; Matsuda, Takayoshi; Fujikura, Yukiko; Saito, Miyuki; Ikari, Masaomi; Watanabe, Megumi; Terada, Takaho; Shirouzu, Mikako; Yoshida, Mayumi; Hirota, Hiroshi; Tanaka, Akiko; Hayashizaki, Yoshihide; Güntert, Peter; Kigawa, Takanori; Yokoyama, Shigeyuki

    2005-01-01

    Among the many PWWP-containing proteins, the largest group of homologous proteins is related to hepatoma-derived growth factor (HDGF). Within a well-conserved region at the extreme N-terminus, HDGF and five HDGF-related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin-associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF-related protein-3 (HRP-3) by NMR spectroscopy. The structure consists of a five-stranded β-barrel with a PWWP-specific long loop connecting β2 and β3 (PR-loop), followed by a helical region including two α-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the β1/β2 loop (β-β arch) and the β3/β4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity. PMID:15689505

  20. Hepatitis B virus X protein mutants exhibit distinct biological activities in hepatoma Huh7 cells

    SciTech Connect

    Liu Xiaohong; Zhang Shuhui; Lin Jing; Zhang Shunmin; Feitelson, Mark A.; Gao Hengjun; Zhu Minghua

    2008-09-05

    The role of the hepatitis B virus X protein (HBx) in hepatocarcinogenesis remains controversial. To investigate the biological impact of hepatitis B virus x gene (HBx) mutation on hepatoma cells, plasmids expressing the full-length HBx or HBx deletion mutants were constructed. The biological activities in these transfectants were analyzed by a series of assays. Results showed that HBx3'-20 and HBx3'-40 amino acid deletion mutants exhibited an increase in cellular proliferation, focus formation, tumorigenicity, and invasive growth and metastasis through promotion of the cell cycle from G0/G1 to the S phase, when compared with the full-length HBx. In contrast, HBx3'-30 amino acid deletion mutant repressed cell proliferation by blocking in G1 phase. The expression of P53, p21{sup WAF1}, p14{sup ARF}, and MDM2 proteins was regulated by expression of HBx mutants. In conclusions, HBx variants showed different effects and functions on cell proliferation and invasion by regulation of the cell cycle progression and its associated proteins expression.

  1. Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination

    PubMed Central

    Baude, Annika; Aaes, Tania Løve; Zhai, Beibei; Al-Nakouzi, Nader; Oo, Htoo Zarni; Daugaard, Mads; Rohde, Mikkel; Jäättelä, Marja

    2016-01-01

    We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage. PMID:26721387

  2. Enhancement of esculetin on Taxol-induced apoptosis in human hepatoma HepG2 cells

    SciTech Connect

    Kuo, H.-C.; Lee, H.-J.; Hu, C.-C.; Shun, H.-I; Tseng, T.-H. . E-mail: tht@csmu.edu.tw

    2006-01-15

    The potential use of low dose chemotherapy has been appealing since lower dosages are more attainable during cancer therapy and cause less toxicity in patients. Combination therapy of Taxol, a promising frontline chemotherapy agent, with natural anti-tumor agents that are considerably less toxic with a capability of activating additional apoptotic signals or inhibiting survival signals may provide a rational molecular basis for novel chemotherapeutic strategies. Esculetin, a well-known lipoxygenase inhibitor, showed an inhibitory effect on the cell cycle progression of HL-60 cells in our previous study. In this report, the effects of a concomitant administration of esculetin and Taxol were investigated in human hepatoma HepG2 cells. Firstly, esculetin alone could exert an antiproliferation effect together with an inhibitory effect on the activation of ERKs and p38 MAPK. As compared to the treatment with Taxol only, a co-administration with esculetin and Taxol could result in a further enhancement of apoptosis as revealed by DNA fragmentation assay and Annexin-V-based assay. Meanwhile, immunoblotting analysis also showed that the co-administration of esculetin and Taxol could increase the expression of Bax and the cytosolic release of cytochrome C and enhance the expression of Fas and Fas ligand while the activation of caspase-8 and caspase-3 was also increased. Finally, the ERK cascade was proven to be involved in the enhancement of esculetin on the Taxol-induced apoptosis.

  3. Extraction of tumor-specific antigen from cells and plasma membranes of line-10 hepatoma.

    PubMed

    Leonard, E J; Richardson, A K; Hardy, A S; Rapp, H J

    1975-07-01

    Tumor-specific antigen was extracted with 3 M KCl from line-10 guinea pig hepatoma cells. The yield of antigenic activity, estimated by production of delayed cutaneous hypersensitivity reactions in line-10 immune guinea pigs, was 10-30% of the antigen present in intact cells. By ultracentrifugation criteria, the extracted antigen was soluble. Gel filtration, ion exchange chromatography, and salting-out studies showed that the antigen was heterogeneous in size and net charge. The possibility that 3 M KCl extracted a homogeneous population of molecules associating into polymers of various sizes at low ionic strength was ruled out by heterogeneity on Sephadex G-200 chromatography at high ionic strength. After osmotic lysis of sucrose-loaded line-10 cells, whole plasma membranes or large membrane fragments were obtained in a yield of about 20%. The isolation procedure did not cause detectable loss of membrane antigenic activity. The membranes had 33 skin test U/mg membrane protein, compared to the intact cell value of 1.7 skin test U/mg cell protein. Extracts of plasma membranes had 10-20% of the antigenic activity of the starting membrane material. In contrast to the wide variety of proteins liberated from intact cells, much of the protein extracted from the membranes was in the molecular weight range above 250,000. PMID:169367

  4. 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. PMID:15651921

  5. Metabolic Flux Distribution during Defatting of Steatotic Human Hepatoma (HepG2) Cells

    PubMed Central

    Yarmush, Gabriel; Santos, Lucas; Yarmush, Joshua; Koundinyan, Srivathsan; Saleem, Mubasher; Nativ, Nir I.; Schloss, Rene S.; Yarmush, Martin L.; Maguire, Timothy J.; Berthiaume, Francois

    2016-01-01

    Methods that rapidly decrease fat in steatotic hepatocytes may be helpful to recover severely fatty livers for transplantation. Defatting kinetics are highly dependent upon the extracellular medium composition; however, the pathways involved are poorly understood. Steatosis was induced in human hepatoma cells (HepG2) by exposure to high levels of free fatty acids, followed by defatting using plain medium containing no fatty acids, or medium supplemented with a cocktail of defatting agents previously described before. We measured the levels of 28 extracellular metabolites and intracellular triglyceride, and fed the data into a steady-state mass balance model to estimate strictly intracellular fluxes. We found that during defatting, triglyceride content decreased, while beta-oxidation, the tricarboxylic acid cycle, and the urea cycle increased. These fluxes were augmented by defatting agents, and even more so by hyperoxic conditions. In all defatting conditions, the rate of extracellular glucose uptake/release was very small compared to the internal supply from glycogenolysis, and glycolysis remained highly active. Thus, in steatotic HepG2 cells, glycolysis and fatty acid oxidation may co-exist. Together, these pathways generate reducing equivalents that are supplied to mitochondrial oxidative phosphorylation. PMID:26742084

  6. Single-walled carbon nanohorn (SWNH) aggregates inhibited proliferation of human liver cell lines and promoted apoptosis, especially for hepatoma cell lines

    PubMed Central

    Zhang, Jinqian; Sun, Qiang; Bo, Jian; Huang, Rui; Zhang, Mengran; Xia, Zhenglin; Ju, Lili; Xiang, Guoan

    2014-01-01

    Single-walled carbon nanohorns (SWNHs) may be useful as carriers for anticancer drugs due to their particular structure. However, the interactions between the material itself and cancerous or normal cells have seldom been studied. To address this problem, the effects of raw SWNH material on the biological functions of human liver cell lines were studied. Our results showed that unmodified SWNHs inhibited mitotic entry, growth, and proliferation of human liver cell lines and promoted their apoptosis, especially in hepatoma cell lines. Individual spherical SWNH particles were found inside the nuclei of human hepatoma HepG2 cells and the lysosomes of normal human liver L02 cells, implying that SWNH particles could penetrate into human liver cells_and the different interacted mechanisms on human normal cell lines compared to hepatoma cell lines. Further research on the mechanisms and application in treatment of hepatocellular carcinoma with SWNHs is needed. PMID:24523586

  7. Gli2 silencing enhances TRAIL-induced apoptosis and reduces tumor growth in human hepatoma cells in vivo

    PubMed Central

    Zhang, Da-wei; Li, Hai-yan; Lau, Wan-yee; Cao, Liang-qi; Li, Yue; Jiang, Xiao-feng; Yang, Xue-wei; Xue, Ping

    2014-01-01

    Our previous studies have showed that Gli2 played a predominant role in proliferation and apoptosis resistance to TRAIL in hepatoma cells. The purpose of this study was to explore whether Gli2 silencing enhances efficiency of TRAIL for hepatoma in vivo. SMMC-7721-shRNA cells were implanted subcutaneously into nude mices and TRAIL was injected into the peritoneal space. TUNEL assay was used to detect apoptosis of tumor cells. The expression of Gli2, c-FLIPL, c-FLIPS, and Bcl-2 protein was determined by immunohistochemistry, respectively. Apoptosis and the level of caspases proteins in SMMC-7721 and HepG2 cells were detected by Flow cytometry and Western blot. Transcriptional activity of c-FLIP induced by Gli2 was measured by luciferase reporter gene assay. The results showed that lower volumes and weights of tumor were found in mice xenografted with SMMC-7721-shRNA cells as compared with control cells in the presence of TRAIL (P < 0.05). TUNEL assay showed significantly higher apoptosis index (AI) in the SMMC-7721-shRNA group than in the control groups (P < 0.05). There were remarkable positive correlations between Gli2 and c-FLIPL, c-FLIPS, Bcl-2 protein expression. Over-expression of c-FLIP or Bcl-2 in HepG2 cells attenuated TRAIL-induced apoptosis via suppression of caspase-8 or caspase-9 activity, respectively. Luciferase reporter gene assay found a regulatory sequence by which Gli2 activated transcription between -1007 to -244 in the c-FLIP promoter region. This study demonstrates that Gli2 showed regulatory activity on transcription of c-FLIP gene, and Gli2 silencing enhances TRAIL-induced apoptosis via down-regulation of c-FLIP and Bcl-2 in human hepatoma cells in vivo. PMID:25535898

  8. Combined gene therapy of endostatin and interleukin 12 with polyvinylpyrrolidone induces a potent antitumor effect on hepatoma

    PubMed Central

    Li, Pei-Yuan; Lin, Ju-Sheng; Feng, Zuo-Hua; He, Yu-Fei; Zhou, He-Jun; Ma, Xin; Cai, Xiao-Kun; Tian, De-An

    2004-01-01

    AIM: To study the antitumor effect of combined gene therapy of endostatin and interleukin 12 (IL-12) with polyvinylpyrrolidone (PVP) on mouse transplanted hepatoma. METHODS: Mouse endostatin eukaryotic plasmid (pSecES) with a mouse Igκ signal sequence inside and mouse IL-12 eukaryotic plasmid (pmIL-12) were transfected into BHK-21 cells respectively. Endostatin and IL-12 were assayed by ELISA from the supernant and used to culture endothelial cells and spleen lymphocytes individually. Proliferation of the latter was evaluated by MTT. H22 cells were inoculated into the leg muscle of mouse, which was injected intratumorally with pSecES/PVP, pmIL-12/PVP or pSecES + pmIL-12/PVP repeatedly. Tumor weight, serum endostatin and serum IL-12 were assayed. Tumor infiltrating lymphocytes, tumor microvessel density and apoptosis of tumor cells were also displayed by HE staining, CD31 staining and TUNEL. RESULTS: Endostatin and IL-12 were secreted after transfection, which could inhibit the proliferation of endothelial cells or promote the proliferation of spleen lymphocytes. Tumor growth was highly inhibited by 91.8% after injection of pSecES + pmIL-12/PVP accompanied by higher serum endostatin and IL-12, more infiltrating lymphocytes, fewer tumor vessels and more apoptosis cells compared with injection of pSecES/PVP, pmIL-12/PVP or vector/PVP. CONCLUSION: Mouse endostatin gene and IL-12 gene can be expressed after intratumoral injection with PVP. Angiogenesis of hepatoma can be inhibited synergisticly, lymphocytes can be activated to infiltrate, and tumor cells are induced to apoptosis. Hepatoma can be highly inhibited or eradiated. PMID:15259064

  9. Distribution of [sup 65]Zn labeled alpha-fetoprotein during proliferation of the BW7756 murine hepatoma

    SciTech Connect

    Keenan, J.F.

    1990-01-01

    The radiolabeling of alpha-fetoprotein (AFP) with [sup 65]Zn for the determination of its biodistribution was studied in mice bearing the BW7756 murine hepatoma as compared to that found with normal mice. AFP is an oncofetal protein of about 70,000 daltons associated with pregnancy and certain cancers (e.g., hepatoma). The AFP was purified from mouse amniotic fluid (MAF) using polyacrylamide gel electrophoresis (PAGE) and higher performance liquid chromatography (HPLC). The biological activity of AFP was maintained through the separation procedures and the purity was determined using double immunodiffusion (DID), immunoelectrophoresis (IEP) and sodium dodecyl sulfate electrophoresis (SDS). The labeling procedures included removal of intrinsic metal with EDTA, incubation with radiotracer ([sup 65]Zn) and buffer, followed by removal of unbound [sup 65]Zn using gel filtration chromatography. The results correlated well with Zn fluctuations recorded by other techniques (RIXRF, radiotracer [sup 65]Zn). Large amounts of [sup 65]Zn-AFP were localized in the liver, spleen and tumor with significant elevations above normal in the log growth phase (day 14-18). [sup 65]Zn-AFP levels in the skin, pancreas, brain and thyroid decreased as the tumor mass increased. Tumor [sup 65]Zn-AFP uptake increased with time but leveled off in the late log phase (day 21) due to tumor necrosis. In light of the results of this investigation, and previous work stating that AFP binds Zn with a higher affinity than does albumin, it is suggestive that the Zn fluctuations observed in the earlier hepatoma studies were due to the in vivo binding of Zn to AFP. These results confirm the thesis that intrinsic labeling (replacement of naturally bound ligands with radioactive analogs) does not alter the biochemical integrity as non-intrinsic labeling (e.g., Iodine) may.

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

    SciTech Connect

    Yang, Wei; Sun, Ting; Cao, Jianping; Liu, Fenju; Tian, Ye; Zhu, Wei

    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 in 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.

  11. Hepatoma SK Hep-1 Cells Exhibit Characteristics of Oncogenic Mesenchymal Stem Cells with Highly Metastatic Capacity

    PubMed Central

    Zhang, Yanling; Zhang, Yanhong; Tschudy-Seney, Benjamin; Ramsamooj, Rajen; Wan, Yu-Jui Yvonne; Theise, Neil D.; Zern, Mark A.; Duan, Yuyou

    2014-01-01

    Background SK Hep-1 cells (SK cells) derived from a patient with liver adenocarcinoma have been considered a human hepatoma cell line with mesenchymal origin characteristics, however, SK cells do not express liver genes and exhibit liver function, thus, we hypothesized whether mesenchymal cells might contribute to human liver primary cancers. Here, we characterized SK cells and its tumourigenicity. Methods and Principal Findings We found that classical mesenchymal stem cell (MSC) markers were presented on SK cells, but endothelial marker CD31, hematopoietic markers CD34 and CD45 were negative. SK cells are capable of differentiate into adipocytes and osteoblasts as adipose-derived MSC (Ad-MSC) and bone marrow-derived MSC (BM-MSC) do. Importantly, a single SK cell exhibited a substantial tumourigenicity and metastatic capacity in immunodefficient mice. Metastasis not only occurred in circulating organs such as lung, liver, and kidneys, but also in muscle, outer abdomen, and skin. SK cells presented greater in vitro invasive capacity than those of Ad-MSC and BM-MSC. The xenograft cells from subcutaneous and metastatic tumors exhibited a similar tumourigenicity and metastatic capacity, and showed the same relatively homogenous population with MSC characteristics when compared to parental SK cells. SK cells could unlimitedly expand in vitro without losing MSC characteristics, its tumuorigenicity and metastatic capacity, indicating that SK cells are oncogenic MSC with enhanced self-renewal capacity. We believe that this is the first report that human MSC appear to be transformed into cancer stem cells (CSC), and that their derivatives also function as CSCs. Conclusion Our findings demonstrate that SK cells represent a transformation mechanism of normal MSC into an enhanced self-renewal CSC with metastasis capacity, SK cells and their xenografts represent a same relative homogeneity of CSC with substantial metastatic capacity. Thus, it represents a novel mechanism of

  12. Osmotic regulation of the heat shock response in H4IIE rat hepatoma cells.

    PubMed

    Schliess, F; Wiese, S; Haussinger, D

    1999-09-01

    The influence of cell hydration on the heat shock response was investigated in H4IIE hepatoma cells at the levels of HSP70 expression, MAP kinase activation, induction of c-jun and the MAP kinase phosphatase MKP-1, heat resistance, and development of tolerance/sensitization to arsenite after a priming heat treatment. Induction of HSP70, MKP-1, and c-jun by heat was delayed, but more pronounced or sustained, under hyperosmotic conditions compared with normo- and hypo-osmotically exposed cells. Anisosmolarity per se was ineffective to induce HSP70; some expression of the mRNAs for MKP-1 and c-jun in response to hyperosmolarity was found, but was small compared with the response to heat. Heat-induced activation of JNK-1 was increased under hyperosmotic conditions and more sustained than the JNK-activity induced by hyperosmolarity at 37 degrees C. A prominent Erk-2 activation was found immediately after heat shock under hypo- and normo-osmotic conditions, but Erk-2 activation was weak in hyperosmolarity-exposed cells. Despite anisosmotic alterations of the heat shock response at the molecular level, the heat resistance of H4IIE cells toward heat shock was not affected by ambient osmolarity. However, an osmolarity-dependent sensitization to arsenite was induced by a priming heat shock. The osmodependence of the H4IIE cell response to heat differs from that recently found in primary rat hepatocytes. The data are discussed in terms of cellular adaption mechanisms and their physiological relevance. PMID:10463947

  13. Cortactin and Exo70 mediated invasion of hepatoma carcinoma cells by MMP-9 secretion.

    PubMed

    Zhao, Gang; Zhang, Hongyi; Huang, Ziming; Lv, Liping; Yan, Fan

    2016-05-01

    This study was aimed to evaluate the regulation mechanism of cortactin (CTTN) on matrix metalloproteinases 9 (MMP-9) and its relations with Exo70 in invasion of hepatoma carcinoma (HCC) cells. The expression levels of CTTN, Exo70 and MMP-9 were detected in normal hepatocytes and various HCC cells by real-time PCR. Then the migration and invasion ability of these cells was revealed by scratch and invasion assay. The effects of CTTN on MMP-9 and the ability of migration and invasion were evaluated by silence and overexpress CTTN. During this process, the expression of CTTN was detected by Western blot, the activity and concentration of MMP-9 in supernatant of culture medium was detected by zymography and ELISA assay. Besides, Exo70 was also inhibited to reveal its effects on MMP-9 and the migration and invasion ability of LM3. Increased expression of CTTN, MMP-9, Exo70, reduced scratch area and increased puncture cell numbers were found in HCC cells (p < 0.05). The expression of CTTN was significantly correlated with Exo70 and the migration and invasion ability of HCC (p < 0.05). In addition, the activity and concentration of MMP-9 were significantly affected by the expression level of CTTN, while the expression of MMP-9 was not influenced. Besides, Exo70-si also exhibited significantly inhibition effects on the activity and concentration of MMP-9 and puncture cell numbers (p < 0.05). A synergistic reaction may exhibited on CTTN and Exo70, which could mediate the secretion of MMPs thereby regulate tumor invasion. PMID:27025610

  14. Tetracycline Derivative Minocycline Inhibits Autophagy and Inflammation in Concanavalin-A-Activated Human Hepatoma Cells

    PubMed Central

    Desjarlais, Michel; Pratt, Jonathan; Lounis, Amine; Mounier, Catherine; Haidara, Khadidja; Annabi, Borhane

    2014-01-01

    Inhibition of soluble matrix metalloproteinase (MMP) activity is among the non-antibiotic cellular effects exerted by the anti-inflammatory tetracycline derivative minocycline. The impact of minocycline on the signal transduction functions of membrane-bound MMPs is however unknown. We assessed minocycline in a concanavalin-A (ConA)-activated human HepG2 hepatoma cell model, a condition known to increase the expression of membrane type-1 MMP (MT-MMP) and to trigger inflammatory and autophagy processes. We found that minocycline inhibited ConA-induced formation of autophagic acidic vacuoles, green fluorescent microtubule-associated protein 1 light chain 3 (GFP-LC3) puncta formation, gene and protein expression of autophagy biomarker BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), invasion biomarker MT1-MMP, and inflammation biomarker cyclooxygenase (COX)-2. Gene silencing of MT1-MMP abrogated ConA-induced formation of autophagic acidic vacuoles and ConA-induced expressions of BNIP3 and COX-2. Minocycline was also shown to inhibit ConA-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation as well as gene expression of NANOS1, a biomarker believed to colocalize with MT1-MMP and the specific silencing of which further inhibited ConA-induced STAT3 phosphorylation. Collectively, our data demonstrate that part of minocycline’s effects on autophagy could be exerted through the inhibition of MT1-MMP signaling functions, which contribute to the autophagy and inflammatory phenotype of ConA-activated HepG2 cells. PMID:24634581

  15. Heavy metal interference with growth hormone signalling in trout hepatoma cells RTH-149.

    PubMed

    Marchi, Barbara; Burlando, Bruno; Panfoli, Isabella; Dondero, Francesco; Viarengo, Aldo; Gallo, Gabriella

    2005-04-01

    We have studied the effects of heavy metals (Hg2+, Cu2+, Cd2+) on growth hormone (GH) activation of tyrosine kinase and Ca2+ signaling in the trout (Oncorhynchus mykiss) hepatoma cell line RTH-149. Molecular cloning techniques using primer designed on Oncorhynchus spp. growth hormone receptor (GHR) genes allowed to isolate a highly homologous cDNA fragment from RTH-149 mRNA. Thereafter, cells were analysed by Western blotting or, alternatively, with Ca2+ imaging using fura-2/AM. Exposure of cells to ovine GH alone produced a stimulation of the JAK2/STAT5 pathway and intracellular free Ca2+ variations similar to what has been observed in mammalian models. Cell pre-exposure to Cu2+, Hg2+ or Cd2+ affected cell response to GH by enhancing (Cu2+) or inhibiting (Cd2+) the phosphorylation of JAK2 and STAT5. Heavy metals induced the activation of the MAP kinase p38, and pre-exposure to Hg2+ or Cu2+ followed by GH enhanced the effect of metal alone. Image analysis of fura2-loaded cells indicated that pre-treatment with Hg2+ prior to GH produced a considerable increase of the [Ca2+]i variation produced by either element, while using Cu2+ or Cd2+ the result was similar but much weaker. Data suggest that heavy metals interfere with GH as follows: Hg2+ is nearly ineffective on JAK/STAT and strongly synergistic on Ca2+ signaling; Cu2+ is activatory on JAK/STAT and slightly activatory on Ca2+; Cd2+ is strongly inhibitory on JAK/STAT and slightly activatory on Ca2+; heavy metals could partially activate STAT via p38 independently from GH interaction. PMID:15954744

  16. HRP-3 protects the hepatoma cells from glucose deprivation-induced apoptosis

    PubMed Central

    Cai, Hao; Jiang, Deke; Qi, Fang; Xu, Jianfeng; Yu, Long; Xiao, Qianyi

    2015-01-01

    Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. It is important for HCC cells to resist to apoptosis caused by adverse energy pressure in microenvironment during the HCC tumorigenesis. HRP-3, a member of hepatoma-derived growth factor (HDGF)-related proteins (HRP) family, was shown to be highly up-regulated in HCC tissues and play an important role in HCC pathogenesis based on our previous research. The aim of the study was to investigate the HRP-3’s role in HCC cells endurance against energy pressure. Method: The HRP-3 expression level in primary rat hepatocytes and human HCC cell lines were examined when changing the extracellular glucose concentration. To assess biological function of HRP-3 during glucose deprivation, HRP-3 stable knockdown and control clones of SMMC-7721 and SK-hep1 were constructed for further analysis including cellular morphology observation, apoptotic sub G1 peak analysis and the mTOR-mediated phosphorylation of S6K1 detection in the absence of glucose. Results: Expression level of HRP-3 protein was highly up-regulated both in primary rat hepatocytes and HCC cells as prolonging the stimulation of glucose deprivation. Both morphology and sub-G1 phase analyses indicated that stable knockdown of HRP-3 sensitized HCC cells to glucose deprivation-induced cell apoptosis. Furthermore, silence of HRP-3 prevented the de-phosphorylation of S6K1 induced by glucose deprivation, which was an essential molecular event for HCC cell survival in energy pressure. Conclusions: We propose that glucose deprivation-induced HRP-3 up-regulation potentially plays a major role in protecting HCC cells against apoptosis caused by energy pressure. PMID:26823754

  17. 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. PMID:26434531

  18. The regulatory role of hepatoma-derived growth factor as an angiogenic factor in the eye

    PubMed Central

    LeBlanc, Michelle E.; Wang, Weiwen; Chen, Xiuping; Ji, Yanli; Shakya, Akhalesh; Shen, Chen; Zhang, Chenming; Gonzalez, Vivianne; Brewer, Megan; Ma, Jian-xing; Wen, Rong; Zhang, Fangliang

    2016-01-01

    Purpose Hepatoma-derived growth factor (HDGF) is a mitogen that promotes endothelial proliferation and neuronal survival. Using a unique technology of ligandomics, we recently identified HDGF as a retinal endothelial binding protein. The purpose of this study is to examine the role of HDGF in regulating ocular vasculature and the expression of HDGF in the retina. Methods HDGF expression in the retinal was analyzed with western blot and immunohistochemistry. Angiogenic activity was investigated in human retinal microvascular endothelial cells (HRMVECs) with in vitro endothelial proliferation, migration, and permeability assays. In vivo angiogenic activity was quantified with a corneal pocket assay. The Evans blue assay and western blot using anti-mouse albumin were performed to detect the capacity of HDGF to induce retinal vascular leakage. Results Immunohistochemistry revealed that HDGF is expressed in the retina with a distinct pattern. HDGF was detected in retinal ganglion cells and the inner nuclear layer but not in the inner plexiform layer, suggesting that HDGF is expressed in the nucleus, but not in the cytoplasm, of retinal neurons. In contrast to family member HDGF-related protein 3 (HRP-3) that has no expression in photoreceptors, HDGF is also present in the outer nuclear layer and the inner and outer segments of photoreceptors. This suggests that HDGF is expressed in the nucleus as well as the cytoplasm of photoreceptors. In vitro functional assays showed that HDGF induced the proliferation, migration, and permeability of HRMVECs. Corneal pocket assay indicated that HDGF directly stimulated angiogenesis in vivo. Intravitreal injection of HDGF significantly induced retinal vascular leakage. Conclusions These results suggest that HDGF is an angiogenic factor that regulates retinal vasculature in physiologic and pathological conditions. Identification of HDGF by ligandomics and its independent characterization in this study also support the validity of this

  19. [In vitro targeting effect of lactoferrin modified PEGylated liposomes for hepatoma cells].

    PubMed

    Wei, Min-yan; Zou, Qi; Wu, Chuan-bin; Xu, Yue-hong

    2015-10-01

    A lactoferrin-containing PEGylated liposome system (Lf-PLS) was developed and tested in vitro as a hepatoma-targeting drug delivery system. PEGylated liposomes (PLS) were successfully prepared using the thin film hydration method with peglipid post insertion. Lf was covalently conjugated onto the carboxyl terminal of DSPE-PEG2000-COOH on liposomes. Coumarin-6 was used to trace Lf-PLS with fluorescence. The cellular uptake of this system was carried out in asialoglycoprotein receptor (ASGPR) positive HepG2 cells via confocal microscopy and flow cytometry. The Lf-PLS liposome was observed as spherical or oval vesicles with the particle size around 130 nm, zeta potential about -30 mV and encapsulation efficiency more than 80%. The confocal microscopy images and flow cytometry data demonstrated that Lf-PLS resulted in significantly higher cell association by ASGPR positive HepG2 cells compared to PLS. The association between Lf-PLS and cells were dependent on the concentration, time and temperature, which was inhibited by pre-incubation with excessive free Lf. The results suggest that Lf-PLS has a good targeting effect on HepG2 cells in vitro. The targeting mechanism may be related to the specific binding of Lf and ASGPR on HepG2 cells, which guides Lf-PLS to the cell surface to induce an active endocytosis process. All these results demonstrated that Lf-PLS might be a potential drug delivery system in targeting hepatocellular carcinoma, which deserves more research on its targeting ability, antitumor efficiency, and metabolism in vivo for treatment of hepatomacellular carcinoma. PMID:26837173

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

    SciTech Connect

    Chi, Hsiang-Cheng; Liao, Chen-Hsin; Huang, Ya-Hui; Wu, Sheng-Ming; Tsai, Chung-Ying; Liao, Chia-Jung; Tseng, Yi-Hsin; Lin, Yang-Hsiang; Chen, Cheng-Yi; Chung, I-Hsiao; Wu, Tzu-I; Chen, Wei-Jan; Lin, Kwang-Huei

    2013-09-13

    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 that 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.

  1. Biosynthesis of high molecular weight polylactosamine-type glycopeptides in rat Zajdela hepatoma ascites cells.

    PubMed

    Saunier, B; Goulut, C; Nato, F; Bourrillon, R

    1989-05-10

    The first steps of the biosynthetic pathway of high molecular weight polylactosamine-type glycopeptides from rat Zajdela hepatoma cells were studied by pulse-chase experiments, biochemical analysis and by inhibition of N-glycosylation. It is clear that this process involves firstly the transfer of a lipid-linked high-mannose oligosaccharide precursor to a protein moiety in a similar way to that of N-linked glycopeptides of a more common size range according to the classical 'cycle of dolichol'. In the presence of enzymes which are inhibitors of the processing of high-mannose oligosaccharide chains, this class of oligosaccharides was considerably increased, whereas polylactosamine chains and lower complex N-linked glycopeptides were concomitantly decreased in the same kinetics and the same ratio. As expected in the presence of N-methyldeoxynojirimycin, which is an alpha-glucosidase inhibitor, high-mannose oligosaccharides remained glycosylated and are mostly of the Glc1-3Man9GlcNAc type. In the presence of swainsonine, which is an alpha-mannosidase (EC 3.2.1.24) inhibitor, these chains were devoid of glucose residues. In addition, some chains displayed hybrid structures. It appears, therefore, that the first steps of the biosynthesis of polylactosamine-type and N-linked oligosaccharides of a more common size range proceed similarly and that differences between their biosynthetic pathways occur during the elongation phase, which leads to their final respective structures. Glycopeptides prepared from the cell surface by mild trypsin treatment as well as from entire cells, previously treated or not by processing inhibitors, display the same gel filtration patterns indicating that modifications in protein glycosylation do not prevent glycoprotein insertion into the cell membrane. PMID:2713399

  2. Expression of Hepatoma-derived growth factor family members in the adult central nervous system

    PubMed Central

    El-Tahir, Heba M; Dietz, Frank; Dringen, Ralf; Schwabe, Kerstin; Strenge, Karen; Kelm, Sørge; Abouzied, Mekky M; Gieselmann, Volkmar; Franken, Sebastian

    2006-01-01

    Background Hepatoma-derived growth factor (HDGF) belongs to a polypeptide family containing five additional members called HDGF related proteins 1–4 (HRP-1 to -4) and Lens epithelial derived growth factor. Whereas some family members such as HDGF and HRP-2 are expressed in a wide range of tissues, the expression of others is very restricted. HRP-1 and -4 are only expressed in testis, HRP-3 only in the nervous system. Here we investigated the expression of HDGF, HRP-2 and HRP-3 in the central nervous system of adult mice on the cellular level by immunohistochemistry. In addition we performed Western blot analysis of various brain regions as well as neuronal and glial cell cultures. Results HDGF was rather evenly expressed throughout all brain regions tested with the lowest expression in the substantia nigra. HRP-2 was strongly expressed in the thalamus, prefrontal and parietal cortex, neurohypophysis, and the cerebellum, HRP-3 in the bulbus olfactorius, piriform cortex and amygdala complex. HDGF and HRP-2 were found to be expressed by neurons, astrocytes and oligodendrocytes. In contrast, strong expression of HRP-3 in the adult nervous system is restricted to neurons, except for very weak expression in oligodendrocytes in the brain stem. Although the majority of neurons are HRP-3 positive, some like cerebellar granule cells are negative. Conclusion The coexpression of HDGF and HRP-2 in glia and neurons as well as the coexpression of all three proteins in many neurons suggests different functions of members of the HDGF protein family in cells of the central nervous system that might include proliferation as well as cell survival. In addition the restricted expression of HRP-3 point to a special function of this family member for neuronal cells. PMID:16430771

  3. Chylomicron remnant-vitamin A metabolism by the human hepatoma cell line HepG2

    SciTech Connect

    Lenich, C.M.

    1985-01-01

    The binding and metabolism of (/sup 3/H) vitamin A-containing chylomicron remnants (CMR) by the human hepatoma cell line Hep G2 was studied. Mesenteric lymph chylomicrons (CM) were collected from (/sup 3/H) retinol-fed rats and incubated with lipoprotein-lipase to obtain CMR. At 4/sup 0/C, specific CMR binding was inhibited by excess unlabeled CMR. Specific binding predominated at low concentrations and approached saturation while total binding continued to increase over an extensive concentration range (0.45-32 ..mu..g triglyceride/ml). CMR uptake at 37/sup 0/C was greater than that of CM and at least 100 times more efficient than the fluid-phase pinocytosis of sucrose. CMR binding increased as the extent of lipolysis obtained by incubation with lipoprotein-lipase increased. Addition of human apolipoprotein E enhanced both CMR and CM binding. After internalization, Hep G2 cells hydrolyzed CMR-(/sup 3/H)retinyl esters and radiolabeled metabolites accumulated as a function of time and temperature. As a function of the concentration of (/sup 3/H) VA initially cell-bound, retinol and retinyl esters accumulated as the major cell-associated metabolites. By contrast, retinol was the major metabolite in the medium only at low VA concentrations as other more polar metabolites accumulated at higher concentrations (> 110 pmol VA/mg cell protein). The accumulation of CMR-VA metabolites in the medium was reduced when cells were preincubated in retinol-supplemented media. Also, the specific activity of retinol in the medium closely resembled that in the cell indicating that CMR-VA mixed with the cellular store prior to its secretion.

  4. 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

  5. Effect of isoorientin on intracellular antioxidant defence mechanisms in hepatoma and liver cell lines.

    PubMed

    Yuan, Li; Wang, Jing; Wu, Wanqiang; Liu, Qian; Liu, Xuebo

    2016-07-01

    Isoorientin (ISO) is considered one of the most important flavonoid-like compounds responsible for health benefits, including the prevention of liver damage as well as antioxidant, anti-inflammatory, and anti-nociceptive activities. Our previous study showed that ISO inhibited the proliferation of hepatoma cells through increasing intracellular ROS levels. Interestingly, ISO protects rat liver cells against hydrogen peroxide-induced oxidation stress by decreasing intracellular ROS levels. Why are there different effects of ISO on ROS in different physiological and pathophysiological circumstances? The present study investigated the effect of ISO on mitochondrial respiratory chain complexes and phase II detoxifying enzyme activities in human hepatoblastoma cancer cells (HepG2), buffalo rat liver cells (BRL-3A) and human liver cancer cells (HL-7702). The results showed that intracellular ROS levels and the protein expression of the respiratory chain complexes was significantly (p<0.01) higher in the HepG2 cells than in the BRL-3A and HL-7702 cells. Additionally, ISO notably (p<0.01) increased ROS levels in the HepG2 cells, while no significance was found in the BRL-3A and HL-7702 cells. Furthermore, in the HepG2 cells, the protein expression of the respiratory chain complexes and the phase II detoxifying enzyme activities and GSH content were decreased by ISO (p<0.01), while ISO, in a certain range, enhanced the expression of the protein complexes and the phase II detoxifying enzyme activities and GSH content in BRL-3A and HL-7702 cells. All of these results demonstrated, for the first time, that ISO possesses a notable hepatoprotective effect, which might be mediated through the respiratory chain complexes and phase II detoxifying enzyme activities. PMID:27261613

  6. Zinc protoporphyrin IX enhances chemotherapeutic response of hepatoma cells to cisplatin

    PubMed Central

    Liu, Yang-Sui; Li, Huan-Song; Qi, Dun-Feng; Zhang, Jun; Jiang, Xin-Chun; Shi, Kui; Zhang, Xiao-Jun; Zhang, Xin-Hui

    2014-01-01

    AIM: To investigate the effect of zinc protoporphyrin IX on the response of hepatoma cells to cisplatin and the possible mechanism involved. METHODS: Cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was determined by a flow cytometric assay. Western blotting was used to measure protein expression. Heme oxygenase (HO)-1 activity was measured by determining the level of bilirubin generated in isolated microsomes. Reactive oxygen species (ROS) production was monitored by flow cytometry. Caspase-3 activity was measured with a colorimetric assay kit. Mice were inoculated with 1 × 107 tumor cells subcutaneously into the right flanks. All mice were sacrificed 6 wk after the first treatment and tumors were weighed and measured. RESULTS: Overexpression of HO-1 in HepG2 cell line was associated with increased chemoresistance to cis-diaminedichloroplatinum (cisplatin; CDDP) compared to other cell lines in vitro. Inhibition of HO-1 expression or activity by zinc protoporphyrin IX (ZnPP IX) markedly augmented CDDP-mediated cytotoxicity towards all liver cancer cell lines in vitro and in vivo. In contrast, induction of HO-1 with hemin increased resistance of tumor cells to CDDP-mediated cytotoxicity in vitro and in vivo. Furthermore, cells treated with ZnPP IX plus CDDP exhibited marked production of intracellular ROS and caspase-3 activity, which paralleled the incidence of cell apoptosis, whereas hemin decreased cellular ROS and caspase-3 activity induced by CDDP. CONCLUSION: ZnPP IX increases cellular sensitivity and susceptibility of liver cancer cell lines to CDDP and this may represent a mechanism of increasing ROS. PMID:25024611

  7. Effect of O-4-ethoxyl-butyl-berbamine in combination with pegylated liposomal doxorubicin on advanced hepatoma in mice

    PubMed Central

    Fang, Bai-Jun; Yu, Mei-Li; Yang, Shao-Guang; Liao, Lian-Ming; Liu, Jie-Wen; Zhao, Robert -C-H

    2004-01-01

    AIM: To study the synergistic effects of calmodulin (CaM) antagonist O-4-ethoxyl-butyl-berbamine (EBB) and pegylated liposomal doxorubicin (PLD) on hepatoma-22 (H22) in vivo. METHODS: Hepatoma model was established in 50 Balb/c mice by inoculating H22 cells (2.5 × 106) subcutaneously into the right backs of the mice. These mice were divided into 5 groups, and treated with saline only, PLD only, doxorubicin (Dox) only, PLD plus EBB and Dox plus EBB, respectively. In the treatment groups, mice were given 5 intravenous of PLD or Dox on days 0, 3, 6, 9 and 12. The first dosage of PLD or Dox was 4.5 mg/kg, the other 4 injections was 1 mg/kg. EBB (5 mg/kg) was coadministered with PLD or Dox in the corresponding groups. The effect of drugs on the life spans of hepatoma-bearing mice and tumor response to the drugs were recorded. Dox levels in the hepatoma cells were measured by a fluorescence assay. Light microscopy was performed to determine the histopathological changes in the major organs of these tumor-bearing mice. The MTT method was used to analyze the effect of Dox or PLD alone, Dox in combination with EBB, or PLD in combination with EBB on the growth of H22 cells in an in vitro experiment. RESULTS: EBB (5 mg/kg) significantly augmented the antitumor activity of Dox or PLD, remarkably prolonged the median survival time. The median survival time was 18.2 d for control group, but 89.2 d for PLD + EBB group and 70.1 d for Dox + EBB group, respectively. However, Dox alone did not show any remarkable antitumor activity, and the median survival time was just 29.7 d. Addition of EBB to Dox or PLD significantly increased the level of Dox in H22 cells in vivo. Moreover, EBB diminished liver toxicity of Dox and PLD. In vitro, EBB reduced the IC50 value of Dox or PLD on H22 cells from 0.050 ± 0.006 mg/L and 0.054 ± 0.004 mg/L to 0.012 ± 0.002 mg/L and 0.013 ± 0.002 mg/L, respectively (P < 0.01). CONCLUSION: EBB and liposomization could improve the therapeutic efficacy of

  8. Development of intravenous lipid emulsion of tanshinone IIA and evaluation of its anti-hepatoma activity in vitro.

    PubMed

    Chu, Ting; Zhang, Qing; Li, Hui; Ma, Wei-cong; Zhang, Na; Jin, Hui; Mao, Sheng-jun

    2012-03-15

    The purpose of this study was to develop a lipid emulsion of tanshinone IIA (Tan IIA-LE) for intravenous administration and to investigate its feasibility for future clinical practice. The formulation was optimized using central composite design-response surface methodology (CCD-RSM), and the homogenization process was investigated systematically. The Tan IIA-LE was evaluated in terms of stability, safety and in vitro anti-hepatoma activity. The formulation of Tan IIA-LE is composed of 0.05% (w/v) Tan IIA, 20% (w/v) soybean oil-MCT mixture (1:1, w/w), 1.2% (w/v) soybean lecithin, 0.3% (w/v) F68 and 2.2% (w/v) glycerol, a high pressure homogenization at 100 MPa for 3 cycles was selected as the optimal homogenization process. The Tan IIA-LE was light-sensitive but stable for at least 12 months at room temperature in dark. The safety study demonstrated that the Tan IIA-LE did not cause venous irritation or obvious acute toxicity. Furthermore, the Tan IIA-LE displayed significant anti-tumor activity against human hepatoma cell lines in vitro. Overall, the Tan IIA-LE developed in this study was suggested to be a suitable and safe dosage form of Tan IIA for intravenous administration and has potential in liver cancer therapy in future. PMID:22226873

  9. Chaga mushroom (Inonotus obliquus) induces G0/G1 arrest and apoptosis in human hepatoma HepG2 cells

    PubMed Central

    Youn, Myung-Ja; Kim, Jin-Kyung; Park, Seong-Yeol; Kim, Yunha; Kim, Se-Jin; Lee, Jin Seok; Chai, Kyu Yun; Kim, Hye-Jung; Cui, Ming-Xun; So, Hong Seob; Kim, Ki-Young; Park, Raekil

    2008-01-01

    AIM: To investigate the anti-proliferative and apoptotic effects of Chaga mushroom (Inonotus obliquus) water extract on human hepatoma cell lines, HepG2 and Hep3B cells. METHODS: The cytotoxicity of Chaga extract was screened by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Morphological observation, flow cytometry analysis, Western blot were employed to elucidate the cytotoxic mechanism of Chaga extract. RESULTS: HepG2 cells were more sensitive to Chaga extract than Hep3B cells, as demonstrated by markedly reduced cell viability. Chaga extract inhibited the cell growth in a dose-dependent manner, which was accompanied with G0/G1-phase arrest and apoptotic cell death. In addition, G0/G1 arrest in the cell cycle was closely associated with down-regulation of p53, pRb, p27, cyclins D1, D2, E, cyclin-dependent kinase (Cdk) 2, Cdk4, and Cdk6 expression. CONCLUSION: Chaga mushroom may provide a new therapeutic option, as a potential anticancer agent, in the treatment of hepatoma. PMID:18203281

  10. Characterization of naturally acquired multiple-drug resistance of Yoshida rat ascites hepatoma AH66 cell line.

    PubMed

    Miyamoto, K; Wakabayashi, D; Minamino, T; Nomura, M; Wakusawa, S; Nakamura, S

    1996-01-01

    Characteristics of multiple-drug resistance of rat ascites hepatoma AH66, a cell line induced by dimethylaminoazobenzene and established as a transplantable tumor, were compared with those of AH66F, a drug sensitive line obtained from AH66. The AH66 cell line was resistant to vinblastine, adriamycin, SN-38 an active form of camptothesine, etoposide, and clorambucil by 10-fold or more than the AH66F cell line. The resistance of AH66 cells to vinblastine, adriamycin, and SN-38 was closely related to P-glycoprotein overexpression in the plasma membrane, because the resistance was significantly inhibited by verapamil. AH66 cells contained much glutahione and had a high activity of glutathione S-transferase P-form (GST-P), compared with AH66F cells, and resistance to clorambucil was decreased by treatment with buthionine sulfoximine, an inhibitor of glutathione synthesis. AH66 cells have a similar topoisomerase I activity, but about 6 times lower topoisomerase II activity than AH66F cells. Therefore, the resistance to etoposide and a part of the resistance to adriamycin of AH66 cells seems to depend upon this low topoisomerase II activity. These results, show that the AH66 cell line has high multiple-drug resistance compared with the AH66F cell line, by several mechanisms. Consequently, the AH66 and AH66F cell lines are useful to study naturally acquired multiple-drug resistance of hepatomas. PMID:8702243

  11. Clinacanthus nutans (Burm. f.) Lindau Ethanol Extract Inhibits Hepatoma in Mice through Upregulation of the Immune Response.

    PubMed

    Huang, Danmin; Guo, Wenjie; Gao, Jing; Chen, Jun; Olatunji, Joshua Opeyemi

    2015-01-01

    Clinacanthans nutans (Burm. f.) Lindau is a popular medicinal vegetable in Southern Asia, and its extracts have displayed significant anti-proliferative effects on cancer cells in vitro. However, the underlying mechanism for this effect has yet to be established. This study investigated the antitumor and immunomodulatory activity of C. nutans (Burm. f.) Lindau 30% ethanol extract (CN30) in vivo. CN30 was prepared and its main components were identified using high-performance liquid chromatography (HPLC) and mass spectrometry (LC/MS/MS). CN30 had a significant inhibitory effect on tumor volume and weight. Hematoxylin and eosin (H & E) staining and TUNEL assay revealed that hepatoma cells underwent significant apoptosis with CN30 treatment, while expression levels of proliferation markers PCNA and p-AKT were significantly decreased when treated with low or high doses of CN30 treatment. Western blot analysis of PAPR, caspase-3, BAX, and Bcl2 also showed that CN30 induced apoptosis in hepatoma cells. Furthermore, intracellular staining analysis showed that CN30 treatment increased the number of IFN-γ⁺ T cells and decreased the number of IL-4⁺ T cells. Serum IFN-γ and interleukin-2 levels also significantly improved. Our findings indicated that CN30 demonstrated antitumor properties by up-regulating the immune response, and warrants further evaluation as a potential therapeutic agent for the treatment and prevention of cancers. PMID:26393569

  12. SP1 and USF differentially regulate ADAMTS1 gene expression under normoxic and hypoxic conditions in hepatoma cells.

    PubMed

    Turkoglu, Sumeyye Aydogan; Kockar, Feray

    2016-01-01

    ADAM metallopeptidase with thrombospondin type I motif, 1 (ADAMTS1) that has both antiangiogenic and aggrecanase activity was dysregulated in many pathophysiologic circumstances. However, there is limited information available on the transcriptional regulation of ADAMTS1 gene. Therefore, this study mainly aimed to identify regulatory regions important for the regulation of ADAMTS1 gene under normoxic and hypoxic conditions in human hepatoma cells (HEP3B). Cultured HEP3B cells were exposed to normal oxygen condition, and Cobalt chloride (CoCl2) induced the hypoxic condition, which is an HIF-1 inducer. The cocl2-induced hypoxic condition led to the induced ADAMTS1 mRNA and protein expression in Hepatoma cells. Differential regulation of SP1 and USF transcription factors on ADAMTS1 gene expression was determined by transcriptional activity, mRNA and protein level of ADAMTS1 gene. Ectopic expression of SP1 and USF transcription factors resulted in the decrease in ADAMTS1 transcriptional activity of all promoter constructs consistent with mRNA and protein level in normoxic condition. However, overexpression of SP1 and USF led to the increase of ADAMTS1 gene expressions at mRNA and protein level in hypoxic condition. On the other hand, C/EBPα transcription factor didn't show any statistically significant effect on ADAMTS1 gene expression at mRNA, protein and transcriptional level under normoxic and hypoxic condition. PMID:26299656

  13. Anticancer effect of the extracts from Polyalthia evecta against human hepatoma cell line (HepG2)

    PubMed Central

    Machana, Sasipawan; Weerapreeyakul, Natthida; Barusrux, Sahapat

    2012-01-01

    Objective To investigate the anticancer activity of Polyalthia evecta (P. evecta) (Pierre) Finet & Gagnep against human hepatoma cell line (HepG2). Methods The anticancer activity was based on (a) the cytotoxicity against human hepatoma cells (HepG2) assessed using a neutral red assay and (b) apoptosis induction determined by evaluation of nuclei morphological changes after DAPI staining. Preliminary phytochemical analysis of the crude extract was assessed by HPLC analysis. Results The 50% ethanol-water crude leaf extract of P. evecta (EW-L) showed greater potential anticancer activity with high cytotoxicity [IC50 = (62.8 ± 7.3)µg/mL] and higher selectivity in HepG2 cells than normal Vero cells [selective index (SI) = 7.9]. The SI of EW-L was higher than the positive control, melphalan (SI = 1.6) and the apoptotic cells (46.4 ± 2.6) % induced by EW-L was higher than the melphalan (41.6 ± 2.1)% (P<0.05). The HPLC chromatogram of the EW-L revealed the presence of various kinds of polyphenolics and flavonoids in it. Conclusions P. evecta is a potential plant with anticancer activity. The isolation of pure compounds and determination of the bioactivity of individual compounds will be further performed. PMID:23569932

  14. Activation of AMPK/MnSOD signaling mediates anti-apoptotic effect of hepatitis B virus in hepatoma cells

    PubMed Central

    Li, Lei; Hong, Hong-Hai; Chen, Shi-Ping; Ma, Cai-Qi; Liu, Han-Yan; Yao, Ya-Chao

    2016-01-01

    AIM: To investigate the anti-apoptotic capability of the hepatitis B virus (HBV) in the HepG2 hepatoma cell line and the underlying mechanisms. METHODS: Cell viability and apoptosis were measured by MTT assay and flow cytometry, respectively. Targeted knockdown of manganese superoxide dismutase (MnSOD), AMP-activated protein kinase (AMPK) and hepatitis B virus X protein (HBx) genes as well as AMPK agonist AICAR and antagonist compound C were employed to determine the correlations of expression of these genes. RESULTS: HBV markedly protected the hepatoma cells from growth suppression and cell death in the condition of serum deprivation. A decrease of superoxide anion production accompanied with an increase of MnSOD expression and activity was found in HepG2.215 cells. Moreover, AMPK activation contributed to the up-regulation of MnSOD. HBx protein was identified to induce the expression of AMPK and MnSOD. CONCLUSION: Our results suggest that HBV suppresses mitochondrial superoxide level and exerts an anti-apoptotic effect by activating AMPK/MnSOD signaling pathway, which may provide a novel pharmacological strategy to prevent HCC. PMID:27158203

  15. Human hepatoma cells rich in P-glycoprotein are sensitive to aclarubicin and resistant to three other anthracyclines.

    PubMed Central

    Lehne, G.; De Angelis, P.; Clausen, O. P.; Rugstad, H. E.

    1996-01-01

    Drug resistance is a major obstacle to successful chemotherapy of primary liver cancer, which is associated with high expression of the multidrug resistance (MDR) gene product P-glycoprotein (Pgp), a multidrug efflux transporter. The most effective single agents in treatment of primary liver carcinoma belong to the anthracycline family, yet several anthracyclines are known to be substrates for Pgp. In the present study, we compared four anthracyclines with respect to cell growth inhibition, intracellular accumulation and cellular efflux using the HB8065/R human hepatoma cell line which is rich in Pgp, and the Pgp-poor parental line HB8065/S. The anthracyclines were also administered in conjunction with the Pgp-modifying agents verapamil and SDZ PSC 833 to assess modulation of resistance. The HB8065/R cells were sensitive to aclarubicin (ACL) and highly resistant to epirubicin (EPI), doxorubicin (DOX) and daunorubicin (DNR). SDZ PSC 833 enhanced accumulation, decreased efflux and increased cytotoxicity of EPI, DOX and DNR in the HB8065/R cells, but none of these effects was seen with ACL. In conclusion, ACL is apparently not transported by Pgp and retains its activity in a multidrug-resistant human hepatoma cell line; such properties can be exploited for clinical purposes. Images Figure 5 PMID:8956784

  16. Celecoxib induces apoptosis via a mitochondria‑dependent pathway in the H22 mouse hepatoma cell line.

    PubMed

    Shao, Dan; Kan, Mujie; Qiao, Ping; Pan, Yue; Wang, Zheng; Xiao, Xuanang; Li, Jing; Chen, Li

    2014-10-01

    Celecoxib is a potent nonsteroidal anti-inflammatory drug that has demonstrated promise in cancer chemoprevention and treatment. The present study was conducted to gain insight into the molecular mechanism by which celecoxib induces apoptosis in the H22 mouse hepatoma cell line. The effect of celecoxib on the viability of H22 mouse hepatoma cells was assessed with sulforhodamine B assay. Apoptosis and mitochondrial membrane potential were detected by a flow cytometric assay. The protein expression levels of Bax, Bcl‑2, cytochrome c, caspase-3, caspase-9, apoptosis-inducing factor (AIF), peroxisome proliferator-activated receptor (PPAR)γ and nuclear factor (NF)-κB were determined by western blot analysis. The data demonstrated that celecoxib reduced the percentage of viable H22 cells in a dose- and time-dependent manner, which was associated with cell apoptosis. Furthermore, celecoxib induced apoptosis via the loss of the mitochondrial transmembrane potential (ΔΨm), the release of cytochrome c and AIF, and the activation of caspase-9 and caspase-3. Celecoxib also increased the abundance of the pro-apoptotic protein Bax and reduced the levels of the anti-apoptotic protein Bcl-2. The data demonstrated that celecoxib induced apoptosis in mouse liver cancer cells via the mitochondria-dependent pathway rather than the PPARγ/NF-κB signaling pathway, which indicates that celecoxib may be an effective agent in the clinical management of hepatocellular carcinoma. PMID:25109418

  17. Carnosic acid induces autophagic cell death through inhibition of the Akt/mTOR pathway in human hepatoma cells.

    PubMed

    Gao, Qilong; Liu, Huaimin; Yao, Yamin; Geng, Liang; Zhang, Xinfeng; Jiang, Lifeng; Shi, Bian; Yang, Feng

    2015-05-01

    The therapeutic goal of cancer treatment is now geared towards triggering tumour-selective cell death with autophagic cell death being required for the chemotherapy of apoptosis-resistant cancer. In this study, Carnosic acid (CA), a polyphenolic diterpene isolated from Rosemary (Rosemarinus officinalis), significantly induced autophagic cell death in HepG2 cells. Ca treatment caused the formation of autophagic vacuoles produced an increasing ratio of LC3-II to LC3-I in a time- and dose-dependent manner but had no effect on the levels of autophagy-related protein ATG6 and ATG13 expression. Autophagy inhibitors, 3-methyladenine (3-MA), chloroquine and bafilomycin A1, or ATG genes silencing in HepG2 cells significantly inhibited CA-induced autophagic cell death. The CA treatment decreased the levels of phosphorylated Akt and mTOR without any effects on PI3K or PTEN. Most importantly, overexpression of Akt and knockdown of PTEN attenuated autophagy induction in CA-treated cells. Taken together, our results indicated that CA induced autophagic cell death through inhibition of the Akt/mTOR pathway in human hepatoma cells. These findings suggest that CA has a great potential for the treatment of hepatoma via autophagic induction. PMID:25178877

  18. 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

  19. Inhibition of apolipoprotein B and triglyceride secretion in human hepatoma cells (HepG2).

    PubMed

    Haghpassand, M; Wilder, D; Moberly, J B

    1996-07-01

    Apolipoprotein B (apoB), the major protein component of triglyceride-rich lipoproteins, is assembled into a lipoprotein particle via a complex, multistep process. Recent studies indicate that triglyceride-rich lipoprotein assembly requires the activity of the heterodimeric protein, microsomal triglyceride transfer protein (MTP). We identified a novel inhibitor of apolipoprotein B secretion using the human hepatoma cell line, HepG2. CP-10447, a derivative of the hypnotic drug methaqualone (Quaalude), inhibited apoB secretion from HepG2 cells with an IC50 of approximately 5 microM. CP-10447 also inhibited apoB secretion from Caco-2 cells, a model of intestinal lipoprotein production. In experiments using [3H]glycerol as a precursor for triglyceride synthesis, CP-10447 (20 microM) inhibited radiolabeled triglyceride secretion by approximately 83% (P < 0.0001) in HepG2 cells and 76% (P < 0.05) in Caco-2 cells with no effect on radiolabel incorporation into cellular triglyceride, indicating that CP-10447 inhibited triglyceride secretion without affecting triglyceride synthesis. RNA solution hybridization assay indicated that CP-10447 did not affect apoB or apoA-I mRNA levels. Pulse-chase experiments in HepG2 cells confirmed that CP-10447 inhibited the secretion of apoB (not its synthesis) without affecting secretion of total proteins or albumin and suggested that CP-10447 stimulates the early intracellular degradation of apoB in the endoplasmic reticulum (ER). Further studies demonstrated that CP-10447 is a potent inhibitor of human liver microsomal triglyceride transfer activity (IC50 approximately 1.7 microM) in an in vitro assay containing artificial liposomes and partially purified human MTP. These data suggest that CP-10447 may inhibit apoB and triglyceride secretion by inhibiting MTP activity and stimulating the early ER degradation of apoB. CP-10447 should provide a useful tool for further study of the mechanisms of apoB secretion and triglyceride

  20. The Limonoids TS3 and Rubescin E Induce Apoptosis in Human Hepatoma Cell Lines and Interfere with NF-κB Signaling

    PubMed Central

    Lange, Nicole; Tontsa, Armelle Tsamo; Wegscheid, Claudia; Mkounga, Pierre; Nkengfack, Augustin Ephrem; Sass, Gabriele; Tiegs, Gisa

    2016-01-01

    Hepatocellular carcinoma (HCC) is extremely resistant towards pharmacological therapy. To date, the multi-kinase inhibitor Sorafenib is the only available therapeutic agent with the potential to prolong patient survival. Using the human hepatoma cell lines HepG2 and Huh7, we analyzed anti-cancer activities of 6 purified havanensin type limonoids isolated from the traditional African medicinal plant Trichilia rubescens Oliv. Our results show that two of the compounds, TR4 (TS3) and TR9 (Rubescin E) reduced hepatoma cell viability, but not primary hepatocyte viability, at TC50s of 5 to 10 μM. These were significantly lower than the TC50s for Sorafenib, the histone deacetylase inhibitor SAHA or 5-Fluoruracil. In comparison, TR3 (Rubescin D), a limonoid isolated in parallel and structurally highly similar to TR4 and TR9, did not interfere with hepatoma cell viability. Both, TR4 and TR9, but not TR3, induced apoptosis in hepatoma cells and interfered with NF-κB activation. TR4 as well as TR9 significantly supported anti-cancer activities of Sorafenib. In summary, the limonoids TR4 and TR9 exhibit anti-cancer activities and support Sorafenib effects in vitro, having the potential to support future HCC therapy. PMID:27518192

  1. Expression of alpha fetoprotein messenger RNA in BEL-7404 human hepatoma cells and effect of L-4-oxalysine on the expression

    PubMed Central

    Wang, Xing-Wang; Xu, Bin

    1998-01-01

    AIM: To investigate alpha-fetoprotein (AFP) mRNA expression in BEL-7404 human hepatoma cells and the effect of L-4-oxalysine (OXL) on the expression. METHODS: Bel-7404 human hepatoma cells were maintained in RPMI 1640 media. Human AFP cDNA probe was labelled with digoxigenin-11-dUTP by the random primer labelling method. The expression of AFP mRNA in Bel-7404 cells was determined by an in situ hybridization technique with digoxigenin-labelled human AFP cDNA probe. The positive intensities of AFP mRNA in cells were analyzed by microspectrophotometer and expressed as absorbance at 470 nm. For the experiment with OXL, cells were incubated with various concentrations of the agent for 72 h. RESULTS: Essentially all the hepatoma cells contained AFP mRNA in the cytoplasm, although in various amounts. The specificity of the hybridization reaction was confirmed by control experiments in which the use of Rnase-treated BEL-7404 cells, non-AFP producing cells (HL-60 human leukemia cells) or a nonspecific cDNA probe resulted in negative hybridization. When the cells were treated with OXL (25, 50 mg/L), the content of AFP mRNA in the cytoplasm was decreased with the inhibition percentages of 34.3% and 70.1%, respectively (P < 0.05). CONCLUSION: AFP mRNA was expressed in BEL-7404 human hepatoma cells and OXL suppressed AFP mRNA expression in the cells. PMID:11819302

  2. 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 ...

  3. Core-shell nanoparticles based on pullulan and poly(β-amino) ester for hepatoma-targeted codelivery of gene and chemotherapy agent.

    PubMed

    Liu, Yuanyuan; Wang, Yan; Zhang, Cong; Zhou, Ping; Liu, Yang; An, Tong; Sun, Duxin; Zhang, Ning; Wang, Yinsong

    2014-01-01

    This study designs a novel nanoparticle system with core-shell structure based on pullulan and poly(β-amino) ester (PBAE) for the hepatoma-targeted codelivery of gene and chemotherapy agent. Plasmid DNA expressing green fluorescent protein (pEGFP), as a model gene, was fully condensed with cationic PBAE to form the inner core of PBAE/pEGFP polycomplex. Methotrexate (MTX), as a model chemotherapy agent, was conjugated to pullulan by ester bond to synthesize polymeric prodrug of MTX-PL. MTX-PL was then adsorbed on the surface of PBAE/pEGFP polycomplex to form MTX-PL/PBAE/pEGFP nanoparticles with a classic core-shell structure. MTX-PL was also used as a hepatoma targeting moiety, because of its specific binding affinity for asialoglycoprotein receptor (ASGPR) overexpressed by human hepatoma HepG2 cells. MTX-PL/PBAE/pEGFP nanoparticles realized the efficient transfection of pEGFP in HepG2 cells and exhibited significant inhibitory effect on the cell proliferation. In HepG2 tumor-bearing nude mice, MTX-PL/PBAE/pEGFP nanoparticles were mainly distributed in the tumor after 24 h postintravenous injection. Altogether, this novel codelivery system with a strong hepatoma-targeting property achieved simultaneous delivery of gene and chemotherapy agent into tumor at both cellular and animal levels. PMID:25289563

  4. Exo70 is transcriptionally up-regulated by hepatic nuclear factor 4α and contributes to cell cycle control in hepatoma cells

    PubMed Central

    Zhao, Yujie; Hou, Jihuan; Mi, Panying; Mao, Liyuan; Xu, Liang; Zhang, Youyu; Xiao, Li; Cao, Hanwei; Zhang, Wenqing; Zhang, Bing; Song, Gang; Hu, Tianhui; Zhan, Yan-yan

    2016-01-01

    Exo70, a member of the exocyst complex, is involved in cell exocytosis, migration, invasion and autophagy. However, the expression regulation and function of Exo70 in hepatocellular carcinoma are still poorly understood. In this study, we found Exo70 expression in human hepatoma cells was greatly reduced after knocking down hepatic nuclear factor 4α (HNF4α), the most important and abundant transcription factor in liver. This regulation occurred at the transcriptional level but not post-translational level. HNF4α transactivated Exo70 promoter through directly binding to the HNF4α-response element in this promoter. Cell cycle analysis further revealed that down-regulation of HNF4α and Exo70 was essential to berberine-stimulated G2/M cell cycle arrest in hepatoma cells. Moreover, knocking down either Exo70 or HNF4α induced G2/M phase arrest of hepatoma cells. Exo70 acted downstream of HNF4α to stimulate G2/M transition via increasing Cdc2 expression. Together, our results identify Exo70 as a novel transcriptional target of HNF4α to promote cell cycle progression in hepatoma, thus provide a basis for the development of therapeutic strategies for hepatocellular carcinoma. PMID:26848864

  5. The Limonoids TS3 and Rubescin E Induce Apoptosis in Human Hepatoma Cell Lines and Interfere with NF-κB Signaling.

    PubMed

    Lange, Nicole; Tontsa, Armelle Tsamo; Wegscheid, Claudia; Mkounga, Pierre; Nkengfack, Augustin Ephrem; Loscher, Christine; Sass, Gabriele; Tiegs, Gisa

    2016-01-01

    Hepatocellular carcinoma (HCC) is extremely resistant towards pharmacological therapy. To date, the multi-kinase inhibitor Sorafenib is the only available therapeutic agent with the potential to prolong patient survival. Using the human hepatoma cell lines HepG2 and Huh7, we analyzed anti-cancer activities of 6 purified havanensin type limonoids isolated from the traditional African medicinal plant Trichilia rubescens Oliv. Our results show that two of the compounds, TR4 (TS3) and TR9 (Rubescin E) reduced hepatoma cell viability, but not primary hepatocyte viability, at TC50s of 5 to 10 μM. These were significantly lower than the TC50s for Sorafenib, the histone deacetylase inhibitor SAHA or 5-Fluoruracil. In comparison, TR3 (Rubescin D), a limonoid isolated in parallel and structurally highly similar to TR4 and TR9, did not interfere with hepatoma cell viability. Both, TR4 and TR9, but not TR3, induced apoptosis in hepatoma cells and interfered with NF-κB activation. TR4 as well as TR9 significantly supported anti-cancer activities of Sorafenib. In summary, the limonoids TR4 and TR9 exhibit anti-cancer activities and support Sorafenib effects in vitro, having the potential to support future HCC therapy. PMID:27518192

  6. Hepatitis B virus X protein mutant HBxΔ127 promotes proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT

    SciTech Connect

    Liu, Fabao; You, Xiaona; Chi, Xiumei; Wang, Tao; Ye, Lihong; Niu, Junqi; Zhang, Xiaodong

    2014-02-07

    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 migration 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.

  7. Lipid metabolism enzyme 5-LOX and its metabolite LTB4 are capable of activating transcription factor NF-{kappa}B in hepatoma cells

    SciTech Connect

    Zhao, Yu; Wang, Wenhui; Wang, Qi; Zhang, Xiaodong; Ye, Lihong

    2012-02-24

    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 MK886 (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.

  8. Percutaneous Ethanol Injection of Unresectable Medium-to-Large-Sized Hepatomas Using a Multipronged Needle: Efficacy and Safety

    SciTech Connect

    Ho, C.S. Kachura, J.R.; Gallinger, S.; Grant, D.; Greig, P.; McGilvray, I.; Knox, J.; Sherman, M.; Wong, F.; Wong, D.

    2007-04-15

    Fine needles with an end hole or multiple side holes have traditionally been used for percutaneous ethanol injection (PEI) of hepatomas. This study retrospectively evaluates the safety and efficacy of PEI of unresectable medium-to-large (3.5-9 cm) hepatomas using a multipronged needle and with conscious sedation. Twelve patients, eight men and four women (age 51-77 years; mean: 69) received PEI for hepatomas, mostly subcapsular or exophytic in location with average tumor size of 5.6 cm (range: 3.5-9.0 cm). Patients were consciously sedated and an 18G retractable multipronged needle (Quadrafuse needle; Rex Medical, Philadelphia, PA) was used for injection under real-time ultrasound guidance. By varying the length of the prongs and rotating the needle, the alcohol was widely distributed within the tumor. The progress of ablation was monitored by contrast-enhanced ultrasound, computed tomography (CT) or magnetic resonance imaging (MRI) after each weekly injection and within a month after the final (third) injection and 3 months thereafter. An average total of 63 mL (range: 20-154 ml) of alcohol was injected per patient in an average of 2.3 sessions. Contrast-enhanced CT, ultrasound, or MRI was used to determine the degree of necrosis. Complete necrosis was noted in eight patients (67%), near-complete necrosis (90-99%) in two (16.7%), and partial success (50-89%) in two (16.7%). Follow-up in the first 9 months showed local recurrence in two patients and new lesions in another. There was no mortality. One patient developed renal failure, liver failure, and localized perforation of the stomach. He responded to medical treatment and surgery was not required for the perforation. One patient had severe postprocedural abdominal pain and fever, and another had transient hyperbilirubinemia; both recovered with conservative treatment. PEI with a multipronged needle is a new, safe, and efficacious method in treating medium-to-large-sized hepatocellular carcinoma under conscious

  9. Effect of Endotoxin from Serratia marcescens on the Permeability of Vessels in Hepatomas and Carrageenin Granulomas of Rats

    PubMed Central

    Oswald, N. T. A.; Cater, D. B.

    1969-01-01

    Serratia marcescens polysaccharide (endotoxin) was injected i.v. into rats bearing carrageenin-induced granulomas and transplanted hepatomas, and the vascular-permeability changes were studied by injecting Pelikan ink i.v. at various times after the endotoxin (or saline in the controls). When the ink was given at 0 or 1 hr. there was little difference between the treated rats and the controls. But when the ink was given 2 or more hr. after the endotoxin there was a marked increase of the carbon-lined capillaries and venules, compared with controls, in tumour, granuloma, glomeruli, adrenal cortex, lung, lymph-nodes and sometimes heart. The significance of these changes is discussed in relation to the Shwartzman phenomenon, Arthus reaction, and adrenaline-induced vascular spasm producing anoxia. ImagesFigs. 2-5Figs. 6-9 PMID:4304259

  10. The effect of interferon-{alpha} on the expression of cytochrome P450 3A4 in human hepatoma cells

    SciTech Connect

    Flaman, Anathea S.; Gravel, Caroline; Hashem, Anwar M.; Tocchi, Monika; Li Xuguang

    2011-06-01

    Interferon {alpha} (IFN{alpha}) is used to treat malignancies and chronic viral infections. It has been found to decrease the rate of drug metabolism by acting on cytochrome P450 enzymes, but no studies have investigated the consequences of IFN{alpha} treatment on the CYP3A4 isoform, responsible for the metabolism of a majority of drugs. In this study, we have examined the effect of IFN{alpha} on CYP3A4 catalytic activity and expression in human hepatoma cells. We found that IFN{alpha} inhibits CYP3A4 activity and rapidly down-regulates the expression of CYP3A4, independent of de novo protein synthesis. Pharmacologic inhibitors and a dominant-negative mutant expression plasmid were used to dissect the molecular pathway required for CYP3A4 suppression, revealing roles for Jak1 and Stat1 and eliminating the involvement of the p38 mitogen-activated and extracellular regulated kinases. Treatment of hepatoma cells with IFN{alpha} did not affect the nuclear localization or relative abundance of Sp1 and Sp3 transcription factors, suggesting that the suppression of CYP3A4 by IFN{alpha} does not result from inhibitory Sp3 out-competing Sp1. To our knowledge, this is the first report that IFN{alpha} down-regulates CYP3A4 expression largely through the JAK-STAT pathway. Since IFN{alpha} suppresses CYP3A4 expression, caution is warranted when IFN{alpha} is administered in combination with CYP3A4 substrates to avoid the occurrence of adverse drug interactions.

  11. Antimutagenicity of supercritical CO2 extracts of Terminalia catappa leaves and cytotoxicity of the extracts to human hepatoma cells.

    PubMed

    Ko, Ting-Fu; Weng, Yih-Ming; Lin, Shwu-Bin; Chiou, Robin Y-Y

    2003-06-01

    Natural antimutagens may prevent cancer and are therefore of great interest to oncologists and the public at large. Phytochemicals are potent antimutagen candidates. When the Ames test was applied to examine the antimutagenic potency of supercritical carbon dioxide (SC-CO(2)) extracts of Terminalia catappa leaves at a dose of 0.5 mg/plate, toxicity and mutagenicity were not detected. The antimutagenic activity of SC-CO(2) extracts increased with decreases of temperature (60, 50, and 40 degrees C) and pressure (4000, 3000, and 2000 psi) used for extraction. The most potent antimutagenicity was observed in extracts obtained at 40 degrees C and 2000 psi. At a dose of 0.5 mg of extract/plate, approximately 80% of the mutagenicity of benzo[a]pyrene (B[a]P, with S-9) and 46% of the mutagenicity of N-methyl-N '-nitroguanidine (MNNG, without S-9) were inhibited. Media supplemented with SC-CO(2) extracts at a range of 0-500 microg/mL were used to cultivate human hepatoma (Huh 7) and normal liver (Chang liver) cells. The viability of the cells was assayed by measuring cellular acid phosphatase activity. A dose-dependent growth inhibition of both types of cells was observed. The SC-CO(2) extracts were more cytotoxic to Huh 7 cells than to Chang liver cells. The observation that SC-CO(2) extracts of T. catappa leaves did not induce mutagenicity at the doses tested while exhibiting potent antimutagenicity and were more cytotoxic to human hepatoma cells than to normal liver cells is of merit and warrants further investigation. PMID:12769525

  12. Combination of hypoxia and RNA-interference targeting VEGF induces apoptosis in hepatoma cells via autocrine mechanisms.

    PubMed

    Raskopf, Esther; Vogt, Annabelle; Decker, Georges; Hirt, Sarah; Daskalow, Katjana; Cramer, Thorsten; Standop, Jens; Gonzalez-Carmona, Maria-Angeles; Sauerbruch, Tilman; Schmitz, Volker

    2012-09-01

    Control of VEGF signaling is an intense objective of pre-clinical and clinical studies in HCC disease with steadily increasing clinical application. Despite its emerging role, several aspects of anti-VEGF based treatments are poorly investigated, like the impact on tumor cells themselves, such as the effect on intracellular signaling and apoptosis induction in hepatoma cells. Effects of siRNA-VEGF on VEGF, VEGF-receptor expression and VEGF-A signaling such as AKT and JNK phosphorylation were determined under normoxic or hypoxic conditions in murine hepatoma cells. Apoptosis induction was analyzed by SubG1-fraction, JC1-staining and caspase-8 activation. VEGF receptor expression was analysed by semiquantitative real time PCR. Independent of oxygen status, siRNA-VEGF reduced VEGF levels resulting in decreased AKT and increased JNK phosphorylation in Hepa129 cells. The VEGF-receptors neuropilin-1 (Nrp1) and neuropilin-2 (Nrp2) were downregulated following siRNA-VEGF treatment or hypoxia induction respectively. Functionally, hypoxia significantly increased the apoptosis rate (as analyzed by SubG1-fraction, JC1-staining and JNKphosphorylation) which was further stimulated by siRNA-VEGF treatment. Our data indicate that antitumoral efficacy of an anti-VEGF based treatment with siRNA is partly based on negative autocrine feedback mechanisms which are even enhanced under hypoxic conditions. This observation helps to understand why antitumoral efficacy can be maintained despite of counteracting stimulation of tumoral VEGF secretion due to hypoxia. The direct impact on tumor cells further underscores the attractiveness of an anti-VEGF based siRNA treatment. PMID:21605070

  13. The X protein of hepatitis B virus activates hepatoma cell proliferation through repressing melanoma inhibitory activity 2 gene

    SciTech Connect

    Xu, Yilin; Yang, Yang; Cai, Yanyan; Liu, Fang; Liu, Yingle; Zhu, Ying; Wu, Jianguo

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer We demonstrated that HBV represses MIA2 gene expression both invitro and in vivo. Black-Right-Pointing-Pointer The X protein of HBV plays a major role in such regulation. Black-Right-Pointing-Pointer Knock-down of MIA2 in HepG2 cells activates cell growth and proliferation. Black-Right-Pointing-Pointer HBx activates cell proliferation, over-expression of MIA2 impaired such regulation. Black-Right-Pointing-Pointer HBx activates hepatoma cell proliferation through repressing MIA2 expression. -- Abstract: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer deaths globally. Chronic hepatitis B virus (HBV) infection accounts for over 75% of all HCC cases; however, the molecular pathogenesis of HCC is not well understood. In this study, we found that the expression of the newly identified gene melanoma inhibitory activity 2 (MIA2) was reduced by HBV infection invitro and invivo, and that HBV X protein (HBx) plays a major role in this regulation. Recent studies have revealed that MIA2 is a potential tumor suppressor, and that, in most HCCs, MIA2 expression is down-regulated or lost. We found that the knock-down of MIA2 in HepG2 cells activated cell growth and proliferation, suggesting that MIA2 inhibits HCC cell growth and proliferation. In addition, the over-expression of HBx alone induced cell proliferation, whereas MIA2 over-expression impaired the HBx-mediated induction of proliferation. Taken together, our results suggest that HBx activates hepatoma cell growth and proliferation through repression of the potential tumor suppressor MIA2.

  14. On-line monitoring of adhesion and proliferation of cultured hepatoma cells using optical waveguide lightmode spectroscopy (OWLS).

    PubMed

    Hug, T S; Prenosil, J E; Maier, P; Morbidelli, M

    2002-01-01

    Monitoring of cell adhesion, cell spreading, and cell proliferation opens attractive perspectives in the on-line control of monolayer cell cultures in toxicity tests, in bioreactors as used for the serial production of skin grafts, or in extracorporeal liver devices. In this study the hepatoma Hep G2 cell adhesion and proliferation was monitored using an integrated optical method, optical waveguide lightmode spectroscopy (OWLS). This method is based upon refractive index measurements within a 100-nm thin layer above a Si(Ti)O(2) surface on which the cells were cultured and exposed to cytotoxic and cytostatic agents. The OWLS signal was proportional to cell density during the spreading period (4 h), and in long-term experiments (46 h) the OWLS signal correlated on a logarithmic scale with cell density. After administration of the protein synthesis inhibitor cycloheximide (4 microg/mL) to fully spread hepatoma cells, cell growth was arrested and change of the OWLS signal became noticeable within 6 h after drug administration. For exposure to increasing concentrations of the anticancer drug cyclophosphamide (2.5-20 mM) a concentration-dependent reduction of the OWLS signal was found. For cycloheximide and cyclophospamide the OWLS signal was also confirmed by cell viability measurements using the neutral red assay, the thiazolylblue tetrazoliumbromide assay, total protein measurements, and cell morphology. It was demonstrated that the OWLS signal detects minor changes in cell adhesion, which serve as indicators of metabolic state and growth behavior. OWLS is thus a quantitative tool to characterize impaired cell growth mediated by culture medium, by extracellular matrix, or after exposure to a toxin. PMID:12467478

  15. 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

  16. Effects of dimethyl sulphoxide on the synthesis of plasma proteins in the human hepatoma HepG2. Induction of an acute-phase-like reaction.

    PubMed Central

    Iwasa, F; Galbraith, R A; Sassa, S

    1988-01-01

    Effects of dimethyl sulphoxide (Me2SO) on the synthesis of plasma proteins by the human hepatoma cell line HepG2 were examined. Me2SO treatment resulted in decreased synthesis of albumin and alpha-fetoprotein, and in increased synthesis of haptoglobin. Plasma-protein profiles induced by Me2SO treatment were very similar to those seen in acute-phase reactions. PMID:3140793

  17. 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. PMID:27383887

  18. Occurrence of alpha 1-2-fucosylation in membrane glycoproteins of Morris hepatoma 7777 but not in liver. Aberrant type of fucosylation in a malignant tissue.

    PubMed

    Nuck, R; Orthen, B; Reutter, W

    1992-09-15

    A comparative study was undertaken to characterize the linkages of L-fucose in N-glycans of plasma membrane glycoproteins from Morris hepatoma 7777, host liver and kidney cortex, as well as from rat serum. After in-vivo radiolabelling of rats with L-[6-3H]fucose, the asparagine-linked carbohydrate chains were released from delipidated plasma membrane glycoproteins, as well as from serum glycoproteins, by enzymic digestion with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum. They were then converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride. Two specific alpha-L-fucosidases from almond emulsin and from Aspergillus niger, combined with affinity HPLC on immobilized Aleuria aurantia lectin were used to study the linkage of L-fucose in the oligosaccharide chains. Fucose alpha 1-2 linked to galactose, was present only in the plasma membrane of hepatoma 7777 (18% of total L-[3H]fucose in N-glycans), but was not expressed in host liver, kidney cortex and serum. None of the investigated sources contained an appreciable amount of fucose alpha 1-3/4 linked to N-acetyl-D-glucosamine. All the radioactively labelled oligosaccharides from host liver, kidney cortex and serum, but only 82% of these oligosaccharides from hepatoma, contained alpha-fucosyl residues linked at the C6 position of the proximal N-acetyl-D-glucosamine. PMID:1396674

  19. Regulation of albumin gene expression in hepatoma cells of fetal phenotype: dominant inhibition of HNF1 function and role of ubiquitous transcription factors.

    PubMed Central

    Rollier, A; DiPersio, C M; Cereghini, S; Stevens, K; Tronche, F; Zaret, K; Weiss, M C

    1993-01-01

    Two widely used hepatoma cell lines, mouse BW1J and human HepG2, express gene products characteristic of fetal hepatocytes, including serum albumin, whereas reporter genes driven by the albumin promoter are expressed at very low levels compared with highly differentiated hepatoma cells. We have investigated the low albumin promoter activity in BW1J cells to understand differences in liver gene regulation between fetal and adult cells. Addition of the albumin upstream enhancer, or any other fragment of the albumin gene, failed to modify expression of the transfected promoter in BW1J cells. Analysis of cis elements of the albumin promoter showed that, in contrast to highly differentiated H4II cells, in BW1J cells the activity largely depends on ubiquitous transcription factors. Both BW1J and HepG2 cells produce the liver-enriched transcription factor HNF1; dimerization and DNA binding properties are identical to those of liver HNF1, yet the protein fails to show the anticipated transcriptional stimulatory activity. A transfected HNF1 expression vector strongly trans-activates the albumin promoter in HepG2 but only weakly in BW1J cells, and in hybrids (BW1J x Fao), inefficient HNF1 function is dominant. We conclude that hepatoma cells of the fetal phenotype are deficient in the use of HNF1 to drive transcription of the albumin gene and that they harbor a dominant modulator of HNF1 function. Images PMID:8443410

  20. Synthesis of cholic-acid-carrying polymer and in-vitro evaluation of hepatoma-targeting nanoparticles decorated with the polymer.

    PubMed

    Zhang, Jiantao; Yu, Changjun; Jiang, Guoqiang

    2016-06-01

    The specific interaction between bile acids and the bile acids transporters provides a promising way for hepatoma-targeted drug delivery. We synthesized an amphipathic polymer containing cholic acid (CA), the main bile acids in body, and prepared CA-functionalized nanoparticles to target hepatoma cells. Poly-[3-(4-vinylbenzonate)-7, 12-dihydroxy-5-cholan-24-oic acid] (PVBCA) was synthesized by introducing methyl cholate onto polyvinyl benzoate polymer backbone, and was characterized by (1)H-NMR, FT-IR, and GFC. PVBCA can be incorporated onto PLGA nanoparticles surface via the emulsion-solvent evaporation procedure, resulting in the nanoparticles carrying CA moieties on their surface. The binding of CA moieties to the bile acids' transporters on the cell membrane enhances the cellular uptake of the nanoparticles significantly. The SMMC-7721 cell uptake of PVBCA-decorated nanoparticles increases with amount of incorporated PVBCA and is 2- to 2.8-fold higher than that of the normal PLGA nanoparticles. By exclusion of specific endocytosis pathways using chemical inhibitors, we found that the uptake mechanism of PVBCA-decorated nanoparticles was mainly attributed to clathrin-and-caveolae-independent endocytosis, which was distinct from that of PLGA nanoparticles. The present study provides a simple and versatile method for hepatoma-targeted delivery of nanoparticles. PMID:27045998

  1. Elimination of Cancer Stem-Like “Side Population” Cells in Hepatoma Cell Lines by Chinese Herbal Mixture “Tien-Hsien Liquid”

    PubMed Central

    Yao, Chih-Jung; Yeh, Chi-Tai; Lee, Liang-Ming; Chuang, Shuang-En; Yeh, Chuan-Feng; Chao, Wan-Ju; Lai, Tung-Yuan; Lai, Gi-Ming

    2012-01-01

    There are increasing pieces of evidence suggesting that the recurrence of cancer may result from a small subpopulation of cancer stem cells, which are resistant to the conventional chemotherapy and radiotherapy. We investigated the effects of Chinese herbal mixture Tien-Hsien Liquid (THL) on the cancer stem-like side population (SP) cells isolated from human hepatoma cells. After sorting and subsequent culture, the SP cells from Huh7 hepatoma cells appear to have higher clonogenicity and mRNA expressions of stemness genes such as SMO, ABCG2, CD133, β-catenin, and Oct-4 than those of non-SP cells. At dose of 2 mg/mL, THL reduced the proportion of SP cells in HepG2, Hep3B, and Huh7 cells from 1.33% to 0.49%, 1.55% to 0.43%, and 1.69% to 0.27%, respectively. The viability and colony formation of Huh7 SP cells were effectively suppressed by THL dose-dependently, accompanied with the inhibition of stemness genes, e.g., ABCG2, CD133, and SMO. The tumorigenicity of THL-treated Huh7 SP cells in NOD/SCID mice was also diminished. Moreover, combination with THL could synergize the effect of doxorubicin against Huh7 SP cells. Our data indicate that THL may act as a cancer stem cell targeting therapeutics and be regarded as complementary and integrative medicine in the treatment of hepatoma. PMID:23097677

  2. Improved targeting of 5-[125I/131I]iodo-2‧-deoxyuridine to rat hepatoma by using lipiodol emulsion

    NASA Astrophysics Data System (ADS)

    Yu, Hung-Man; Yeh, Hsin-Pei; Chang, Tien-Kui; Huang, Kuang-Liang; Chuang, Kuo-Tang; Liu, Ren-Shen; Wang, Shyh-Jen; Hwang, Jeng-Jong; Chi, Kwan-Hwa; Chen, Fu-Du; Lin, Wuu-Jyh; Chen, Chin-Hsiung; Wang, Hsin-Ell

    2006-12-01

    This study aims to assess whether emulsion of [ 125/131I]IUdR and lipiodol (IUdR/LP) can improve delivery of IUdR into hepatoma. MethodsIn vitro release profile of IUdR from IUdR/LP to serum was performed. IUdR/LP was injected into N1-S1 hepatoma-bearing SD rat via hepatic artery and IUdR/normal saline (IUdR/NS) was used for comparison. Biodistribution, autoradiography, imaging and tumor DNA incorporation assay were performed. The radioactive metabolites in plasma and urine were analyzed. Radiation doses to tumor and organs were estimated. ResultsIUdR released from lipiodol into serum was fast. There were longer retention, more DNA incorporation and higher radiation dose of IUdR in the tumor by using IUdR/LP. IUdR/LP deposited deep in the hepatomas. Only free iodide was found in the plasma and urine after injection of IUdR/LP. ConclusionsHepatic artery injection of IUdR/LP emulsion could definitely enhance the tumor cell uptake and incorporation to DNA of *IUdR, prolong the tumor retention time and increase radiation dose to tumor. IUdR/LP may be an effective therapeutic agent for the treatment of hepatic tumors.

  3. Nano-cerium-element-doped titanium dioxide induces apoptosis of Bel 7402 human hepatoma cells in the presence of visible light

    PubMed Central

    Wang, Long; Mao, Jian; Zhang, Gao-Hua; Tu, Ming-Jing

    2007-01-01

    AIM: To investigate the apoptotic effect of photoexcited titanium dioxide (TiO2) nanoparticles in the presence of visible light on human hepatoma cell line (Bel 7402) and to study the underlying mechanism. METHODS: Cerium-element-doped titanium dioxide nanoparticles were prepared by impregnation method. Bel 7402 human hepatoma cells were cultured in RPMI 1640 medium in a humidified incubator with 50 mL/L CO2 at 37°C. A 15 W fluorescent lamp with continuous wavelength light was used as light source in the photocatalytic test. Fluorescence morphology and agarose gel eletrophoresis pattern were performed to analyze apoptotic cells. RESULTS: The Ce (IV)-doped TiO2 nanoparticles displayed their superiority. The adsorption edge shifted to the 400-450 nm region. With visible light illuminated for 10 min, 10 μg/cm3 Ce (IV)-doped TiO2 induced micronuclei and significant apoptosis in 4 and 24 h, respectively. Hochest 33 258 staining of the fixed cells revealed typical apoptotic structures (apoptotic bodies), agarose gel electrophoresis showed typical DNA ladder pattern in treated cells but not in untreated ones. CONCLUSION: Ce (IV) doped TiO2 nanoparticles can induce apoptosis of Bel 7402 human hepatoma cells in the presence of visible light. PMID:17663520

  4. Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells

    SciTech Connect

    Amacher, S.L.; Goodman, L.J.; Bravo, D.A.; Wong, K.Y.; Goldfine, I.D.; Hawley, D.M.; Firestone, G.L. )

    1989-10-01

    Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by (125I) insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways.

  5. Glyco-nanoparticles with sheddable saccharide shells: a unique and potent platform for hepatoma-targeting delivery of anticancer drugs.

    PubMed

    Chen, Wei; Zou, Yan; Meng, Fenghua; Cheng, Ru; Deng, Chao; Feijen, Jan; Zhong, Zhiyuan

    2014-03-10

    Reduction-sensitive shell-sheddable glyco-nanoparticles were designed and developed based on poly(ε-caprolactone)-graft-SS-lactobionic acid (PCL-g-SS-LBA) copolymer for efficient hepatoma-targeting delivery of doxorubicin (DOX). PCL-g-SS-LBA was prepared by ring-opening copolymerization of ε-caprolactone and pyridyl disulfide carbonate followed by postpolymerization modification with thiolated lactobionic acid (LBA-SH) via thiol-disulfide exchange reaction. The dynamic light scattering (DLS) and transmission electron microscopy (TEM) showed that PCL-g-SS-LBA was self-assembled into monodisperse nanoparticles (SS-GNs) with a mean diameter of about 80 nm. SS-GNs while remaining stable under physiological conditions (37 °C, pH 7.4) were prone to rapid shell-shedding and aggregation in the presence of 10 mM dithiothreitol (DTT). DOX was loaded into SS-GNs with a decent loading content of 12.0 wt %. Notably, in vitro release studies revealed that about 80.3% DOX was released from DOX-loaded SS-GNs in 24 h under a reductive condition while low drug release (<21%) was observed for DOX-loaded PCL-g-LBA nanoparticles (reduction-insensitive control) under otherwise the same condition and for DOX-loaded SS-GNs under a nonreductive condition. The flow cytometry and confocal microscopy observations indicated that SS-GNs were efficiently taken up by asialoglycoprotein receptor (ASGP-R)-overexpressing HepG2 cells likely via a receptor-mediated endocytosis mechanism and DOX was released into the nuclei of cells following 4 h incubation. MTT assays showed that DOX-loaded SS-GNs exhibited a high antitumor activity toward HepG2 cells, which was comparable to free DOX and about 18-fold higher than their reduction-insensitive counterparts, while blank SS-GNs were nontoxic up to a tested concentration of 1.0 mg/mL. These shell-sheddable glyco-nanoparticles are promising for hepatoma-targeting chemotherapy. PMID:24460130

  6. Carvacrol and rosemary oil at higher concentrations induce apoptosis in human hepatoma HepG2 cells

    PubMed Central

    Melušová, Martina; Jantová, Soňa

    2014-01-01

    Natural essential oils are volatile herbal complex compounds which manifest cytotoxic effects on living cells depending on their type and concentration but usually they are not genotoxic. Our previous studies showed that carvacrol (CA) and rosemary essential oil (RO) induced growth inhibition of both human cell lines HepG2 and BHNF-1, with hepatoma HepG2 cells being more sensitive to either compound tested. Cytotoxic concentrations of CA and RO induced the formation of DNA strand breaks. Further ex vivo studies showed that extracts prepared from hepatocytes of CA- and RO-supplemented rats did not increase incision repair activity compared to extracts from liver cells of control animals. Therefore, the aim of this work was to determine the effect of cytotoxic concentrations of CA and RO on the cell cycle and the ability of both natural volatiles to induce DNA fragmentation and apoptotic death of human hepatoma HepG2 cells. These effects were measured after 24 h incubation of HepG2 cells with CA and RO using three independent methods – flow cytometry, internucleosomal DNA fragmentation (electrophoresis) and micronucleus assay. Evaluation of morphological changes and formation of micronuclei in HepG2 cells showed no increase in the number of micronuclei in cells treated by CA and RO compared to control cells. On the other hand, CA and RO induced morphological changes typical for apoptosis in concentration-dependent manner. The presence of necrosis was negligible. Both natural compounds caused shrinking of cytoplasmic membrane and formation of apoptotic bodies. In addition, the highest concentrations of CA and RO induced internucleosomal DNA fragmentation (formation of DNA ladder) in HepG2 cells. Cell cycle analysis revealed the accumulation of cells in the G1 phase, which was accompanied by a reduction in the number of cells in the S phase after 24 h exposure to the substances tested. The cell division was thus slowed down or stopped and this process resulted in

  7. Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cells

    PubMed Central

    Liu, Yitong; Flynn, Thomas J.; Xia, Menghang; Wiesenfeld, Paddy L.; Ferguson, Martine S.

    2016-01-01

    A human hepatoma cell line (HuH-7) was evaluated as a metabolically competent cell model to investigate cytochrome P450 3A4 (CYP3A4) inhibition, induction, and hepatotoxicity. First, CYP3A4 gene expression and activity were determined in HuH-7 cells under three culture conditions: 1-week culture, 3-week culture, or 1% dimethyl sulfoxide (DMSO) treatment. HuH-7 cells treated with DMSO for 2 weeks after confluence expressed the highest CYP3A4 gene expression and activity compared to the other two culture conditions. Furthermore, CYP3A4 activity in DMSO-treated HuH-7 cells was compared to that in a human hepatoma cell line (HepG2/C3A) and human bipotent progenitor cell line (HepaRG), which yielded the following ranking: HepaRG > DMSO-treated HuH-7 >> HepG2/C3A cells. The effects of three known CYP3A4 inhibitors were evaluated using DMSO-treated HuH-7 cells. CYP3A4 enzyme inhibition in HuH-7 cells was further compared to human recombinant CYP3A4, indicating similar potency for reversible inhibitors (IC50 within 2.5 fold), but different potency for the irreversible inhibitor. Next, induction of CYP3A4 activity was compared between DMSO-treated HuH-7 and HepaRG cells using two known inducers. DMSO-treated HuH-7 cells yielded minimal CYP3A4 induction compared to that in the HepaRG cells after 48-h treatments. Finally, the cytotoxicity of five known hepatotoxicants was evaluated in DMSO-treated HuH-7 cells, HepG2/C3A, and HepaRG cells, and significant differences in cytotoxic sensitivity were observed. Overall, DMSO-treated HuH-7 cells are a valuable model for medium- or high-throughput screening of chemicals for CYP3A4 inhibition and hepatotoxicity. PMID:26377104

  8. Carvacrol and rosemary oil at higher concentrations induce apoptosis in human hepatoma HepG2 cells.

    PubMed

    Melušová, Martina; Jantová, Soňa; Horváthová, Eva

    2014-12-01

    Natural essential oils are volatile herbal complex compounds which manifest cytotoxic effects on living cells depending on their type and concentration but usually they are not genotoxic. Our previous studies showed that carvacrol (CA) and rosemary essential oil (RO) induced growth inhibition of both human cell lines HepG2 and BHNF-1, with hepatoma HepG2 cells being more sensitive to either compound tested. Cytotoxic concentrations of CA and RO induced the formation of DNA strand breaks. Further ex vivo studies showed that extracts prepared from hepatocytes of CA- and RO-supplemented rats did not increase incision repair activity compared to extracts from liver cells of control animals. Therefore, the aim of this work was to determine the effect of cytotoxic concentrations of CA and RO on the cell cycle and the ability of both natural volatiles to induce DNA fragmentation and apoptotic death of human hepatoma HepG2 cells. These effects were measured after 24 h incubation of HepG2 cells with CA and RO using three independent methods - flow cytometry, internucleosomal DNA fragmentation (electrophoresis) and micronucleus assay. Evaluation of morphological changes and formation of micronuclei in HepG2 cells showed no increase in the number of micronuclei in cells treated by CA and RO compared to control cells. On the other hand, CA and RO induced morphological changes typical for apoptosis in concentration-dependent manner. The presence of necrosis was negligible. Both natural compounds caused shrinking of cytoplasmic membrane and formation of apoptotic bodies. In addition, the highest concentrations of CA and RO induced internucleosomal DNA fragmentation (formation of DNA ladder) in HepG2 cells. Cell cycle analysis revealed the accumulation of cells in the G1 phase, which was accompanied by a reduction in the number of cells in the S phase after 24 h exposure to the substances tested. The cell division was thus slowed down or stopped and this process resulted in cell

  9. Transcriptional regulation of the apolipoprotein F (ApoF) gene by ETS and C/EBPα in hepatoma cells.

    PubMed

    Shen, Xue-Bin; Huang, Ling; Zhang, Shao-Hong; Wang, De-Ping; Wu, Yun-Li; Chen, Wan-Nan; Xu, Shang-Hua; Lin, Xu

    2015-05-01

    Apolipoprotein F (ApoF) inhibits cholesteryl ester transfer protein (CETP) activity and plays an important role in lipid metabolism. In the present study, the full-length human ApoF promoter was cloned, and the molecular mechanism of the regulation of ApoF was investigated. The ApoF promoter displayed higher activities in hepatoma cell lines, and the -198 nt to +79 nt promoter region contained the maximum promoter activity. In the promoter region of -198 nt to -2 nt there were four putative binding sites for transcription factors ETS-1/ETS-2 (named EBS-1 to EBS-4) and one for C/EBP. Mutation of EBS-2, EBS4 and the C/EBP binding site abolished the promoter activity, and ETS-1/ETS-2 and C/EBPα could interact with corresponding binding sites. In addition, overexpression of ETS-1/2 or C/EBPα enhanced, while dominant-negative mutants of ETS-1/2 and knockdown of C/EBPα decreased, ApoF promoter activities. ETS-1 and C/EBPα associated physically, and acted synergistically to activate ApoF transcription. These results demonstrated combined activation of the ApoF promoter by liver-enriched and ubiquitous transcription factors. Direct interactions between C/EBPα and ETS-1 were important for high liver-specific expression of ApoF. PMID:25726912

  10. Matrine-induced autophagy regulated by p53 through AMP-activated protein kinase in human hepatoma cells.

    PubMed

    Xie, Shan-Bu; He, Xing-Xing; Yao, Shu-Kun

    2015-08-01

    Matrine, one of the main extract components of Sophora flavescens, has been shown to exhibit inhibitory effects on some tumors through autophagy. However, the mechanism underlying the effect of matrine remains unclear. The cultured human hepatocellular carcinoma cell line HepG2 and SMMC‑7721 were treated with matrine. Signal transduction and gene expression profile were determined. Matrine stimulated autophagy in SMMC‑7721 cells in a mammalian target of rapamycin (mTOR)-dependent manner, but in an mTOR-independent manner in HepG2 cells. Next, in HepG2 cells, autophagy induced by matrine was regulated by p53 inactivation through AMP-activated protein kinase (AMPK) signaling transduction, then AMPK suppression switched autophagy to apoptosis. Furthermore, the interferon (IFN)-inducible genes, including interferon α-inducible protein 27 (IFI27) and interferon induced transmembrane protein 1 (IFITM1), which are downstream effector of p53, might be modulated by matrine-induced autophagy. In addition, we found that the p53 protein isoforms, p53β, p53γ, ∆133p53, and ∆133p53γ, due to alternative splicing of intron 9, might be regulated by the p53-mediated autophagy. These results show that matrine induces autophagy in human hepatoma cells through a novel mechanism, which is p53/AMPK signaling pathway involvement in matrine-promoted autophagy. PMID:26034977

  11. Hydrotropic polymeric mixed micelles based on functional hyperbranched polyglycerol copolymers as hepatoma-targeting drug delivery system.

    PubMed

    Zhang, Xuejiao; Zhang, Xinge; Yu, Peien; Han, Yucai; Li, Yangguang; Li, Chaoxing

    2013-01-01

    Mixed copolymer nanoparticles (NPs) self-assembled from β-cyclodextrin-grafted hyperbranched polyglycerol (HPG-g-CD) and lactobionic acid (LA)-grafted hyperbranched polyglycerol (HPG-g-LA) were applied as carriers for a hydrophobic antitumor drug, paclitaxel (PTX), achieving hepatocellular carcinoma-targeted delivery. The resulting NPs exhibited high drug loading capacity and substantial stability in aqueous solution. In vitro drug release studies demonstrated a controlled drug release profile with increased release at acidic pH. Remarkably, tumor proliferation assays showed that PTX-loaded mixed copolymer NPs inhibited asialoglycoprotein (ASGP) receptor positive HepG2 cell proliferation in a concentration-dependent manner in comparison with ASGP receptor negative BGC-823 cells. Moreover, the competition assay demonstrated that the small molecular LA inhibited the cellular uptake of the PTX-loaded mixed copolymer NPs, indicating the ASGP receptor-mediated endocytosis in HepG2 cells. In addition, the intracellular uptake tests by confocal laser scanning microscopy showed that the mixed copolymer NPs were more efficiently taken up by HepG2 cells compared with HPG-g-CD NPs. These results suggest a feasible application of the mixed copolymer NPs as nanocarriers for hepatoma-targeted delivery of potent antitumor drugs. PMID:23132353

  12. Gentiopicroside and sweroside from Veratrilla baillonii Franch. induce phosphorylation of Akt and suppress Pck1 expression in hepatoma cells.

    PubMed

    Huang, Xian-Ju; Li, Jun; Mei, Zhi-Yi; Chen, Guoxun

    2016-06-01

    The use of phytochemicals and herbal medicines has accompanied human history. Advances in modern biomedical sciences have allowed us to investigate the functional mechanisms of herbal medicines and phytochemicals. Veratrilla baillonii Franch. has long been used as a medicinal herb in southwestern China. Here, we analyzed the effects of an ethanol extract from V. baillonii (VBFE) on the expression levels of the cytosolic form of the phosphoenolpyruvate carboxykinase gene (Pck1) mRNA and components of the insulin signalling cascade in HL1C hepatoma cells. Compared with the insulin control, VBFE treatment inhibited the expression of Pck1 mRNA in a dose-dependent manner. This was associated with the phosphorylation of Akt and Erk1/2 in a time-dependent manner. Further analysis of the purified components of VBFE indicated that gentiopicroside and sweroside from VBFE, alone and in combination, suppressed Pck1 expression and induced Akt and Erk1/2 phosphorylation. In conclusion, gentiopicroside and sweroside suppress Pck1 expression and induce phosphorylation of components in the insulin signalling cascade. This is the first study to demonstrate that gentiopicroside and sweroside show insulin-mimicking effects on the regulation of Pck1 expression. Further studies are warranted to explore the potential of gentiopicroside and sweroside in the control of blood glucose in animals. PMID:27248905

  13. Antiproliferation of berberine is mediated by epigenetic modification of constitutive androstane receptor (CAR) metabolic pathway in hepatoma cells.

    PubMed

    Zhang, Lei; Miao, Xiao-Jie; Wang, Xin; Pan, Hai-Hui; Li, Pu; Ren, Hong; Jia, Yong-Rui; Lu, Chuang; Wang, Hong-Bing; Yuan, Lan; Zhang, Guo-Liang

    2016-01-01

    Constitutive androstane receptor (CAR) regulates hepatic xenobiotic and energy metabolism, as well as promotes cell growth and hepatocarcinogenesis. Berberine is an ancient multipotent alkaloid drug which derived from Coptis chinensis plants. Here we report that berberine is able to be cellular uptake and accessible to chromatin in human hepatoma HepG2 cells. Berberine induces more apoptosis, cell cycle arrest, but less ROS production in CAR overexpressed mCAR-HepG2 cells. Moreover, berberine inhibits expressions of CAR and its target genes CYP2B6 and CYP3A4. Furthermore, berberine enhances DNA methylation level in whole genome but reduces that in promoter regions CpG sites of CYP2B6 and CYP3A4 genes under the presence of CAR condition. These results indicated that the antiproliferation of berberine might be mediated by the unique epigenetic modifying mechanism of CAR metabolic pathway, suggesting that berberine is a promising candidate in anticancer adjuvant chemotherapy, due to its distinct pharmacological properties in clinic. PMID:27311637

  14. 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

  15. Quercetin modulates NF-kappa B and AP-1/JNK pathways to induce cell death in human hepatoma cells.

    PubMed

    Granado-Serrano, Ana Belén; Martín, María Angeles; Bravo, Laura; Goya, Luis; Ramos, Sonia

    2010-01-01

    Quercetin, a dietary flavonoid, has been shown to possess anticarcinogenic properties, but the precise molecular mechanisms of action are not thoroughly elucidated. The aim of this study was to investigate the regulatory effect of quercetin (50 microM) on two main transcription factors (NF-kappa B and AP-1) related to survival/proliferation pathways in a human hepatoma cell line (HepG2) over time. Quercetin induced a significant time-dependent inactivation of the NF-kappa B pathway consistent with a downregulation of the NF-kappa B binding activity (from 15 min onward). These features were in concert with a time-dependent activation (starting at 15 min and maintained up to 18 h) of the AP-1/JNK pathway, which played an important role in the control of the cell death induced by the flavonoid and contributed to the regulation of survival/proliferation (AKT, ERK) and death (caspase-3, p38, unbalance of Bcl-2 proapoptotic and antiapoptotic proteins) signals. These data suggest that NF-kappa B and AP-1 play a main role in the tight regulation of survival/proliferation pathways exerted by quercetin and that the sustained JNK/AP-1 activation and inhibition of NF-kappa B provoked by the flavonoid induced HepG2 death. PMID:20358477

  16. Procyanidins from Nelumbo nucifera Gaertn. Seedpod induce autophagy mediated by reactive oxygen species generation in human hepatoma G2 cells.

    PubMed

    Duan, Yuqing; Xu, Hui; Luo, Xiaoping; Zhang, Haihui; He, Yuanqing; Sun, Guibo; Sun, Xiaobo

    2016-04-01

    In this study, autophagic effect of procyanidins from lotus (Nelumbo nucifera Gaertn.) seedpod (LSPCs) on human hepatoma G2 (HepG2) cells, and the inherent correlation between autophagic levels and reactive oxygen species (ROS) generation were investigated. The results showed that LSPCs increased monodansylcadaverine (MDC) fluorescence intensity and LC3-I/LC3-II conversion in HepG2 cells. In addition, the typically autophagic characteristics (autophagosomes and autolysosomes) were observed in LSPCs-treated cells, but not found in the cells treated with autophagy inhibitor 3-methyladenine (3-MA). Furthermore, the elevated ROS level was in line with the increasing of autophagy activation caused by LSPCs, however, both 3-MA and the ROS scavenger N-acetylcyteine (NAC) inhibitors effectively suppressed the autophagy and ROS generation triggered by LSPCs. As a result, these results indicated that LSPCs induced HepG2 cell autophagy in a time- and dose-dependent manner, and promoted reactive oxygen species (ROS) generation on HepG2 cells. Moreover, we found that LSPCs caused DNA damage, S phase arrest and the decrement of mitochondria membrane potential (MMP) which were associated with ROS generation. In summary, our findings demonstrated that the LSPCs-induced autophagy and autophagic cell death were triggered by the ROS generation in HepG2 cells, which might be associated with ROS generation through the mitochondria-dependent signaling way. PMID:27044822

  17. Human hepatoma cell lines on gas foaming templated alginate scaffolds for in vitro drug-drug interaction and metabolism studies.

    PubMed

    Stampella, A; Rizzitelli, G; Donati, F; Mazzarino, M; de la Torre, X; Botrè, F; Giardi, M F; Dentini, M; Barbetta, A; Massimi, M

    2015-12-25

    Liver in vitro systems that allow reliable prediction of major human in vivo metabolic pathways have a significant impact in drug screening and drug metabolism research. In the present study, a novel porous scaffold composed of alginate was prepared by employing a gas-in-liquid foaming approach. Galactose residues were introduced on scaffold surfaces to promote cell adhesion and to enhance liver specific functions of the entrapped HepG2/C3A cells. Hepatoma cells in the gal-alginate scaffold showed higher levels of liver specific products (albumin and urea) and were more responsive to specific inducers (e.g. dexamethasone) and inhibitors (e.g. ketoconazole) of the CYP3A4 system than in conventional monolayer culture. HepG2/C3A cells were also more efficient in terms of rapid elimination of testosterone, used as a model substance, at rates comparable to those of in vivo excretion. In addition, an improvement in metabolism of testosterone, in terms of phase II metabolite formation, was also observed when the more differentiated HepaRG cells were used. Together the data suggest that hepatocyte/gas templated alginate-systems provide an innovative high throughput platform for in vitro drug metabolism and drug-drug interaction studies, with broad fields of application, and might provide a valid tool for minimizing animal use in preclinical testing of human relevance. PMID:26456671

  18. RhoC is essential for TGF-{beta}1-induced invasive capacity of rat ascites hepatoma cells

    SciTech Connect

    Mukai, M.; Endo, H.; Iwasaki, T.; Tatsuta, M.; Togawa, A.; Nakamura, H.; Inoue, M. . E-mail: inoue-ma2@mc.pref.osaka.jp

    2006-07-21

    Transforming growth factor-{beta}1 (TGF-{beta}1) is a multifunctional growth factor that plays a role in cell proliferation, differentiation, extracellular matrix production, apoptosis, and cell motility. We show here that TGF-{beta}1 increased the invasiveness of MM1 cells, which are a highly invasive clone of rat ascites hepatoma cells. Both mRNA and protein levels of RhoC but not RhoA in TGF-{beta}1-treated MM1 cells increased. In parallel with this increase in expression, RhoC activity was induced by TGF-{beta}1 treatment. When RhoC was overexpressed in MM1 cells, the invasive capacity increased. The RhoC-overexpressing cells formed more nodules than did mock cells when injected into rat peritoneum. Furthermore, when RhoC expression was reduced by transfection with shRNA/RhoC, the invasiveness of MM1 cells decreased with concomitant suppression of RhoC expression. Thus, the induced expression of RhoC by TGF-{beta}1 in MM1 cells plays a critical role in TGF-{beta}1-induced cell migration.

  19. Silencing clusterin gene transcription on effects of multidrug resistance reversing of human hepatoma HepG2/ADM cells.

    PubMed

    Zheng, Wenjie; Sai, Wenli; Yao, Min; Gu, Hongbin; Yao, Yao; Qian, Qi; Yao, Dengfu

    2015-05-01

    Abnormal clusterin (CLU) expression is associated with multidrug resistance (MDR) of hepatocellular carcinoma (HCC). In the present study, the CLU expression was analyzed in human hepatoma cells and chemoresistant counterpart HepG2/ADM cells. Compared with L02 cells, the overexpression of cellular CLU was identified in HepG2, HepG2/ADM, SMMC7721, Hep3B ,and PLC cells and relatively lower expression in Bel-7404, SNU-739, and MHCC97H cells. Specific short hairpin RNAs (shRNAs) to silence CLU gene transcription were designed, and the most effective sequences were screened. After the HepG2/ADM cells transfected with shRNA-1, the inhibition of CLU expression was 73.68 % at messenger RNA (mRNA) level by real-time quantitative RT-PCR with obvious enhancement in cell chemosensitivity, increasing apoptosis induced by doxorubicin using fluorescence kit, and Rh-123 retention qualified with flow cytometry. Knockdown CLU also significantly decreased the drug efflux pump activity through the depression of MDR1/P-glycoprotein (q = 11.739, P < 0.001). Moreover, silencing CLU led to downregulation of β-catenin (q = 13.544, P = 0.001), suggesting that downregulation of CLU might be a key point to reverse multidrug resistance of HepG2/ADM cells. PMID:25600802

  20. Composition of Lycium barbarum polysaccharides and their apoptosis-inducing effect on human hepatoma SMMC-7721 cells

    PubMed Central

    Zhang, Qian; Lv, Xiaoling; Wu, Tao; Ma, Qian; Teng, Anguo; Zhang, Ying; Zhang, Min

    2015-01-01

    Background Lycium barbarum polysaccharide (LBP) is a natural functional component that has a variety of biological activities. The molecular structures and apoptosis-inducing activities on human hepatoma SMMC-7721 cells of two LBP fractions, LBP-d and LBP-e, were investigated. Results The results showed that LBP-d and LBP-e both consist of protein, uronic acid, and neutral sugars in different proportions. The structure of LBP was characterized by gas chromatography, periodate oxidation, and Smith degradation. LBP-d was composed of eight kinds of monosaccharides (fucose, ribose, rhamnose, arabinose, xylose, mannose, galactose, and glucose), while LBP-e was composed of six kinds of monosaccharides (fucose, rhamnose, arabinose, mannose, galactose, and glucose). LBP-d and LBP-e blocked SMMC-7721 cells at the G0/G1 and S phases with an inhibition ratio of 26.70 and 45.13%, respectively, and enhanced the concentration of Ca2+ in the cytoplasm of SMMC-7721. Conclusion The contents of protein, uronic acid, and galactose in LBP-e were much higher than those in LBP-d, which might responsible for their different bioactivities. The results showed that LBP can be provided as a potential chemotherapeutic agent drug to treat cancer. PMID:26563650

  1. Screening of α-Tocopherol Transfer Protein Sensitive Genes in Human Hepatoma Cells (HepG2).

    PubMed

    Qu, Yang-Hua; Fu, Jun-Cai; Liu, Kun; Zuo, Zhao-Yun; Jia, Hui-Na; Ma, Yong; Luo, Hai-Ling

    2016-01-01

    α-Tocopherol transfer protein (α-TTP) is a ~32 kDa protein expressed mainly in hepatocytes. The major function of the protein is to bind specifically to α-tocopherol and, together, the complex transfers from late lysosomes to the cell membrane. A previous study indicated that some factors might be required in the transferring process. However, there is little information available about the potential transferring factors. In addition, there remains much to learn about other physiological processes which α-TTP might participate in. Thus, in this study a human α-TTP eukaryotic expression vector was successfully constructed and expressed in human hepatoma cells (HepG2). The sensitive genes related to α-TTP were then screened by microarray technology. Results showed that expression of the vector in HepG2 cells led to the identification of 323 genes showing differential expression. The differentially expressed transcripts were divided into four main categories, including (1) cell inflammation; (2) cell cycle and cell apoptosis; (3) cell signaling and gene regulation; and (4) cellular movement. A few cellular movement related transcripts were selected and verified by quantitative real-time PCR. Expressions of some were significantly increased in α-TTP-expressed group, which indicated that these factors were likely to play a role in the transferring process. PMID:27355945

  2. pH-Responsive Hyaluronic Acid-Based Mixed Micelles for the Hepatoma-Targeting Delivery of Doxorubicin

    PubMed Central

    Wu, Jing-Liang; Tian, Gui-Xiang; Yu, Wen-Jing; Jia, Guang-Tao; Sun, Tong-Yi; Gao, Zhi-Qin

    2016-01-01

    The tumor targetability and stimulus responsivity of drug delivery systems are crucial in cancer diagnosis and treatment. In this study, hepatoma-targeting mixed micelles composed of a hyaluronic acid–glycyrrhetinic acid conjugate and a hyaluronic acid-l-histidine conjugate (HA–GA/HA–His) were prepared through ultrasonic dispersion. The formation and characterization of the mixed micelles were confirmed via 1H-NMR, particle size, and ζ potential measurements. The in vitro cellular uptake of the micelles was evaluated using human liver carcinoma (HepG2) cells. The antitumor effect of doxorubicin (DOX)-loaded micelles was investigated in vitro and in vivo. Results indicated that the DOX-loaded HA–GA/HA–His micelles showed a pH-dependent controlled release and were remarkably absorbed by HepG2 cells. Compared with free DOX, the DOX-loaded HA–GA/HA–His micelles showed a higher cytotoxicity to HepG2 cells. Moreover, the micelles effectively inhibited tumor growth in H22 cell-bearing mice. These results suggest that the HA–GA/HA–His mixed micelles are a good candidate for drug delivery in the prevention and treatment of hepatocarcinoma. PMID:27043540

  3. Hg2+ signaling in trout hepatoma (RTH-149) cells: involvement of Ca2+-induced Ca2+ release.

    PubMed

    Burlando, Bruno; Bonomo, Marco; Fabbri, Elena; Dondero, Francesco; Viarengo, Aldo

    2003-09-01

    Mercury is a non-essential heavy metal affecting intracellular Ca2+ dynamics. We studied the effects of Hg2+ on [Ca2+]i in trout hepatoma cells (RTH-149). Confocal imaging of fluo-3-loaded cells showed that Hg2+ induced dose-dependent, sustained [Ca2+]i transient, triggered intracellular Ca2+ waves, stimulated Ca2+-ATPase activity, and promoted InsP3 production. The effect of Hg2+ was reduced by the Ca2+ channel blocker verapamil and totally abolished by extracellular GSH, but was almost unaffected by cell loading with the heavy metal chelator TPEN or esterified GSH. In a Ca2+-free medium, Hg2+ induced a smaller [Ca2+]i transient, that was unaffected by TPEN, but was abolished by U73122, a PLC inhibitor, and by cell loading with GDP-betaS, a G protein inhibitor, or heparin, a blocker of intracellular Ca2+ release. Data indicate that Hg2+ induces Ca2+ entry through verapamil-sensitive channels, and intracellular Ca2+ release via a G protein-PLC-InsP3 mechanism. However, in cells loaded with heparin and exposed to Hg2+ in the presence of external Ca2+, the [Ca2+]i rise was maximally reduced, indicating that the global effect of Hg2+ is not a mere sum of Ca2+ entry plus Ca2+ release, but involves an amplification of Ca2+ release operated by Ca2+ entry through a CICR mechanism. PMID:12887976

  4. Ligand-independent tyrosine kinase signalling in RTH 149 trout hepatoma cells: comparison among heavy metals and pro-oxidants.

    PubMed

    Burlando, Bruno; Magnelli, Valeria; Panfoli, Isabella; Berti, Elena; Viarengo, Aldo

    2003-01-01

    Tyrosine phosphorylation depends on the activity of receptor and non-receptor tyrosine kinases and promote cell growth, differentiation and apoptosis. Different stressors are known to stimulate tyrosine kinase activities and this could explain a wide spectrum of effects that these agents produce on different organisms. We studied the effects of heavy metals and pro-oxidants on tyrosine kinase signalling in trout hepatoma cells (RTH 149) by Western immunoblotting. Use of antiphosphotyrosine showed that Hg(2+) and Cu(2+)in the microM range, and H(2)O(2) in the mM range, induced tyrosine phosphorylation. The effect of Cu(2+)was prevented by pre-incubation with genistein, while those of Hg(2+)and H(2)O(2) were only decreased, probably due to tyrosine kinase stimulation coupled to phosphatase inhibition. Phosphospecific antibodies against the three types of MAPKs showed that ERK is activated by heavy metals only, while p38 and SAPK/JNK are activated by H(2)O(2), Hg(2+), and Cu(2+) plus low H(2)O(2). Cell pre-incubation with p38 inhibitors indicated that ERK activation by H(2)O(2) is prevented by concomitant activation of p38. Phosphospecific STAT antibodies revealed activation by H(2)O(2) only. In conclusion, fish cell exposure to heavy metals and pro-oxidants produce specific tyrosine kinase responses, involving cross talk and redox modulatory effects. PMID:12876385

  5. Antiproliferation of berberine is mediated by epigenetic modification of constitutive androstane receptor (CAR) metabolic pathway in hepatoma cells

    PubMed Central

    Zhang, Lei; Miao, Xiao-Jie; Wang, Xin; Pan, Hai-Hui; Li, Pu; Ren, Hong; Jia, Yong-Rui; Lu, Chuang; Wang, Hong-Bing; Yuan, Lan; Zhang, Guo-Liang

    2016-01-01

    Constitutive androstane receptor (CAR) regulates hepatic xenobiotic and energy metabolism, as well as promotes cell growth and hepatocarcinogenesis. Berberine is an ancient multipotent alkaloid drug which derived from Coptis chinensis plants. Here we report that berberine is able to be cellular uptake and accessible to chromatin in human hepatoma HepG2 cells. Berberine induces more apoptosis, cell cycle arrest, but less ROS production in CAR overexpressed mCAR-HepG2 cells. Moreover, berberine inhibits expressions of CAR and its target genes CYP2B6 and CYP3A4. Furthermore, berberine enhances DNA methylation level in whole genome but reduces that in promoter regions CpG sites of CYP2B6 and CYP3A4 genes under the presence of CAR condition. These results indicated that the antiproliferation of berberine might be mediated by the unique epigenetic modifying mechanism of CAR metabolic pathway, suggesting that berberine is a promising candidate in anticancer adjuvant chemotherapy, due to its distinct pharmacological properties in clinic. PMID:27311637

  6. Effects of barley β-glucan on radiation damage in the human hepatoma cell line HepG2.

    PubMed

    Ghavami, Laleh; Goliaei, Bahram; Taghizadeh, Bita; Nikoofar, Alireza

    2014-12-01

    Damage to normal tissue is an obstacle to radiotherapy of cancer. We have tested whether barley β-glucan can enhance radioprotection in the human hepatoma cell line HepG2. The cytotoxicity of β-glucan was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. A clonogenic assay was used to study the sensitivity of cells to β-glucan, ionizing radiation (2-8Gy), and the combination of both treatments. Acridine Orange/ethidium bromide staining was used to examine induction of apoptosis by β-glucan, radiation (6Gy), and the combination. DNA strand breaks were assessed by the comet assay. The MTT assay showed that treatment with β-glucan was not cytotoxic. Indeed, a slight increase in cell viability was observed. Pre-treatment with β-glucan, 1μg/ml, for 72h protected HepG2 cells against radiation, as indicated by increased surviving fraction, reduced apoptosis, and fewer DNA strand breaks. These results show that barley β-glucan is a radioprotective agent. PMID:25435350

  7. Effect of Mesenchymal Stem Cells and a Novel Curcumin Derivative on Notch1 Signaling in Hepatoma Cell Line

    PubMed Central

    Abdel Aziz, Mohamed Talaat; Khaled, Hussien Mostafa; El Hindawi, Ali; Roshdy, Nagwa Kamal; Rashed, Laila A.; Hassouna, Amira A.; Taha, Fatma; Ali, Walaa Ibrahim

    2013-01-01

    This study was conducted to evaluate the effect of mesenchymal stem cells (MSCs) and a novel curcumin derivative (NCD) on HepG2 cells (hepatoma cell line) and to investigate their effect on Notch1 signaling pathway target genes. HepG2 cells were divided into HepG2 control group, HepG2 cells treated with MSC conditioned medium (MSCs CM), HepG2 cells treated with a NCD, HepG2 cells treated with MSCs CM and NCD, and HepG2 cells treated with MSCs CM (CM of MSCs pretreated with a NCD). Expression of Notch1, Hes1, VEGF, and cyclin D1 was assessed by real-time, reverse transcription-polymerase chain reaction (RT-PCR) in HepG2 cells. In addition, HepG2 proliferation assay was performed in all groups. Notch1 and its target genes (Hes1 and cyclin D1) were downregulated in all treated groups with more suppressive effect in the groups treated with both MSCs and NCD. Also, treated HepG2 cells showed significant decrease in cell proliferation rate. These data suggest that modulation of Notch1 signaling pathway by MSCs and/or NCD can be considered as a therapeutic target in HCC. PMID:24024180

  8. Generation of a human hepatoma cell line supporting efficient replication of a lamivudine resistant hepatitis B virus.

    PubMed

    Zhang, Yijun; Zhang, Yongmei; Kang, Yaoyue; Wang, Jinyu; Liu, Hongyan; Zhu, Haoxiang; Qin, Yanli; Mao, Richeng; Lin, Xu; Lu, Mengji; Zhang, Jiming

    2014-06-01

    Emergence of lamivudine (LAM) resistance causes treatment failure in patients with chronic hepatitis B and compromise the efficacy of subsequent salvage therapies with other nucleot(s)ide analogs (NAs). Establishment of cell-based assays supporting LAM-resistant hepatitis B virus (HBV) replication will not only provide tools for investigating the replication property, but also screening for antiviral agents efficiently inhibiting the replication of LAM-resistant HBV variants. Accordingly, a human hepatoma (HepG2)-derived cell line was established by stable transfection of a plasmid containing a 1.2 unit length of HBV genome harboring rtL180M and rtM204V mutations that confer LAM resistance. In addition to support efficient viral genome replication, the cell line also produces high levels of HBV virions and subviral particles. As expected, HBV DNA replication in this cell line is completely resistant to lamivudine, but sensitive to adefovir (ADV), entecavir (ETV) and tenofovir (TDF). The cell line is suitable for screening for antiviral agents that inhibit LAM-resistant HBV replication and inhibitors of HBsAg biosynthesis and secretion, which may reduce HBsAg antigenemia and ultimately help to restore host antiviral immune response against HBV and cure chronic HBV infection. PMID:24583110

  9. Ultrasound-targeted microbubble destruction mediated herpes simplex virus-thymidine kinase gene treats hepatoma in mice

    PubMed Central

    2010-01-01

    Objective The purpose of the study was to explore the anti-tumor effect of ultrasound -targeted microbubble destruction mediated herpes simplex virus thymidine kinase (HSV-TK) suicide gene system on mice hepatoma. Methods Forty mice were randomly divided into four groups after the models of subcutaneous transplantation tumors were estabilished: (1) PBS; (2) HSV-TK (3) HSV-TK+ ultrasound (HSV-TK+US); (4) HSV-TK+ultrasound+microbubbles (HSV-TK+US+MB). The TK protein expression in liver cancer was detected by western-blot. Applying TUNEL staining detected tumor cell apoptosis. At last, the inhibition rates and survival time of the animals were compared among all groups. Results The TK protein expression of HSV-TK+MB+US group in tumor-bearing mice tissues were significantly higher than those in other groups. The tumor inhibitory effect of ultrasound-targeted microbubble destruction mediated HSV-TK on mice transplantable tumor was significantly higher than those in other groups (p < 0.05), and can significantly improve the survival time of tumor-bearing mice. Conclusion Ultrasound-targeted microbubble destruction can effectively transfect HSV-TK gene into target tissues and play a significant inhibition effect on tumors, which provides a new strategy for gene therapy in liver cancer. PMID:21176239

  10. Screening of α-Tocopherol Transfer Protein Sensitive Genes in Human Hepatoma Cells (HepG2)

    PubMed Central

    Qu, Yang-Hua; Fu, Jun-Cai; Liu, Kun; Zuo, Zhao-Yun; Jia, Hui-Na; Ma, Yong; Luo, Hai-Ling

    2016-01-01

    α-Tocopherol transfer protein (α-TTP) is a ~32 kDa protein expressed mainly in hepatocytes. The major function of the protein is to bind specifically to α-tocopherol and, together, the complex transfers from late lysosomes to the cell membrane. A previous study indicated that some factors might be required in the transferring process. However, there is little information available about the potential transferring factors. In addition, there remains much to learn about other physiological processes which α-TTP might participate in. Thus, in this study a human α-TTP eukaryotic expression vector was successfully constructed and expressed in human hepatoma cells (HepG2). The sensitive genes related to α-TTP were then screened by microarray technology. Results showed that expression of the vector in HepG2 cells led to the identification of 323 genes showing differential expression. The differentially expressed transcripts were divided into four main categories, including (1) cell inflammation; (2) cell cycle and cell apoptosis; (3) cell signaling and gene regulation; and (4) cellular movement. A few cellular movement related transcripts were selected and verified by quantitative real-time PCR. Expressions of some were significantly increased in α-TTP-expressed group, which indicated that these factors were likely to play a role in the transferring process. PMID:27355945

  11. Two new isoforms of the human hepatoma-derived growth factor interact with components of the cytoskeleton.

    PubMed

    Nüße, Jessica; Mirastschijski, Ursula; Waespy, Mario; Oetjen, Janina; Brandes, Nadine; Rebello, Osmond; Paroni, Federico; Kelm, Sørge; Dietz, Frank

    2016-05-01

    Hepatoma-derived growth factor (HDGF) is involved in diverse, apparently unrelated processes, such as cell proliferation, apoptosis, DNA-repair, transcriptional control, ribosome biogenesis and cell migration. Most of the interactions of HDGF with diverse molecules has been assigned to the hath region of HDGF. In this study we describe two previously unknown HDGF isoforms, HDGF-B and HDGF-C, generated via alternative splicing with structurally unrelated N-terminal regions of their hath region, which is clearly different from the well described isoform, HDGF-A. In silico modeling revealed striking differences near the PHWP motif, an essential part of the binding site for glycosaminoglycans and DNA/RNA. This observation prompted the hypothesis that these isoforms would have distinct interaction patterns with correspondingly diverse roles on cellular processes. Indeed, we discovered specific associations of HDGF-B and HDGF-C with cytoskeleton elements, such as tubulin and dynein, suggesting previously unknown functions of HDGF in retrograde transport, site directed localization and/or cytoskeleton organization. In contrast, the main isoform HDGF-A does not interact directly with the cytoskeleton, but via RNA with messenger ribonucleoprotein (mRNP) complexes. In summary, the discovery of HDGF splice variants with their discrete binding activities and subcellular distributions opened new avenues for understanding its biological function and importance. PMID:26845719

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

    SciTech Connect

    Niklas, Jens; Noor, Fozia; 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 these 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.

  13. Induction of hepatoma carcinoma cell apoptosis through activation of the JNK-nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-ROS self-driven death signal circuit.

    PubMed

    Zeng, Ke-Wu; Song, Fang-Jiao; Wang, Ying-Hong; Li, Ning; Yu, Qian; Liao, Li-Xi; Jiang, Yong; Tu, Peng-Fei

    2014-10-28

    As an efficient method for inducing tumor cell apoptosis, ROS can be constantly formed and accumulated in NADPH oxidase overactivated-cells, resulting in further mitochondrial membrane damage and mitochondria-dependent apoptosis. In addition, JNK mitogen-activated protein kinase (JNK MAPK) signal also acts as a vital candidate pathway for inducing tumor cell apoptosis by targeting mitochondrial death pathway. However, the relationship between NADPH oxidase-ROS and JNK MAPK signal still remains unclear. Here, we discovered a novel self-driven signal circuit between NADPH oxidase-ROS and JNK MAPK, which was induced by a cytotoxic steroidal saponin (ASC) in hepatoma carcinoma cells. NADPH oxidase-dependent ROS production was markedly activated by ASC and directly led to JNK MAPK activation. Moreover, antioxidant, NADPH oxidase inhibitor and specific knock-out for p47 subunit of NADPH oxidase could effectively block NADPH oxidase-ROS-dependent JNK activation, suggesting that NADPH oxidase is an upstream regulator of JNK MAPK. Conversely, a specific JNK inhibitor could inhibit ASC-induced NADPH oxidase activation and down-regulate ROS levels as well, indicating that JNK might also regulate NADPH oxidase activity to some extent. These observations indicate that NADPH oxidase and JNK MAPK activate each other as a signal circuit. Furthermore, drug pretreatment experiments with ASC showed this signal circuit operated continuously via a self-driven mode and finally induced apoptosis in hepatoma carcinoma cells. Taken together, we provide a proof for inducing hepatoma carcinoma cell apoptosis by activating the JNK-NADPH oxidase-ROS-dependent self-driven signal circuit pathway. PMID:25064608

  14. Protein kinase activity of RNA polymerase I purified from a rat hepatoma: probable function of Mr 42,000 and 24,600 polypeptides.

    PubMed Central

    Rose, K M; Stetler, D A; Jacob, S T

    1981-01-01

    RNA polymerase I was purified to homogeneity from Morris hepatoma 3924A. Purified RNA polymerase I contained a protein kinase activity but comigrated with the polymerase in nondenaturing gels. RNA polymerase II, purified from the same hepatoma, lacked protein kinase activity. Analysis of the subunit composition of the RNA polymerase I showed the presence of eight polypeptides: S1, Mr 190,000; S2, Mr 120,000; S3, Mr 62,000; S4, Mr 42,000; S5, Mr 24,600; S6, Mr 21,000; S7, Mr 19,500; and S8, Mr 17,500. Antibodies prepared against purified polymerase I specifically inhibited RNA synthesis catalyzed by RNA polymerase I. When subunits of the enzyme were covalently linked to diazobenzyloxymethyl paper, complexes between the antibody preparation and S1-S6 were visualized. No immune complexes were formed between RNA polymerase I antibodies and RNA polymerase II subunits. The antibody preparation was able to inhibit both the protein phosphorylation catalyzed by RNA polymerase I and that catalyzed by a nuclear kinase (NII) purified from the same hepatoma. The two polypeptides of the nuclear kinase--Mr 42,000 and 24,600 (identical in size to S4 and S5 of polymerase I)--formed visible complexes with the RNA polymerase I antibodies. Both S4 and S5 of the polymerase contained an ATP binding site, a property associated with protein phosphorylation and also exhibited by the polypeptides of the purified kinase. These data suggest that polypeptides of Mr 42,000 and 24,600 associated with polymerase I are responsible for its kinase activity. Images PMID:6942406

  15. 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

  16. Differential effects of phorbol ester on growth and protein kinase C isoenzyme regulation in human hepatoma Hep3B cells.

    PubMed Central

    Hsu, S L; Chou, Y H; Yin, S C; Liu, J Y

    1998-01-01

    PMA has both mitogenic and antiproliferative effects on human hepatoma Hep3B cells. In response to low PMA concentration (10 nM), Hep3B cells displayed an increasing proliferation potentiation. At high PMA concentration (1 microM) Hep3B cells exhibited modest cytostatic effects. Determinations of protein kinase C (PKC) activity in PMA-treated cells revealed that alterations in PKC activity are associated with proliferative capacity. The decrease in PKC activity mediated by a high dose of PMA was accompanied by cell growth inhibition. Increases in PKC activity mediated by a low dose of PMA were consistent with proliferation stimulation. Immunoblot analysis showed that there are at least six PKC isoenzymes: alpha, delta, epsilon, mu, zeta and iota/lambda, constitutively expressed in Hep3B cells. Cellular fractionation and immunocytochemical staining results demonstrated that both 10 nM and 1 microM PMA treatments induced a marked translocation of PKC-alpha from cytosol to membrane or nuclear fraction within 5-30 min. At the same time PKC-delta and epsilon were translocated from the membrane to nuclear fraction. In addition, prolonged treatment with 1 microM PMA, but not with 10 nM PMA, selectively mediated the down-regulation of these three PKC isoenzymes. The distinct effects of different concentrations of PMA on cell proliferation and PKC-alpha, delta and epsilon isoenzyme modulation support the involvement of these three PKC isotypes in the mechanism of action of Hep3B cells in cell growth events. PMID:9639562

  17. Myricetin, quercetin, (+)-catechin and (-)-epicatechin protect against N-nitrosamines-induced DNA damage in human hepatoma cells.

    PubMed

    Delgado, M E; Haza, A I; García, A; Morales, P

    2009-10-01

    The aim of this study was to investigate the protective effect of myricetin, quercetin, (+)-catechin and (-)-epicatechin, against N-nitrosodibutylamine (NDBA) and N-nitrosopiperidine (NPIP)-induced DNA damage in human hepatoma cells (HepG2). DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single-cell gel electrophoresis or Comet assay. (+)-Catechin at the lowest concentration (10 microM) showed the maximum reduction of DNA strand breaks (23%), the formation of endonuclease III (Endo III, 19-21%) and formamidopyrimidine-DNA glycosylase (Fpg, 28-40%) sensitive sites induced by NDBA or NPIP. (-)-Epicatechin also decreased DNA strand breaks (10 microM, 20%) and the oxidized pyrimidines/purines (33-39%) induced by NDBA or NPIP, respectively. DNA strand breaks induced by NDBA or NPIP were weakly reduced by myricetin at the lowest concentration (0.1 microM, 10-19%, respectively). Myricetin also reduced the oxidized purines (0.1 microM, 17%) and pyrimidines (0.1 microM, 15%) induced by NDBA, but not the oxidized pyrimidines induced by NPIP. Quercetin did not protect against NDBA-induced DNA damage, but it reduced the formation of Endo III and Fpg sensitive sites induced by NPIP (0.1 microM, 17-20%, respectively). In conclusion, our results indicate that (+)-catechin and (-)-epicatechin at the concentrations tested protect human derived cells against oxidative DNA damage effects of NDBA and NPIP. However, myricetin at the concentrations tested only protects human cells against oxidative DNA damage induced by NDBA and quercetin against oxidative DNA damage induced by NPIP. PMID:19628030

  18. Gene expression profiling and differentiation assessment in primary human hepatocyte cultures, established hepatoma cell lines, and human liver tissues

    SciTech Connect

    Olsavsky, Katy M.; Page, Jeanine L.; Johnson, Mary C.; Zarbl, Helmut; Strom, Stephen C.; Omiecinski, Curtis J. . E-mail: cjo10@psu.edu

    2007-07-01

    Frequently, primary hepatocytes are used as an in vitro model for the liver in vivo. However, the culture conditions reported vary considerably, with associated variability in performance. In this study, we characterized the differentiation character of primary human hepatocytes cultured using a highly defined, serum-free two-dimensional sandwich system, one that configures hepatocytes with collagen I as the substratum together with a dilute extracellular matrix (Matrigel{sup TM}) overlay combined with a defined serum-free medium containing nanomolar levels of dexamethasone. Gap junctional communication, indicated by immunochemical detection of connexin 32 protein, was markedly enhanced in hepatocytes cultured in the Matrigel sandwich configuration. Whole genome expression profiling enabled direct comparison of liver tissues to hepatocytes and to the hepatoma-derived cell lines, HepG2 and Huh7. PANTHER database analyses were used to identify biological processes that were comparatively over-represented among probe sets expressed in the in vitro systems. The robustness of the primary hepatocyte cultures was reflected by the extent of unchanged expression character when compared directly to liver, with more than 77% of the probe sets unchanged in each of the over-represented categories, representing such genes as C/EBP{alpha}, HNF4{alpha}, CYP2D6, and ABCB1. In contrast, HepG2 and Huh7 cells were unchanged from the liver tissues for fewer than 48% and 55% of these probe sets, respectively. Further, hierarchical clustering of the hepatocytes, but not the cell lines, shifted from donor-specific to treatment-specific when the probe sets were filtered to focus on phenobarbital-inducible genes, indicative of the highly differentiated nature of the hepatocytes when cultured in a highly defined two-dimensional sandwich system.

  19. XPC is essential for nucleotide excision repair of zidovudine-induced DNA damage in human hepatoma cells

    SciTech Connect

    Wu Qiangen; Beland, Frederick A.; Chang, Ching-Wei; Fang Jialong

    2011-03-01

    Zidovudine (3'-azido-3'-dexoythymidine, AZT), a nucleoside reverse transcriptase inhibitor, can be incorporated into DNA and cause DNA damage. The mechanisms underlying the repair of AZT-induced DNA damage are unknown. To investigate the pathways involved in the recognition and repair of AZT-induced DNA damage, human hepatoma HepG2 cells were incubated with AZT for 2 weeks and the expression of DNA damage signaling pathways was determined using a pathway-based real-time PCR array. Compared to control cultures, damaged DNA binding and nucleotide excision repair (NER) pathways showed significantly increased gene expression. Further analysis indicated that AZT treatment increased the expression of genes associated with NER, including XPC, XPA, RPA1, GTF2H1, and ERCC1. Western blot analysis demonstrated that the protein levels of XPC and GTF2H1 were also significantly up-regulated. To explore further the function of XPC in the repair of AZT-induced DNA damage, XPC expression was stably knocked down by 71% using short hairpin RNA interference. In the XPC knocked-down cells, 100 {mu}M AZT treatment significantly increased [{sup 3}H]AZT incorporation into DNA, decreased the total number of viable cells, increased the release of lactate dehydrogenase, induced apoptosis, and caused a more extensive G2/M cell cycle arrest when compared to non-transfected HepG2 cells or HepG2 cells transfected with a scrambled short hairpin RNA sequence. Overall, these data indicate that XPC plays an essential role in the NER repair of AZT-induced DNA damage.

  20. Tumor growth inhibition and nutritional effect of D-amino acid solution in AH109A hepatoma-bearing rats.

    PubMed

    Sasamura, T; Matsuda, A; Kokuba, Y

    1998-02-01

    We examined the inhibitional and nutritional effects of total parenteral nutrition (TPN) containing D-amino acids (D-phenylalanine, D-Phe; D-valine, D-Val; D-leucine, D-Leu; D-methionine, D-Met) on tumor growth in AH109A hepatoma-bearing rats. Five experimental groups were examined: a control amino acid solution group (control group), D-Phe group, D-Val group, D-Leu group and D-Met group. The analysis of tumor volume and weight revealed significant tumor growth inhibition in the D-Val group as compared with the control group. In the D-Val group, decreases of DNA and protein contents in the tumor tissues were also observed. The D-Leu and D-Met groups showed a tendency toward tumor growth inhibition. The protein content in the liver tissues of these two groups was significantly higher as compared with the control group. The DNA content in the liver tissue was also significantly higher in the D-Met group. The body weight including the tumor (on the final day of TPN) was significantly lower in the D-Val group as compared with the control group, but there was no significant difference in the groups for body weights not including tumors (carcass body weight). The hematocrit and hemoglobin values, indicators of anemia, were significantly higher in the D-Val group as compared with the control group. From these results, regarding tumor growth inhibition, the D-Val solution had the strongest inhibitory effect with no negative influence on the host, and improvement of nutritional status was also suggested in the rats that received the D-Leu or D-Met solutions. PMID:9591236

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

    SciTech Connect

    Mylonis, Ilias; Lakka, Achillia; Tsakalof, Andreas; Simos, George

    2010-07-16

    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 including 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.

  2. Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma

    NASA Technical Reports Server (NTRS)

    Baracos, V. E.; DeVivo, C.; Hoyle, D. H.; Goldberg, A. L.

    1995-01-01

    Rats implanted with Yoshida ascites hepatoma (YAH) show a rapid and selective loss of muscle protein due mainly to a marked increase (63-95%) in the rate of protein degradation (compared with rates in muscles of pair-fed controls). To define which proteolytic pathways contribute to this increase, epitrochlearis muscles from YAH-bearing and control rats were incubated under conditions that modify different proteolytic systems. Overall proteolysis in either group of rats was not affected by removal of Ca2+ or by blocking the Ca(2+)-dependent proteolytic system. Inhibition of lysosomal function with methylamine reduced proteolysis (-12%) in muscles from YAH-bearing rats, but not in muscles of pair-fed rats. When ATP production was also inhibited, the remaining accelerated proteolysis in muscles of tumor-bearing rats fell to control levels. Muscles of YAH-bearing rats showed increased levels of ubiquitin-conjugated proteins and a 27-kDa proteasome subunit in Western blot analysis. Levels of mRNA encoding components of proteolytic systems were quantitated using Northern hybridization analysis. Although their total RNA content decreased 20-38%, pale muscles of YAH-bearing rats showed increased levels of ubiquitin mRNA (590-880%) and mRNA for multiple subunits of the proteasome (100-215%). Liver, kidney, heart, and brain showed no weight loss and no change in these mRNA species. Muscles of YAH-bearing rats also showed small increases (30-40%) in mRNA for cathepsins B and D, but not for calpain I or heat shock protein 70. Our findings suggest that accelerated muscle proteolysis and muscle wasting in tumor-bearing rats result primarily from activation of the ATP-dependent pathway involving ubiquitin and the proteasome.

  3. 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

  4. Acetaminophen induces a caspase-dependent and Bcl-XL sensitive apoptosis in human hepatoma cells and lymphocytes.

    PubMed

    Boulares, A Hamid; Zoltoski, Anna J; Stoica, Bogdan A; Cuvillier, Olivier; Smulson, Mark E

    2002-01-01

    Acetaminophen is a widely used analgesic and antipyretic drug that exhibits toxicity at high doses to the liver and kidneys. This toxicity has been attributed to cytochrome P-450-generated metabolites which covalently modify target proteins. Recently, acetaminophen, in its unmetabolized form, has been shown to affect a variety of cells and tissues, for instance, testicular and lymphoid tissues and lymphocyte cell lines. The effects on cell viability of acetaminophen at a concentration comparable to that achieved in plasma during acetaminophen toxicity have now been examined with a hepatoma cell line SK-Hep1, primary human peripheral blood lymphocytes and human Jurkat T cells. Acetaminophen reduced cell viability in a time-dependent manner. Staining of cells with annexin-V also revealed that acetaminophen induced, after 8 hr of treatment, a loss of the asymmetry of membrane phospholipids, which is an early event associated with apoptosis. Acetaminophen triggered the release of cytochrome c from mitochondria into the cytosol, activation of caspase-3, 8, and 9, cleavage of poly(ADP-ribose) polymerase, and degradation of lamin B1 and DNA. Whereas cleavage of DNA into internucleosomal fragments was apparent in acetaminophen treated SK-Hep1 and primary lymphocytes, DNA was only degraded to 50-kb fragments in treated Jurkat cells. Overexpression of the antiapoptotic protein Bcl-XL prevented these various apoptotic events induced by acetaminophen in Jurkat cells. Caspase-8 activation was a postmictochondrial event and occurred in a Fas-independent manner. These results demonstrate that acetaminophen induces caspases-dependent apoptosis with mitochondria as a primary target. These results also reiterate the potential role of apoptosis in acetaminophen hepatic and extrahepatic toxicity. PMID:12005112

  5. Endotoxin-stimulated macrophages decrease bile acid uptake in WIF-B cells, a rat hepatoma hybrid cell line.

    PubMed

    Sturm, E; Zimmerman, T L; Crawford, A R; Svetlov, S I; Sundaram, P; Ferrara, J L; Karpen, S J; Crawford, J M

    2000-01-01

    Endotoxemia leads to cytokine-mediated alterations of the hepatocellular sodium-taurocholate-cotransporting polypeptide (ntcp). We hypothesized that stimulated macrophages are essential transducers for down-regulating hepatocellular bile salt uptake in response to endotoxin (lipopolysaccharide [LPS]) exposure. Using an in vitro model, we exposed mouse macrophages (IC-21 cell line) to LPS for 24 hours. Concentrations of cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 increased 10.6-fold, 12.5-fold, and 444-fold, respectively, in LPS-conditioned IC-21 medium (CM) versus unconditioned IC-21 medium (UM). WIF-B rat hepatoma hybrid cells were incubated with either CM or UM or treated directly with medium containing recombinant TNF-alpha, IL-1beta, and IL-6. [(3)H]Taurocholate ([(3)H]TC) uptake decreased in WIF-B cells exposed to either TNF-alpha (54% of control), IL-1beta (78%), IL-6 (55%) as single additives, or in triple combination (TCC) (43%). A virtually identical decrease was observed after exposing WIF-B cells to CM (52%, P <.001). LPS had no direct effect on [(3)H]TC uptake. CM treatment did not decrease L-alanine transport in WIF-B cells. Blocking antibodies against TNF-alpha, IL-1beta, and IL-6 restored the diminished [(3)H]TC uptake in cells exposed to TCC and CM to 87% and 107% of controls, respectively. Northern blotting revealed that ntcp messenger RNA (mRNA) expression was significantly reduced in WIF-B cells after exposure to CM, and in primary rat hepatocytes exposed to CM or TNF-alpha (68%, 14%, and 29% of control, respectively). We conclude that macrophages and their ability to secrete the cytokines TNF-alpha, IL-1beta, and IL-6 may be essential in mediating the endotoxin-induced cholestatic effect of decreased hepatocellular bile salt uptake. PMID:10613737

  6. Newcastle disease virus chimeras expressing the Hemagglutinin- Neuraminidase protein of mesogenic strain exhibits an enhanced anti-hepatoma efficacy.

    PubMed

    He, Jinjiao; Pan, Ziye; Tian, Guiyou; Liu, Xin; Liu, Yunye; Guo, Xiaochen; An, Ying; Song, Liying; Wu, Hongsong; Cao, Hongwei; Yu, Dan; Che, Ruixiang; Xu, Pengfei; Rasoul, Lubna M; Li, Deshan; Yin, Jiechao

    2016-08-01

    Newcastle disease virus (NDV) is an intrinsically tumor-specific virus, many researchers have reported that lentogenic NDV is a safe and effective agent for human cancer therapy. It had been demonstrated that the amino acid sequence of the fusion protein cleavage site is a major factor in the pathogenicity and anti-tumor efficacy of rNDV. However, the role of Hemagglutinin-Neuraminidase (HN) gene that contributes to virulence and anti-tumor efficacy remains undefined. To assess the role of HN gene in virus pathogenicity and anti-tumor efficacy, a reverse genetic system was developed using the lentogenic NDV Clone30 strain to provide backbone for gene exchange. Chimeric virus (rClone30-Anh(HN)) created by exchange of the HN gene of lentogenic strain Clone30 with HN gene of mesogenic strain produce no significant changes in virus pathogenicity as assessed by conducting the mean death time (MDT) and intracerebral pathogenicity index (ICPI) assays. In vitro, infection with chimeras could induce the formation of syncytium relative significantly in HepG2 cells. Furthermore, chimeras was shown to induce the cell apoptosis via MTT and Annexin V-PI assays, reduce mitochondrial membrane potential and increase the mRNA transcription level of caspase 3. In vivo, ICR mice carrying tumor of hepatoma H22 cells were treated via intratumoral injection of chimeric virus. The treatment of chimera shows an obvious suppression in tumor volume. These results suggest that it could be an ideal approach to enhance the antitumor ability of Newcastle disease virus and highlighted the potential therapeutic application of rClone30-Anh(HN) as a viral vector to deliver foreign genes for treatment of cancers. PMID:27164362

  7. Analysis of the genotoxic potential of low concentrations of Malathion on the Allium cepa cells and rat hepatoma tissue culture.

    PubMed

    Bianchi, Jaqueline; Mantovani, Mario Sérgio; Marin-Morales, Maria Aparecida

    2015-10-01

    Based on the concentration of Malathion used in the field, we evaluated the genotoxic potential of low concentrations of this insecticide on meristematic and F1 cells of Allium cepa and on rat hepatoma tissue culture (HTC cells). In the A. cepa, chromosomal aberrations (CAs), micronuclei (MN), and mitotic index (MI) were evaluated by exposing the cells at 1.5, 0.75, 0.37, and 0.18mg/mL of Malathion for 24 and 48hr of exposure and 48hr of recovery time. The results showed that all concentrations were genotoxic to A. cepa cells. However, the analysis of the MI has showed non-relevant effects. Chromosomal bridges were the CA more frequently induced, indicating the clastogenic action of Malathion. After the recovery period, the higher concentrations continued to induce genotoxic effects, unlike the observed for the lowest concentrations tested. In HTC cells, the genotoxicity of Malathion was evaluated by the MN test and the comet assay by exposing the cells at 0.09, 0.009, and 0.0009mg/5mL culture medium, for 24hr of exposure. In the comet assay, all the concentrations induced genotoxicity in the HTC cells. In the MN test, no significant induction of MN was observed. The genotoxicity induced by the low concentrations of Malathion presented in this work highlights the importance of studying the effects of low concentrations of this pesticide and demonstrates the efficiency of these two test systems for the detection of genetic damage promoted by Malathion. PMID:26456612

  8. 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. PMID:24763554

  9. c-Ha-ras down regulates the alpha-fetoprotein gene but not the albumin gene in human hepatoma cells.

    PubMed Central

    Nakao, K; Lawless, D; Ohe, Y; Miyao, Y; Nakabayashi, H; Kamiya, H; Miura, K; Ohtsuka, E; Tamaoki, T

    1990-01-01

    We studied the effects of transfection of the normal c-Ha-ras gene, rasGly-12, and its oncogenic mutant, rasVal-12, on expression of the alpha-fetoprotein (AFP) and albumin genes in a human hepatoma cell line, HuH-7. The mutant and, to a lesser extent, the normal ras gene caused reduction of the AFP mRNA but not the albumin mRNA level in transfected HuH-7 cells. Cotransfection experiments with a rasVal-12 expression plasmid and a chloramphenicol acetyltransferase reporter gene fused to AFP regulatory sequences showed that rasVal-12 suppressed the activity of enhancer and promoter regions containing A + T-rich sequences (AT motif). In contrast, rasVal-12 did not affect the promoter activity of the albumin and human hepatitis B virus pre-S1 genes even though these promoters contain homologous A + T-rich elements. ras transfection appeared to induce phosphorylation of nuclear proteins that interact with the AFP AT motif, since gel mobility analysis revealed the formation of slow-moving complexes which was reversed by phosphatase treatment. However, similar changes in complex formation were observed with the albumin and hepatitis B surface antigen pre-S1 promoters. Therefore, this effect alone cannot explain the specific down regulation of the AFP promoter and enhancer activity. ras-mediated suppression of the AFP gene may reflect the process of developmental gene regulation in which AFP gene transcription is controlled by a G-protein-linked signal transduction cascade triggered by external growth stimuli. Images PMID:1690841

  10. Elevated Expression of Hepatoma Up-Regulated Protein Inhibits γ-Irradiation-Induced Apoptosis of Prostate Cancer Cells.

    PubMed

    Hassan, Mohamed; El Khattouti, Abdelouahid; Ejaeidi, Ahmed; Ma, Tangeng; Day, William A; Espinoza, Ingrid; Vijayakumar, Srinivasan; Gomez, Christian R

    2016-06-01

    Despite progression in diagnosis and treatment, prostate cancer (PCa) still represents the main cause of cancer-related mortality and morbidity in men. Although radiation therapy offers clinical benefit over other therapeutic modalities, the success of this therapeutic modality is commonly hampered by the resistance of advanced tumors. So far, the mechanisms governing tumor resistance to radiotherapy are not discussed in detail. Here, we demonstrate for the first time that the resistance of PCa to radiation therapy is attributed to elevated expression of Hepatoma Up-Regulated Protein (HURP). In PCa cells, the induction of HURP expression suppresses γ-irradiation-induced apoptosis. γ-irradiation-induced apoptosis of PCa cells is associated with expression of E2F1, p53, p21 proteins together with the phosphorylation of apoptosis signal-regulating kinase1 (ASK1), c-jun-N-terminal kinase (JNK) and Ataxia-telangiectasia mutated (ATM) and histone family member X (H2AX). Whereas, the induction of HURP expression is able to suppress γ-irradiation-induced effects on E2F1, p53, p21, ATM, ASK1, JNK and ATM, and H2AX. Also, inhibition of γ-irradiation-induced- cytochrome c release, cleavage of caspase-9, caspase-3, PARP, and reactive oxygen species (ROS) were noted in PCa cells induced for HURP expression. The observed radio-resistance of PCa is thought to be the consequence of HURP-mediated destabilization of p53 and ATM proteins that are essential for the modulation of γ-irradiation-induced apoptosis. Thus, based on our findings, PCa resistance to radiation therapy results from the deregulation of ASK1/ JNK; ATM/ H2AX; ATM/p53 and checkpoint kinase 2 (Chk2)/ E2F-1 in response to the elevated expression of HURP. J. Cell. Biochem. 117: 1308-1318, 2016. © 2015 Wiley Periodicals, Inc. PMID:26505164

  11. Effect of methoxychlor on Ca²⁺ homeostasis and apoptosis in HA59T human hepatoma cells.

    PubMed

    Horng, Chi-Ting; Chou, Chiang-Ting; Tseng, Hui-Wen; Cheng, Jin-Shiung; Chang, Hong-Tai; Chang, Po-Min; Chen, I-Li; Hung, Ming-Chi; Tsai, Yi-Jen; Tsai, Peng-Chih; Liang, Wei-Zhe; Kuo, Chun-Chi; Kuo, Daih-Huang; Ho, Chin-Man; Lin, Jia-Rong; Shieh, Pochuen; Jan, Chung-Ren

    2015-02-28

    Methoxychlor, an organochlorine pesticide, is thought to be an endocrine disrupter that affects Ca²⁺ homeostasis and cell viability in different cell models. This study explored the action of methoxychlor on cytosolic free Ca²⁺ concentrations ([Ca²⁺]i) and apoptosis in HA59T human hepatoma cells. Fura-2, a Ca²⁺-sensitive fluorescent dye, was applied to measure [Ca²⁺]i. Methoxychlor at concentrations of 0.1-1 μM caused a [Ca²⁺]i rise in a concentration-dependent manner. Removal of external Ca²⁺ abolished methoxychlor's effect. Methoxychlor-induced Ca²⁺ influx was confirmed by Mn²⁺-induced quench of fura-2 fluorescence. Methoxychlor-induced Ca²⁺ entry was inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators. Methoxychlor killed cells at concentrations of 10-130 μM in a concentration-dependent fashion. Chelation of cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not prevent methoxychlor's cytotoxicity. Methoxychlor (10 and 50 μM) induced apoptosis concentration-dependently as determined by using Annexin V/propidium iodide staining. Together, in HA59T cells, methoxychlor induced a [Ca²⁺]i rise by inducing Ca²⁺ entry via protein kinase C-sensitive Ca²⁺-permeable channels, without causing Ca²⁺ release from stores. Methoxychlor also induced apoptosis that was independent of [Ca²⁺]i rises. PMID:25687486

  12. The metabolomic profile of gamma-irradiated human hepatoma and muscle cells reveals metabolic changes consistent with the Warburg effect

    PubMed Central

    Wang, Min; Keogh, Adrian; Treves, Susan; Idle, Jeffrey R.

    2016-01-01

    The two human cell lines HepG2 from hepatoma and HMCL-7304 from striated muscle were γ-irradiated with doses between 0 and 4 Gy. Abundant γH2AX foci were observed at 4 Gy after 4 h of culture post-irradiation. Sham-irradiated cells showed no γH2AX foci and therefore no signs of radiation-induced double-strand DNA breaks. Flow cytometry indicated that 41.5% of HepG2 cells were in G2/M and this rose statistically significantly with increasing radiation dose reaching a plateau at ∼47%. Cell lysates from both cell lines were subjected to metabolomic analysis using Gas Chromatography-Mass Spectrometry (GCMS). A total of 46 metabolites could be identified by GCMS in HepG2 cell lysates and 29 in HMCL-7304 lysates, most of which occurred in HepG2 cells. Principal Components Analysis (PCA) showed a clear separation of sham, 1, 2 and 4 Gy doses. Orthogonal Projection to Latent Structures-Discriminant Analysis (OPLS-DA) revealed elevations in intracellular lactate, alanine, glucose, glucose 6-phosphate, fructose and 5-oxoproline, which were found by univariate statistics to be highly statistically significantly elevated at both 2 and 4 Gy compared with sham irradiated cells. These findings suggested upregulation of cytosolic aerobic glycolysis (the Warburg effect), with potential shunting of glucose through aldose reductase in the polyol pathway, and consumption of reduced Glutathione (GSH) due to γ-irradiation. In HMCL-7304 myotubes, a putative Warburg effect was also observed only at 2 Gy, albeit a lesser magnitude than in HepG2 cells. It is anticipated that these novel metabolic perturbations following γ-irradiation of cultured cells will lead to a fuller understanding of the mechanisms of tissue damage following ionizing radiation exposure. PMID:26823999

  13. Simulation of oxygen carrier mediated oxygen transport to C3A hepatoma cells housed within a hollow fiber bioreactor.

    PubMed

    Sullivan, Jesse P; Gordon, Jason E; Palmer, Andre F

    2006-02-01

    A priori knowledge of the dissolved oxygen (O2) concentration profile within a hepatic hollow fiber (HF) bioreactor is important in developing an effective bioartificial liver assist device (BLAD). O2 provision is limiting within HF bioreactors and we hypothesize that supplementing a hepatic HF bioreactor's circulating media with bovine red blood cells (bRBCs), which function as an O2 carrier, will improve oxygenation. The dissolved O2 concentration profile within a single HF (lumen, membrane, and representative extra capillary space (ECS)) was modeled with the finite element method, and compared to experimentally measured data obtained on an actual HF bioreactor with the same dimensions housing C3A hepatoma cells. Our results (experimental and modeling) indicate bRBC supplementation of the circulating media leads to an increase in O2 consumed by C3A cells. Under certain experimental conditions (pO2,IN) = 95 mmHg, Q = 8.30 mL/min), the addition of bRBCs at 5% of the average in vivo human red blood cell concentration (% hRBC) results in approximately 50% increase in the O2 consumption rate (OCR). By simply adjusting the operating conditions (pO2,IN) = 25 mmHg, Q = 1.77 mL/min) and increasing bRBC concentration to 25% hRBC the OCR increase is approximately 10-fold. However, the improved O2 concentration profile experienced by the C3A cells could not duplicate the full range of in vivo O2 tensions (25-70 mmHg) typically experienced within the liver sinusoid with this particular HF bioreactor. Nonetheless, we demonstrate that the O2 transport model accurately predicts O2 consumption within a HF bioreactor, thus setting up the modeling framework for improving the design of future hepatic HF bioreactors. PMID:16161160

  14. Forced expression of PDX-1 gene makes hepatoma cells to acquire glucose-responsive insulin secretion while maintaining hepatic characteristic.

    PubMed

    Hashimoto, H; Higuchi, Y; Kawai, K

    2015-01-01

    Evidence shows that forced expression of the PDX1 gene converts hepatoma cells, mouse liver epithelial cells (MLECs) and HepaRG cells, into insulin—producing cells, β—cells, or islets of Langerhans. However, no reports have investigated the characteristics of mouse or human hepatocytes introduced with the PDX1 gene over prolonged observation periods. In this study, we immunohistologically and molecularly investigated the alternative processes induced by PDX1 gene introduction in mouse and human hepatocytes over prolonged observation periods using immunocytochemistry, immunofluorescence, polymerase chain reaction (PCR), Western blotting, and flow cytometry (FCM) analysis. Immunocytochemical and immunofluorescent observations showed that MLECs and HepaRG cells on 2 and 21 days after introduction of the PDX1 gene comprised cells double—positive for insulin and albumin. Additionally, they showed MAFA expression and glucose—responsive insulin secretion with glucokinase expression. However mouse embryonic fibroblasts introduced with PDX1—GFP could not acquire glucose—responsive insulin secretion and glucokinase expression. Subsequently, we hypothesized that the number of albumin—positive MLECs and HepaRG cells would decrease after introduction of PDX1 due to the conversion of MLECs and HepaRG cells into insulin—producing cells. However, FCM analysis indicated that the number of albumin—positive MLECs and HepaRG cells was not altered by the introduction of PDX1. We thought that MLECs and HepaRG cells introduced with the PDX1 gene could acquire a functional insulin secretory capacity without conversion to β—cells, or islets of Langerhans, and the acquisition could need glucokinase expression. PMID:25817342

  15. Segetoside I, a plant-derived bisdesmosidic saponin, induces apoptosis in human hepatoma cells in vitro and inhibits tumor growth in vivo.

    PubMed

    Firempong, Caleb Kesse; Zhang, Hui Yun; Wang, Yan; Chen, Jingjing; Cao, Xia; Deng, Wenwen; Zhou, Jie; Wang, Qiang; Tong, Shan-Shan; Yu, Jiangnan; Xu, Ximing

    2016-08-01

    Segetoside I is a plant-derived bisdesmosidic saponin from Vaccaria segetalis (Neck) with reported anticancer activities. This development has raised an interest in the therapeutic potential of segetoside I. Here, we report the in vitro and in vivo antitumor activities of segetoside I against some selected cancer cell lines (HepG2, human hepatoma; H22, mouse hepatoma; MCF-7, breast cancer; U251, gliocoma; BGC, HGC & SGC, gastric cancinoma; Lovo-1,colon cancer). MTT bioassay analysis showed that HepG2 cells were the most sensitive to segetoside I compared with other cancer cell lines, with lower toxicity in healthy mouse embryonic fibroblast cells. Segetoside I pretreatment of HepG2 resulted in apoptotic induction, dose-dependent DNA fragmentation, inhibition of cell migration, up-regulation of Bax and down-regulation of Bcl-2, which indicated that an apoptotic signaling event could have been initiated. The segetoside I also suppressed hepato-tumour growth in mice with virtually no cytotoxicity and prolonged animal survival, making it a strong oncology drug agent. These findings showed that segetoside I exhibited its antitumor activity via apoptotic induction and significantly support the possible application of the antitumor agent as a potential chemotherapeutic candidate worthy of further investigations. PMID:27180010

  16. Chemically induced oxidative stress increases polyamine levels by activating the transcription of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase in human hepatoma HUH7 cells.

    PubMed

    Smirnova, Olga A; Isaguliants, Maria G; Hyvonen, Mervi T; Keinanen, Tuomo A; Tunitskaya, Vera L; Vepsalainen, Jouko; Alhonen, Leena; Kochetkov, Sergey N; Ivanov, Alexander V

    2012-09-01

    Biogenic polyamines spermine and spermidine participate in numerous cellular processes including transcription, RNA processing and translation. Specifically, they counteract oxidative stress, an alteration of cell redox balance involved in generation and progression of various pathological states including cancer. Here, we investigated how chemically induced oxidative stress affects polyamine metabolism, specifically the expression and activities of enzymes catalyzing polyamine synthesis (ornithine decarboxylase; ODC) and degradation (spermidine/spermine-N(1)-acetyltransferase; SSAT), in human hepatoma cells. Oxidative stress induced the up-regulation of ODC and SSAT gene transcription mediated by Nrf2, and in case of SSAT, also by NF-κB transcription factors. Activation of transcription led to the elevated intracellular activities of both enzymes. The balance in antagonistic activities of ODC and SSAT in the stressed hepatoma cells was shifted towards polyamine biosynthesis, which resulted in increased intracellular levels of putrescine, spermidine, and spermine. Accumulation of putrescine is indicating for accelerated degradation of polyamines by SSAT - acetylpolyamine oxidase (APAO) pathway generating toxic products that promote carcinogenesis, whereas accelerated polyamine synthesis via activation of ODC is favorable for proliferation of cells including those sub-lethally damaged by oxidative stress. PMID:22579641

  17. CdTe quantum dots with daunorubicin induce apoptosis of multidrug-resistant human hepatoma HepG2/ADM cells: in vitro and in vivo evaluation

    NASA Astrophysics Data System (ADS)

    Zhang, Gen; Shi, Lixin; Selke, Matthias; Wang, Xuemei

    2011-06-01

    Cadmium telluride quantum dots (Cdte QDs) have received significant attention in biomedical research because of their potential in disease diagnosis and drug delivery. In this study, we have investigated the interaction mechanism and synergistic effect of 3-mercaptopropionic acid-capped Cdte QDs with the anti-cancer drug daunorubicin (DNR) on the induction of apoptosis using drug-resistant human hepatoma HepG2/ADM cells. Electrochemical assay revealed that Cdte QDs readily facilitated the uptake of the DNR into HepG2/ADM cells. Apoptotic staining, DNA fragmentation, and flow cytometry analysis further demonstrated that compared with Cdte QDs or DNR treatment alone, the apoptosis rate increased after the treatment of Cdte QDs together with DNR in HepG2/ADM cells. We observed that Cdte QDs treatment could reduce the effect of P-glycoprotein while the treatment of Cdte QDs together with DNR can clearly activate apoptosis-related caspases protein expression in HepG2/ADM cells. Moreover, our in vivo study indicated that the treatment of Cdte QDs together with DNR effectively inhibited the human hepatoma HepG2/ADM nude mice tumor growth. The increased cell apoptosis rate was closely correlated with the enhanced inhibition of tumor growth in the studied animals. Thus, Cdte QDs combined with DNR may serve as a possible alternative for targeted therapeutic approaches for some cancer treatments.

  18. Effects of dietary phenolics and botanical extracts on hepatotoxicity-related endpoints in human and rat hepatoma cells and statistical models for prediction of hepatotoxicity.

    PubMed

    Liu, Yitong; Flynn, Thomas J; Ferguson, Martine S; Hoagland, Erica M; Yu, Liangli Lucy

    2011-08-01

    Toxicity assessment of botanical materials is difficult because they are typically complex mixtures of phytochemicals. In the present study, 16 phenolics were tested in both human (HepG2/C3A) and rat (MH1C1) hepatoma cells using a battery of eight toxicity endpoints. Cluster analysis was used to group the phenolics into four clusters for each cell type. Comparison of overall and individual liver activity of phenolics on both human and rat hepatoma cell lines showed significant differences for some endpoints. However, the cluster membership was similar across both cell types with the majority of phenolics clustering with the solvent control group (cluster 1). Each cell type produced a cluster of compounds with reported in vivo liver toxicity (cluster 2). Five herbal extracts were prepared and then tested as above. Using the cluster model developed with the phenolics, in the HepG2/C3A cells green tea was assigned to cluster 2 and the remaining four extracts to cluster 1. In the MH1C1 cells, green tea and thyme were assigned to cluster 2, cinnamon to cluster 4, and juniper berry and peppermint to cluster 1. The data suggest that this in vitro model may be useful for identifying hepatotoxic phenolics and botanical preparations rich in phenolics. PMID:21569817

  19. Establishment by adriamycin exposure of multidrug-resistant rat ascites hepatoma AH130 cells showing low DT-diaphorase activity and high cross resistance to mitomycins.

    PubMed

    Wakusawa, S; Nakamura, S; Miyamoto, K

    1997-01-01

    A resistant subline (AH130/5A) selected from rat hepatoma AH130 cells after exposure to adriamycin (ADM) showed remarkable resistance to multiple antitumor drugs, including mitomycin C (MMC) and porfiromycin (PFM). PFM, vinblastine (VLB), and ADM accumulated in AH130/5A far less than in the parent AH130 (AH130/P) cells. AH130/5A cells showed overexpression of P-glycoprotein (PGP), an increase in glutathione S-transferase activity, and a decrease in DT-diaphorase and glutathione peroxidase activity. The resistance to MMC and VLB of AH130/5A cells was partly reversed by H-87, an inhibitor of PGP. Buthionine sulfoximine, an inhibitor of glutathione synthase, did not affect the action of MMC. tert-Butylhydroquinone induced DT-diaphorase activity, increased PFM uptake, and enhanced the growth-inhibitory action of MMC in AH130/5A cells. Dicumarol, an inhibitor of DT-diaphorase, decreased PFM uptake and reduced the growth-inhibitory action of MMC in AH130/P cells. These results indicated that the adriamycin treatment of hepatoma cells caused multifactorial multidrug resistance involving a decrease in DT-diaphorase activity. PMID:9045901

  20. Determination of malondialdehyde by liquid chromatography as the 2,4-dinitrophenylhydrazone derivative: a marker for oxidative stress in cell cultures of human hepatoma HepG2.

    PubMed

    Mateos, Raquel; Goya, Luis; Bravo, Laura

    2004-06-01

    Malondialdehyde (MDA) is considered a presumptive biomarker for lipid peroxidation in live organisms and cultured cells. The present study adapts an accurate and reproducible method to measure MDA by high-performance liquid chromatography (HPLC) as its 2,4-dinitrophenylhydrazone derivative in human hepatoma HepG2 cells in culture. Since MDA is assumed to increase in conditions of cellular oxidative stress, two compounds that induce pharmacological oxidative stress in cell cultures, hydrogen peroxide (H(2)O(2)) and tert-butyl hydroperoxide (t-BOOH), have been used in HepG2 cells. The results report a significant increase in the content of MDA derivative after treatment with 200 and 500microM t-BOOH for 3h, while H(2)O(2) in doses up to 500microM failed to evoke a similar response, indicating a stronger lipid peroxidation of t-BOOH to HepG2 cells than H(2)O(2). Thus, MDA can be used as a reliable biomarker for cellular oxidative stress in human hepatoma HepG2. PMID:15113537

  1. Insulin-like growth factor-1 prevents miR-122 production in neighbouring cells to curtail its intercellular transfer to ensure proliferation of human hepatoma cells

    PubMed Central

    Basu, Sudarshana; Bhattacharyya, Suvendra N.

    2014-01-01

    miRNAs are 20–22 nt long post-transcriptional regulators in metazoan cells that repress protein expression from their target mRNAs. These tiny regulatory RNAs follow tissue and cell-type specific expression pattern, aberrations of which are associated with various diseases. miR-122 is a liver-specific anti-proliferative miRNA that, we found, can be transferred via exosomes between human hepatoma cells, Huh7 and HepG2, grown in co-culture. Exosomal miR-122, expressed and released by Huh7 cells and taken by miR-122 deficient HepG2 cells, was found to be effective in repression of target mRNAs and to reduce growth and proliferation of recipient HepG2 cells. Interestingly, in a reciprocal process, HepG2 secretes Insulin-like Growth Factor 1 (IGF1) that decreases miR-122 expression in Huh7 cells. Our observations suggest existence of a reciprocal interaction between two different hepatic cells with distinct miR-122 expression profiles. This interaction is mediated via intercellular exosome-mediated miR-122 transfer and countered by a reciprocal IGF1-dependent anti-miR-122 signal. According to our data, human hepatoma cells use IGF1 to prevent intercellular exosomal transfer of miR-122 to ensure its own proliferation by preventing expression of growth retarding miR-122 in neighbouring cells. PMID:24813441

  2. Size-dependent cytotoxicity of Fe3O4 nanoparticles induced by biphasic regulation of oxidative stress in different human hepatoma cells

    PubMed Central

    Xie, Yuexia; Liu, Dejun; Cai, Chenlei; Chen, Xiaojing; Zhou, Yan; Wu, Liangliang; Sun, Yongwei; Dai, Huili; Kong, Xianming; Liu, Peifeng

    2016-01-01

    The application of Fe3O4 nanoparticles (NPs) has made great progress in the diagnosis of disease and in the drug delivery system for cancer therapy, but the relative mechanisms of potential toxicity induced by Fe3O4 have not kept pace with its development in the application, which has hampered its further clinical application. In this article, we used two kinds of human hepatoma cell lines, SK-Hep-1 and Hep3B, to investigate the cytotoxic effects and the involved mechanisms of small Fe3O4 NPs with different diameters (6 nm, 9 nm, and 14 nm). Results showed that the size of NPs effectively influences the cytotoxicity of hepatoma cells: 6 nm Fe3O4 NPs exhibited negligible cytotoxicity and 9 nm Fe3O4 NPs affected cytotoxicity via cellular mitochondrial dysfunction and by inducing necrosis mediated through the mitochondria-dependent intracellular reactive oxygen species generation. Meanwhile, 14 nm Fe3O4 NPs induced cytotoxicity by impairing the integrity of plasma membrane and promoting massive lactate dehydrogenase leakage. These results explain the detailed mechanism of different diameters of small Fe3O4 NPs-induced cytotoxicity. We anticipate that this study will provide different insights into the cytotoxicity mechanism of Fe3O4 NPs, so as to make them safer to use in clinical application. PMID:27536098

  3. Steroid-regulated intracellular signals involved in proliferation of rat epithelial cells. II. Glucocorticoid regulation of phosphoprotein maturation in rat hepatoma cells

    SciTech Connect

    Vallerga, A.K.

    1988-01-01

    Cultured BDS1 rat hepatoma cells, growth-arrested in the presence of serum and glucocorticoid, were induced to synchronously enter the cell cycle upon removal of steroid from the medium. Analysis of total RNA isolated from the proliferating cells revealed a peak of transcript levels at 0.5 hours for c-fos, at 2 hours for c-myc and at 8 hours for both c-ras{sup Ha} and c-ras{sup Ki}. The onset of DNA synthesis, as measured by ({sup 3}H)thymidine incorporation, occurred after an 8 hour time lag and peaked at 16 hours after the removal of dexamethasone. The induction of DNA polymerase alpha activity occurred during the onset of DNA synthesis and peaked at 24 hours. Cytoplasmic extracts from non-growing BDS1 cells did not contain an inhibitory activity that could suppress the activity of DNA polymerase alpha. A high molecular weight (M{sub r}210,000) DNA polymerase alpha protein was present in proliferating but not in quiescent cell extracts. In M1.54 rat liver hepatoma cells that contain mouse mammary tumor provirus (MMTV), the phosphorylated viral precursor polypeptide (Pr74), is cleaved posttranslationally into p24, after a 4 hour exposure to glucocortocoid. Twenty hours after hormone withdrawal, p24 is degraded whereas Pr74 remained ten-fold over its basal level.

  4. 3-Bromopyruvic acid, a hexokinase II inhibitor, is an effective antitumor agent on the hepatoma cells : in vitro and in vivo findings.

    PubMed

    Gong, Lei; Wei, Yuhua; Yu, Xin; Peng, Jirun; Leng, Xisheng

    2014-06-01

    Over-expressed in cancer cells, hexokinase II (HK II) forms a mitochondrial complex, which promotes cancer survival. 3- Bromopyruvic acid (3-BrPA) dissociates HK II from this complex, causing cell death, and thus, having an anti-tumor effect. The design of this study was to first analyze the expression of HK II in the hepatoma cell line, BEL-7402, then investigate the effects of 3-Br-PA on these cells, and finally, discuss its potential for clinical usage. HK II expression was detected in BEL-7402 cells by immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). In vitro treatment of cells with 3-BrPA significantly inhibited their growth, as evaluated by MTT assay and adenosine triphosphate-tumor chemosensitivity assay (ATP-TCA). To analyze the in vivo function and safety of this drug, a tumor model was established by subcutaneously implanting hepatic cancer cells into nude mice. 3-BrPA treatment (50 mg/kg ip. daily, 6 days/week for three weeks) was effective in the animal model by attenuating tumor growth and causing tumor necrosis. Toxic signs were not observed. The acute toxicity study provided an LD50 of 191.7 mg/kg for 3-BrPA. Taken together, our in vitro and in vivo analyses suggest that 3-BrPA exerts anti-hepatoma effects, and may be an effective pharmacological agent for the treatment of hepatocellular carcinoma. PMID:24738957

  5. Size-dependent cytotoxicity of Fe3O4 nanoparticles induced by biphasic regulation of oxidative stress in different human hepatoma cells.

    PubMed

    Xie, Yuexia; Liu, Dejun; Cai, Chenlei; Chen, Xiaojing; Zhou, Yan; Wu, Liangliang; Sun, Yongwei; Dai, Huili; Kong, Xianming; Liu, Peifeng

    2016-01-01

    The application of Fe3O4 nanoparticles (NPs) has made great progress in the diagnosis of disease and in the drug delivery system for cancer therapy, but the relative mechanisms of potential toxicity induced by Fe3O4 have not kept pace with its development in the application, which has hampered its further clinical application. In this article, we used two kinds of human hepatoma cell lines, SK-Hep-1 and Hep3B, to investigate the cytotoxic effects and the involved mechanisms of small Fe3O4 NPs with different diameters (6 nm, 9 nm, and 14 nm). Results showed that the size of NPs effectively influences the cytotoxicity of hepatoma cells: 6 nm Fe3O4 NPs exhibited negligible cytotoxicity and 9 nm Fe3O4 NPs affected cytotoxicity via cellular mitochondrial dysfunction and by inducing necrosis mediated through the mitochondria-dependent intracellular reactive oxygen species generation. Meanwhile, 14 nm Fe3O4 NPs induced cytotoxicity by impairing the integrity of plasma membrane and promoting massive lactate dehydrogenase leakage. These results explain the detailed mechanism of different diameters of small Fe3O4 NPs-induced cytotoxicity. We anticipate that this study will provide different insights into the cytotoxicity mechanism of Fe3O4 NPs, so as to make them safer to use in clinical application. PMID:27536098

  6. Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5' region.

    PubMed Central

    Tratner, I; Nahon, J L; Sala-Trepat, J M; Venetianer, A

    1987-01-01

    We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines. Images PMID:2439898

  7. Aquaporin 9 is down-regulated in hepatocellular carcinoma and its over-expression suppresses hepatoma cell invasion through inhibiting epithelial-to-mesenchymal transition.

    PubMed

    Zhang, Wen-Guang; Li, Chuan-Fei; Liu, Min; Chen, Xiao-Feng; Shuai, Kai; Kong, Xin; Lv, Lin; Mei, Zhe-Chuan

    2016-08-10

    Aquaporin 9 (AQP9) is the main aquaglyceroporin in the liver. Few studies have been performed regarding the role of AQP9 in hepatocellular carcinoma (HCC). Here, we report the expression and function of AQP9 in HCC tissues and cell lines. We found that AQP9 mRNA and protein levels were down-regulated in HCC tissues and human hepatoma cell lines compared to the para-cancer normal liver tissues and normal hepatocyte line, respectively. In a human HCC SMMC-7721 cell line, over-expression of AQP9 suppressed cell invasion in vitro and xenograft tumor growth in vivo. AQP9 over-expression increased the expression of E-cadherin and decreased the expression of N-cadherin in SMMC-7721 cells and xenografted tumors, which was correlated with decreased levels of phosphoinositide 3-kinase (PI3K) and p-Akt. Conversely, using siRNA to knock down AQP9 over-expression could reverse the phenotype caused by AQP9 over-expression. Our findings suggest that AQP9 is down-regulated in hepatocellular carcinoma and its over-expression suppresses hepatoma cell invasion through inhibiting epithelial-to-mesenchymal transition. PMID:27216981

  8. The effect of aromatic hydrocarbon receptor on the phenotype of the Hepa 1c1c7 murine hepatoma cells in the absence of dioxin.

    PubMed

    Wang, Feng; Zhang, Ruixue; Shi, Shengli; Hankinson, Oliver

    2007-01-01

    The aromatic hydrocarbon receptor (AhR) mediates biological responses to certain exogenous ligands, such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and has also been demonstrated to modulate the cell cycle and differentiated state of several cell lines independently of exogenous ligands. In this study, we used DNA microarray analysis to elucidate the profile of genes responsive to the expression of unliganded AhR by re-introducing AhR into an AhR-deficient mouse derivative (c19) of the mouse hepatoma cell line Hepa 1c1c7. 22 gene products were up-regulated and 8 were down-regulated two-fold or more in c19 cells infected with a retroviral vector expressing mouse AhR. Surprisingly, expression of genes involved in cell proliferation or differentiation were not affected by introduction of AhR. AhR also did not restore expression of the albumin gene in c19 cells. Introduction of AhR into c12, a similar AhR-defective mouse hepatoma cell line, also did not restore albumin expression, and furthermore, did not lead to changes in cellular morphology or cell cycle parameters. These observations fail to support the notion that unliganded AhR regulates proliferation and differentiation of liver-derived cells. PMID:19936078

  9. The Effect of Aromatic Hydrocarbon Receptor on the Phenotype of the Hepa 1c1c7 Murine Hepatoma Cells in the Absence of Dioxin

    PubMed Central

    Wang, Feng; Zhang, Ruixue; Shi, Shengli; Hankinson, Oliver

    2007-01-01

    The aromatic hydrocarbon receptor (AhR) mediates biological responses to certain exogenous ligands, such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and has also been demonstrated to modulate the cell cycle and differentiated state of several cell lines independently of exogenous ligands. In this study, we used DNA micorarray analysis to elucidate the profile of genes responsive to the expression of unliganded AhR by re-introducing AhR into an AhR-deficient mouse derivative (c19) of the mouse hepatoma cell line Hepa1c1c7. 22 gene products were up-regulated and 8 were down-regulated two-fold or more in c19 cells infected with a retroviral vector expressing mouse AhR. Surprisingly, expression of genes involved in cell proliferation or differentiation were not affected by introduction of AhR. AhR also did not restore expression of the albumin gene in c19 cells. Introduction of AhR into c12, a similar AhR-defective mouse hepatoma cell line, also did not restore albumin expression, and furthermore, did not lead to changes in cellular morphology or cell cycle parameters. These observations fail to support the notion that unliganded AhR regulates proliferation and differentiation of liver-derived cells. PMID:19936078

  10. Chemotherapeutic efficacy of the protein-doxorubicin conjugates on multidrug resistant rat hepatoma cell line in vitro.

    PubMed Central

    Ohkawa, K.; Hatano, T.; Tsukada, Y.; Matsuda, M.

    1993-01-01

    In vitro studies were initiated to study the antitumour effect of protein-doxorubicin (DXR) conjugate on the growth of the multidrug resistant rat ascites hepatoma cell line, AH66DR. The 50% inhibitory concentration (IC50) for DXR in AH66DR cell line was 16 mumol l-1 (AH66 parental cell line, AH66P, IC50 was 0.08 mumol l-1). Treatment of AH66P and AH66DR cells with various concentrations of DXR or conjugates at equivalent concentrations of DXR was performed. The two types of conjugates used were bovine serum albumin (BSA)-DXR conjugate and immunoglobulin G (IgG)-DXR conjugate. Both of these conjugates showed potent dose-dependent inhibition of cell growth against AH66DR cells as compared with the cells treated with DXR or other controls. The IC50 for BSA-DXR and IgG-DXR conjugates in AH66DR cell line was 0.05 (equivalent DXR) mumol l-1 and 0.07 (equivalent DXR) mumol l-1, respectively. These values were similar to that of the AH66P treated with DXR. Cellular uptake and accumulation of DXR or BSA-DXR conjugate was also quantitated in both cell lines. The cellular concentration of DXR in AH66DR cells was 2-fold lower than that of AH66P cells throughout the experiment. In contrast, by the treatment of AH66DR cells with BSA-DXR conjugate, the intracellular drug concentration increased as a function of time up to 24 h (639.1 +/- 41.8, equivalent DXR, ng 10(-5) cells) and reached the same drug level as AH66P cells treated with DXR (617.9 +/- 17.3 ng-5 cells). Ammonium chloride treatment inhibited the effects of the conjugates but did not inhibit the free drugs. Intracellular DXR was effluxed rapidly from AH66DR cells, but BSA-DXR conjugate remained in the cells at relatively high concentration for a long time. These results indicate that by chemically modifying DXR, such as by conjugation of the drug with proteins, it may be possible to overcome multidrug resistance. Images Figure 2 PMID:8431358