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Sample records for hepatoma

  1. Studies on responsiveness of hepatoma cells to catecholamines. IV. Lack of adrenergic activation of phosphorylase in rat ascites hepatoma cells.

    PubMed

    Miyamoto, K; Yanaoka, T; Sanae, F; Wakusawa, S; Koshiura, R

    1986-10-01

    Glycogen phosphorylase a activity in 7 rat ascites hepatoma cell lines treated with adrenergic agents, phenylephrine, epinephrine and isoproterenol, was investigated as compared with that in freshly isolated rat hepatocytes. Basal phosphorylase activities in hepatoma cells except AH7974 cells were lower than that in hepatocytes. Phosphorylase in hepatoma cells was not activated by any of the agents, while the enzyme activity in hepatocytes was clearly increased in a dose- and time-dependent manner. Phosphorylase in hepatocytes was sensitive to glucagon, but it was found to be insensitive to glucagon in all hepatoma cells. The present results suggest that rat ascites hepatoma cells may escape the glycogenolytic regulation by catecholamines and glucagon.

  2. [Dynamic monitoring risk of anti-hepatoma new drug development].

    PubMed

    Zhang, Jing; Fan, Wei; Li, Hong-Fa; Man, Shu-Li; Liu, Zhen; Gao, Wen-Yuan

    2014-10-01

    Risk monitoring of new Chinese patent anti-hepatoma drugs is tracking recognized risks and residual risks, identifying emerging risk and ensure the implementation of the plan, estimating the process of reducing effectiveness. The paper is mainly through understanding the status of Chinese patent anti-hepatoma drugs, the content, characteristic and analysis method of dynamic risk monitoring, and then select the risk control indicators, collect risk information. Finally, puts forward the thought of anti-hepatoma drugs listed evaluation in our country, and try to establish the model of dynamic risk management of anti-hepatoma drugs.

  3. Studies on responsiveness of hepatoma cells to catecholamines. VI. Characteristics of adrenoceptors and adenylate cyclase response in rat ascites hepatoma cells and human hepatoma cells.

    PubMed

    Sanae, F; Kohei, K; Nomura, M; Miyamoto, K

    1992-06-01

    Alpha 1, alpha 2- and beta-Adrenoceptor densities and catecholamine responsiveness in established hepatoma cells, rat ascites hepatoma AH13, AH66, AH66F, AH109A, AH130 and AH7974 cells and human hepatocellular carcinoma HLF and HepG2 cells, were compared with those in normal rat hepatocytes and Chang liver cells. Alpha 1-Adrenoceptor densities measured by [3H]prazosin bindings were not detected in all hepatoma cell lines. Alpha 2-Adrenoceptor densities measured by [3H]clonidine bindings were also barely detected in hepatoma cell lines except for AH130 cells and HepG2 cells. Regarding beta-adrenoceptor, AH109A, AH130 and AH7974 cells had much more [125I]iodocyanopindolol binding sites than normal rat hepatocytes, although we could not detect the binding in HepG2 cells. Adenylate cyclase of normal rat hepatocyte and Chang liver cells were stimulated by beta 2-adrenergic agonist salbutamol, while the cyclase in hepatoma cells had no beta 2-adrenergic response but a beta 1-type response. These findings indicate that the characteristics of adrenergic response in hepatoma cell lines is very different from that in normal hepatocytes, suggesting a participation in the hepatocarcinogenesis and/or the autonomous proliferation of hepatoma cells.

  4. Trout hepatoma--a preliminary report

    USGS Publications Warehouse

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

    1961-01-01

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

  5. Intracellularly Swollen Polypeptide Nanogel Assists Hepatoma Chemotherapy

    PubMed Central

    Shi, Bo; Huang, Kexin; Ding, Jianxun; Xu, Weiguo; Yang, Yu; Liu, Haiyan; Yan, Lesan; Chen, Xuesi

    2017-01-01

    Nowadays, chemotherapy is one of the principal modes of treatment for tumor patients. However, the traditional formulations of small molecule drugs show short circulation time, low tumor selectivity, and high toxicity to normal tissues. To address these problems, a facilely prepared, and pH and reduction dual-responsive polypeptide nanogel was prepared for selectively intracellular delivery of chemotherapy drug. As a model drug, doxorubicin (DOX) was loaded into the nanogel through a sequential dispersion and dialysis technique, resulting in a high drug loading efficiency (DLE) of 96.7 wt.%. The loading nanogel, defined as NG/DOX, exhibited a uniform spherical morphology with a mean hydrodynamic radius of 58.8 nm, pH and reduction dual-triggered DOX release, efficient cell uptake, and cell proliferation inhibition in vitro. Moreover, NG/DOX exhibited improved antitumor efficacy toward H22 hepatoma-bearing BALB/c mouse model compared with free DOX·HCl. Histopathological and immunohistochemical analyses were implemented to further confirm the tumor suppression activity of NG/DOX. Furthermore, the variations of body weight, histopathological morphology, bone marrow cell micronucleus rate, and white blood cell count verified that NG/DOX showed excellent safety in vivo. With these excellent properties in vitro and in vivo, the pH and reduction dual-responsive polypeptide nanogel exhibits great potential for on-demand intracellular delivery of antitumor drug, and holds good prospect for future clinical application. PMID:28255361

  6. Lipoprotein binding to cultured human hepatoma cells.

    PubMed Central

    Krempler, F; Kostner, G M; Friedl, W; Paulweber, B; Bauer, H; Sandhofer, F

    1987-01-01

    Binding of various 125I-lipoproteins to hepatic receptors was studied on cultured human hepatoma cells (Hep G2). Chylomicrons, isolated from a chylothorax, chylomicron remnants, hypertriglyceridemic very low-density lipoproteins, normotriglyceridemic very low-density lipoproteins (NTG-VLDL), their remnants, low-density lipoproteins (LDL), and HDL-E (an Apo E-rich high-density lipoprotein isolated from the plasma of a patient with primary biliary cirrhosis) were bound by high-affinity receptors. Chylomicron remnants and HDL-E were bound with the highest affinity. The results, obtained from competitive binding experiments, are consistent with the existence of two distinct receptors on Hep G2 cells: (a) a remnant receptor capable of high-affinity binding of triglyceride-rich lipoproteins and HDL-E, but not of Apo E free LDL, and (b) a LDL receptor capable of high-affinity binding of LDL, NTG-VLDL, and HDL-E. Specific binding of Apo E-free LDL was completely abolished in the presence of 3 mM EDTA, indicating that binding to the LDL receptor is calcium dependent. Specific binding of chylomicron remnants was not inhibited by the presence of even 10 mM EDTA. Preincubation of the Hep G2 cells in lipoprotein-containing medium resulted in complete suppression of LDL receptors but did not affect the remnant receptors. Hep G2 cells seem to be a suitable model for the study of hepatic receptors for lipoprotein in man. Images PMID:3038957

  7. 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.

  8. 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.

  9. INHIBITORY EFFECT OF CHITOSAN OLIGOSACCHARIDE ON HUMAN HEPATOMA CELLS IN VITRO.

    PubMed

    Liu, Likun; Xin, Yi; Liu, Jia; Zhang, Ershao; Li, Weiling

    2017-01-01

    Chitosan oligosaccharide, the degradation products of chitin, was reported to have a wide range of physiological functions and biological activities. In this study, we explored the inhibitory effect of Chitosan oligosaccharide on human hepatoma cells. MTT assay was applied to detect cell viability of the human hepatoma cells treated with Chitosan oligosaccharide. Flow cytometric analysis was used to investigate the apoptosis of the human hepatoma cells treated with Chitosan oligosaccharide. We employed western blot to investigate the underlying mechanisms involved in the apoptosis. Our data indicated that chitosan oligosaccharide dose-dependently inhibited the growth of hepatoma cells and induced apoptosis. On the molecular level, chitosan oligosaccharide decreased Bcl-2 and increased Caspase-3 expression which may be related to the apoptosis of hepatoma cells. Our results provide an experimental basis for the clinical development of Chitosan oligosaccharide as a novel anti-hepatoma drug.

  10. 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.

  11. [Experimental study on electrical impedance properties of human hepatoma cells].

    PubMed

    Fang, Yun; Tang, Zhiyuan; Zhang, Qian; Zhao, Xin; Ma, Qing

    2014-10-01

    The AC impedance of human hepatoma SMMC-7721 cells were measured in our laboratory by Agilent 4294A impedance analyzer in the frequency range of 0.01-100 MHz. And then the effect of hematocrit on electrical impedance characteristics of hepatoma cells was observed by electrical impedance spectroscopy, Bode diagram, Nyquist diagram and Nichols diagram. The results showed that firstly, there is a frequency dependence, i.e., the increment of real part and the imaginary part of complex electrical impedance (δZ', δZ"), the increment of the amplitude modulus of complex electrical impedance (δ[Z *]) and phase angle (δθ) were all changed with the increasing frequency. Secondly, it showed cell volume fraction (CVF) dependence, i. e. , the increment of low-frequency limit (δZ'0, δ[Z*] 0), peak (δZ"(p), δθ(p)), area and radius (Nyquist diagram, Nichols diagram) were all increased along with the electric field frequency. Thirdly, there was the presence of two characteristic frequencies: the first characteristic frequency (f(c1)) and the second characteristic frequency (f(c2)), which were originated respectively in the polarization effects of two interfaces that the cell membrane and extracellular fluid, cell membrane and cytoplasm. A conclusion can be drawn that the electrical impedance spectroscopy is able to be used to observe the electrical characteristics of human hepatoma cells, and therefore this method can be used to investigate the electrophysiological mechanisms of liver cancer cells, and provide research tools and observation parameters, and it also has important theoretical value and potential applications for screening anticancer drugs.

  12. 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.

  13. Synergistic inhibitory effect of hyperbaric oxygen combined with sorafenib on hepatoma cells.

    PubMed

    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

    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. 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. 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. 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.

  14. 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

  15. 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

  16. Inhibitory effect of coffee on hepatoma proliferation and invasion in culture and on tumor growth, metastasis and abnormal lipoprotein profiles in hepatoma-bearing rats.

    PubMed

    Miura, Yutaka; Ono, Kanako; Okauchi, Rieko; Yagasaki, Kazumi

    2004-02-01

    We have already reported that instant coffee powder (ICP) and ICP-loaded rat sera could suppress proliferation and invasion of rat ascites hepatoma cell line of AH109A in vitro. In this report, we examined the mechanisms for suppression of tumor cell proliferation and invasion by ICP, and the effect of ICP on in vivo tumor growth, metastasis and abnormal lipoprotein profiles in hepatoma-bearing rats. ICP, when directly added to the culture media, induced cell cycle arrest (elongation of S phase) at a lower concentration (0.3 mg/mL) and apoptosis at a higher concentration (0.6-1.2 mg/mL). ICP and ICP-loaded rat sera showed reactive oxygen species (ROS)-scavenging property and canceled the enhancement of invasive activity of hepatoma cells induced by ROS in vitro. These results suggest that ICP suppresses the proliferation by inducing cell cycle arrest and apoptosis, and the invasion by scavenging ROS and that ICP could retain these properties after their gastrointestinal absorption. The hepatoma-bearing rats were fed with a 20% casein diet (20C) or 20C supplemented with 0.1%, ICP for 14 d. Dietary ICP significantly reduced solid tumor growth and tended to reduce hepatoma metastases to lung and lymphatic nodes, suggesting that ICP could suppress tumor cell proliferation and invasion in vivo. In addition, dietary ICP significantly increased serum high-density lipoprotein (HDL)-cholesterol and tended to reduce very low-density and low-density lipoprotein (VLDL+LDL)-cholesterol, resulting in amelioration of abnormal lipoprotein profiles occurred in hepatoma-bearing rats. In conclusion, ICP has the ability to induce cell cycle arrest and apoptosis in hepatoma cells and to suppress tumor cell invasion by reducing oxidative stresses in vitro, and it could also exhibit these effects in vivo, leading to the inhibition of tumor growth and metastases.

  17. Fibronectin synthesized by a human hepatoma cell line

    SciTech Connect

    Glasgow, J.E.; Colman, R.W.

    1984-07-01

    Fibronectin is a family of immunologically similar glycoproteins which mediate a variety of cell-cell and cell-substratum interactions. It is a constituent of the extracellular matrix of connective tissue and circulates in plasma. When suspension and adherent cultures of a human hepatoma cell line (SK-HEP-1) were incubated in serum-free medium, the resulting conditioned medium contained material which was specifically immunoprecipitated by antisera to human plasma fibronectin. By double immunodiffusion, a component in the conditioned culture medium was shown to form a line of identity with fibronectin in human plasma and to migrate as an alpha 2- to beta-globulin during immunoelectrophoresis. Human fibronectin was quantified in conditioned medium by electroimmunodiffusion, and was found to increase for at least three days at about 0.1 micrograms/10(6) cells/day. Adherent cultures of SK-HEP-1 cells were incubated with L-(/sup 35/S)methionine to label newly synthesized proteins. Labeled fibronectin in conditioned medium or in cell extracts comigrated with fibronectin in human plasma as shown by autoradiography following crossed-immunoelectrophoresis. Fibronectin was demonstrated in the extra-cellular matrix of adherent SK-HEP-1 cultures by immunofluorescence. It was shown previously that SK-HEP-1 cells synthesize alpha 1-protease inhibitor, one of the products of normal hepatocytes. The finding that these hepatoma cells also synthesize fibronectin supports the concept that the hepatocyte may be one source of circulating fibronectin, a possibility consistent with the established role of this cell type in blood plasma protein synthesis.

  18. 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

  19. Involvement of anoikis-resistance in the metastasis of hepatoma cells.

    PubMed

    Cao, Lili; Han, Lihui; Zhang, Zhiyong; Li, Jie; Qu, Zhonghua; Du, Juan; Liang, Xiaohong; Liu, Yugang; Liu, Hua; Shi, Yongyu; Liu, Suxia; Gao, Lifen; Sun, Wensheng

    2009-04-15

    Acquisition of anoikis-resistance is a pre-requisite for cancer cell metastasis. We have demonstrated that hepatoma cells could resist anoikis by a synoikis-like survival style. In this study, we further suggest that acquisition of anoikis-resistance confer cancer cells more capacity for invasiveness, evading from cancer therapeutic agents and escaping from host immune attacks. We investigated the response of anoikis-resistant hepatoma cells to TNF-related apoptosis-inducing ligand (TRAIL), a typical immune surveillant molecule as well as a potential anticancer agent. Our data indicated that detached hepatoma cells not only resist TRAIL-induced apoptosis, but also domesticate TRAIL to exert a stealth "tumor counterattack" effect. These results reveal that acquisition of anoikis-resistance may act as a selective pressure to superimpose on hepatoma cells more metastatic potential for the development of cancer.

  20. CD4-independent, productive human immunodeficiency virus type 1 infection of hepatoma cell lines in vitro.

    PubMed Central

    Cao, Y Z; Friedman-Kien, A E; Huang, Y X; Li, X L; Mirabile, M; Moudgil, T; Zucker-Franklin, D; Ho, D D

    1990-01-01

    Five hepatoma cell lines, including CZHC/8571, PLC/PRF/5, Hep3B, HepG2, and HUH7, were inoculated with three diverse isolates of human immunodeficiency virus type 1 (HIV-1). Productive infection was noted in all hepatoma cell lines, and expression of viral p24 antigen lasted for over 3 months, but its level decreased in proportion to the number of viable cells. HIV-1 antigens were also found in the cells by immunohistochemical staining and radioimmunoprecipitation assay, as were viral RNA by in situ hybridization and HIV-1-like particles by electron microscopy. Virus yield assays were also positive on supernatant fluids collected from hepatoma cultures inoculated with HIV-1. Despite their susceptibility to infection, all five hepatoma cell lines were negative for CD4 by immunofluorescence and for CD4 mRNA by slot-blot hybridization. In addition, HIV-1 infection of hepatoma cell lines was not blocked by anti-CD4 monoclonal antibody or soluble CD4. Together, these findings clearly demonstrate that all five hepatoma cell lines were susceptible to productive infection by HIV-1 in vitro via a CD4-independent mechanism. Images PMID:2159530

  1. Studies on responsiveness of hepatoma cells to catecholamines. II. Comparison of beta-adrenergic responsiveness of rat ascites hepatoma cells with cultured normal rat liver cells.

    PubMed

    Miyamoto, K; Matsunaga, T; Takemoto, N; Sanae, F; Koshiura, R

    1985-05-01

    The pharmacological properties of beta-adrenoceptors in rat ascites hepatoma cells were compared with those in normal rat liver cells which were cultured for 24 hr after collagenase digestion. Adenylate cyclases in the homogenates of cultured normal rat liver cells and rat ascites hepatoma cells, AH44, AH66, AH109A, AH130 and AH7974, were all activated by isoproterenol or NaF to different degrees. The enzyme in rat liver cells was activated by several beta 2-agonists but those in all hepatoma cells hardly responded. Furthermore, salbutamol, a beta 2-partial agonist, antagonized the cyclase activation by isoproterenol in AH130 cells. The Kact value of isoproterenol for the activation of adenylate cyclase in AH130 cells was smaller than that in rat liver cells. A comparison of the Ki values of beta-antagonists for the inhibition of isoproterenol-stimulated cyclase activity shows that while the Ki values of propranolol and butoxamine in AH130 cells were similar to those in rat liver cells, a significant difference was observed in the values for beta 1-selective antagonists between AH130 cells and rat liver cells. The Ki values of metoprolol and atenolol for AH130 cells were 137- and 90-fold lower, respectively, than for normal rat liver cells. From these findings, it is strongly suggested that beta-adrenoceptors in rat ascites hepatoma cells including AH130 cells have similar properties to the mammalian beta 1-receptor.

  2. Hepatoma polarization limits CD81 and hepatitis C virus dynamics

    PubMed Central

    Harris, H J; Clerte, C; Farquhar, M J; Goodall, M; Hu, K; Rassam, P; Dosset, P; Wilson, G K; Balfe, P; IJzendoorn, S C; Milhiet, P E; McKeating, J A

    2013-01-01

    Many viruses target the polarized epithelial apex during host invasion. In contrast, hepatitis C virus (HCV) engages receptors at the basal surface of hepatocytes in the polarized liver parenchyma. Hepatocyte polarization limits HCV entry by undefined mechanism(s). Given the recent reports highlighting a role for receptor mobility in pathogen entry, we studied the effect(s) of hepatocyte polarization on viral receptor and HCV pseudoparticle (HCVpp) dynamics using real-time fluorescence recovery after photobleaching and single particle tracking. Hepatoma polarization reduced CD81 and HCVpp dynamics at the basal membrane. Since cell polarization is accompanied by changes in the actin cytoskeleton and CD81 links to actin via its C-terminus, we studied the dynamics of a mutant CD81 lacking a C-terminal tail (CD81ΔC) and its effect(s) on HCVpp mobility and infection. CD81ΔC showed an increased frequency of confined trajectories and a reduction of Brownian diffusing molecules compared to wild-type protein in non-polarized cells. However, these changes were notobserved in polarized cells. HCVpp showed a significant reduction in Brownian diffusion and infection of CD81ΔC expressing non-polarized cells. In summary, these data highlight the dynamic nature of CD81 and demonstrate a role for CD81 lateral diffusion to regulate HCV infection in a polarization-dependent manner. PMID:23126643

  3. Anti-hepatoma activity of resveratrol in vitro

    PubMed Central

    Sun, Zhong-Jie; Pan, Cheng-En; Liu, Hong-Shan; Wang, Guo-Jun

    2002-01-01

    AIM: To study the anti-tumor effect of resveratrol alone and the synergistic effects of resveratrol with 5-FU on the growth of H22 cells line in vitro METHODS: The number of cells was measured by MTT method,the morphological changes of H22 cells were investigated under microscopy and electron microscopyq. RESULTS: Resveratrol inhibited the growth of hepatoma cells line H22 in a dose- and time-dependent manner. IC50 of the resveratrol on H22 cells was 6.57 mg·L-1. The synergistic anti-tumor effects of resveratrol with 5-FU increased to a greater extent than for H22 cells treated with 5-FU alone (70.2% vs 28.4%)(P < 0.05). Under microscope and electron microscope, characteristics of apoptosis such as typical apoptotic bodies were commonly found in tumor cells in the drug-treated groups. CONCLUSION: Resveratrol can suppresses the growth of H22 cells in vitro, its anti-tumor activity may occur through the induction of apoptosis. PMID:11833076

  4. Overexpression of c-Jun contributes to sorafenib resistance in human hepatoma cell lines

    PubMed Central

    Haga, Yuki; Nakamura, Masato; Nakamoto, Shingo; Sasaki, Reina; Takahashi, Koji; Wu, Shuang; Yokosuka, Osamu

    2017-01-01

    Background Despite recent advances in treatment strategies, it is still difficult to cure patients with hepatocellular carcinoma (HCC). Sorafenib is the only approved multiple kinase inhibitor for systemic chemotherapy in patients with advanced HCC. The majority of advanced HCC patients are resistant to sorafenib. The mechanisms of sorafenib resistance are still unknown. Methods The expression of molecules involved in the mitogen-activated protein kinase (MAPK) signaling pathway in human hepatoma cell lines was examined in the presence or absence of sorafenib. Apoptosis of human hepatoma cells treated with sorafenib was investigated, and the expression of Jun proto-oncogene (c-Jun) was measured. Results The expression and phosphorylation of c-Jun were enhanced in human hepatoma cell lines after treatment with sorafenib. Inhibiting c-Jun enhanced sorafenib-induced apoptosis. The overexpression of c-Jun impaired sorafenib-induced apoptosis. The expression of osteopontin, one of the established AP-1 target genes, was enhanced after treatment with sorafenib in human hepatoma cell lines. Conclusions The protein c-Jun plays a role in sorafenib resistance in human hepatoma cell lines. The modulation and phosphorylation of c-Jun could be a new therapeutic option for enhancing responsiveness to sorafenib. Modulating c-Jun may be useful for certain HCC patients with sorafenib resistance. PMID:28323861

  5. Celecoxib suppresses hepatoma stemness and progression by up-regulating PTEN

    PubMed Central

    Kuo, Hsiao-Mei; Liu, Li-Fen; Hu, Tsung-Hui; Sun, Cheuk-Kwan; Kung, Mei-Lang; Lin, Shih-Wei; Wang, E-Ming; Ma, Yi-Ling; Cheng, Kwan-Hung; Lai, Kwok Hung; Wen, Zhi-Hong; Hsu, Ping-I; Tai, Ming-Hong

    2014-01-01

    Celecoxib, a COX-2 inhibitor and non-steroidal anti-inflammatory drug, can prevent several types of cancer, including hepatocellular carcinoma (HCC). Here we show that celecoxib suppressed the self-renewal and drug-pumping functions in HCC cells. Besides, celecoxib depleted CD44 + /CD133 + hepatic cancer stem cells (hCSC). Prostaglandin E2 (PGE2) and CD133 overexpression did not reverse the celecoxib-induced depletion of hCSC. Also, celecoxib inhibited progression of rat Novikoff hepatoma. Moreover, a 60-day celecoxib program increased the survival rate of rats with hepatoma. Histological analysis revealed that celecoxib therapy reduced the abundance of CD44 + /CD133 + hCSCs in hepatoma tissues. Besides, the hCSCs depletion was associated with elevated apoptosis and blunted proliferation and angiogenesis in hepatoma. Celecoxib therapy activated peroxisome proliferator-activated receptor γ (PPARγ) and up-regulated PTEN, thereby inhibiting Akt and disrupting hCSC expansion. PTEN gene delivery by adenovirus reduced CD44/CD133 expression in vitro and hepatoma formation in vivo. This study suggests that celecoxib suppresses cancer stemness and progression of HCC via activation of PPARγ/PTEN signaling. PMID:24721996

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

    PubMed

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

    2013-10-10

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

  7. 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.

  8. Processing of proinsulin by transfected hepatoma (FAO) cells.

    PubMed

    Vollenweider, F; Irminger, J C; Gross, D J; Villa-Komaroff, L; Halban, P A

    1992-07-25

    Rat hepatoma (FAO) cells were stably transfected with the gene encoding either rat proinsulin II (using the DOL retroviral vector) or human proinsulin (using the RSV retroviral vector). Using the DOL vector, production of insulin immunoreactive material was stimulated up to 30-fold by dexamethasone (5 x 10(-7) M). For both proinsulins, fractional release of immunoreactive material relative to cellular content was high, in keeping with the absence of any storage compartment for secretory proteins in these cells. Pulse-chase experiments showed kinetics of release of newly synthesized products in keeping with release via the constitutive pathway. High performance liquid chromatography analysis showed immunoreactivity in the medium distributed between three peaks. For rat proinsulin II, the first coeluted with intact proinsulin; the second coeluted with des-64,65 split proinsulin (the product of endoproteolytic attack between the insulin A-chain and C-peptide followed by trimming of C-terminal basic residues by carboxypeptidase); the third (and minor peak) coeluted with native (fully processed) insulin. For human proinsulin, by contrast, the second peak coeluted with des-31,32 split proinsulin (split and trimmed at the B-chain/C-peptide junction). Analysis of cellular extracts showed intact proinsulin as the major product. The generation of the putative conversion intermediates and insulin was not due to proteolysis of proinsulin after its release but rather to an intracellular event. The data suggest that proinsulin, normally processed in secretory granules and released via the regulated pathway, may also be processed, albeit less efficiently, by the constitutive pathway conversion machinery. The comparison of the sites preferentially cleaved in rat II or human proinsulin suggests cleavage by endoprotease(s) with a preference for R/KXR/KR as substrate.

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

    PubMed

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

    2015-12-28

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

  10. Methionine adenosyltransferase II beta subunit gene expression provides a proliferative advantage in human hepatoma.

    PubMed

    Martínez-Chantar, Maria L; García-Trevijano, Elena R; Latasa, M Ujue; Martín-Duce, Antonio; Fortes, Puri; Caballería, Juan; Avila, Matías A; Mato, José M

    2003-04-01

    Of the 2 genes (MAT1A, MAT2A) encoding methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, MAT1A, is expressed in liver, whereas MAT2A is expressed in extrahepatic tissues. In liver, MAT2A expression associates with growth, dedifferentiation, and cancer. Here, we identified the beta subunit as a regulator of proliferation in human hepatoma cell lines. The beta subunit has been cloned and shown to lower the K(m) of methionine adenosyltransferase II alpha2 (the MAT2A product) for methionine and to render the enzyme more susceptible to S-adenosylmethionine inhibition. Methionine adenosyltransferase II alpha2 and beta subunit expression was analyzed in human and rat liver and hepatoma cell lines and their interaction studied in HuH7 cells. beta Subunit expression was up- and down-regulated in human hepatoma cell lines and the effect on DNA synthesis determined. We found that beta subunit is expressed in rat extrahepatic tissues but not in normal liver. In human liver, beta subunit expression associates with cirrhosis and hepatoma. beta Subunit is expressed in most (HepG2, PLC, and Hep3B) but not all (HuH7) hepatoma cell lines. Transfection of beta subunit reduced S-adenosylmethionine content and stimulated DNA synthesis in HuH7 cells, whereas down-regulation of beta subunit expression diminished DNA synthesis in HepG2. The interaction between methionine adenosyltransferase II alpha2 and beta subunit was demonstrated in HuH7 cells. Our findings indicate that beta subunit associates with cirrhosis and cancer providing a proliferative advantage in hepatoma cells through its interaction with methionine adenosyltransferase II alpha2 and down-regulation of S-adenosylmethionine levels.

  11. 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

  12. [Regularity of drugs compatibility of anti-hepatoma traditional Chinese medicine ancient prescriptions and risk evaluation of anti-hepatoma new drug research and development].

    PubMed

    Zhang, Jing; Li, Hong-Fa; Fan, Wei; Liu, Zhen; Man, Shu-Li; Si, Shu-Yong; Gao, Wen-Yuan

    2014-10-01

    Traditional Chinese ancient prescriptions have been used for treatment of liver cancer for a long history and the scientific and rational compatibility is a great wealth for modern research and development (R&D) of new drugs. The research and development of new drugs are often accompanied with a large investment, a long cycle and a high risk, especially for the anti-tumor drugs R&D which are facing more risks and lower successful rate. In this research, the regularity of compatibility of drugs was analyzed from 124 anti-hepatoma ancient prescriptions by computer program. The results can offer help to the R&D of anti-hepatoma new drugs and reduce the risk of drug screening. In addition, we surveyed 22 companies in this field from six provinces such as Beijing, Shanghai, Tianjin and so on and obtained 240 risk assessment questionaires. Then we used qualitative analysis method to interpret the greatest impacts for the risks in the process of R&D, production and sales of anti-hepatoma new drugs. The study provides a basis for anti-liver cancer drugs R&D researchers, who can take effective measures to reduce the R&D risks and improve successful rate.

  13. Decreased Mitochondrial OGG1 Expression is Linked to Mitochondrial Defects and Delayed Hepatoma Cell Growth

    PubMed Central

    Lee, Young-Kyoung; Youn, Hwang-Guem; Wang, Hee-Jung; Yoon, Gyesoon

    2013-01-01

    Many solid tumor cells exhibit mitochondrial respiratory impairment; however, the mechanisms of such impairment in cancer development remain unclear. Here, we demonstrate that SNU human hepatoma cells with declined mitochondrial respiratory activity showed decreased expression of mitochondrial 8-oxoguanine DNA glycosylase/lyase (mtOGG1), a mitochondrial DNA repair enzyme; similar results were obtained with human hepatocellular carcinoma tissues. Among several OGG1-2 variants with a mitochondrial- targeting sequence (OGG1-2a, -2b, -2c, -2d, and -2e), OGG1-2a was the major mitochondrial isoform in all examined hepatoma cells. Interestingly, hepatoma cells with low mtOGG1 levels showed delayed cell growth and increased intracellular reactive oxygen species (ROS) levels. Knockdown of OGG1-2 isoforms in Chang-L cells, which have active mitochondrial respiration with high mtOGG1 levels, significantly decreased cellular respiration and cell growth, and increased intracellular ROS. Overexpression of OGG1-2a in SNU423 cells, which have low mtOGG1 levels, effectively recovered cellular respiration and cell growth activities, and decreased intracellular ROS. Taken together, our results suggest that mtOGG1 plays an important role in maintaining mitochondrial respiration, thereby contributing to cell growth of hepatoma cells. PMID:23677377

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

    USDA-ARS?s Scientific Manuscript database

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

  15. Studies on anti-hepatoma activity of Annona squamosa L. pericarp extract.

    PubMed

    Chen, Ya-Yun; Cao, Yu-Zhu; Li, Fu-Qiang; Zhu, Xiao-Li; Peng, Chen-Xiao; Lu, Jia-Hui; Chen, Jian-Wei; Li, Xiang; Chen, Yong

    2017-05-01

    This study investigated the anti-hepatoma activity of different extracts from A. squamosa pericarps, phytochemistry of the ethyl acetate (EtOAc) fraction and possible anti-hepatoma mechanism of active constituents. The anti-hepatoma activity of different extracts from A. squamosa pericarps were evaluated by MTT assay against SMMC-7721 cells in vitro and verified by using H22 xenografts bearing mice. Phytochemical investigation of the active pericarp extract was carried out. The pro-apoptosis and cycle arrest effects of active constituents were observed by fluorescent microscope and flow cytometry. Western blot assay was conducted to find the possible anti-hepatoma mechanisms of active constituents. The result showed that EtOAc extract was the active fraction. Two ent-kaurane diterpenoids, named ent-kauran-16-en-19-oic acid and ent-kauran-15-en-19-oic acid, were isolated from the active EtOAc fraction. The pro-apoptosis and G1 phase arrest effects of these diterpenoids were found. Western blot assay showed that ent-kauran-16-en-19-oic acid could activate caspase-3,-8,-9, up-regulate of Bax and down-regulate of Bcl-2. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Experimental study of simultaneous photodynamic and hyperthermic treatment of Ehrlich ascites carcinoma and A22 hepatoma

    NASA Astrophysics Data System (ADS)

    Luksiene, Zivile

    1997-12-01

    Combined treatment of PDT and hyperthermia was examined on Ehrlich ascites tumor cell viability and A22 hepatoma tumor growth inhibition. Histological evaluations of tumors after different treatments have shown tumor necrosis, congestion of blood vessels and hemorrhage. The most drastic damages were observed after simultaneous PDT and hyperthermia action. At these experimental conditions tumor growth for 5 days was absolutely inhibited.

  17. Comparison of cell-surface glycoproteins of rat hepatomas and embryonic rat liver.

    PubMed Central

    van Beek, W. P.; Emmelot, P.; Homburg, C.

    1977-01-01

    Cell-surface glycoprotein of 3 rat hepatoma strains and late-embryonic liver was metabolically labelled in vivo with [3H]- or [14C]-fucose. Trypsinization of the cells and exhaustive pronase digestion of combined hepatoma-liver trypsinates followed by gel filtration over Sephadex-Biogel mixtures, yielded elution profiles that contained more early-eluting (high-mol.-wt.) glycopeptides for hepatomas than for liver. At least 3 factors were identified which acted to augment the fraction of early-eluting tumour glycopeptides: (a) increase of neuraminidase-sensitive sialic acid, (b) increase of neuraminidase-insensitive sialic acid that was sensitive to mild HCl hydrolysis, and (c) presence of sugar sulphate groups contributing to a restricted extent, relative to possible unknown factor(s). Whether (a), (b) or (c) operated depended on the hepatoma strain or its mode of growth. Notwithstanding these differences in the nature of the increase in early-eluting glycopeptides, the increase itself appears not to be due to growth per se, nor to an embryonic expression, but rather may serve as a marker of tumourigenicity. PMID:199223

  18. Beta/sub 1/-adrenoceptors in rat hepatoma, desensitization by isoproterenol and phorbol-myristate-acetate

    SciTech Connect

    Garcia-Sainz, J.A.; Alcantara, R.; Hernandez-Sotomayor, S.M.T.; Mas-Oliva, J.

    1989-01-01

    The beta-adrenergic responsiveness of hepatocytes obtained from hypothyroid rats and of a transplantable hepatoma cell line (AS-30D) were studied by measuring the accumulation of cyclic AMP. The potency order for agonists in hepatocytes was: isoproterenol > epinephrine >> norepinephrine whereas in the hepatoma cells the potency order was: isoproterenol > norepinephrine /equivalent to/ epinephrine. The effect of isoproterenol was antagonized in hepatocytes by low concentrations of ICI 118551 and only partially by concentrations of atenolol as high as 100 ..mu..M. In hepatome cells the effect of isoproterenol was inhibited by both antagonists with the potency order atenolol > ICI 118551. These data indicate that in hepatocytes the effect is mediated by beta/sub 2/-adrenoceptors whereas in hepatoma cells it is through beta/sub 1/-adrenoceptors. Preincubation of hepatoma cells with isoproterenol or phorbol-myristate-acetate diminished the subsequent beta-adrenergic responsiveness of the cells. Interestingly, when both isoproterenol and phorbol-myristate-acetate were present during the preincubation the beta-adrenergic desensitization observed was bigger than that induced by any of these agents alone.

  19. Berberine Inhibits Human Hepatoma Cell Invasion without Cytotoxicity in Healthy Hepatocytes

    PubMed Central

    Pan, Xuediao; Yang, Zhicheng; Zang, Linquan

    2011-01-01

    Conventional chemotherapy fails to cure metastatic hepatoma mainly due to its high hepatotoxicity. Many plant-derived agents have been accepted to effectively inhibit hepatoma cell invasion. However, the investigation that whether effectual plant-derived agents against invasive hepatoma cells exert unexpected cytotoxicity in healthy hepatocytes has been ignored. This study demonstrated that berberine exhibited significant cytotoxicity in HepG2 cells mainly through upregulation of reactive oxygen species (ROS) production but was ineffective in normal Chang liver cells. Berberine exerted anti-invasive effect on HepG2 cells through suppression of matrix metalloproteinase-9 (MMP-9) expression. Moreover, berberine could significantly inhibit the activity of PI3K-AKT and ERK pathways. Combination treatment of ERK pathway inhibitor PD98059 or AKT pathway inhibitor LY294002 and berberine could result in a synergistic reduction on MMP-9 expression along with an inhibition of cell invasion. Enhancement of ROS production by berberine had no influence on its suppressive effects on the activity of PI3K-AKT and ERK pathways, as well as MMP-9 expression and HepG2 cell invasion. In conclusion, our results suggest that berberine may be a potential alternative against invasive hepatoma cells through PI3K-AKT and ERK pathways-dependent downregulation of MMP-9 expression. This study also provides a previously neglected insight into the investigation of plant-derived agents-based therapy against tumor invasion with the consideration of damage to healthy cells. PMID:21738655

  20. Anti-hepatoma activities of ethyl acetate extract from Ampelopsis sinica root.

    PubMed

    Wang, Jia-Zhi; Huang, Bi-Sheng; Cao, Yan; Chen, Ke-Li; Li, Juan

    2017-03-13

    Ampelopsis sinica root (ASR) is a known hepatoprotective folk traditional Chinese medicine. The anti‑hepatoma activity of ethyl acetate extract from A. sinica root (ASRE) in vitro and in vivo and its possible mechanism were explored. This study was designed to investigate cytotoxicity by MTT assay, induction of apoptosis via Hoechst 33258 staining, scanning electron microscopy and bivariate flow cytometric analysis (Annexin V-FITC/PI), inflammation and apoptosis related genes expression by RT-PCR and p53 protein expression by immunofluorescence assay in HepG2 cells. Then, the antitumor activity in vivo was detected by hepatoma H22 xenograft tumor in mice. The results showed that ASRE had powerful anti‑hepatoma activity in vitro without obvious toxicity on normal cells and could induce HepG2 cell apoptosis. The mechanism may be associated with downregulation of inflammatory cytokines including cyclooxygenase-2, 5-lipoxygenase and FLAP, increase of the ratio of bax/bcl-2, activation caspase-3 and inhibition of survivin, and increased expression of p53 protein. Furthermore, the HPLC assay showed the main compounds of ASRE were gallic acid, catechin and gallic acid ethyl ester. In animal experiments, ASR ethanol extract decreased the tumor weights of hepatoma H22 tumor-bearing mice. Therefore, ASR may be a potential therapeutic agent in the treatment of hepatocellular carcinoma.

  1. Structure and properties of an under-sulfated heparan sulfate proteoglycan synthesized by a rat hepatoma cell line

    PubMed Central

    1984-01-01

    A rat hepatoma cell line was shown to synthesize heparan sulfate and chondroitin sulfate proteoglycans. Unlike cultured hepatocytes, the hepatoma cells did not deposit these proteoglycans into an extracellular matrix, and most of the newly synthesized heparan sulfate proteoglycans were secreted into the culture medium. Heparan sulfate proteoglycans were also found associated with the cell surface. These proteoglycans could be solubilized by mild trypsin or detergent treatment of the cells but could not be displaced from the cells by incubation with heparin. The detergent-solubilized heparan sulfate proteoglycan had a hydrophobic segment that enabled it to bind to octyl- Sepharose. This segment could conceivably anchor the molecule in the lipid interior of the plasma membrane. The size of the hepatoma heparan sulfate proteoglycans was similar to that of proteoglycans isolated from rat liver microsomes or from primary cultures of rat hepatocytes. Ion-exchange chromatography on DEAE-Sephacel indicated that the hepatoma heparan sulfate proteoglycans had a lower average charge density than the rat liver heparan sulfate proteoglycans. The lower charge density of the hepatoma heparan sulfate can be largely attributed to a reduced number of N-sulfated glucosamine units in the polysaccharide chain compared with that of rat liver heparan sulfate. Hepatoma heparan sulfate proteoglycans purified from the culture medium had a considerably lower affinity for fibronectin-Sepharose compared with that of rat liver heparan sulfate proteoglycans. Furthermore, the hepatoma proteoglycan did not bind to the neoplastic cells, whereas heparan sulfate from normal rat liver bound to the hepatoma cells in a time-dependent reaction. The possible consequences of the reduced sulfation of the heparan sulfate proteoglycan produced by the hepatoma cells are discussed in terms of the postulated roles of heparan sulfate in the regulation of cell growth and extracellular matrix formation. PMID

  2. Andrographolide inhibits hepatoma cells growth and affects the expression of cell cycle related proteins.

    PubMed

    Shen, Kai-Kai; Liu, Tian-Yu; Xu, Chong; Ji, Li-Li; Wang, Zheng-Tao

    2009-09-01

    The present study is aimed to investigate the toxic effects of andrographolide (Andro) on hepatoma cells and elucidate its preliminary mechanisms. After cells were treated with different concentrations of Andro (0-50 micromol x L(-1)) for 24 h, cell viability was evaluated with 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, after hepatoma cells (Hep3B and HepG2) were treated with different concentrations of Andro (0-30 micromol x L(-1)) for 14 d, the number of colony formation was accounted under microscope. Cell cycle related proteins such as Cdc-2, phosphorylated-Cdc-2, Cyclin B and Cyclin D1 were detected with Western blotting assay and the cell cycle was analyzed by flow cytometry using propidium iodide staining. MTT results showed that Andro induced growth inhibition of hepatoma cells in a concentration-dependent manner but had no significant effects on human normal liver L-02 cells. Andro dramatically decreased the colony formation of hepatoma cells in the concentration-dependent manner. Moreover, Andro induced a decrease of Hep3B cells at the G0-G1 phase and a concomitant accumulation of cells at G2-M phase. At the molecular level, Western blotting results showed that Andro decreased the expression of Cdc-2, phosphorylated-Cdc-2, Cyclin D1 and Cyclin B proteins in a time-dependent manner, which are all cell cycle related proteins. Taken together, the results demonstrated that Andro specifically inhibited the growth of hepatoma cells and cellular cell cycle related proteins were possibly involved in this process.

  3. 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.

  4. 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

  5. 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.

  6. Effect of dietary soy protein on tumor necrosis factor productivity in macrophages from nephritic and hepatoma-bearing rats.

    PubMed

    Komatsu, Wataru; Nagata, Junko; Kaneko, Masaharu; Yamada, Tatsuhiko; Moriya, Daisuke; Miura, Yutaka; Yagasaki, Kazumi

    2008-12-01

    The present study investigated the effect of dietary soy protein isolate (SPI) on tumor necrosis factor-alpha (TNF) productivity in peritoneal macrophages from nephritic and hepatoma-bearing rats. Dietary SPI significantly inhibited the elevated production of TNF by lipopolysaccharide-stimulated macrophages in nephritic and hepatoma-bearing rats compared with dietary casein, while it exerted no influence on the TNF productivity in normal rats. Removal of the minor components contained in SPI by ethanol extraction could significantly or partially restore the reduced TNF production caused by SPI in nephritic and hepatoma-bearing rats, respectively. These results suggest that dietary SPI could suppress the enhanced productivity of TNF associated with the progression of nephritis and hepatoma, and some factors existing in the ethanol extract of SPI are suggested to be involved in suppressing TNF productivity by macrophages.

  7. 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.

  8. Dichloroacetate Enhances Adriamycin-Induced Hepatoma Cell Toxicity In Vitro and In Vivo by Increasing Reactive Oxygen Species Levels

    PubMed Central

    Huang, Gang; Liu, Jianjun; Sheng, Shile; Wang, Hongjian; Qin, Wenxin

    2014-01-01

    A unique bioenergetic feature of cancer, aerobic glycolysis is considered an attractive therapeutic target for cancer therapy. Recently, dichloroacetate (DCA), a small-molecule metabolic modulator, was shown to reverse the glycolytic phenotype, induce reactive oxygen species (ROS) generation, and trigger apoptosis in various tumor cells. In this work, the capacity of DCA to enhance Adriamycin (ADM) efficacy in hepatoma cells by modulating glucose metabolism and redox status was evaluated. Two human hepatoma (HCC-LM3 and SMMC-7721) and a normal liver (LO2) cell lines were treated with DCA or ADM alone, or in combination. Exposure of hepatoma cells to DCA/ADM combination resulted in significantly decreased cell viability and increased percentage of apoptotic cells as well as intracellular ROS levels, in comparison with treatment with DCA or ADM alone. However, simultaneous treatment with the thiol antioxidant N-acetylcysteine (NAC, 10 mmol/L) reduced the elevated ROS levels and protected hepatoma cells from the cytotoxic effects of DCA/ADM combination. L-buthionine-[S,R]-sulfoximine, an inhibitor of glutathione synthesis, enhanced hepatoma cell sensitivity to DCA/ADM combination. Interestingly, treatment with DCA/ADM combination did not significantly increase cytotoxicity in normal hepatocytes in comparison with the drugs administered individually. Finally, DCA reduced tumor growth and enhanced ADM efficacy on HCC-LM3 hepatoma in mice. Overall, our data suggest that DCA enhances ADM cytotoxicity in hepatoma cells by increasing intracellular ROS levels and provide a strong biochemical rationale for the use of DCA in combination with ADM for treatment of hepatoma. PMID:24728083

  9. Sorafenib suppresses growth and survival of hepatoma cells by accelerating degradation of enhancer of zeste homolog 2.

    PubMed

    Wang, Shanshan; Zhu, Yu; He, Hongyong; Liu, Jing; Xu, Le; Zhang, Heng; Liu, Haiou; Liu, Weisi; Liu, Yidong; Pan, Deng; Chen, Lin; Wu, Qian; Xu, Jiejie; Gu, Jianxin

    2013-06-01

    Enhancer of zeste homolog 2 (EZH2) is a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes that regulate cancer cell growth and survival. It is overexpressed in hepatocellular carcinoma (HCC) with a clinical significance that remains obscure. Sorafenib, a multikinase inhibitor, has been used as a first-line therapeutic drug and shown clinical efficiency for advanced-stage HCC patients. In the present study, we found that sorafenib lowered the protein level of EZH2 through accelerating proteasome-mediated EZH2 degradation in hepatoma cells. Overexpression of EZH2 reversed sorafenib-induced cell growth arrest, cell cycle arrest, and cell apoptosis dependent on histone methyltransferase activity in hepatoma cells. More importantly, shRNA-mediated EZH2 knockdown or EZH2 inhibition with 3-deazaneplanocin A treatment promoted sorafenib-induced hepatoma cell growth arrest and apoptosis. Sorafenib altered the hepatoma epigenome by reducing EZH2 and H3K27 trimethylation. These results revealed a novel therapeutic mechanism underlying sorafenib treatment in suppressing hepatoma growth and survival by accelerating EZH2 degradation. Genetic deletion or pharmacological ablation of EZH2 made hepatoma cells more sensitive to sorafenib, which helps provide a strong framework for exploring innovative combined therapies for advanced-stage HCC patients.

  10. The combinational effect of vincristine and berberine on growth inhibition and apoptosis induction in hepatoma cells.

    PubMed

    Wang, Ling; Wei, Dandan; Han, Xiaojuan; Zhang, Wei; Fan, Chengzhong; Zhang, Jie; Mo, Chunfen; Yang, Ming; Li, Junhong; Wang, Zhe; Zhou, Qin; Xiao, Hengyi

    2014-04-01

    The use of vincristine, a known antitumor agent, in hepatoma therapy is limited particularly because of its toxic effect. Meanwhile, berberine has drawn increasing attention to its antineoplastic effect in recent years. In view of the advantages of combinational drug treatment reported in anti-cancer chemotherapy, we evaluated the effects of co-treatment of vincristine and berberine on hepatic carcinoma cell lines in this study. We find that combinational usage of these two drugs can significantly induce cell growth inhibition and apoptosis even under a concentration of vincristine barely showing cytotoxicity in the same cells when used alone. The underlying mechanism about this combinational effect was addressed in this study by monitoring the signals related to mitochondrial function, apoptotic pathway and endoplasmic reticulum stress. Our results suggest a new value of berberine as a potential adjuvant agent in cancer chemotherapy and provide a hopeful approach for developing hepatoma therapy by utilizing the combinational effect of vincristine and berberine.

  11. 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

  12. [Antineoplastic effect of hydrogel prospidin on Seidel ascites hepatoma used as a model].

    PubMed

    Bychkovskiĭ, P M; Iurkshtovich, T L; Kladiev, A A; Revtovich, M Iu

    2012-01-01

    Antineoplastic effect of hydrogel dextran phosphate, hydrogel prospidin, and prospidin in an injectable preparation has been assessed using Seidel ascites hepatoma as a model. Injectable and hydrogel prospidin in doses from 250, 500 to 1000 mg/kg and hydrogel phosphate dextran in doses of 500 and 1000 mg/kg were administered to rats intraperitoneally in a single dose in a volume of 1 or 2 ml per each 100 g of animal body weight. The study has shown that irrespective of rats with Seidel ascites hepatoma and significantly increase in the dosage of prospidin preparations and hydrogel dextran phosphate results in a longer average life expectancy of rats Compared with its injectable variant, hydrogel prospidin appears to produce more than twice as high antineoplastic effect, and is found to provide prolonged therapeutic effects, as well as cure of animals in more than 60 % of cases.

  13. Efficient replication systems for hepatitis C virus using a new human hepatoma cell line.

    PubMed

    Kato, Nobuyuki; Mori, Kyoko; Abe, Ken-ichi; Dansako, Hiromichi; Kuroki, Misao; Ariumi, Yasuo; Wakita, Takaji; Ikeda, Masanori

    2009-12-01

    Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a serious global health problem. Cell culture-based persistent HCV RNA replication systems and infectious HCV production systems are widely used in HCV research. However, persistent HCV production systems have been developed only for HuH-7 hepatoma cells. Here we found a new human hepatoma cell line, Li23, that enables persistent HCV production and anti-HCV reagent assay. Li23's cDNA expression profile differed from HuH-7's, although the two cells had similar liver-specific expression profiles. We used HCV RNA with a specific combination of adaptive mutations to develop an HCV replicon system and genome-length HCV RNA replicating systems including a reporter assay system. Finally, Li23-derived cells persistently produced infectious virus of an HCV strain. Li23-derived cells are potentially useful for understanding the HCV life cycle and for finding antiviral targets.

  14. Cyclooxygenase-2 Is a Target of MicroRNA-16 in Human Hepatoma Cells

    PubMed Central

    Agra Andrieu, Noelia; Motiño, Omar; Mayoral, Rafael; Llorente Izquierdo, Cristina; Fernández-Alvarez, Ana; Boscá, Lisardo; Casado, Marta; Martín-Sanz, Paloma

    2012-01-01

    Cyclooxygenase-2 (COX-2) expression has been detected in human hepatoma cell lines and in human hepatocellular carcinoma (HCC); however, the contribution of COX-2 to the development of HCC remains controversial. COX-2 expression is higher in the non-tumoral tissue and inversely correlates with the differentiation grade of the tumor. COX-2 expression depends on the interplay between different cellular pathways involving both transcriptional and post-transcriptional regulation. The aim of this work was to assess whether COX-2 could be regulated by microRNAs in human hepatoma cell lines and in human HCC specimens since these molecules contribute to the regulation of genes implicated in cell growth and differentiation. Our results show that miR-16 silences COX-2 expression in hepatoma cells by two mechanisms: a) by binding directly to the microRNA response element (MRE) in the COX-2 3′-UTR promoting translational suppression of COX-2 mRNA; b) by decreasing the levels of the RNA-binding protein Human Antigen R (HuR). Furthermore, ectopic expression of miR-16 inhibits cell proliferation, promotes cell apoptosis and suppresses the ability of hepatoma cells to develop tumors in nude mice, partially through targeting COX-2. Moreover a reduced miR-16 expression tends to correlate to high levels of COX-2 protein in liver from patients affected by HCC. Our data show an important role for miR-16 as a post-transcriptional regulator of COX-2 in HCC and suggest the potential therapeutic application of miR-16 in those HCC with a high COX-2 expression. PMID:23226427

  15. 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.

  16. 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

  17. Rat hepatoproliferin revealed the status of a complete hepatomitogen in human hepatoma cells.

    PubMed

    Oosthuizen, M M J; Ndaba, N; Myburgh, J A

    2005-01-01

    Hepatoproliferin (HPF), a liver regeneration factor isolated from rat hepatocytes, was assessed for its mitogenic status in the human hepatoma cell line PLC/PRF-5. HPF was able to enhance hepatoma cell growth on its own without the aid of the established complete mitogens EGF and TGF-alpha or the hepato-priming factor TNF-alpha. HPF therefore acted as a complete hepatomitogen and had no co-mitogenic properties since it did not augment proliferation when combined with EGF or TGF-alpha but showed only an additive effect in the presence of TGF-alpha. Rat HPF was phylogenetically unrestricted, because it was found active in human cells. When each of the established growth factors (GFs) was used alone, the hepatoma cells responded with the same kind of response profile, namely a bi-phasic bell-shaped dose-dependent response due to stimulation at low levels and inhibition at higher levels. However, hepatocyte growth factor (HGF) was an exception since it did not induce a growth response in hepatoma cells. On the contrary HPF, on its own, showed a progressive enhanced linear dose response at the levels used for the GFs (ie 1.0-15 ng/5 x 10(5) cells). The comparative potency (CP) (dpm x 10(3)/microg DNA/ng GF) of HPF (CP = 13) was in the same range as for the complete hepatomitogens EGF (CP = 12) and TGF-alpha (CP = 14), revealing that HPF has indeed the status of a complete mitogen.

  18. 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

  19. Intracellular glutathione regulates Andrographolide-induced cytotoxicity on hepatoma Hep3B cells.

    PubMed

    Ji, Lili; Shen, Kaikai; Liu, Jun; Chen, Ying; Liu, Tianyu; Wang, Zhengtao

    2009-01-01

    Andrographolide (ANDRO), a diterpenoid lactone isolated from the traditional herbal plant Andrographis paniculata, was reported to induce apoptosis in hepatoma Hep3B cells in our previous study (Ji LL, Liu TY, Liu J, Chen Y, Wang ZT. Andrographolide inhibits human hepatoma-derived Hep3B cells growth through the activation of c-Jun N-terminal kinase. Planta Med 2007; 73: 1397-1401). The present investigation was carried out to observe whether cellular reduced glutathione (GSH) plays important roles in ANDRO-induced apoptosis. ANDRO initially increased intracellular GSH levels which then decreased later, while inhibition of cellular GSH synthesis by L-Buthionine-(S,R)-sulfoximine (BSO) augmented ANDRO-induced cytotoxicity and apoptosis in Hep3B cells. On the other hand, the thiol antioxidant dithiothreitol (DTT) rescued ANDRO-depleted cellular GSH, and abrogated ANDRO-induced cytotoxicity and apoptosis. Furthermore, BSO pretreatment augmented ANDRO-decreased expression of antioxidant protein thioredoxin 1 (Trx1), while DTT reversed this decrease. Further results showed that ANDRO increased the activity of the GSH-related antioxidant enzyme glutathione peroxidase (GPx) and the production of intracellular reactive oxygen species (ROS). Taken together, this study demonstrates that the intracellular redox system plays important roles in regulating the cytotoxicity of ANDRO on hepatoma Hep3B cells.

  20. Expression of rat class I major histocompatibility complex (MHC) alloantigens and hepatocytes and hepatoma cells

    SciTech Connect

    Hunt, J.M.; Desai, P.A.; Chakraborty, S.

    1986-03-05

    Altered expression of Class I MHC alloantigens has been reported for murine tumors, and may be associated with the tumorigenic phenotype of tumor cells. To characterize MHC Class I alloantigen expression on a chemically-induced transplantable rat hepatoma cell line, 17X, derived from a (WF x F344) F/sub 1/ rat, polyvalent anti-F344 and anti-WF rat alloantisera were first used to immunoprecipitate the rat RT1.A Class I MHC alloantigens expressed on primary (WF x F344) F/sub 1/ hepatocyptes in short-term monolayer cultures. Two-dimensional isoelectric focusing and SDS-PAGE of immunoprecipitates from /sup 35/S-methionine-labeled (WF x F344) F/sub 1/ hepatocytes clearly resolved the RT1.A/sup u/ (WF) and RT1.A/sup LvI/ (F344) parental alloantigens. Identical radiolabeling and immunoprecipitation failed to detect either parental alloantigen on the 17X hepatoma cells. However, indirect immunofluorescence and immunoblot analyses demonstrated the presence of parental alloantigens on the 17X cells. Immunization of F344 rats but not of WF rats with 17X cells resulted in antibodies cytotoxic for normal (WF X F344) F/sub 1/ spleen cells in the presence of complement. These findings indicate that a combination of detection techniques will be necessary to characterize altered alloantigen expression on rat hepatoma cells.

  1. [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.

  2. HAF, hepatoma aggregation factor produced by Streptomyces sp. strain No. A-6143.

    PubMed

    Suzuki, K; Nakano, N; Nagatomi, Y; Tominaga, H; Nakazono, N; Itai, M; Uyeda, M; Shibata, M

    1990-08-01

    We searched for a new cell aggregation factor for hepatoma AH109A cells, and found one we called HAF in the culture filtrate of Streptomyces sp. strain No. A-6143 isolated from a soil sample. HAF was purified by salting-out with ammonium sulfate. DEAE-cellulose column chromatography, gel filtration on Sephadex G-100, and hydroxylapatite column chromatography, HAF was glycoprotein which had a molecular weight of about 73,000. HAF was stable from pH 6 to 8 at 37 degrees C and up to 40 degrees C at pH 8.0 and the aggregation activity of HAF was maximum around pH 8 at 30 degrees C. The activity was not influenced by some saccharides, but it was inhibited by EDTA and EGTA: moreover HAF activity was restored by the addition of calcium ions. HAF aggregated hepatoma AH136B and COS-7 cells as well as hepatoma AH109A cells, but it was inert to other cancer cells and human erythrocytes. These properties proved that HAF is completely different from other aggregation factors for cancer cells so far reported.

  3. CEA and AFP expression in human hepatoma cells transfected with antisense IGF-I gene

    PubMed Central

    Zhang, Li; Li, Shu-Nong; Wang, Xiao-Ning

    1998-01-01

    AIM: To determine whether antisense insulin-like growth factor-I (IGF-I) gene can modulate CEA and AFP expression in human hepatoma cells (HepG2). METHODS: Transfection of HepG2 cells was accomplished using Lipofectin reagent. Northern blot analysis confirmed the antisense IGF-I RNA of the transfected cells. CEA and AFP levels were measured using radioimmunoassay. RESULTS: Human hepatoma cell lines (HepG2) were transfected with antisense IGF-I gene. Northern blot analysis confirmed that antisense IGF-I RNA was expressed in the transfected cells. The effect of antisense IGF-I gene on CEA and AFP expression was demonstrated by the fact that the CEA and AFP levels in the supernatant of transfected cell culture were significantly lower as compared with the parent cells, [CEA 7.0 μg/L ± 0.76 μg/L and 3.29 μg/L ± 1.80 μg/L (P < 0.05) and AFP 53.63 μg/L ± 6.02 μg/L and 9.0 μg/L ± 5.26 μg/L (P < 0.01), respectively]. CONCLUSION: The malignant potentiality of the transfected cells was partially suppressed.Antisense IGF-I gene can modulate the expression of CEA and AFP in human hepatoma cell lines (HepG2) PMID:11819225

  4. 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

  5. Target-specific delivery of siRNA into hepatoma cells' cytoplasm by bifunctional carrier peptide.

    PubMed

    Liu, Xiaoxuan; Zhu, Lin; Ma, Jingjing; Qiao, Xinxiao; Zhu, Dunwan; Liu, Lanxia; Leng, Xigang

    2017-02-01

    RNA interference (RNAi) is among the most potential approach for the therapy of hepatocellular carcinoma and the major barrier hindering siRNA therapeutics is the low efficiency of delivery to the desired cells. The current study aimed at developing a novel peptide for more efficient hepatoma targeted siRNA delivery, by combining luteinizing hormone-releasing hormone with hepatoma targeting specificity and MPG(△NLS) with cytoplasm-delivery tendency. The developed bifunctional peptide LHRH-MPG(△NLS) and siRNA were mixed together and resulted in LHRH-MPG(△NLS)/siRNA polyplexes through self-assembly. The polyplexes were characterized by agarose gel retardation and dynamic light scatting analysis. Hepatoma targeting specificity was analyzed with the GE IN Cell Analyzer 2000 High-Content Cellular Analysis System after cell transfection, and the effect of RNA interference was detected by RT-PCR. The results demonstrated that LHRH-MPG(△NLS) was able to assemble with siRNA to form stable and nano-sized peptide/siRNA polyplexes, which could inhibit the expression of the target gene and was essentially non-cytotoxic, as compared with the commercial transfection reagent lipofectamine 2000.

  6. Dual role of acetaminophen in promoting hepatoma cell apoptosis and kidney fibroblast proliferation

    PubMed Central

    YU, YUNG-LUEN; YIANG, GIOU-TENG; CHOU, PEI-LUN; TSENG, HSU-HUNG; WU, TSAI-KUN; HUNG, YU-TING; LIN, PEI-SHIUAN; LIN, SHU-YU; LIU, HSIAO-CHUN; CHANG, WEI-JUNG; WEI, CHYOU-WEI

    2014-01-01

    Acetaminophen (APAP), is a safe analgesic and antipyretic drug at therapeutic dose, and is widely used in the clinic. However, high doses of APAP can induce hepatotoxicity and nephrotoxicity. Most studies have focused on high-dose APAP-induced acute liver and kidney injury. So far, few studies have investigated the effects of the therapeutic dose (1/10 of the high dose) or of the low dose (1/100 of the high dose) of APAP on the cells. The aim of this study was to investigate the cellular effects of therapeutic- or low-dose APAP treatment on hepatoma cells and kidney fibroblasts. As expected, high-dose APAP treatment inhibited while therapeutic and low-dose treatment did not inhibit cell survival of kidney tubular epithelial cells. In addition, therapeutic-dose treatment induced an increase in the H2O2 level, activated the caspase-9/-3 cascade, and induced cell apoptosis of hepatoma cells. Notably, APAP promoted fibroblast proliferation, even at low doses. This study demonstrates that different cellular effects are exerted upon treatment with different APAP concentrations. Our results indicate that treatment with the therapeutic dose of APAP may exert an antitumor activity on hepatoma, while low-dose treatment may be harmful for patients with fibrosis, since it may cause proliferation of fibroblasts. PMID:24682227

  7. Tumor-targeted gene therapy using Adv-AFP-HRPC/IAA prodrug system suppresses growth of hepatoma xenografted in mice.

    PubMed

    Dai, M; Liu, J; Chen, D-E; Rao, Y; Tang, Z-J; Ho, W-Z; Dong, C-Y

    2012-02-01

    Clinical efficacy of current therapies for hepatocellular carcinoma (HCC) treatment is limited. Indole-3-acetic acid (IAA) is non-toxic for mammalian cells. Oxidative decarboxylation of IAA by horseradish peroxidase (HRP) leads to toxic effects of IAA. The purpose of this study was to investigate the effects of a novel gene-targeted enzyme prodrug therapy with IAA on hepatoma growth in vitro and in vivo mouse hepatoma models. We generated a plasmid using adenovirus to express HRP isoenzyme C (HRPC) with the HCC marker, alpha-fetoprotein (AFP), as the promoter (pAdv-AFP-HRPC). Hepatocellular cells were infected with pAdv-AFP-HRPC and treated with IAA. Cell death was detected using MTT assay. Hepatoma xenografts were developed in mice by injection of mouse hepatoma cells. The size and weight of tumors and organs were evaluated. Cell death in tumors was assessed using hematoxylin and eosin-stained tissue sections. HRPC expression in tissues was detected using Reverse Transcriptase-Polymerase Chain Reaction. IAA stimulated death of hepatocellular cells infected with pAdv-AFP-HRPC, in a dose- and time-dependent manner, but not in control cells. Growth of hepatoma xenografts, including the size and weight, was inhibited in mice treated with pAdv-AFP-HRPC and IAA, compared with that in control group. pAdv-AFP-HRPC/IAA treatment induced cell death in hepatoma xenografts in mice. HRPC gene expressed only in hepatoma, but not in other normal organs of mice. pAdv-AFP-HRPC/IAA treatment did not cause any side effects on normal organs. These findings suggest that pAdv-AFP-HRPC/IAA enzyme/prodrug system may serve as a strategy for HCC therapy.

  8. 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

  9. Studies on chemical constituents and anti-hepatoma effects of essential oil from Annona squamosa L. pericarps.

    PubMed

    Chen, Ya-Yun; Peng, Chen-Xiao; Hu, Yan; Bu, Chen; Guo, Shu-Chen; Li, Xiang; Chen, Yong; Chen, Jian-Wei

    2017-06-01

    Annona squamosa L. fruit played great anti-neoplastic activities. Its pericarps were discarded as waste. In this study, essential oil extracted from A. squamosa L. pericarps (APEO) was obtained by hydrodistillation and analysed by GC-MS. Furthermore, the anti-hepatoma activities and the underlying mechanism of the oil were firstly described. A total of 59 compounds were identified by Gas chromatography-mass spectrometry (GC-MS). The major compound in the oil was (-)-spathulenol (32.51%). The APEO demonstrated anti-hepatoma activity against SMMC-7721 hepatoma cell line with IC50 lower than 55 μg/mL. At the same time, nucleus shrinkage or broken were found in cells incubated with APEO through fluorescent microscope. In addition, pro-apoptosis and cell cycle arrest effects were confirmed by flow cytometry analysis.

  10. Isolation and characterization of cAMP-resistant mutants of the H-4 rat hepatoma cells

    SciTech Connect

    Liu, A.Y.; Lin, Z.

    1987-05-01

    H-4 rat hepatoma cells were mutagenized with ethyl methane-sulfonate and the frequency of emergence of cAMP resistant mutant cells were evaluated by cloning the EMS-treated cells in a semi-solid agar medium that contained either 1-3 mM 8-bromo-cAMP plus 1 mM 3-isobutyl-1-methyl xanthine or 5 ..mu..g/ml cholera toxin plus 1 mM IBMX. cAMP resistant mutants emerged at a frequency of 8 x 10/sup -5/. 15 colonies were isolated, recloned, grown in mass culture, and cell extracts were prepared. Analysis of cAMP-dependent protein kinase demonstrated that: (1) the type II enzyme is the only cAMP-dependent protein kinase detected in extracts of the hepatoma cells; (2) of the 15 cAMP resistant clonal cell lines examined, only one (H/sub 4/M/sub 18/) was found to be devoid of cAMP-dependent protein kinase activity. In another cell line (H/sub 4/M/sub 10/) the activity was 30% of that of the parental H-4 cells; (3) there was an increase (130-300%) in cAMP-dependent protein kinase activity in 13/15 of the mutant cell lines over that of the parental H-4 cells. Analysis of cAMP-phosphodiesterase demonstrated significant increases (150-370%) in the enzyme activity in extracts of the mutants over that of the H-4 parental line. Their results suggest that while a deficiency in cAMP-dependent protein kinase may confer resistance to the hepatoma cells against the cytostatic effects of 8-bromo-cAMP and cholera toxin, other events such as overexpression of phosphodiesterase may contribute to this phenotype.

  11. 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.

  12. 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

  13. Drug Transporter Expression and Activity in Human Hepatoma HuH-7 Cells.

    PubMed

    Jouan, Elodie; Le Vée, Marc; Denizot, Claire; Parmentier, Yannick; Fardel, Olivier

    2016-12-28

    Human hepatoma cells may represent a valuable alternative to the use of human hepatocytes for studying hepatic drug transporters, which is now a regulatory issue during drug development. In the present work, we have characterized hepatic drug transporter expression, activity and regulation in human hepatoma HuH-7 cells, in order to determine the potential relevance of these cells for drug transport assays. HuH-7 cells displayed notable multidrug resistance-associated protein (MRP) activity, presumed to reflect expression of various hepatic MRPs, including MRP2. By contrast, they failed to display functional activities of the uptake transporters sodium taurocholate co-transporting polypeptide (NTCP), organic anion-transporting polypeptides (OATPs) and organic cation transporter 1 (OCT1), and of the canalicular transporters P-glycoprotein and breast cancer resistance protein (BCRP). Concomitantly, mRNA expressions of various sinusoidal and canalicular hepatic drug transporters were not detected (NTCP, OATP1B1, organic anion transporter 2 (OAT2), OCT1 and bile salt export pump) or were found to be lower (OATP1B3, OATP2B1, multidrug and toxin extrusion protein 1, BCRP and MRP3) in hepatoma HuH-7 cells than those found in human hepatocytes, whereas other transporters such as OAT7, MRP4 and MRP5 were up-regulated. HuH-7 cells additionally exhibited farnesoid X receptor (FXR)- and nuclear factor erythroid 2-related factor 2 (Nrf2)-related up-regulation of some transporters. Such data indicate that HuH-7 cells, although expressing rather poorly some main hepatic drug transporters, may be useful for investigating interactions of drugs with MRPs, notably MRP2, and for studying FXR- or Nrf2-mediated gene regulation.

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

    PubMed

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

    2017-07-01

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

  15. Drug Transporter Expression and Activity in Human Hepatoma HuH-7 Cells

    PubMed Central

    Jouan, Elodie; Le Vée, Marc; Denizot, Claire; Parmentier, Yannick; Fardel, Olivier

    2016-01-01

    Human hepatoma cells may represent a valuable alternative to the use of human hepatocytes for studying hepatic drug transporters, which is now a regulatory issue during drug development. In the present work, we have characterized hepatic drug transporter expression, activity and regulation in human hepatoma HuH-7 cells, in order to determine the potential relevance of these cells for drug transport assays. HuH-7 cells displayed notable multidrug resistance-associated protein (MRP) activity, presumed to reflect expression of various hepatic MRPs, including MRP2. By contrast, they failed to display functional activities of the uptake transporters sodium taurocholate co-transporting polypeptide (NTCP), organic anion-transporting polypeptides (OATPs) and organic cation transporter 1 (OCT1), and of the canalicular transporters P-glycoprotein and breast cancer resistance protein (BCRP). Concomitantly, mRNA expressions of various sinusoidal and canalicular hepatic drug transporters were not detected (NTCP, OATP1B1, organic anion transporter 2 (OAT2), OCT1 and bile salt export pump) or were found to be lower (OATP1B3, OATP2B1, multidrug and toxin extrusion protein 1, BCRP and MRP3) in hepatoma HuH-7 cells than those found in human hepatocytes, whereas other transporters such as OAT7, MRP4 and MRP5 were up-regulated. HuH-7 cells additionally exhibited farnesoid X receptor (FXR)- and nuclear factor erythroid 2-related factor 2 (Nrf2)-related up-regulation of some transporters. Such data indicate that HuH-7 cells, although expressing rather poorly some main hepatic drug transporters, may be useful for investigating interactions of drugs with MRPs, notably MRP2, and for studying FXR- or Nrf2-mediated gene regulation. PMID:28036031

  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. High permissivity of human HepG2 hepatoma cells for influenza viruses.

    PubMed

    Ollier, Laurence; Caramella, Anne; Giordanengo, Valérie; Lefebvre, Jean-Claude

    2004-12-01

    Human HepG2 hepatoma cells are highly permissive for influenza virus type A and type B, even without the addition of trypsin, and they exhibit a marked cytopathic effect. This property greatly facilitates the primary isolation of influenza viruses. Virus replication was significantly reduced by the plasmin(ogen)-specific inhibitor tranexamic acid, and this suggests a potential role played by the plasminogen/tissue plasminogen activator complex at the surface of HepG2 cells. This might represent a new approach for study of the interrelations of this complex with influenza viruses.

  18. High Permissivity of Human HepG2 Hepatoma Cells for Influenza Viruses

    PubMed Central

    Ollier, Laurence; Caramella, Anne; Giordanengo, Valérie; Lefebvre, Jean-Claude

    2004-01-01

    Human HepG2 hepatoma cells are highly permissive for influenza virus type A and type B, even without the addition of trypsin, and they exhibit a marked cytopathic effect. This property greatly facilitates the primary isolation of influenza viruses. Virus replication was significantly reduced by the plasmin(ogen)-specific inhibitor tranexamic acid, and this suggests a potential role played by the plasminogen/tissue plasminogen activator complex at the surface of HepG2 cells. This might represent a new approach for study of the interrelations of this complex with influenza viruses. PMID:15583326

  19. Effects of chemotherapeutic agents on alpha-fetoprotein secretion and growth of human hepatoma cell lines in vitro.

    PubMed Central

    Muraoka, A.; Tokiwa, T.; Sato, J.

    1989-01-01

    The effects of various chemotherapeutic agents on alpha-fetoprotein (AFP) secretion and growth of human hepatocellular carcinoma and hepatoblastoma cell lines were investigated in vitro. It was found that there was a high correlation between hepatoma cell number and AFP secretion after treatment and that the amount of AFP secreted per cell per 72 h was not affected with therapeutically achievable concentrations. These results suggest that serum AFP level in patients with hepatomas does not correlate with the size of whole tumour but with that of viable tumour mass, and that AFP-secreting capacity of tumour cells in the mass is kept unchanged after chemotherapy. PMID:2469455

  20. A novel protocol for the subcellular fractionation of C3A hepatoma cells using sucrose density gradient centrifugation.

    PubMed

    Srinivas, Kitambi Satish; Chandrasekar, Gayathri; Srivastava, Renu; Puvanakrishnan, Rengarajulu

    2004-07-30

    In this paper, we describe a method to obtain a relatively pure mitochondrial and microsomal fractions by subcellular fractionation of human hepatoma cell line C3A using sucrose as the hypoosmotic medium. The cells were subjected to osmotic stress with sucrose and homogenized. Osmolarity was then restored to the cells and the organelles were separated by density gradient centrifugation. The protein profiles were examined by SDS-PAGE and the purity was analysed by marker enzymes and Western blotting. Our results indicate a good separation of mitochondrial and microsomal fractions from human hepatoma C3A cells.

  1. Mitochondria as an important target in heavy metal toxicity in rat hepatoma AS-30D cells.

    PubMed

    Belyaeva, Elena A; Dymkowska, Dorota; Wieckowski, Mariusz R; Wojtczak, Lech

    2008-08-15

    The mechanisms of toxic effects of divalent cations of three heavy metals Hg, Cd and Cu in rat ascites hepatoma AS-30D cells cultivated in vitro were compared. It was found that the toxicity of these ions, applied in the micromolar range (10-500 microM), decreased from Hg(2+) (most toxic) to Cu(2+) (least toxic). Hg(2+) and Cd(2+) produced a high percentage of cell death by both necrosis and apoptosis, whereas Cu(2+) at concentrations up to 500 microM was weakly effective. Hg(2+) at concentration of 10 microM appeared slightly uncoupling (i.e., stimulated resting state respiration and decreased the mitochondrial transmembrane potential), whereas it exerted a strong inhibitory effect on the respiratory chain and rapid dissipation of the membrane potential at higher concentrations. Cu(2+) had inhibitory effect on cell respiration only at 500 microM concentration and after incubation of 48 h but produced a significant uncoupling effect at lower concentrations. Cu(2+) induced an early and sharp increase of intracellular production of reactive oxygen species (ROS). The action of Hg(2+) and Cd(2+) on ROS generation was biphasic. They stimulated ROS generation within the cells at low concentrations and at short incubation times but decreased ROS generation at higher concentrations and at longer incubation. It is concluded that mitochondria are an important target for toxic effects of Hg(2+), Cd(2+) and Cu(2+) in AS-30D rat hepatoma cells.

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

    PubMed Central

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

    2016-01-01

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

  3. 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.

  4. 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.

  5. Glucocorticoid Modulation of Mitochondrial Function in Hepatoma Cells Requires the Mitochondrial Fission Protein Drp1

    PubMed Central

    Hernández-Alvarez, María Isabel; Paz, José C.; Sebastián, David; Muñoz, Juan Pablo; Liesa, Marc; Segalés, Jessica; Palacín, Manuel

    2013-01-01

    Abstract Aims: Glucocorticoids, such as dexamethasone, enhance hepatic energy metabolism and gluconeogenesis partly through changes in mitochondrial function. Mitochondrial function is influenced by the balance between mitochondrial fusion and fission events. However, whether glucocorticoids modulate mitochondrial function through the regulation of mitochondrial dynamics is currently unknown. Results: Here, we report that the effects of dexamethasone on mitochondrial function and gluconeogenesis in hepatoma cells are dependent on the mitochondrial fission protein dynamin-related protein 1 (Drp1). Dexamethasone increased routine oxygen consumption, maximal respiratory capacity, superoxide anion, proton leak, and gluconeogenesis in hepatoma cells. Under these conditions, dexamethasone altered mitochondrial morphology, which was paralleled by a large increase in Drp1 expression, and reduced mitofusin 1 (Mfn1) and Mfn2. In vivo dexamethasone treatment also enhanced Drp1 expression in mouse liver. On the basis of these observations, we analyzed the dependence on the Drp1 function of dexamethasone effects on mitochondrial respiration and gluconeogenesis. We show that the increase in mitochondrial respiration and gluconeogenesis induced by dexamethasone are hampered by the inhibition of Drp1 function. Innovation: Our findings provide the first evidence that the effects of glucocorticoids on hepatic metabolism require the mitochondrial fission protein Drp1. Conclusion: In summary, we demonstrate that the mitochondrial effects of dexamethasone both on mitochondrial respiration and on the gluconeogenic pathway depend on Drp1. Antioxid. Redox Signal. 19, 366–378. PMID:22703557

  6. Targeting NFKB by autophagy to polarize hepatoma-associated macrophage differentiation.

    PubMed

    Chang, Chih-Peng; Su, Yu-Chi; Lee, Pei-Huan; Lei, Huan-Yao

    2013-04-01

    Tumor-associated macrophages (TAMs) have been linked to promoting tumor progression by stimulating angiogenesis, cell growth and inflammation. NFKB activity in TAMs may mediate inflammation-associated tumor formation. However, most isolated TAMs from established tumors express a M2 phenotype with less NFKB activation and show a strong immunosuppressive phenomenon. How tumors affect the dynamic of NFKB activity in TAMs, and hence maintain their pro-tumor M2 phenotype is still poorly understood. We recently found that hepatoma-derived toll-like receptor 2 (TLR2)-related ligands are capable of stimulating M2 macrophage differentiation via controlling NFKB RELA/p65 protein homeostasis by selective autophagy. TLR2 signal induces NFKB RELA cytosolic ubiquitination and leads to its degradation by SQSTM1/p62-mediated autophagy. Inhibition of autophagy will rescue NFKB activity and shape the phenotype of hepatoma-polarized M2 macrophages. This suggests that autophagy might play a role in manipulating TAM functions and tumor-associated immune responses. Our study also demonstrates that autophagy can directly control a transcriptional factor in addition to its regulatory molecules. This finding uncovers a new role of autophagy in controlling cellular functions.

  7. Targeting NFKB by autophagy to polarize hepatoma-associated macrophage differentiation

    PubMed Central

    Chang, Chih-Peng; Su, Yu-Chi; Lee, Pei-Huan; Lei, Huan-Yao

    2013-01-01

    Tumor-associated macrophages (TAMs) have been linked to promoting tumor progression by stimulating angiogenesis, cell growth and inflammation. NFKB activity in TAMs may mediate inflammation-associated tumor formation. However, most isolated TAMs from established tumors express a M2 phenotype with less NFKB activation and show a strong immunosuppressive phenomenon. How tumors affect the dynamic of NFKB activity in TAMs, and hence maintain their pro-tumor M2 phenotype is still poorly understood. We recently found that hepatoma-derived toll-like receptor 2 (TLR2)-related ligands are capable of stimulating M2 macrophage differentiation via controlling NFKB RELA/p65 protein homeostasis by selective autophagy. TLR2 signal induces NFKB RELA cytosolic ubiquitination and leads to its degradation by SQSTM1/p62-mediated autophagy. Inhibition of autophagy will rescue NFKB activity and shape the phenotype of hepatoma-polarized M2 macrophages. This suggests that autophagy might play a role in manipulating TAM functions and tumor-associated immune responses. Our study also demonstrates that autophagy can directly control a transcriptional factor in addition to its regulatory molecules. This finding uncovers a new role of autophagy in controlling cellular functions. PMID:23360732

  8. 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.

  9. Simultaneous expression of salivary and pancreatic amylase genes in cultured mouse hepatoma cells.

    PubMed Central

    Darlington, G J; Tsai, C C; Samuelson, L C; Gumucio, D L; Meisler, M H

    1986-01-01

    The tissue-specific expression of two types of mouse amylase genes does not overlap in vivo; the Amy-1 locus is transcribed in the parotid gland and the liver, while expression of Amy-2 is limited to the pancreas. We identified a mouse hepatoma cell line, Hepa 1-6, in which both amylase genes can be simultaneously expressed. Amy-1 is constitutively active in these cells and is inducible by dexamethasone at the level of mRNA. We demonstrated that the liver-specific promoter of Amy-1 is utilized by the dexamethasone-treated hepatoma cells, and that glucocorticoid consensus sequences are present upstream of this promoter. Amy-2 is not detectable constitutively, but can be activated if the cells are cultured in serum-free medium containing dexamethasone. Expression of Amy-2 in a nonpancreatic cell type has not previously been observed. We speculate that induction of Amy-1 and activation of Amy-2 may involve different regulatory mechanisms. Hepa 1-6 cells provide an experimental system for molecular analysis of these events. Images PMID:2431276

  10. Quercetin induces oxidative stress and potentiates the apoptotic action of 2-methoxyestradiol in human hepatoma cells.

    PubMed

    Chang, Yuh-Fang; Hsu, Yi-Chiung; Hung, Hui-Fang; Lee, Hui-Ju; Lui, Wing-Yiu; Chi, Chin-Wen; Wang, Jane-Jen

    2009-01-01

    Hepatocellular carcinoma (HCC) is the leading cause of cancer mortality in Asia. This study evaluated the growth inhibition effect of quercetin and 2-methoxyestradiol in vitro in human HCC cell lines. Combination treatment enhanced the cytotoxic effect in HA22T/VGH and HepG2 cell lines as compared with quercetin or 2-methoxyestradiol alone. The cell population of sub-G0/G1 phase and the level of annexin V binding were increased synergistically after combination treatment with quercetin and 2-methoxyestradiol in both cell lines. Moreover, quercetin combined with 2-methoxyestradiol increased superoxide levels, mitochondrial superoxide dismutase (MnSOD) in mRNA, protein levels, and SOD activity. Finally, we also found the mitochondrial membrane potential was decreased after combination treatment. The changes of reactive oxygen species and mitochondrial disruption were likely to be involved in the mechanism for the synergistic cytotoxicity effects of combination treatment in human hepatoma cells. These results provided a basis for further study of the potential usage of quercetin combination with hormonal agents for the treatment of human hepatoma.

  11. Melatonin, a novel selective ATF-6 inhibitor, induces human hepatoma cell apoptosis through COX-2 downregulation.

    PubMed

    Bu, Li-Jia; Yu, Han-Qing; Fan, Lu-Lu; Li, Xiao-Qiu; Wang, Fang; Liu, Jia-Tao; Zhong, Fei; Zhang, Cong-Jun; Wei, Wei; Wang, Hua; Sun, Guo-Ping

    2017-02-14

    To clarify the mechanisms involved in the critical endoplasmic reticulum (ER) stress initiating unfolded protein response pathway modified by melatonin. Hepatoma cells, HepG2, were cultured in vitro. Flow cytometry and TUNEL assay were used to measure HepG2 cell apoptosis. Western blotting and quantitative reverse transcription-polymerase chain reaction methods were used to determine the protein and messenger RNA levels of ER stress and apoptosis related genes' expression, respectively. Tissue microarray construction from patients was verified by immunohistochemical analysis. In the present study, we first identified that melatonin selectively blocked activating transcription factor 6 (ATF-6) and then inhibited cyclooxygenase-2 (COX-2) expression, leading to enhanced liver cancer cell apoptosis under ER stress condition. Dramatically increased CCAAT-enhancer-binding protein homologous protein level, suppressed COX-2 and decreased Bcl-2/Bax ratio by melatonin or ATF-6 siRNA contributed the enhanced HepG2 cell apoptosis under tunicamycin (an ER stress inducer) stimulation. In clinical hepatocellular carcinoma patients, the close relationship between ATF-6 and COX-2 was further confirmed. These findings indicate that melatonin as a novel selective ATF-6 inhibitor can sensitize human hepatoma cells to ER stress inducing apoptosis.

  12. Melatonin, a novel selective ATF-6 inhibitor, induces human hepatoma cell apoptosis through COX-2 downregulation

    PubMed Central

    Bu, Li-Jia; Yu, Han-Qing; Fan, Lu-Lu; Li, Xiao-Qiu; Wang, Fang; Liu, Jia-Tao; Zhong, Fei; Zhang, Cong-Jun; Wei, Wei; Wang, Hua; Sun, Guo-Ping

    2017-01-01

    AIM To clarify the mechanisms involved in the critical endoplasmic reticulum (ER) stress initiating unfolded protein response pathway modified by melatonin. METHODS Hepatoma cells, HepG2, were cultured in vitro. Flow cytometry and TUNEL assay were used to measure HepG2 cell apoptosis. Western blotting and quantitative reverse transcription-polymerase chain reaction methods were used to determine the protein and messenger RNA levels of ER stress and apoptosis related genes’ expression, respectively. Tissue microarray construction from patients was verified by immunohistochemical analysis. RESULTS In the present study, we first identified that melatonin selectively blocked activating transcription factor 6 (ATF-6) and then inhibited cyclooxygenase-2 (COX-2) expression, leading to enhanced liver cancer cell apoptosis under ER stress condition. Dramatically increased CCAAT-enhancer-binding protein homologous protein level, suppressed COX-2 and decreased Bcl-2/Bax ratio by melatonin or ATF-6 siRNA contributed the enhanced HepG2 cell apoptosis under tunicamycin (an ER stress inducer) stimulation. In clinical hepatocellular carcinoma patients, the close relationship between ATF-6 and COX-2 was further confirmed. CONCLUSION These findings indicate that melatonin as a novel selective ATF-6 inhibitor can sensitize human hepatoma cells to ER stress inducing apoptosis. PMID:28246472

  13. [Partial purification and certain properties of gamma-glutamyltransferase from rat liver and hepatoma].

    PubMed

    Loginov, V A; Chernov, N N; Berezov, T T

    1980-07-01

    gamma-Glutamyltransferase (GGT) from the liver and transplantable hepatoma of rats was purified 130- and 170-fold, respectively. The properties of the enzymatic preparations obtained were studied. The optimum pH for the transferase reaction between L-gamma-glutamyl-p-nitroanilide (5 mM) and glycyl-glycine (50 mM) was 8.0--8.2, while that for the auto-transferase reaction (without glycyl-glycine) amounted to 9.3--9.5. The isoelectric points of GGT from the liver correlated with the pH 3.9, 4.2 and 4.4 whereas those of hepatoma enzyme with the pH 5.7, 6.0 and 7.0 GGT obtained from both sources were equally specific for various acceptors at the pH 8.1 and 7.0. It has been shown that metotrexate (0.9 mM) and folic acid (3.5 mM) inhibited both enzymes by 50%.

  14. 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.

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

    PubMed

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

    2015-06-19

    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.

  16. 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.

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

    PubMed

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

    2016-11-15

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

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

    PubMed

    Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

    2016-02-28

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

  19. Role of ROS-mediated autophagy in radiation-induced bystander effect of hepatoma cells.

    PubMed

    Wang, Xiangdong; Zhang, Jianghong; Fu, Jiamei; Wang, Juan; Ye, Shuang; Liu, Weili; Shao, Chunlin

    2015-05-01

    Autophagy plays a crucial role in cellular response to ionizing radiation, but it is unclear whether autophagy can modulate radiation-induced bystander effect (RIBE). Here, we investigated the relationship between bystander damage and autophagy in human hepatoma cells of HepG2. HepG2 cells were treated with conditioned medium (CM) collected from 3 Gy γ-rays irradiated hepatoma HepG2 cells for 4, 12, or 24 h, followed by the measurement of micronuclei (MN), intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and protein expressions of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 in the bystander HepG2 cells. In some experiments, the bystander HepG2 cells were respectively transfected with LC3 small interfering RNA (siRNA), Beclin-1 siRNA or treated with 1% dimethyl sulfoxide (DMSO). Additional MN and mitochondrial dysfunction coupled with ROS were induced in the bystander cells. The expressions of protein markers of autophagy, LC3-II/LC3-I and Beclin-1, increased in the bystander cells. The inductions of bystander MN and overexpressions of LC3 and Beclin-1 were significantly diminished by DMSO. However, when the bystander cells were transfected with LC3 siRNA or Beclin-1 siRNA, the yield of bystander MN was significantly enhanced. The elevated ROS have bi-functions in balancing the bystander effects. One is to cause MN and the other is to induce protective autophagy.

  20. Harmine suppresses homologous recombination repair and inhibits proliferation of hepatoma cells

    PubMed Central

    Zhang, Lei; Zhang, Fan; Zhang, Wenjun; Chen, Lu; Gao, Neng; Men, Yulong; Xu, Xiaojun; Jiang, Ying

    2015-01-01

    To avoid cell cycle arrest or apoptosis, rapidly proliferating cancer cells have to promote DNA double strand break (DSB) repair to fix replication stress induced DSBs. Therefore, developing drugs blocking homologous recombination (HR) and nonhomologous end joining (NHEJ) – 2 major DSB repair pathways – holds great potential for cancer therapy. Over the last few decades, much attention has been paid to explore drugs targeting DSB repair pathways for cancer therapy. Here, using 2 well-established reporters for analyzing HR and NHEJ efficiency, we found that both HR and NHEJ are elevated in hepatoma cell lines Hep3B and HuH7 compared with normal liver cell lines Chang liver and QSG-7701. Our further study found that Harmine, a natural compound, negatively regulates HR but not NHEJ by interfering Rad51 recruitment, resulting in severe cytotoxicity in hepatoma cells. Furthermore, NHEJ inhibitor Nu7441 markedly sensitizes Hep3B cells to the anti-proliferative effects of Harmine. Taken together, our study suggested that Harmine holds great promise as an oncologic drug and combination of Harmine with a NHEJ inhibitor might be an effective strategy for anti-cancer treatment. PMID:26382920

  1. [Aitongxiao recipe regulated survivin and Bcl- 2 in rats' transplanted hepatoma carcinoma cell].

    PubMed

    Wang, Shu-jie; Wei, Ai-ling; Zhang, Yong-qin

    2012-12-01

    To study the main mechanisms of Aitongxiao Recipe (ATXR) for anti-tumor at the molecular level, and to clarify different efficient drugs' roles in anti-tumor, thus in-depth explaining the objectivity and substance of "cancer toxic" theory. Walker-256 tumor strain was used for Wistar rat transplanted liver cancer modeling. After successful modeling rats were randomly divided into 5 groups, i. e., the ATXP group, the qi regulating and blood circulating group (as the assembled I group), the heat clearing and detoxification group (as the assembled II group), the body resistance strengthening and cultivating group (as the assembled III group), and the model group, 10 in each group. Corresponding medication was given to rats in each group for 14 successive days. Finally rats were sacrificed and the tumor mass was taken out. The apoptosis rate and the cell cycle of tumor cells were detected by flow cytometry Annexin V/PI. The protein and mRNA expressions of Bcl-2 and survivin were detected using immunohistochemistry and real-time fluorescent quantitative PCR. (1) The apoptosis of hepatoma carcinoma cells could be obviously promoted in the ATXP group. The cell cycle could also be affected, making major cells arrest at G0/G1 phase. The proliferation of hepatoma carcinoma cells was effectively prevented. The efficacy in the assembled II group was in line with that in the ATXP group with no statistical difference (P>0.05). It was also effective in the assembled III group, but its efficacy was not as good as that in the former two groups, showing statistical difference (P<0.01). (2) ATXP could obviously down-regulate the protein and mRNA expressions of Bcl-2 and survivin in hepatoma carcinoma cells. Drugs for heat clearing and detoxification showed significant effects on down-regulating the protein and mRNA expressions of Bcl-2 and survivin in hepatoma carcinoma cells. Their effects were similar to that of ATXP (P>0.05). The effects of drugs for body resistance strengthening

  2. 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

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

    PubMed

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

    2012-11-01

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

  4. 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.

  5. A position-dependent silencer plays a major role in repressing. alpha. -fetoprotein expression in human hepatoma

    SciTech Connect

    Nakabayashi, Hidekazu; Hashimoto, Tomoko; Miyao, Yasuyoshi; Tjong, Kuokoewang; Chan, J.; Tamaoki, Taiki )

    1991-12-01

    A large percentage of human hepatomas produce {alpha}-fetoprotein (AFP), but the levels of AFP expression vary greatly among hepatomas. To understand the molecular basis for this variation. The authors analyzed transcriptional regulatory activities associated with the 5{prime}-flanking region of the AFP gene in two human hepatoma cell lines, HuH-7 and huH-1/cl-2, which produce a high and a low level of AFP, respectively. They found that the low level of AFP production in huH-1/cl-2 is due to the action of at least two silencer regions located between the enhancer and the promoter of the AFP gene. In contrast, no silencer activity is expressed in HuH-7. They identified 5{prime}-CTTCATAACTAATACTT-3{prime} to be a core sequence responsible for the negative regulatory activity. This sequence is repeated four times in a strong, distal silencer region, Sd, whereas one copy is present in a weak, proximal silencer region, Sp. The silencer reduces transcriptional initiation by blocking enhancer activation of the AFP promoter in a position-dependent manner. The silencer functions in the presence of positive transcription factors and may play a key role in developmental repression as well as variable expression of the AFP gene in hepatomas.

  6. Inhibition of IGF receptor signaling and hepatoma cell growth by an antibody to ligand oligopeptide of receptor.

    PubMed

    Kong, Jing; Diao, Zhenyu; Deng, Xiaozhao

    2008-02-01

    Research on insulin-like growth factor (IGF) system have shown it to be potent mitogen for hepatoma cells and made it an attractive therapeutic target. But little strategy has been reported to date on targeting and sequestrating IGF against hepatoma. This study is based on the capability of ligand oligopeptide (LOP) to recognize IGF receptor with high efficiency, which is over-expressed on some hepatoma cells. We have been hypothesizing that antibody to LOP would mimic the extracellular ligand-binding domain of IGF receptor and inhibit receptor signaling and cell proliferation. Gene encoding for LOP [E5 (EPFRSPDLALETYG)] of IGF receptor was inserted into HBc carrier for expression in Escherichia coli. The monoclonal antibody (mAb) specific LOP potently inhibited signal transduction mediated by the IGF-IR interaction with IGF-I. Furthermore, it exhibited 47% inhibitory rate of soft agar colony formation and also induced apoptosis. These results indicate an anti-hepatoma potential of the mAb to an LOP of IGF receptor could block the activation of receptor and downstream signaling pathways, and suppress the biological effects mediated by receptor.

  7. Generation of fluorescently labeled cell lines, C3A hepatoma cells, and human adult skin fibroblasts to study coculture models.

    PubMed

    Samluk, Anna; Zakrzewska, Karolina Ewa; Pluta, Krzysztof Dariusz

    2013-07-01

    Hepatic/nonhepatic cell cocultures are widely used in studies on the role of homo- and heterotypic interactions in liver physiology and pathophysiology. In this article, for the first time, establishment of the coculture model employing hepatoma C3A cells and human skin fibroblasts, stably expressing fluorescent markers, is described. Suitability of the model in studying coculture conditions using fluorescence microscopy and flow cytometry was examined. C3A cells spontaneously formed island-like growth patterns surrounded by fibroblasts. The "islands" size and resulting intensity of the homo- and heterotypic interactions can easily be tuned by applying various plated cells ratios. We examined the capability of the hepatoma cells to produce albumin in hepatic/nonhepatic cell cocultures. The enzyme-linked immunosorbent assay (ELISA) tests showed that greater number of fibroblasts in coculture, resulting in smaller sizes of hepatoma "islands," and thus, a larger heterotypic interface, promoted higher albumin synthesis. The use of fluorescently labeled cells in flow cytometry measurements enabled us to separately gate two cell populations and to evaluate protein expression only in/on cells of interest. Flow cytometry confirmed ELISA results indicating the highest albumin production in hepatoma cells cocultured with the greatest number of fibroblasts and the inhibited protein synthesis in coculture with osteosarcoma cells.

  8. Combined effect of honokiol and rosiglitazone on cell growth inhibition through enhanced G0/G1 phase arrest in hepatoma cells.

    PubMed

    Chen, Hsiao-Chi; Hsu, Hui-Tzu; Weng, Jing-Wen; Chang, Yuh-Fang; Hsia, Cheng-Yuan; Lee, Hsin-Chen; Chi, Chin-Wen

    2016-08-01

    Honokiol, a derivative extracted from the stem and bark of Magnolia officinalis, has been reported to have anticancer effects in hepatoma cells. Recently, it was found that honokiol acted as not only a retinoid X receptor (RXR) agonist but also as a peroxisome proliferator-activated receptor gamma (PPARγ) agonist. Additionally, honokiol is capable of activating PPARγ/RXR heterodimers synergistically in the presence of rosiglitazone in 3T3-L1 adipocyte and HLE human hepatoma cells. Furthermore, synthetic PPARγ agonist thiazolidinediones exhibited growth inhibition effects in hepatoma cells through PPARγ-dependent and PPARγ-independent pathways. However, the combined effects of treatment with honokiol and PPARγ agonist are unclear in hepatoma cells. In this study, sulforhodamine B assay, flow cytometry, and Western blot analysis were used to examine the combined effects of honokiol and PPARγ agonist (rosiglitazone) treatment on growth inhibition in SK-Hep1 and Mahlavu hepatoma cells. Honokiol or rosiglitazone treatment in hepatoma cells induced growth inhibition at high dose by sulforhodamine B assay. Moreover, we found that combined treatment with honokiol and rosiglitazone showed more effective growth inhibition in hepatoma cells than treatment with honokiol or rosiglitazone alone. Also, treatment with honokiol and rosiglitazone induced cell cycle arrest in the G0/G1 phase; increased p21; and decreased cyclin D1, cyclin E1, and Rb expression in SK-Hep1 hepatoma cells. Honokiol combined with rosiglitazone showed more effective growth inhibition in hepatoma cells mediated through the regulation of G0/G1 phase-related proteins p21, cyclin D1, cyclin E1, and Rb and cell cycle progression. Copyright © 2016. Published by Elsevier Taiwan LLC.

  9. Effects of cumene hydroperoxide on the Ca(2+)-induced Ca2+ efflux from mitochondria and on the viability of hepatoma cells.

    PubMed

    Teplova, V V; Kudin, A P; Evtodienko YuV

    1998-01-01

    Effects of cumene hydroperoxide on the Ca(2+)-induced Ca2+ efflux from mitochondria isolated from rat liver and Zaidelja hepatoma were compared. Cumene hydroperoxide at micromolar concentrations (0.3-10 microM) prevented the closing of the permeability transition pore in the inner mitochondrial membrane and, therefore, potentiated the Ca(2+)-induced Ca2+ efflux. This response was 10-100 times greater in hepatoma mitochondria than in rat liver mitochondria. Micromolar concentrations of cumene hydroperoxide induced the death of the hepatoma cells in vitro.

  10. The effects of propofol on the growth behavior of hepatoma xenografts in Balb/c mice.

    PubMed

    Liu, Yi; Zhang, Na; Cao, Quanjun; Cui, Xuejie; Zhou, Qiaoling; Yang, Chengxiang

    2017-06-01

    Studies on the effects of propofol on the growth of hepatoma xenografts in Balb/c mice. In an effort to establish a hepatoma-xenograft model of BALB/C mice, human hepatocellular carcinoma cells SMMC-7721 were inoculated subcutaneously into BALB/C mice. Forty mice were randomly divided into five different groups (n=8): control group (C group), Intralipid group (Y group), low dose (50mg/kg) propofol group (P1 group), medium dose (100mg/kg) propofol group (P2 group) and high dose (150mg/kg) propofol group (P3 group). The tumor volume was measured before treatment and every 3days after treatment (T0d-T18d, T0 represents time point before treatment, T3d-T18d represent time points every 3days after treatment for a total of 18 days). All mice were sacrificed 19days after drug withdrawal. The tumor masses were extracted, weighed, and the tumor inhibition rate of propofol was calculated. The protein levels of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) in the xenografted tumors were analyzed by immunohistochemistry staining. No statistical significance in the tumor volume at T0d (before treatment), T3d (3days after treatment), and T6d (6days after treatment) among the five groups (P>0.05) could be determined. Compared to group C, the tumor volumes in the P1, P2, and P3 groups were found to be significantly decreased in size upon increasing the propofol dosages (P<0.05). There was no statistical significance at time points T9d-T18d in group Y compared to group C (P>0.05). The tumor weights in the P1, P2, and P3 groups were found to be significantly lower as the propofol dosages increased (P<0.05), with no statistical significance determined in group Y (P>0.05). MMP-2 and VEGF protein levels were found to be significantly lower in the P1, P2, and P3 groups as the propofol dosages increased (P<0.05), with no statistical significance in group Y (P>0.05). Within a certain range, propofol was found to inhibit tumor growth and expression of MMP

  11. Stimulation of Hepatoma Cell Invasiveness and Metastatic Potential by Proteins Secreted From Irradiated Nonparenchymal Cells

    SciTech Connect

    Zhou Leyuan; Wang Zhiming; Gao Yabo; Wang Lingyan; Zeng Zhaochong

    2012-11-01

    Purpose: To determine whether factors secreted by irradiated liver nonparenchymal cells (NPCs) may influence invasiveness and/or metastatic potential of hepatocellular carcinoma (HCC) cells and to elucidate a possible mechanism for such effect. Methods and Materials: Primary rat NPCs were cultured and divided into irradiated (10-Gy X-ray) and nonirradiated groups. Forty-eight hours after irradiation, conditioned medium from irradiated (SR) or nonirradiated (SnonR) cultures were collected and added to sublethally irradiated cultures of the hepatoma McA-RH7777 cell line. Then, hepatoma cells were continuously passaged for eight generations (RH10Gy-SR and RH10Gy-SnonR). The invasiveness and metastatic potential of McA-RH7777, RH10Gy-SnonR, and RH10Gy-SR cells were evaluated using an in vitro gelatinous protein (Matrigel) invasion and an in vivo metastasis assay. In addition, SR and SnonR were tested using rat cytokine antibody arrays and enzyme-linked immunosorbent assay (ELISA). Results: In vitro gelatinous protein invasion assay indicated that the numbers of invading cells was significantly higher in RH10Gy-SR (40 {+-} 4.74) than in RH10Gy-SnonR (30.6 {+-} 3.85) cells, and lowest in McA-RH7777 (11.4 {+-} 3.56) cells. The same pattern was observed in vivo in a lung metastasis assay, as evaluated by number of metastatic lung nodules seen with RH10Gy-SR (28.83 {+-} 5.38), RH10Gy-SnonR (22.17 {+-} 4.26), and McA-RH7777 (8.3 {+-} 3.8) cells. Rat cytokine antibody arrays and ELISA demonstrated that metastasis-promoting cytokines (tumor necrosis factor-{alpha} and interleukin-6), circulating growth factors (vascular endothelial growth factor and epidermal growth factor), and metalloproteinases (MMP-2 and MMP-9) were upregulated in SR compared with SnonR. Conclusions: Radiation can increase invasiveness and metastatic potential of sublethally irradiated hepatoma cells, and soluble mediators released from irradiated NPCs promote this potential. Increased secretion of

  12. Establishment of a human hepatoma multidrug resistant cell line in vitro

    PubMed Central

    Zhou, Yuan; Ling, Xian-Long; Li, Shi-Wei; Li, Xin-Qiang; Yan, Bin

    2010-01-01

    AIM: To establish a multidrug-resistant hepatoma cell line (SK-Hep-1), and to investigate its biological characteristics. METHODS: A highly invasive SK-Hep-1 cell line of human hepatocellular carcinoma, also known as malignant hepatoma was incubated with a high concentration of cisplatin (CDDP) to establish a CDDP-resistant cell subline (SK-Hep-1/CDDP). The 50% inhibitory dose (IC50) values and the resistance indexes [(IC50 SK-Hep-1/CDDP)/(IC50 SK-Hep-1)] for other chemotherapeutic agents and the growth curve of cells were all evaluated using cell counting kit-8 assays. The distribution of the cell cycles were detected by flow cytometry. Expression of acquired multidrug resistance P-glycoprotein (MDR1, ABCB1) and multidrug resistance-associated protein 1 (MRP1, ABCC1) was compared with that in parent cells by Western blotting and immunofluorescence combined with laser scanning confocal microscopy. RESULTS: The SK-Hep-1/CDDP cells (IC50 = 70.61 ± 1.06 μg/mL) was 13.76 times more resistant to CDDP than the SK-Hep-1 cells (IC50 = 5.13 ± 0.09 μg/mL), and CDDP-resistant cells also demonstrated cross-resistance to many anti-tumor agents such as doxorubicin, 5-fluorouracil and vincristine. Similar morphologies were determined in both SK-Hep-1 and SK-Hep-1/CDDP groups. The cell cycle distribution of the SK-Hep-1/CDDP cell line exhibited a significantly increased percentage of cells in S (42.2% ± 2.65% vs 27.91% ± 2.16%, P < 0.01) and G2/M (20.67% ± 5.69% vs 12.14% ± 3.36%, P < 0.01) phases in comparison with SK-Hep-1 cells, while the percentage of cells in the G0/G1 phase decreased (37.5% ± 5.05% vs 59.83% ± 3.28%, P < 0.01). The levels of MDR1 and MRP1 were overexpressed in the SK-Hep-1/CDDP cells exhibiting the MDR phenotype. CONCLUSION: Multiple drug resistance of multiple drugs in the human hepatoma cell line SK-Hep-1/CDDP was closely related to the overexpression of MDR1 and MRP1. PMID:20458768

  13. The citrus fruit flavonoid naringenin suppresses hepatic glucose production from Fao hepatoma cells.

    PubMed

    Purushotham, Aparna; Tian, Min; Belury, Martha A

    2009-02-01

    Hepatic gluconeogenesis is the major source of fasting hyperglycemia. Here, we investigated the role of the citrus fruit flavonoid naringenin, in the attenuation of hepatic glucose production from hepatoma (Fao) cells. We show that naringenin, but not its glucoside naringin, suppresses hepatic glucose production. Furthermore, unlike insulin-mediated suppression of hepatic glucose production, incubation of hepatocytes with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor Ly294002 had no effect on the ability of naringenin to suppress hepatic glucose production. Further, naringenin did not increase phosphorylation of Akt at Ser473 or, Thr308, indicating this down-stream target of PI3-kinase is also not a player in naringenin-mediated suppression of hepatic glucose production. Importantly, like the dimethylbiguanide, metformin, naringenin significantly decreased cellular ATP levels without increasing cell cytotoxicity. Together, these results suggest that the aglycone, naringenin, has a role in the attenuation of hyperglycemia and may exert this effect in a manner similar to the drug, metformin.

  14. Isolation of Peroxisomes from Rat Liver and Cultured Hepatoma Cells by Density Gradient Centrifugation.

    PubMed

    Manner, Andreas; Islinger, Markus

    2017-01-01

    Subcellular fractionation is still a valuable technique to unravel organelle-specific proteomes, validate the location of uncharacterized proteins, or to functionally analyze import and metabolism in individual subcellular compartments. In this respect, density gradient centrifugation still represents a very classic, indispensable technique to isolate and analyze peroxisomes. Here, we present two independent protocols for the purification of peroxisomes from either liver tissue or the HepG2 hepatoma cell line. While the former permits the isolation of highly pure peroxisomes suitable for, e.g., subcellular proteomics experiments, the latter protocol yields peroxisomal fractions from considerably less purity but allows to easily modify metabolic conditions in the culture medium or to genetically manipulate the peroxisomal compartment. In this respect, both purification methods represent alternative tools to be applied in experiments investigating peroxisome physiology.

  15. A novel multi-target RNAi adenovirus inhibits hepatoma cell proliferation, migration, and induction of angiogenesis

    PubMed Central

    Pan, Tingting; Cheng, Ya; Ren, Weihua; Jia, Weidong; Ma, Jinliang; Xu, Geliang

    2016-01-01

    The pathogenesis of hepatocellular carcinoma (HCC) is a multi-step process involving many genes. Consequently, single gene targeting therapy has limited efficacy, making combination therapy targeting multiple genes a necessity. Based on our previous findings, we constructed a single vector mediating simultaneous expression of multiple short hairpin RNAs (shRNAs) against human vascular endothelial growth factor receptor 2 (VEGFR2), chemokine C-C motif receptor 1 (CCR1), and epithelial cell adhesion molecule (EpCAM), three genes closely related to HCC progression that act through separate pathways. The shRNA vector efficiently downregulated the mRNA and protein of all three molecules in Huh7 hepatoma cells. The vector also inhibited cell proliferation and migration and reduced angiogenesis. Furthermore, this shRNA vector can be recombined into adenovirus, a gene therapy vector, for better in vivo application. It thus offers a potentially effective future gene therapy approach to treating human liver cancer. PMID:27221035

  16. S-adenosylmethionine: DNA-cytosine 5-methyltransferase from a Novikoff rat hepatoma cell line.

    PubMed Central

    Sneider, T W; Teague, W M; Rogachevsky, L M

    1975-01-01

    Partial purification of DNA methylase from Novikoff rat hepatoma cells is described. Contamination with other proteins persists although the enzyme preparation has a high specific activity and is purified 980-fold over homogenate activity. Evidence suggests, but does not prove, that there may be more than one species of DNA methylase in these cells. The enzyme has two broad pH optima at pH 7.0 and 7.5 and most readily methylates heterologous denatured DNAs although complex reaction kinetics indicate that native DNAs may eventually be methylated to an equal or greater level. The preparation of undermethylated DNA from Novikoff cells is also described. Undermethylated homologous DNA is an 85-fold greater acceptor of methyl groups than fully methylated Novikoff cell DNA. In contrast to other DNA substrates, the enzyme preparation methylates native undermethylated homologous DNA at a 3.5-fold greater than denatured undermethylated homologous DNA. Images PMID:171625

  17. Isoliquiritigenin inhibits cell proliferation and induces apoptosis in human hepatoma cells.

    PubMed

    Hsu, Ya-Ling; Kuo, Po-Lin; Lin, Liang-Tzung; Lin, Chun-Ching

    2005-02-01

    Isoliquiritigenin (4,2',4'-trihydroxychalcone, ISL) is a natural pigment with a simple chalcone structure. In this study, we report the ISL-induced inhibition on the growth of human hepatoma cells (Hep G2) for the first time. The cell growth inhibition achieved by ISL treatment resulted in programmed cell death in a caspase activation-dependent manner, with an IC50 of 10.51 microg/mL. Outcomes of ISL treatment included the up-regulation of IkappaBalpha expression in the cytoplasm, and the decrease of NF-kappaB level as well as its activity in the nucleus. In addition, ISL also suppressed the expression of Bcl-XL and c-IAP1/2 protein, the downstream target molecule of NF-kappaB. These results demonstrated that ISL treatment inhibited the NF-kappaB cell survival-signaling pathway and induced apoptotic cell death in Hep G2 cells.

  18. 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.

  19. Time-course regulation of quercetin on cell survival/proliferation pathways in human hepatoma cells.

    PubMed

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

    2008-04-01

    Quercetin, a dietary flavonoid, has been shown to possess anticarcinogenic properties, but the precise molecular mechanisms of action are not thoroughly elucidated. This study was aimed at investigating the time-course regulation effect of quercetin on survival/proliferation pathways in a human hepatoma cell line (HepG2). Quercetin induced a significant time-dependent inactivation of the major survival signaling proteins, i. e., phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (AKT), extracellular regulated kinase (ERK), protein kinase C-alpha (PKC-alpha), in concert with a time-dependent activation of key death-related signals: c-jun amino-terminal kinase (JNK) and PKC-delta. These data suggest that quercetin exerts a tight regulation of survival/proliferation pathways that requires the integration of different signals and persists over time, being the balance of these regulatory signals what determines the fate of HepG2 cells.

  20. [Retention jaundice caused by central hepatic hepatoma. Treatment with Kron's extra-anatomic biliary prosthesis].

    PubMed

    Partensky, C; Paliard, P; Maurin, T; Bret, P M

    1983-01-01

    A 31 year-old patient presented with a retention jaundice from a centrally located hepatoma invading the hilum. Because of the patient's age and the severity of the pruritus and jaundice, palliative treatment was performed by percutaneous catheterization of the intrahepatic biliary tracts to drain the right and left hepatic canals. As the hilar stenosis could not be overcome, the external drainage was transformed into internal drainage by implantation of a Kron's biliary prosthesis linking the intrahepatic biliary tracts, following segment III hepatotomy, to the duodenal lumen, with burying of the prosthesis in the gastric antrum region. Jaundice was reduced until death of the patients from metastases 6 months later. This case demonstrates that the use of Kron's biliary prosthesis to perform a biliodigestive shunt from intrahepatic biliary pathways is a valid palliative procedure in patients with limited life expectancies.

  1. [Effects of niflumic acid on the proliferation of human hepatoma cells].

    PubMed

    Tian, Jing; Tao, Ling; Cao, Yun-Xin; Dong, Ling; Hu, Yu-Zhen; Yang, An-Gang; Zhou, Shi-Sheng

    2003-04-25

    The purpose of this work was to investigate the effects of niflumic acid (NFA), a chloride channel blocker, on the proliferation of human hepatoma cell line (HHCC). Cell proliferation was analyzed by cell count and MTT assay. Cell cycle analysis was carried out by flow cytometry. [Ca(2+)](i) was determined by laser scanning confocal system. It was found that NFA decreased significantly the cell number and the MTT optical density (OD) of HHCC cells, and that the OD value was reversed after washout of NFA. Compared with control, NFA blocked cell cycle progression in G(1) phase. Extracellular application of NFA (100 micromol/L) induced a rapid decrease in [Ca(2+)](i). These findings demonstrate that blockage of chloride channels by NFA induces growth arrest of HHCC in G(1) phase, which may be due to the inhibition of Ca(2+)/CaM-dependent signaling pathways.

  2. Coexistence of hepatoma with mantle cell lymphoma in a hepatitis B carrier

    PubMed Central

    Lee, Mu-Hsien; Lin, Yu-Ching; Cheng, Hao-Tsai; Chuang, Wen-Yu; Huang, Hsin-Chih; Kao, Hsiao-Wen

    2015-01-01

    The coexistence of hepatocellular carcinoma (HCC) and non-Hodgkin’s lymphoma (NHL) in the liver is rare. Reports show that these patients have cirrhotic livers or hepatitis virus infections before they develop HCC and NHL. We present a patient with hepatitis B virus infection who was transferred to our hospital with a newly detected liver mass; abdominal computed tomography examination showed one hypodense mass of 7 cm in diameter and multiple mesenteric and mediastinal lymph nodes. A liver tumor biopsy showed a hepatoma, and the pathologic findings from an inguinal lymph node excision showed mantle cell lymphoma. An immunohistochemical stain confirmed that the atypical lymphoid cells within the HCC were positive for the CD20, CD5 and cyclin D1 antigens. Taking these findings into account, the hepatic tumor was determined to be a HCC infiltrated by mantle cell lymphoma. PMID:26668520

  3. Coexistence of hepatoma with mantle cell lymphoma in a hepatitis B carrier.

    PubMed

    Lee, Mu-Hsien; Lin, Yu-Ching; Cheng, Hao-Tsai; Chuang, Wen-Yu; Huang, Hsin-Chih; Kao, Hsiao-Wen

    2015-12-07

    The coexistence of hepatocellular carcinoma (HCC) and non-Hodgkin's lymphoma (NHL) in the liver is rare. Reports show that these patients have cirrhotic livers or hepatitis virus infections before they develop HCC and NHL. We present a patient with hepatitis B virus infection who was transferred to our hospital with a newly detected liver mass; abdominal computed tomography examination showed one hypodense mass of 7 cm in diameter and multiple mesenteric and mediastinal lymph nodes. A liver tumor biopsy showed a hepatoma, and the pathologic findings from an inguinal lymph node excision showed mantle cell lymphoma. An immunohistochemical stain confirmed that the atypical lymphoid cells within the HCC were positive for the CD20, CD5 and cyclin D1 antigens. Taking these findings into account, the hepatic tumor was determined to be a HCC infiltrated by mantle cell lymphoma.

  4. Two unusual cases with Wilson's disease: hepatoma and fulminant hepatitis treated with plasma exchange.

    PubMed Central

    Aydinli, Musa; Harmanci, Ozgur; Ersoy, Osman; Iskit, Arzu T.; Ozcebe, Osman; Abbasoglu, Osman; Bayraktar, Yusuf

    2006-01-01

    We report two atypical cases of Wilson's disease. The first case is a 22-year-old male patient with a history of disease for 15 years and diagnosed as Wilson's disease upon investigations. Alpha-fetoprotein level was found elevated and computed tomography showed a 3.5-cm liver mass. Hepatocellular carcinoma was diagnosed. Radiofrequency ablation and liver transplantation were performed successfully. The second case is a 24-year-old female patient who presented with fulminant hepatitis. Urinary copper excretion and ceruloplasmin levels were suggestive of Wilson's disease. Despite chelation therapy, no improvement was observed. Plasma exchange therapy was performed for seven days. Her clinical status improved, and transplantation was no longer needed. To conclude, although hepatoma is rarely seen in Wilson's disease, patients should be examined regularly to diagnose it in a treatable stage. Removal of copper and toxic metabolites with plasma exchange therapy may be a way of treatment for fulminant hepatitis associated with Wilson's disease. PMID:17225847

  5. Influence of lipid environment on insulin binding in cultured hepatoma cells.

    PubMed

    Bruneau, C; Staedel-Flaig, C; Crémel, G; Leray, C; Beck, J P; Hubert, P

    1987-05-18

    The influence of alterations of plasma membrane physico-chemical properties on insulin binding have been characterized in an insulin-sensitive rat hepatoma cell line adapted to grow for several generations in culture medium enriched with linoleic acid (18:2) or with 25-hydroxycholesterol. The cells took up 18:2 and 25-hydroxycholesterol added to the culture medium, without exhibiting any sign of intolerance or intoxication. These compounds respectively increased and decreased membrane fluidity at 37 degrees C. The cells demonstrated extensive changes in insulin binding parameters in response to experimental modifications of their membrane lipid composition. When determined at 4 degrees C, insulin receptors were present in the control cells at 136,000 sites/cell but this fell to 111,000 (P less than 0.05) in cells enriched in 18:2, and rose to 176,000 (P less than 0.001) in hydroxysterol-grown cells. According to a two-site model, the main effect of 18:2 was a significant increase of the number of high-affinity sites with a concomitant decrease of low-affinity sites. The hydroxysterol had the opposite effects on these parameters. The high-affinity insulin binding capacity of the hepatoma cells was affected by lipid supplementation in a similar way, whether it was determined at 4 degrees C or at 37 degrees C. Assuming a negative cooperativity model, 18:2 enhanced the degree of negative cooperativity among the sites, while 25-hydroxycholesterol reduced it. The time-course of insulin-induced receptor down-regulation was accelerated in the cells enriched in polyunsaturated fatty acids, but reduced in cells exposed to 25-hydroxycholesterol. These insulin-binding alterations cannot be directly related to modifications of cellular growth rate, receptor internalization or membrane fluidity per se, and are discussed as being more likely due to membrane lipid composition than to overall cell metabolism modifications.

  6. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

    PubMed Central

    2011-01-01

    Background Cancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44). Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans) -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs. PMID:21669008

  7. Chaperone proteins involved in troglitazone-induced toxicity in human hepatoma cell lines.

    PubMed

    Maniratanachote, Rawiwan; Minami, Keiichi; Katoh, Miki; Nakajima, Miki; Yokoi, Tsuyoshi

    2005-02-01

    Troglitazone (TRO), an effective thiazolidinedione antidiabetic agent, was reported to produce idiosyncratic hepatotoxic effects in some individuals. In contrast, rosiglitazone (RSG), in the same group of agents, has no significant toxic effects and now is widely used. In this study, human hepatoma (HepG2) cell lines were exposed to various doses of TRO as well as RSG (0, 25, 50, and 75 microM) for 48 h. Cell lysates were separated by two-dimensional electrophoresis, and the gels were stained with coomassie brilliant blue to compare the spot profiles. The greatest protein expression at a MW of 75 kDa and isoelectric point of 5 was specifically increased with TRO treatments of 50 and 75 microM. The spot was identified as a mixture of immunoglobulin heavy chain binding protein (BiP) and, to a lesser extent, protein disulfide isomerase-related protein (PDIrp). Immunoblot analyses showed that the BiP protein was dose-dependently increased by TRO treatment and, to a lower degree, by RSG. These effects were also correlated with the high induction of BiP mRNA by TRO (50 and 75 microM) and the lower induction by RSG. However, both treatments showed no significant effects on PDIrp expression. The toxic effects of TRO in relation to the overexpression of BiP were also demonstrated in HLE cells, another human hepatoma cell line. In HLE cells, the inhibition of BiP expression by small interference RNA rendered cells more susceptible to the toxic effects of TRO. These results suggest that the overexpression of BiP is a defense mechanism of the endoplasmic reticulum in response to TRO-induced toxicity.

  8. Inhibiting effect of a hepatoma extract on the mitotic rate of regenerating liver.

    PubMed

    Echave Llanos, J M; Badrán, A F; Moreno, F R

    1986-01-01

    Aqueous tumor extracts were prepared by the homogenization of a fast-growing, undifferentiated, transplantable malignant murine hepatoma in distilled water. After centrifugation, an aliquot of 0.01 ml of the supernatant g body weight was injected intraperitoneally into partially hepatectomized mice. Control animals were injected with saline. Groups of mice were killed at various times in relation to the hepatectomy. Four h before killing the animals were given Colcemid (1 microgram/g body weight). The number of Colcemid-arrested mitoses in the hepatocytes and in the littoral cells, respectively, were counted in 140 microscopic fields. The extract significantly inhibited the mitotic rate in hepatocytes when the injection was given between 22 h before, and up to 26 h after hepatectomy. In the littoral cells, a slight initial stimulation was followed by a slight but significant inhibition which occurred when the injection was given at hepatectomy or until 18 h after hepatectomy. The effect was not modified by exposing the extracts to temperatures of 47 degrees C for 30 min or 22 degrees C for 24 h, but 10 min of boiling destroyed their inhibitory effect. Lyophilization and storing at -18 degrees C for up to 4 weeks did not modify the effect. The mitosis-inhibiting effect was also measurable when the extract was injected subcutaneously. There was an almost linear dose-response curve. The results are discussed in relation to circadian rhythms, the pattern of liver cell proliferation after hepatectomy, and recent similar reports from the literature. The conclusion is drawn that extracts of a hepatoma contain one or more growth-inhibitory factors significantly active on regenerating liver cells, and less significantly on littoral cells.

  9. Effect of dexamethasone, 2-bromopalmitate and clofibrate on L-FABP mediated hepatoma proliferation.

    PubMed

    Rajaraman, G; Burczynski, F J

    2004-09-01

    Cytosolic liver fatty acid binding protein (L-FABP) is involved in many intracellular functions including cellular mitogenesis. We investigated the role of L-FABP and the plasma membrane liver fatty acid binding proteins (L-FABP(pm)) in the modulation of hepatoma growth and proliferation, hypothesizing that agents that affect either the content of, or ligand binding to, L-FABP would affect hepatocellular mitogenesis. L-FABP expressing 1548-rat hepatoma cells were treated with 0.5 microM dexamethasone or 500 microM clofibrate for 4 days to downregulate and upregulate L-FABP expression, respectively. The competitive inhibitor 2-bromopalmitate (BrPA, 600 microM) was used to inhibit ligand binding to L-FABP. The peripherally present plasma membrane fatty acid transporter was inactivated by treating cells with 1:50 rabbit antisera (FABP-Ab) raised against L-FABP. Western blot analysis was used to monitor L-FABP levels while [(3)H]-thymidine incorporation and growth curves were used to monitor hepatocellular proliferation. [(3)H]-Palmitate clearance studies were performed using monolayer cultures. Palmitate clearance in dexamethasone-, BrPA- and FABP-Ab-treated cells was significantly reduced when compared with control (P < 0.05), while clofibrate treatment moderately increased the rate. [(3)H]-Thymidine incorporation by dexamethasone- and BrPA-treated cells was significantly lower than control (P < 0.05), suggesting that hepatocellular proliferation was inhibited. Clofibrate treatment did not statistically affect growth rate. Lowering L-FABP using dexamethasone or interfering with its activity using BrPA significantly affected hepatocellular proliferation. This may be due to the non-availability of long-chain fatty acids or other intracellular mediators that are transported by L-FABP to the nucleus.

  10. The phytoestrogen daidzein affects the antioxidant enzyme system of rat hepatoma H4IIE cells.

    PubMed

    Röhrdanz, Elke; Ohler, Sandra; Tran-Thi, Quynh-Hoa; Kahl, Regine

    2002-03-01

    Phytoestrogens such as the soy isoflavonoid daidzein have potential health benefits. The antioxidant properties of phytoestrogens are considered to be responsible in part for their protective effects. The antioxidant enzyme (AOE) system plays an important role in the defense of cells against oxidative insults. To determine whether flavonoids can exert antioxidative effects not only directly but also indirectly by modulating the AOE system, we investigated the influence of the flavonoid daidzein on the expression of different AOE. Daidzein treatment of hepatoma H4IIE cells increased catalase mRNA expression two- to threefold. Expression levels of copper zinc superoxide dismutase (CuZnSOD) were not affected by exposure to daidzein. Manganese superoxide dismutase (MnSOD) mRNA expression levels decreased slightly and glutathione peroxidase (GPx) levels increased slightly after daidzein exposure. Changes in AOE mRNA expression levels were significant at 300 micromol/L daidzein. To elucidate the mechanisms underlying the strong increase in catalase mRNA, transfection experiments were performed. Transient transfection of hepatoma cells with reporter plasmids containing different parts of the upstream region of the catalase gene showed a significant one- to threefold increase in reporter gene activity after daidzein exposure. This indicates that daidzein can directly activate the rat catalase promoter region. Despite the increase in catalase mRNA, daidzein pretreatment of cells did not protect against oxidative stress resulting from H(2)O(2) exposure. On the contrary, daidzein itself exerted a mild oxidative stress. In conclusion, the changes in the AOE system provoked by daidzein affected the oxidant rather than the antioxidant properties of daidzein.

  11. Mitochondria as an important target in heavy metal toxicity in rat hepatoma AS-30D cells

    SciTech Connect

    Belyaeva, Elena A. Dymkowska, Dorota; Wieckowski, Mariusz R.; Wojtczak, Lech

    2008-08-15

    The mechanisms of toxic effects of divalent cations of three heavy metals Hg, Cd and Cu in rat ascites hepatoma AS-30D cells cultivated in vitro were compared. It was found that the toxicity of these ions, applied in the micromolar range (10-500 {mu}M), decreased from Hg{sup 2+} (most toxic) to Cu{sup 2+} (least toxic). Hg{sup 2+} and Cd{sup 2+} produced a high percentage of cell death by both necrosis and apoptosis, whereas Cu{sup 2+} at concentrations up to 500 {mu}M was weakly effective. Hg{sup 2+} at concentration of 10 {mu}M appeared slightly uncoupling (i.e., stimulated resting state respiration and decreased the mitochondrial transmembrane potential), whereas it exerted a strong inhibitory effect on the respiratory chain and rapid dissipation of the membrane potential at higher concentrations. Cu{sup 2+} had inhibitory effect on cell respiration only at 500 {mu}M concentration and after incubation of 48 h but produced a significant uncoupling effect at lower concentrations. Cu{sup 2+} induced an early and sharp increase of intracellular production of reactive oxygen species (ROS). The action of Hg{sup 2+} and Cd{sup 2+} on ROS generation was biphasic. They stimulated ROS generation within the cells at low concentrations and at short incubation times but decreased ROS generation at higher concentrations and at longer incubation. It is concluded that mitochondria are an important target for toxic effects of Hg{sup 2+}, Cd{sup 2+} and Cu{sup 2+} in AS-30D rat hepatoma cells.

  12. Inhibition of spermidine synthase gene expression by transforming growth factor-beta 1 in hepatoma cells.

    PubMed Central

    Nishikawa, Y; Kar, S; Wiest, L; Pegg, A E; Carr, B I

    1997-01-01

    We screened genes responsive to transforming growth factor-beta (TGF-beta 1) protein in a human hepatoma cell line (Hep3B) using a PCR-mediated differential display technique, in order to investigate the mechanisms involved in TGF-beta-induced growth suppression. We found a gene that was down-regulated by TGF-beta 1 to be completely identical in an approx. 620 bp segment to the gene for the enzyme spermidine synthase, which mediates the conversion of putrescine into spermidine. Both spermidine synthase mRNA expression and its enzyme activity were decreased after TGF-beta 1 treatment of Hep3B cells. The inhibition of spermidine synthase gene expression by TGF-beta 1 protein was also observed in other hepatoma cell lines. The expression of genes for other biosynthetic enzymes in polyamine metabolism (ornithine decarboxylase and S-adenosylmethionine decarboxylase) was also inhibited to the same extent as for spermidine synthase, while the gene expression of spermidine/spermine N1-acetyltransferase, a catabolic enzyme, was relatively resistant to TGF-beta 1. Spermine levels in Hep3B cells were decreased by TGF-beta 1 treatment, although the levels of spermidine and putrescine were unchanged, probably due to compensation by remaining spermidine/spermine N1-acetyltransferase activity. Exogenously added spermidine or spermine, but not putrescine, partially antagonized the growth-inhibitor effects of TGF-beta 1 on Hep3B cells. Our data suggest that down-regulation of gene expression of the enzymes involved in polyamine metabolism, including spermidine synthase, may be associated with the mechanism of TGF-beta-induced growth suppression. PMID:9020892

  13. Mitomycin C induces bystander killing in homogeneous and heterogeneous hepatoma cellular models

    PubMed Central

    Kumari, Ratna; Sharma, Aanchal; Ajay, Amrendra Kumar; Bhat, Manoj Kumar

    2009-01-01

    Background Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide that is particularly refractory to chemotherapy. Several studies have proposed combination chemotherapy regimen for HCC treatment. However, these therapies are not effective in regressing tumor and prolonging survival of patient's suffering from HCC. Therefore, the development of more effective therapeutic tools and new strategies for the treatment of HCC are urgently needed. Over the last decade much attention has been focused on "bystander effect" as a possible therapeutic strategy for the treatment of certain human tumors. Interest in this therapeutic approach originated from numerous reports describing the radiation induced bystander effect. However, the knowledge about chemotherapy induced bystander effect is still limited. Hence, chemotherapy induced bystander phenomenon in hepatoma cells was explored by utilizing Mitomycin C (MMC). Results MMC induced bystander killing was observed only in hepatoma cells and it did not occur in cervical cancer cells. MMC induced bystander killing was transferable via medium. It occurred in co-cultured cells indicating the involvement of secreted as well as membrane bound factors. FasL and TRAIL were detected in the conditioned medium from treated cells. In medium transfer experiment, pre-treatment with EDTA (a broad range protease inhibitor) diminished MMC induced bystander killing. Following drug exposure, expression of Fas and TRAIL receptors increased and treatment with neutralizing antibodies against FasL and TRAIL inhibited bystander killing. Conclusion Our results highlight the therapeutic importance of MMC in the treatment of HCC and implicate role of membrane bound and secreted forms of FasL and TRAIL in MMC induced bystander killing. PMID:19845939

  14. Spatial Analysis of the Home Addresses of Hospital Patients with Hepatitis B Infection or Hepatoma in Shenzhen, China from 2010 to 2012

    PubMed Central

    Hu, Tao; Du, Qingyun; Ren, Fu; Liang, Shi; Lin, Denan; Li, Jiajia; Chen, Yan

    2014-01-01

    Background: Hepatoma associated with hepatitis B infection is a major public health problem in Shenzhen (China) and worldwide. China has the largest number of people infected with the hepatitis B virus (HBV), and many studies have demonstrated that HBV infection is associated with hepatoma development. Shenzhen officials have been attempting to monitor and control these diseases for many years. The methodology and the results of this study may be useful in developing a system to monitor, prevent and control these diseases. Methods: The aim of the study was to analyze HBV infection and hepatoma distribution characteristics and patterns in Shenzhen by combining geographic information system (GIS) technology and spatial analysis. The study used data from patients at the district level from the 2010–2012 population censuses. Results: Only one-third of the patients were female, and 70.7% of all cases were 20–50 years of age. There was no global spatial correlation of the distribution of hepatitis B infections and hepatomas; however, there was a local spatial correlation, and certain sub-districts of the Nanshan district had significant agglomeration effects. Based on incidence density and rate maps, we can conclude that the Shenzhen special zone had a higher incidence density and rate of hepatitis B infections and hepatomas compared with the area outside of the Shenzhen special zone during 2010–2012. Conclusions: This study demonstrated substantial geographic variation in the incidence of hepatitis B infection and hepatoma in Shenzhen. The prediction and control of hepatitis B infections and hepatoma development and interventions for these diseases should focus on disadvantaged areas to reduce disparities. GIS and spatial analysis play an important role in public health risk-reduction programs and may become integral components in the epidemiologic description, analysis and risk assessment of hepatitis B and hepatoma. PMID:24637909

  15. Induction of aldose reductase gene expression in LEC rats during the development of the hereditary hepatitis and hepatoma.

    PubMed

    Takahashi, M; Hoshi, A; Fujii, J; Miyoshi, E; Kasahara, T; Suzuki, K; Aozasa, K; Taniguchi, N

    1996-04-01

    We examined age-related changes in the protein and the mRNA expression of aldose reductase in livers of Long-Evans with a cinnamon-like color (LEC) rats, which develop hereditary hepatitis and hepatoma with aging, using Long-Evans with an agouti color rats as controls. The levels of the protein and mRNA of aldose reductase increased after 20 weeks, at the stage of acute hepatitis, and were maintained at 60 weeks of age, while those of aldehyde reductase seemed to be constant at all ages. The expression of aldose reductase was marked in cancerous lesions in hepatoma-bearing LEC rat liver compared to uninvolved surrounding tissues. These results indicated that elevation of aldose reductase accompanied hepatocarcinogenesis and may be related to the acquisition of immortality of the cancer cells through detoxifying cytotoxic aldehyde compounds.

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

    PubMed

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

    2015-01-01

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

  18. Impact of graphene oxide on viability of Chinese hamster ovary and mouse hepatoma MH-22A cells.

    PubMed

    Batiuskaite, Danute; Grinceviciute, Nora; Snitka, Valentinas

    2015-08-01

    The evaluation of the cyto- and bio-compatibility is a critical step in the development of graphene oxide (GO) as a new promising material for in vivo biomedical applications. In this study, we report the impact of GO, with and without the addition of bovine serum albumin, on healthy (Chinese hamster ovary) and a cancer (mouse hepatoma MH-22A) cells viability and the estimation of the intracellular distribution of GO inside the cells in vitro. The viability tests were performed using a colony formation assay. The intracellular distribution of GO was estimated using Raman spectroscopy and imaging. The viability of both cell lines decreased with increasing concentration of graphene oxide (12.5-50.0 μg/ml): in the case of Chinese hamster ovary cells viability decreased from 44% to 11%, in the case of mouse hepatoma MH-22A cells--from 22% to 3%. These cell lines significantly differed in their response to GO and GO-BSA formulations. The results of viability tests correlate with results of atomic force microscopy and Raman spectroscopy and imaging findings. The GO influence on cell morphology changes, cell structure, cells colony growth dynamics and GO accumulation inside the cells was higher in the case of mouse hepatoma MH-22A cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    PubMed

    Pan, Tao; Zhang, Mei; Zhang, Fang; Yan, Guang; Ru, Yi; Wang, Qinhao; Zhang, Yao; Wei, Xuehui; Xu, Xinyuan; Shen, Lan; Zhang, Jian; Wu, Kaichun; Yao, Libo; Li, Xia

    2017-02-05

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

  20. Troglitazone but not rosiglitazone induces G1 cell cycle arrest and apoptosis in human and rat hepatoma cell lines.

    PubMed

    Bae, Myung-Ae; Rhee, Herman; Song, Byoung J

    2003-03-20

    Rosiglitazone (RSG), an agonist of peroxisome proliferator-activated receptor gamma (PPARgamma), induces minor toxicity in humans relative to another PPARgamma agonist, troglitazone (TRO). In contrast, recent reports suggest that RSG causes growth arrest and apoptosis of normal and cancerous cells. Therefore, in this study, we investigated the relative toxicities of TRO and RSG on three different hepatoma cell lines, and observed that TRO, but not RSG, was cytotoxic. Additionally, we studied the mechanism by which TRO induced damage to HepG2 hepatoma cells. Our results indicated that TRO increased the levels of p53, p27, and p21, while it reduced the levels of cyclin D1 and phospho-Rb in a time-dependent manner. Increased p27 and p21 levels coincided with reduced activities of cell cycle dependent kinases (cdk) such as cdk2- and cyclin A-protein kinases 24 h after TRO treatment. These results demonstrate that TRO, but not RSG, causes G1 arrest of hepatoma cells, most likely through changing the levels of cell cycle regulators. Furthermore, because RSG did not affect the levels of cell cycle regulators, TRO-mediated growth inhibition appears independent of PPARgamma activation.

  1. Chemopreventive action of Lygodium flexuosum extract in human hepatoma PLC/PRF/5 and Hep 3B cells.

    PubMed

    Wills, P J; Asha, V V

    2009-03-18

    Lygodium flexuosum (Lygodiaceae), a medicinal fern used in Indian traditional medicine against liver disorders. The rationale of the study was to examine whether the n-hexane extract from plant Lygodium flexuosum affects apoptosis on human hepatoma PLC/PRF/5 and Hep 3B cells. Chemopreventive activity of the Lygodium flexuosum extract was determined by MTT assay, annexin-V FITC binding to phosphatidyl serine and cleavage of PARP. Subdiploid condition of cells treated with Lygodium flexuosum was analyzed by flow cytometry. Further, used transiently transfected NF-kappaB reporter in PLC/PRF/5 cells to evaluate the inhibitive effect of Lygodium flexuosum extract. Lygodium flexuosum extract inhibited the cell viability and induced apoptosis in hepatoma cells in a concentration dependent manner as evidenced by apoptotic changes such as flipping of phosphatidyl serine, cleavage of PARP. Cell cycle analysis showed the subG1 apoptotic population in cells treated with higher concentrations of the extract. When activated with exogenous TNF-alpha in transfected hepatoma cells it was observed that NF-kappaB dependent gene expression was inhibited by treatment with Lygodium flexuosum extract in PLC/PRF/5 cells dose-dependently. This investigation suggests that the Lygodium flexuosum extract has antiproliferative and apoptotic activity in both cancer cells and has inhibitive role in TNF-alpha induced NF-kappaB activation in PLC/PRF/5 cells confirms the potential of the extract as a chemopreventive agent.

  2. Reverse relationship between malignancy and cyclic AMP-dependent protein kinase activity in Yoshida rat ascites hepatomas.

    PubMed

    Miyamoto, K; Nakamura, S; Nomura, M; Yamamoto, H; Sanae, F; Hidaka, H

    1993-08-31

    Rat ascites hepatoma (AH) cells (10(6) cells/head) inoculated intraperitoneally into rats had host-killing ability (malignancy) in the order AH66F > AH44 > AH13 > AH7974 > AH109A > AH66 > AH130. The life span of the rats after inoculation closely correlated with the activity of cyclic AMP-dependent protein kinase (protein kinase A) in the tumor cells but not the activity of Ca2+/phospholipid-dependent protein kinase (protein kinase C). N-[2-[N-[3-(4-chlorophenyl)-1-methyl-2-propenyl]amino]ethyl]-5- isoquinoline-sulfonamide (H-87), a potent, selective inhibitor of protein kinase A, inhibited in vitro growth of these hepatoma cells with a similar potency and, intraperitoneally injected, prolonged the lives of rats bearing less malignant AH66 cells (with high protein kinase A activity) but did not affect the life span of rats bearing highly malignant AH66F cells (with low protein kinase A activity). On the other hand N-(2-methylpiperazyl)-5-isoquinolinesulfonamide (H-7), an inhibitor of protein kinase C, inhibited AH66F cells more than AH66 cells, but did not influence the life span of rats bearing either hepatoma. From these results it is deduced that protein kinase A may be important in the regulation of malignancy and in vivo proliferation of AH cells.

  3. Identifying apoptosis-evasion proteins/pathways in human hepatoma cells via induction of cellular hormesis by UV irradiation.

    PubMed

    Hsieh, Sen-Yung; Hsu, Chih-Yun; He, Jung-Ru; Liu, Chiung-Liang; Lo, Shao-Jung; Chen, Ying-Ching; Huang, Hui-Yu

    2009-08-01

    Evading apoptosis is pivotal in both of carcinogenesis and resistance to anticancer therapy. We investigated the molecules and pathways of apoptosis evasion in human hepatoma cells by irradiating hepatoma cells with optimized UV (so-called "hormetic responses"). Proteins and pathways related to hormetic responses were identified via proteomic approaches followed by reconstruction of function-networks. Of the 2326 defined protein spots, 42 distinct proteins significantly changed their expression. Eleven hormetic response proteins (HINT1, PHB, CTSD, ANXA1, LGASL1, TPT1, NPM, PRDX2, UCHL1, CERK, and C1QBP) were involved in 5 death-regulatory pathways, including the p53-dependent apoptotic pathway, protein ubiquinization, cellular redox, calcium-mediated signaling pathway, and sphingomyelin-metabolism pathway. Knockdown of HINT1 expression via RNA interference increased tumor cell resistance to apoptosis induction, while silencing NPM, UCHL1, or CERK greatly sensitized tumor cells to apoptosis induction. In conclusion, NPM, UCHL1, and CERK act as apoptosis-evasion proteins that may serve as therapeutic targets for hepatoma. Silencing their expression would increase therapeutic efficacy, thereby reducing the corresponding doses and side-effects of anticancer therapy. This model of induction of cellular hormetic responses to identify apoptosis-evasion molecules/pathways via proteomic approaches can be applied to other modalities of anticancer therapy.

  4. Dihydromyricetin inhibits migration and invasion of hepatoma cells through regulation of MMP-9 expression

    PubMed Central

    Zhang, Qing-Yu; Li, Ran; Zeng, Guo-Fang; Liu, Bin; Liu, Jie; Shu, Yang; Liu, Zhong-Kao; Qiu, Zhi-Dong; Wang, Dong-Jun; Miao, Hui-Lai; Li, Ming-Yi; Zhu, Run-Zhi

    2014-01-01

    AIM: To investigate the effects of dihydromyricetin (DHM) on the migration and invasion of human hepatic cancer cells. METHODS: The hepatoma cell lines SK-Hep-1 and MHCC97L were used in this study. The cells were cultured in RPIM-1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO2 incubator. DHM was dissolved in dimethyl sulfoxide and diluted to various concentrations in medium before applying to cells. MTT assays were performed to measure the viability of the cells after DHM treatment. Wound healing and Boyden transwell assays were used to assess cancer cell motility. The invasive capacity of cancer cells was measured using Matrigel-coated transwell chambers. Matrix metalloproteinase (MMP)-2/9 activity was examined by fluorescence analysis. Western blot was carried out to analyze the expression of MMP-2, MMP-9, p-38, JNK, ERK1/2 and PKC-δ proteins. All data were analyzed by Student’s t tests in GraphPad prism 5.0 software and are presented as mean ± SD. RESULTS: DHM was found to strongly inhibit the migration of the hepatoma cell lines SK-Hep-1 (without DHM, 24 h: 120 ± 8 μmol/L vs 100 μmol/L DHM, 24 h: 65 ± 10 μmol/L, P < 0.001) and MHCC97L (without DHM, 24 h: 126 ± 7 μmol/L vs 100 μmol/L DHM, 24 h: 74 ± 6 μmol/L, P < 0.001). The invasive capacity of the cells was reduced by DHM treatment (SK-Hep-1 cells without DHM, 24 h: 67 ± 4 μmol/L vs 100 μmol/L DHM, 24 h: 9 ± 3 μmol/L, P < 0.001; MHCC97L cells without DHM, 24 h: 117 ± 8 μmol/L vs 100 μmol/L DHM, 24 h: 45 ± 2 μmol/L, P < 0.001). MMP2/9 activity was also inhibited by DHM exposure (SK-Hep-1 cells without DHM, 24 h: 600 ± 26 μmol/L vs 100 μmol/L DHM, 24 h: 100 ± 6 μmol/L, P < 0.001; MHCC97L cells without DHM, 24 h: 504 ± 32 μmol/L vs 100 μmol/L DHM 24 h: 156 ± 10 μmol/L, P < 0.001). Western blot analysis showed that DHM decreased the expression level of MMP-9 but had little effect on MMP-2. Further investigation indicated that DHM markedly

  5. Preparation of a dual cored hepatoma-specific star glycopolymer nanogel via arm-first ATRP approach.

    PubMed

    Lou, Shaofeng; Zhang, Xiuyuan; Zhang, Mingming; Ji, Shenglu; Wang, Weiwei; Zhang, Ju; Li, Chen; Kong, Deling

    2017-01-01

    A reductase-cleavable and thermo-responsive star-shaped polymer nanogel was prepared via an "arm-first" atom transfer radical polymerization approach. The nanogel consists of a thermo- and redox-sensitive core and a zwitterionic copolymer block. The dual sensitive core is composed of poly(N-isopropylacrylamide) that is formed by disulfide crosslinking of N-isopropylacrylamide. The zwitterionic copolymer block contains a poly(sulfobetaine methacrylate) component, a known anti-adsorptive moiety that extends blood circulation time, and a lactose motif of poly(2-lactobionamidoethyl methacrylamide) that specifically targets the asialoglycoprotein receptors (ASGP-Rs) of hepatoma. Doxorubicin (DOX) was encapsulated into the cross-linked nanogels via solvent extraction/evaporation method and dialysis; average diameter of both blank and DOX-loaded nanogels was ~120 nm. The multi-responsiveness of nanogel drug release in different temperatures and redox conditions was assessed. After 24 h, DOX release was only ~20% at 30°C with 0 mM glutathione (GSH), whereas over 90% DOX release was observed at 40°C and 10 mM GSH, evidence of dual responsiveness to temperature and reductase GSH. The IC50 value of DOX-loaded nanogels was much lower in human hepatoma (HepG2) cells compared to non-hepatic HeLa cells. Remarkably, DOX uptake of HepG2 cells differed substantially in the presence and absence of galactose (0.31 vs 1.42 µg/mL after 48 h of incubation). The difference was non-detectable in HeLa cells (1.21 vs 1.57 µg/mL after 48 h of incubation), indicating that the overexpression of ASGP-Rs leads to the DOX-loaded lactosylated nanogels actively targeting hepatoma. Our data indicate that the lactose-decorated star-shaped nanogels are dual responsive and hepatoma targeted, and could be employed as hepatoma-specific anti-cancer drug delivery vehicle for cancer chemotherapy.

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

    PubMed

    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.

  7. 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.

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

    PubMed

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

    2013-11-01

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

  9. Troglitazone-induced intracellular oxidative stress in rat hepatoma cells: a flow cytometric assessment.

    PubMed

    Narayanan, Padma Kumar; Hart, Timothy; Elcock, Fiona; Zhang, Cindy; Hahn, Laura; McFarland, David; Schwartz, Lester; Morgan, D Gwyn; Bugelski, Peter

    2003-03-01

    Troglitazone (TRO), a thiazolidinedione (TZD) peroxisome proliferator-activated receptor gamma agonist, was recently withdrawn from the market because of rare but serious hepatotoxicity. Previous studies investigating the cytotoxicity of TRO in cultured rat hepatocytes have conjectured about the role of oxidative stress in TRO-induced hepatotoxicity. Therefore, we investigated whether TRO induces oxidative stress and, if so, the portion of the TRO molecule responsible for the induction of oxidative stress. Novikoff rat hepatoma (N1S1) cells were incubated with TRO, troglitazone quinone (TQ), thiazolidinedione-phenoxyacetic acid (TD-PAA) or rosiglitazone (RSG). Membrane peroxidation, intracellular glutathione (GSH) content, and cellular viability were monitored simultaneously by multiparameter flow cytometry. TRO and TQ increased membrane peroxidation, decreased intracellular GSH, and decreased cell viability in a concentration-dependent manner. In contrast, TD-PAA and RSG neither increased membrane peroxidation nor induced loss of cell viability. In addition, TRO caused a concentration-dependent increase in intracellular superoxide generation accompanied by a collapse in mitochondrial membrane potential. Multiparameter flow cytometric evaluation of N1S1 cells indicated that the chromane ring of TRO, rather than the TZD moiety, may be responsible for oxidative stress and suggested that a direct effect on mitochondrial physiology may play a role in TRO-mediated hepatotoxicity. Copyright 2003 Wiley-Liss, Inc.

  10. Antiproliferative and apoptotic potential of Daphne gnidium L. root extract on lung cancer and hepatoma cells.

    PubMed

    Chaouki, W; Meddah, B; Hmamouchi, M

    2015-03-01

    Daphne gnidium L. (Thymeleacees) is a famous Moroccan plant with cancer-related ethnobotanical use. Previously, we demonstrated that ethyl acetate extract of D. gnidium had antiproliferative and pro-apoptotic potential on human breast tumor MCF-7 cells. The purpose of this study was to investigate if the antiproliferative effect of this extract was similar for different human cancer cell lines such as A549 lung cancer and SMMC-7721 hepatoma cells. Moreover, this work essentially focused on the intrinsic apoptotic signaling pathway. Antiproliferative activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide on A549 and SMMC-7721 cells. The characterization of the mechanisms involved in this effect was determined by lactate dehydrogenase test, apoptosis assays and western blot analyses. Our present study has shown that this extract strongly inhibited proliferation of A549 (IC50: 213 ± 15 μg/ml) and SMMC-7721 (IC50: 170 ± 13 μLg/ml) cells. The characterization of antiproliferative effect demonstrated that this extract was an apoptosis inducer in both cell lines tested. The results of western blot analyses have shown in SMMC-7721 cells that this extract activated caspase signaling triggered by the modulation of Bcl-2 family proteins. These findings suggest that this natural extract-induced effects may have novel therapeutic applications for the treatment of different cancer types.

  11. Glucocorticoid regulation of amino acid transport in anucleate rat hepatoma (HTC) cells

    PubMed Central

    1981-01-01

    The transport of alpha-aminoisobutyric acid (AIB) by rat hepatoma tissue culture (HTC) cells is rapidly and reversibly inhibited by dexamethasone and other glucocorticoids. To investigate the role of the nucleus in the regulation of transport and to determine whether steroid hormones or steroid-receptor complexes may have direct effects on cytoplasmic or membrane functions, we have examined the regulation of transport by dexamethasone in anucleate HTC cells. Cytoplasts prepared from suspension cultures of HTC cells fully retain active transport of AIB with the same kinetic properties as intact cells. However, the uptake of AIB is not inhibited by dexamethasone or other corticosteroids. Neither is the inhibited rate of transport, manifested by cytoplasts prepared from dexamethasone-treated cells, restored to normal upon removal of the hormone. Anucleate cells exhibit specific, saturable binding of [3H]dexamethasone; however, the binding is reduced compared with that of intact cells. The nucleus is thus required for the glucocorticoid regulation of amino acid transport in HTC cells. PMID:7217203

  12. GLUCOCORTICOID-INDUCED ALTERATION OF THE SURFACE MEMBRANE OF CULTURED HEPATOMA CELLS

    PubMed Central

    Ballard, Philip L.; Tomkins, Gordon M.

    1970-01-01

    Glucocorticoids induce an alteration of the surface of hepatoma tissue culture (HTC) cells as expressed by changes in cell electrophoretic, antigenic, and adhesive properties. The alteration is assayed by the increased adhesiveness of induced cells for a glass surface. The induction process has a lag period of about 3 hr and attains a plateau level after 24–30 hr when 50–80% of the steroid-treated cells are firmly adhered. Less than 10% of untreated cells adhere under the same conditions. Induction is inhibited by actinomycin D and cycloheximide, demonstrates both pH and temperature dependence, and responds to changes in steroid concentration and structure. By contrast, the attachment per se of preinduced cells is not affected by inhibitors of RNA and protein synthesis, fluctuations of temperature and pH, and the presence or absence of the hormone. When the induction process is reversed by removal of steroid or addition of actinomycin D, preinduced adhesiveness is lost with a half-life of 13–24 hr, but in the presence of cycloheximide the loss is accelerated (t1/2 3–5.5 hr). These results suggest that glucocorticoids induce the biosynthesis of a protein which either modifies the cell surface (an enzyme) or is incorporated into surface structures (structural protein). PMID:4327515

  13. 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

  14. Transplantable Subcutaneous Hepatoma 22a Affects Functional Activity of Resident Tissue Macrophages in Periphery

    PubMed Central

    Kisseleva, Ekaterina P.; Krylov, Andrei V.; Stepanova, Olga I.; Lioudyno, Victoria I.

    2011-01-01

    Tumors spontaneously develop central necroses due to inadequate blood supply. Recent data indicate that dead cells and their products are immunogenic to the host. We hypothesized that macrophage tumor-dependent reactions can be mediated differentially by factors released from live or dead tumor cells. In this study, functional activity of resident peritoneal macrophages was investigated in parallel with tumor morphology during the growth of syngeneic nonimmunogenic hepatoma 22a. Morphometrical analysis of tumor necroses, mitoses and leukocyte infiltration was performed in histological sections. We found that inflammatory potential of peritoneal macrophages in tumor-bearing mice significantly varied depending on the stage of tumor growth and exhibited two peaks of activation as assessed by nitroxide and superoxide anion production, 5′-nucleotidase activity and pinocytosis. Increased inflammatory reactions were not followed by the enhancement of angiogenic potential as assessed by Vascular Endothelial Growth Factor mRNA expression. Phases of macrophage activity corresponded to the stages of tumor growth characterized by high proliferative potential. The appearance and further development of necrotic tissue inside the tumor did not coincide with changes in macrophage behavior and therefore indirectly indicated that activation of macrophages was a reaction mostly to the signals produced by live tumor cells. PMID:21760797

  15. Hepatoma-derived growth factor: from the bovine uterus to the in vitro embryo culture.

    PubMed

    Gómez, E; Correia-Álvarez, E; Caamaño, J N; Díez, C; Carrocera, S; Peynot, N; Martín, D; Giraud-Delville, C; Duranthon, V; Sandra, O; Muñoz, M

    2014-10-01

    Early in cow embryo development, hepatoma-derived growth factor (HDGF) is detectable in uterine fluid. The origin of HDGF in maternal tissues is unknown, as is the effect of the induction on developing embryos. Herein, we analyze HDGF expression in day 8 endometrium exposed to embryos, as well as the effects of recombinant HDGF (rHDGF) on embryo growth. Exposure to embryos did not alter endometrial levels of HDGF mRNA or protein. HDGF protein localized to cell nuclei in the luminal epithelium and superficial glands and to the apical cytoplasm in deep glands. After uterine passage, levels of embryonic HDGF mRNA decreased and HDGF protein was detected only in the trophectoderm. In fetal fibroblast cultures, addition of rHDGF promoted cell proliferation. In experiments with group cultures of morulae in protein-free medium containing polyvinyl alcohol, adding rHDGF inhibited blastocyst development and did not affect cell counts when the morulae were early (day 5), whereas it enhanced blastocyst development and increased cell counts when the morulae were compact (day 6). In cultures of individual day 6 morulae, adding rHDGF promoted blastocyst development and increased cell counts. Our experiments with rHDGF indicate that the growth factor stimulates embryonic development and cell proliferation. HDGF is synthesized similarly by the endometrium and embryo, and it may exert embryotropic effects by autocrine and/or paracrine mechanisms.

  16. Reculation of folylpolyglutamate synthetase in extracts of H35 hepatoma cells

    SciTech Connect

    Johnson, T.B.; Galivan, J.; Nair, M.G. )

    1987-05-01

    Folylpolyglutamate synthetase (FPGS) in extracts of H35 hepatoma cells was assayed using 250 {mu}M methotrexate (MTX) as the substrate under conditions where ({sup 3}H)glutamate incorporation was linear with respect to time and rotein concentration. Extracts from confluent cultures with reduced cellular folates exhibited nearly 1.7-fold higher FPGS specific activity than extracts of control cultures (600 pmol/hr/mg). Extracts of rapidly dividing cells (72 hrs) showed nearly a 2.3-fold increase. The addition of reduced exogenous folates such as folinic acid and 5-methyltetrahydrofolate (20 {mu}M, 24 hrs) to confluent cultures of folate-depleted cells typically lowered the FPGS activity in the resultant extracts by 40%, while a 42-hour exclusion of methionine from the media reduced the activity by half. The combination of methionine exclusion and folate addition for 42 hrs resulted in 75% lower FPGS activity vs extracts of control cultures of folate-depleted cells. These data suggest that the change sin the glutamylation rate of MTX in whole cells due to culture conditions such as folate restriction, reduced folate addition, methionine exclusion, and growth state are at least in part a consequence of alterations in FPGS activity. Using MTX or N{sup 10}-propargyl-5,8-dideazafolic acid (CB3717) as the starting substrate under appropriate assay conditions, FPGS from extracts catalyzed the formation of similar polyglutamate products as seen in analogous whole cell experiments.

  17. Glutathione-degradable drug-loaded nanogel effectively and securely suppresses hepatoma in mouse model

    PubMed Central

    Liu, Xingang; Wang, Jianmeng; Xu, Weiguo; Ding, Jianxun; Shi, Bo; Huang, Kexin; Zhuang, Xiuli; Chen, Xuesi

    2015-01-01

    The reduction-responsive polymeric nanocarriers have attracted considerable interest because of a significantly higher concentration of intracellular glutathione in comparison with that outside cells. The smart nanovehicles can selectively transport the antitumor drugs into cells to improve efficacies and decrease side effects. In this work, a facilely prepared glutathione-degradable nanogel was employed for targeting intracellular delivery of an antitumor drug (ie, doxorubicin [DOX]). DOX was loaded into nanogel through a sequential dispersion and dialysis approach with a drug loading efficiency of 56.8 wt%, and the laden nanogel (noted as NG/DOX) showed an appropriate hydrodynamic radius of 56.1±3.5 nm. NG/DOX exhibited enhanced or improved maximum tolerated dose on healthy Kunming mice and enhanced intratumoral accumulation and dose-dependent antitumor efficacy toward H22 hepatoma-xenografted mouse model compared with free drug. In addition, the upregulated antitumor efficacy of NG/DOX was further confirmed by the histopathological and immunohistochemical analyses. Furthermore, the excellent in vivo security of NG/DOX was confirmed by the detection of body weight, histopathology, and biochemical indices of corresponding organs and serum. With controllable large-scale preparation and fascinating in vitro and in vivo properties, the reduction-responsive nanogel exhibited a good prospect for clinical chemotherapy. PMID:26543363

  18. Proteomic analysis of human hepatoma cells expressing methionine adenosyltransferase I/III☆

    PubMed Central

    Schröder, Paul C.; Fernández-Irigoyen, Joaquín; Bigaud, Emilie; Serna, Antonio; Renández-Alcoceba, Rubén; Lu, Shelly C.; Mato, José M.; Prieto, Jesús; Corrales, Fernando J.

    2015-01-01

    Methionine adenosyltransferase I/III (MATI/III) synthesizes S-adenosylmethionine (SAM) in quiescent hepatocytes. Its activity is compromised in most liver diseases including liver cancer. Since SAM is a driver of hepatocytes fate we have studied the effect of re-expressing MAT1A in hepatoma Huh7 cells using proteomics. MAT1A expression leads to SAM levels close to those found in quiescent hepatocytes and induced apoptosis. Normalization of intracellular SAM induced alteration of 128 proteins identified by 2D-DIGE and gel-free methods, accounting for deregulation of central cellular functions including apoptosis, cell proliferation and survival. Human Dead-box protein 3 (DDX3X), a RNA helicase regulating RNA splicing, export, transcription and translation was down-regulated upon MAT1A expression. Our data support the regulation of DDX3X levels by SAM in a concentration and time dependent manner. Consistently, DDX3X arises as a primary target of SAM and a principal intermediate of its antitumoral effect. Based on the parallelism between SAM and DDX3X along the progression of liver disorders, and the results reported here, it is tempting to suggest that reduced SAM in the liver may lead to DDX3X up-regulation contributing to the pathogenic process and that replenishment of SAM might prove to have beneficial effects, at least in part by reducing DDX3X levels. This article is part of a Special Issue entitled: Proteomics: The clinical link. PMID:22270009

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

    PubMed Central

    Midoux, P; Mendes, C; Legrand, A; Raimond, J; Mayer, R; Monsigny, M; Roche, A C

    1993-01-01

    Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes in a sugar dependent manner: i) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected with pSV2Luc/mannosylated polylysine complexes, ii) HeLa cells which do not express membrane lectin specific for galactose were not transfected with pSV2Luc/lactosylated polylysine complexes. The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated polylysine complexes was greatly enhanced either in the presence of chloroquine or in the presence of a fusogenic peptide. A 22-residue peptide derived from the influenza virus hemagglutinin HA2 N-terminal polypeptide that mimics the fusogenic activity of the virus, was selected. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger than that of cells transfected with pSV2Luc/lactosylated polylysine complexes in the presence of chloroquine. Images PMID:8383843

  20. Effects of Lipofectamine 2000/siRNA complexes on autophagy in hepatoma cells.

    PubMed

    Mo, Robert H; Zaro, Jennica L; Ou, Jing-Hsiung James; Shen, Wei-Chiang

    2012-05-01

    Lipofectamine 2000 is commonly used for siRNA transfections. However, few studies have examined cellular responses to this delivery system. The purpose of this study is to evaluate the effect of siRNA transfection using Lipofectamine 2000 on cellular autophagy. Huh7.5 cells, stably transfected to express GFP-LC3, were treated with Lipofectamine 2000/negative control siRNA (NC siRNA) complexes. At different time points after treatment, cells were lysed and analyzed by immunoblotting and fluorescence spectroscopy. Cells were also observed using confocal microscopy. An increase of endogenous LC3 lipidation, GFP-LC3 fluorescence, and autophagosomal puncta was observed in cells treated with Lipofectamine 2000/NC siRNA complexes. The kinetics of the increase of GFP-LC3 fluorescence correlated with the concentration of NC siRNA transfected, where 50, 100, and 200 nM NC siRNA caused a significant increase at 72, 48, and 24 h, respectively, after transfection. A similar effect on the GFP-LC3 signal was also observed for cells treated with Lipofectamine 2000 complexed with two other NC siRNAs. The effects were also confirmed in another hepatoma cell line, H4IIE, by immunoblotting. Lipofectamine 2000-mediated transport of NC siRNAs led to an increase of autophagosomes in a dose- and time-dependent manner. Thus, this effect on cells should be taken into consideration when using this approach for intracellular delivery of siRNA.

  1. Curcumin inhibits ROS formation and apoptosis in methylglyoxal-treated human hepatoma G2 cells.

    PubMed

    Chan, Wen-Hsiung; Wu, Hsin-Jung; Hsuuw, Yan-Der

    2005-05-01

    Methylglyoxal (MG) is a reactive dicarbonyl compound endogenously produced mainly from glycolytic intermediates. Elevated MG levels in diabetes patients are believed to contribute to diabetic complications. MG is cytotoxic through induction of apoptosis. Curcumin, the yellow pigment of Curcuma longa, is known to have antioxidant and anti-inflammatory properties. In the present study, we investigated the effect of curcumin on MG-induced apoptotic events in human hepatoma G2 cells. We report that curcumin prevented MG-induced cell death and apoptotic biochemical changes such as mitochondrial release of cytochrome c, caspase-3 activation, and cleavage of PARP (poly [ADP-ribose] polymerase). Using the cell permeable dye 2',7'-dichlorofluorescein diacetate (DCF-DA) as an indicator of reactive oxygen species (ROS) generation, we found that curcumin abolished MG-stimulated intracellular oxidative stress. The results demonstrate that curcumin significantly attenuates MG-induced ROS formation, and suggest that ROS triggers cytochrome c release, caspase activation, and subsequent apoptotic biochemical changes.

  2. Combination antitumor effect with central nervous system depressants on rat ascites hepatomas.

    PubMed

    Koshiura, R; Miyamoto, K; Sanae, F

    1980-02-01

    Combined effect of twenty-one central nervous system depressants with several antitumor agents was studied in the in vitro and in vivo experimental systems, using rat ascites hepatoma call lines, AH13 and AH44, sensitive and insensitive to alkylating agents, respectively. Reserpine remarkably enhanced the cytotoxic effect of 1-(gamma-chloropropyl)-2-chloromethylpiperidine hydrobromide (CAP-2) both on AH13 and AH44 cells. In the in vivo combined experiments, reserpine also synergistically enhanced the life-prolonging effect of CAP-2 on AH13-bearing rats and, although CAP-2 was not potent on the prolongation of life span of AH44-bearing rats and reserpine was also ineffective at the doses examined, the life span of tumor-bearing rats receiving the combined administration was apparently prolonged compared with control groups. Thus, there was a parallelism between in vitro and in vivo experiments. These findings suggested that the antitumor-enhancing effect of reserpine might be due to the direct action on the tumor cells, and a possible mechanism that reserpine inhibited the DNA damage-repairing activity of the cells was contradictory. Other mechanisms are also discussed.

  3. Extinction of Hepatitis C Virus by Ribavirin in Hepatoma Cells Involves Lethal Mutagenesis

    PubMed Central

    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

  4. 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

  5. Circulating auto-antibody against hepatoma-derived growth factor (HDGF) in patients with ulcerative colitis.

    PubMed

    Nahamura, Hideji; Yoshida, Kenya; Kishima, Yoshihiko; Enomoto, Hirayuki; Uyama, Hirokazu; Kuroda, Toshifumi; Okuda, Yorihide; Hirotani, Tomonori; Ito, Hiroaki; Kawase, Ichiro

    2004-01-01

    Various circulating auto-antibodies have been reported in patients with ulcerative colitis. Hepatoma-derived growth factor (HDGF) is a mitogen, localized dominantly in the nucleus of proliferating cells. In this study, we demonstrated the circulating anti-HDGF auto-antibody and investigated its clinical roles in patients with ulcerative colitis. Anti-HDGF IgG antibodies were measured by the enzyme-linked immunosorbent assay with recombinant HDGF in 20 healthy volunteers and 40 patients with ulcerative colitis. Circulating anti-HDGF antibody was detected in the serum of a patient with total colitis by Western blotting. Anti-HDGF auto-antibodies were detected at 65.6% in the serum of patients with total/left-sided colitis, compared with healthy subjects at 10%. During active stage, the circulating anti-HDGF auto-antibodies were detected at a higher frequency of 78.3% than those in remission stage at 37.5%. Furthermore, the titers during active colitis were higher than those during the remission stage. Anti-HDGF auto-antibodies were not detected in any patients with proctitis. These findings suggest that anti-HDGF auto-antibodies in the serum of patients with ulcerative colitis would help to classify the total/left-sided colitis from proctitis, and the serial measurement of the titer would also be a good marker for the active colitis.

  6. 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.

  7. 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

  8. 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.

  9. The augmented anti-tumor effects of Antrodia camphorata co-fermented with Chinese medicinal herb in human hepatoma cells.

    PubMed

    Li, Shun-Lai; Huang, Zih-Ning; Hsieh, Hsiao-Hui; Yu, Wen-Chun; Tzeng, Win-Yu; Lee, Guo-Yang; Chen, Yi-Peng; Chang, Chia-Yu; Chuu, Jiunn-Jye

    2009-01-01

    Antrodia camphorata, unique fungal specie, has been used as a folk medicine in Taiwan for many years. The purpose of this study was to compare the extracts from the solid-state culture of A. camphorata co-fermented with Chinese medicinal herb (AC-CF) with two other extracts from fruiting bodies (AC-FB) or solid-state culture (AC-SS), for their anti-tumor effects in human hepatoma HepG2 cells. We measured in vitro cell proliferation, percentage of apoptosis, population distribution of cell cycles, Western blot analysis of multiple drugs resistance-1 (MDR-1), and apoptosis-related proteins in HepG2 cells treated with three different preparations of A. camphorate extracts. Our results showed that AC-CF had better anti-proliferation effect on human hepatoma HepG2 cells than AC-FB or AC-SS dose-dependently. In addition, AC-CF in combination with anti-tumor agents (mitomycin C or methotrexate) showed better adjuvant anti-tumor effects than AC-FB or AC-SS. We further demonstrated the augmented adjuvant anti-tumor effects of AC-CF not only through down regulation of MDR-1 expression but also through a COX-2 dependent apoptosis pathway, involving down-regulation of COX-2 and p-AKT and up-regulation of PARP-1. In conclusion, in this study, we have demonstrated a novel strategy of fermenting A. camphorata with Chinese medicinal herb (AC-CF), which augmented their anti-tumor effects in human hepatoma HepG2 cells as compared to the traditional ones (AC-FB or AC-SS).

  10. Inhibitory effects of ganoderma lucidum on tumorigenesis and metastasis of human hepatoma cells in cells and animal models.

    PubMed

    Weng, Chia-Jui; Chau, Chi-Fai; Yen, Gow-Chin; Liao, Jiunn-Wang; Chen, Deng-Hai; Chen, Kuang-Dee

    2009-06-10

    Metastasis is considered to be the major cause of death in patients with cancers, and hepatocellular carcinoma (HCC) is a highly metastatic cancer. Ganoderma lucidum , a well-known mushroom with various biological effects, is a functional food known to contain lucidenic acid. The objectives of this study were to investigate the anti-invasion effect of a lucidenic acid-rich G. lucidum extract (GLE) on human hepatoma HepG2 cells as well as the antiproliferative and antimetastatic effects of GLE in human hepatoma cells implanted into ICR-nu/nu mice. Phorbol-12-myristate-13-acetate (PMA)-induced invasion and matrix metalloproteinase (MMP)-9 expression levels of HepG2 cells were reduced by GLE treatment in a dose-dependent manner. The inhibitory effects of GLE on MMP-9 expression proceeded by inhibiting the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and protein kinase B in the cytosol as well as reducing activator protein-1 and nuclear factor-kappa B levels in the nucleus of HepG2 cells. In a human tumor xenograft model, a dose-response inhibition was observed in the average size, volume, and weight of tumors upon oral administration of GLE. The number of metastatic tumor-bearing mice, the number of affected organs, and the number of tumor foci as well as the MMP-2 and -9 activities in serum of mice were also significantly suppressed by oral administration of GLE. These results suggest that the lucidenic acid-rich GLE could serve as a chemopreventive agent for the tumorigenesis and metastasis of highly invasive hepatoma cells.

  11. Thyromimetic actions of tetrabromobisphenol A (TBBPA) in steatotic FaO rat hepatoma cells.

    PubMed

    Grasselli, E; Cortese, K; Fabbri, R; Smerilli, A; Vergani, L; Voci, A; Gallo, G; Canesi, L

    2014-10-01

    Tetrabromobisphenol A (2,2-bis(3,5-dibromo-4-hydroxyphenyl propane-TBBPA) is the most produced brominated flame retardant, detected in the environment and in biological samples. TBBPA shares structural similarities with thyroid hormones (THs), and it has been shown to interfere with different aspects of TH physiology, this raising concern on its possible effects as an endocrine disruptor in humans and wildlife. THs play a major role in lipid metabolism, with the liver representing one of their main target tissues. At the cellular level, THs act through interactions with TH receptors (TRs), as well as through TR-independent mechanisms. Rat hepatoma FaO cells (a liver cell line defective for functional TRs) overloaded with lipids have been utilized as a model to investigate the anti-steatotic effects of THs in the hepatocyte. In this work, the possible effects of TBBPA in steatotic FaO cells were investigated. Exposure to TBBPA for 24 h reduced triglyceride (TAG) content and the size of lipid droplets (LDs); similar effects were obtained with equimolar doses (10(-6) M) of T3 (3,3',5-L-triiodothyronine). TBBPA and T3 showed common effects on transcription of genes involved in lipid homeostasis. In particular, TBBPA mainly up-regulated mRNA levels for LD-associated oxidative tissue-enriched PAT protein (OXPAT), peroxisome proliferator-activated receptor (PPAR) isoform β/δ, and the mitochondrial uncoupling protein 2 (UCP2). The results demonstrate that TBBPA can decrease lipid accumulation in steatotic cells through stimulation of oxidative pathways. These data identify novel thyromimetic actions of TBBPA at the cellular level.

  12. Partial characterization of mechanism of insulin accumulation in H35 hepatoma cell nuclei.

    PubMed

    Smith, R M; Jarett, L

    1990-06-01

    The mechanism controlling insulin accumulation in nuclei of H35 hepatoma cells was investigated by incubating intact cells with 125I-labeled insulin in the presence or absence of agents that perturb different intracellular sites involved in the processing of ligand-receptor complexes. Purified nuclei were isolated, and nuclear-associated 125I-insulin was determined. Insulin accumulation in the nuclei was time and temperature dependent. Nuclear accumulation was linear and insulin-concentration dependent between 5 and 50 ng insulin/ml. However, pharmacological concentrations of insulin increased the amount of insulin translocated to the nucleus to a far greater extent than it increased total cell-associated insulin. Chloroquine, an acidotrophic agent, increased total cell-associated and intracellular insulin but had no effect on nuclear accumulation. The monovalent ionophores monensin and nigericin inhibited nuclear accumulation of insulin at low concentrations (0.5-5.0 microM) without affecting total insulin binding or intracellular accumulation. At 10 or 25 microM, monensin and nigericin also acted as acidotrophic agents and increased total insulin binding and intracellular accumulation but inhibited nuclear accumulation by a maximum of 50%. Low concentrations of monensin and nigericin were additive; maximal concentrations were not. A 23187 and valinomycin did not affect insulin binding or intracellular and nuclear accumulation of insulin. Neither depletion of ATP by sodium azide, 2,4-dinitrophenol, sodium cyanide, or oligomycin nor disruption of cytoskeletal elements by cytochalasin D or colchicine had any effect on nuclear accumulation of insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. 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.

  14. A domain of methylation change at the albumin locus in rat hepatoma cell variants.

    PubMed Central

    Orlofsky, A; Chasin, L A

    1985-01-01

    A well-differentiated rat hepatoma cell line, Fu5-5, yields variant clones whose rate of secretion of serum albumin ranges from 40 to less than 0.08 micrograms of albumin/mg of cell protein per 48 h. Clones were classified as high producers (10 to 40 micrograms/mg per 48 h), intermediate producers (1 to 10 micrograms/mg per 48 h), low producers (0.1 to 1.0 micrograms/mg per 48 h), and null variants (less than 0.1 micrograms/mg per 48 h). Albumin synthetic rates are proportional to secretion rates and range from 0.9 to less than 0.002% of total protein synthesis as measured by pulse-labeling. Steady-state albumin mRNA levels were measured by filter hybridization of fragmented, end-labeled mRNA and by Northern blotting. Message levels are proportional to albumin synthetic rates except for a high producer in which albumin mRNA is less elevated than the synthetic rate. The extent of methylation was quantitated at each of 24 CpG-containing sites or site clusters at the albumin locus. These sites span a region that contains the albumin gene as well as 10 kilobases of the 5' flank and 1 kilobase of the 3' flank. An 8-kilobase region is described, with boundaries in the 5' flank and in the middle of the gene, within which all 11 sites examined showed a correlation of undermethylation with the high-producer phenotype. In contrast, 12 of 13 sites outside of this region showed no phenotype correlation. Null variants derived from a high producer underwent de novo methylation of this domain. Six independent hybrid clones derived from the cross of a high producer with a null variant showed extinction of albumin production and hypermethylation of the domain. Apparently these cells retain the capacity for the de novo methylation of these specific sites. Images PMID:2984551

  15. Vanadium compounds discriminate hepatoma and normal hepatic cells by differential regulation of reactive oxygen species.

    PubMed

    Wang, Qin; Liu, Tong-Tong; Fu, Ying; Wang, Kui; Yang, Xiao-Gai

    2010-09-01

    Our previous study indicated that vanadium compounds can block cell cycle progression at the G1/S phase in human hepatoma HepG2 cells via a highly activated extracellular signal-regulated protein kinase (ERK) signal. To explore their differential action on normal cells, we investigated the response of an immortalized hepatic cell line, L02 cells. The results demonstrated that a higher concentration of vanadium compounds was needed to inhibit L02 proliferation, which was associated with S and G2/M cell cycle arrest. In addition, in contrast to insignificant reactive oxygen species (ROS) generation in HepG2 cells, all of the vanadium compounds resulted significant increases in both O2.- and H2O2 levels in L02 cells. At the same time, ERK and c-Jun N-terminal kinase (JNK) as well as cell division control protein 2 homolog (Cdc2) were found to be highly phosphorylated, which could be counteracted with the antioxidant N-acetylcysteine (NAC). The current study also demonstrated that both the ERK and the JNK pathways contributed to the cell cycle arrest induced by vanadium compounds in L02 cells. More importantly, it was found that although NAC can ameliorate the cytotoxicity of vanadium compounds in L02 cells, it did not decrease their cytotoxicity in HepG2 cells. It thus shed light on the potential therapeutic applications of vanadium compounds with antioxidants as synergistic agents to reduce their toxicities in human normal cells without affecting their antitumor activities in cancer cells.

  16. Palmitic and linoleic acids induce ER stress and apoptosis in hepatoma cells

    PubMed Central

    2012-01-01

    Objectives Hepatic inflammation and degeneration induced by lipid depositions may be the major cause of nonalcoholic fatty liver disease. In this study, we tried to investigate the effects of saturated and unsaturated fatty acids on hepatoma cell apoptosis. Methods H4IIE liver cells were treated with palmitic acid, linoleic acid, or both with or without the calcium-specific chelator BAPTA-AM after which the expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis, caspase-3 levels, and calcium flux were measured. Results Palmitic or linoleic acid (250 μM) induced H4IIE cell apoptosis, which required calcium flux but not caspase-3. Apoptosis was not observed when cells were co-treated with linoleic acid (125 μM) and palmitic acid (250 μM). Importantly, the release of cytochrome C from mitochondria into cytoplasm during cell apoptosis was specifically detected only when linoleic acid (125 μM), but not palmitic acid (250 μM), was added to the cells. Depletion of intracellular calcium flux by the calcium-specific chelator, BAPTA-AM, abolished linoleic acid-induced apoptosis. Moreover, in the presence of BAPTA-AM, expression of the unfolded protein response (UPR)-associated genes, CHOP, GRP78, and GRP94, was induced by linoleic acid, but not palmitic acid. Conclusions The results suggest that linoleic acid promotes cell apoptosis through the release of cytochrome C, only if the intracellular calcium flux is unperturbed and intact. These results confirm that ER stress contributes to fatty acid-induced liver cell apoptosis. PMID:22221411

  17. Apoptosis of hepatoma cells SMMC-7721 induced by Ginkgo biloba seed polysaccharide

    PubMed Central

    Chen, Qun; Yang, Gui-Wen; An, Li-Guo

    2002-01-01

    AIM: To study the apoptosis of hepatoma cells SMMC-7721 induced by polysaccharide isolated from Ginkgo biloba seed. METHODS: Ginkgo biloba seed polysaccharide (GBSP) was isolated by ethanol fractionation of Ginkgo biloba seed and purified by Sephadex G-200 chromatography. The purity of GBSP was verified by reaction with iodine-potassium iodide and ninhydrin and confirmed by UV spectrophotometer, cellulose acetate membrane electrophoresis and Sepharose 4B gel filtration chromatography. The Scanning Electron Microscope (SEM) and Flow Cytometry (FCM) were used to examine the SMMC-7721 cells with and without GBSP treatment at 500 mg/mL for 36 h. RESULTS: GBSP product obtained was of high purity with the average molecular weight of 1.86 × 105. Quantitative analysis of SMMC-7721 cells in vitro with FCM showed that the percentages of G2-M cells without and with GBSP treatment were 17.01% ± 1.28% and 11.77% ± 1.50% (P < 0.05), the debris ratio of the cells were 0.46% ± 0.12% and 0.06% ± 0.06% (P < 0.01), and the apoptosis ratio of cells was 3.84% ± 0.55% and 9.13% ± 1.48% (P < 0.01) respectively. Following GBSP treatment, microvilli of SMMC-7721 cells appeared thinner and the number of spherical cells increased markedly. Most significantly, the apoptosis bodies were formed on and around the spherical cells treated with GBSP. CONCLUSION: GBSP could potentially induce the apoptosis of SMMC-7721 cells. PMID:12378625

  18. Hepatoma-derived growth factor regulates breast cancer cell invasion by modulating epithelial--mesenchymal transition.

    PubMed

    Chen, San-Cher; Kung, Mei-Lang; Hu, Tsung-Hui; Chen, Hsuan-Yu; Wu, Jian-Ching; Kuo, Hsiao-Mei; Tsai, Han-En; Lin, Yu-Wei; Wen, Zhi-Hong; Liu, Jong-Kang; Yeh, Ming-Hsin; Tai, Ming-Hong

    2012-10-01

    Hepatoma-derived growth factor (HDGF) participates in tumourigenesis but its role in breast cancer is unclear. We set out to elucidate the expression profile and function of HDGF during breast carcinogenesis. Immunoblot and immunohistochemical studies revealed elevated HDGF expression in human breast cancer cell lines and tissues. Nuclear HDGF labelling index was positively correlated with tumour grade, stage and proliferation index, but negatively correlated with survival rate in breast cancer patients. HDGF over-expression was associated with lymph node metastasis and represented an independent prognostic factor for tumour recurrence. Gene transfer studies were performed to elucidate the influence of cellular HDGF level on the malignant behaviour and epithelial-mesenchymal transition (EMT) of breast cancer cells. Adenovirus-mediated HDGF over-expression stimulated the invasiveness and colony formation of MCF-7 cells. Moreover, HDGF over-expression promoted breast cancer cell EMT by E-cadherin down-regulation and vimentin up-regulation. Conversely, HDGF knockdown by RNA interference in MDA-MB-231 cells attenuated the malignant behaviour and elicited EMT reversal by enhancing E-cadherin expression while depleting vimentin expression. Because HDGF is a secreted protein, we evaluated the cellular function of recombinant HDGF and found that exogenously supplied HDGF enhanced the invasiveness of breast cancer cells by down-regulating E-cadherin and up-regulating vimentin at transcriptional and translational levels. In contrast, blockade of HDGF secretion with an HDGF antibody inhibited the malignant behaviours and EMT. Finally, exogenous HDGF partially reversed benzyl isothiocyanate (BITC)-induced EMT suppression. HDGF over-expression may exert a prognostic role for tumour metastasis and recurrence in breast cancer by modulating EMT. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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

    PubMed

    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; Li, Wei

    2016-01-01

    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. 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. 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. 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 new technology for systematic

  20. Forskolin inhibits the Gs-stimulated adenylate cyclase in rat ascites hepatoma AH66F cells.

    PubMed

    Miyamoto, K; Sanae, F; Koshiura, R; Matsunaga, T; Hasegawa, T; Takagi, K; Satake, T

    1989-09-01

    Forskolin increased intracellular cyclic AMP and augmented cyclic AMP formation by prostaglandin E1 (PGE1) in normal rat hepatocytes and ascites hepatoma AH66 cells. However, in AH66F cells which were derived from the AH66 cell line, the diterpene only slightly increased the cyclic AMP level, and dose-dependently inhibited the accumulation caused by PGE1. Forskolin dose-dependently activated adenylate cyclase in these membranes, and the magnitude of activation by forskolin was largest in the following order: hepatocytes, AH66 cells, and AH66F cells. This difference may be based on the number of forskolin-binding sites. The binding affinity of forskolin for each cell membrane was similar. The number and affinity of forskolin-binding sites in these cells were not influenced by 5'-guanylylimidodiphosphate [Gpp(NH)p]. In hepatocytes and AH66 cells, forskolin and other adenylate cyclase activators such as PGE1, GTP, Gpp(NH)p, F-, and Mn2+ synergistically increased the enzyme activity. In AH66F cells, the forskolin-stimulated activity was hardly influenced by the GTP analog, and forskolin diminished the activities induced by the GTP analog in a manner similar to that of diterpene alone. Forskolin (10 microM) also significantly inhibited the activities induced by PGE1, GTP, and F-. The effect of forskolin with Mn2+ was additive in AH66F cells. The data suggest that forskolin promotes the interaction between the stimulatory guanine nucleotide-binding protein and the catalytic unit in the membrane of normal hepatocytes and AH66 cells, but it interferes with the coupling in AH66F cells.

  1. Stimulatory and inhibitory effects of forskolin on adenylate cyclase in rat normal hepatocytes and hepatoma cells.

    PubMed

    Miyamoto, K; Sanae, F; Koshiura, R; Matsunaga, T; Takagi, K; Satake, T; Hasegawa, T

    1989-02-01

    Forskolin synergistically potentiated adenosine 3',5'-cyclic monophosphate formation by prostaglandin E1 (PGE1) in rat normal hepatocytes freshly prepared by collagenase digestion and rat ascites hepatoma AH66 cells, but dose-dependently inhibited the accumulation by PGE1 in AH66F cells. Forskolin activated adenylate cyclase in a dose-dependent manner in homogenates of all cell lines. In normal hepatocytes and AH66 cells, simultaneous addition of forskolin and other adenylate cyclase activators [isoproterenol (IPN), PGE1, guanosine 5'-triphosphate sodium salt (GTP), 5'-guanylylimidodiphosphate sodium salt (Gpp (NH)p), NaF, cholera toxin, islet activating protein and MnCl2] gave greater than additive responses. On the other hand, in AH66F cells, the effect of forskolin on adenylate cyclase was hardly influenced by GTP, but forskolin diminished the activities induced by high concentrations of GTP to that by the diterpene alone. Forskolin also significantly inhibited the PGE1-stimulated and the guanine nucleotide binding regulatory protein-stimulated activities. Because AH66F cells were insensitive to IPN, the combination with forskolin and IPN gave similar activity to that obtained with the diterpene alone. The effect of forskolin on the activation by manganese ion was neither synergistic nor inhibitory but was additive in AH66F cells. These results suggest that forskolin promotes the interaction between the stimulatory guanine nucleotide binding regulatory protein and the catalytic unit in normal hepatocytes and AH66 cells, but in AH66F cells forskolin interferes with the coupling of the two components of adenylate cyclase.

  2. Altered adrenergic response and specificity of the receptors in rat ascites hepatoma AH130.

    PubMed

    Sanae, F; Miyamoto, K; Koshiura, R

    1989-11-15

    Adenylate cyclase activation through adrenergic receptors in rat ascites hepatoma (AH) 130 cells in response to adrenergic drugs was studied, and receptor binding and displacement were compared with those of normal rat hepatocytes. Epinephrine (Epi) and norepinephrine (NE) activated AH130 adenylate cyclase about half as much as isoproterenol (IPN) but equaled IPN after treatment with the alpha-antagonist phentolamine or islet-activating protein (IAP). The three catecholamines in hepatocytes were similar regardless of phentolamine or IAP. These catecholamines activated adenylate cyclase in order of IPN greater than NE greater than Epi in AH130 cells but IPN greater than Epi greater than NE in hepatocytes. We then used the alpha 1-selective ligand [3H]prazosin, the alpha 2-selective ligand [3H]clonidine, and the beta-ligand [125I]iodocyanopindolol [( 125I]ICYP), and found that AH130 cells had few prazosin-binding sites, about eight times as many clonidine-binding sites with high affinity, and many more ICYP-binding sites than in hepatocytes. The dissociation constant (Ki) of the beta 1-selective drug metoprolol by Hofstee plots for AH130 cells was lower than that for hepatocytes. The inhibition of specific ICYP binding by the beta 2-selective agonist salbutamol for AH130 cells gave only one Ki value which was much higher than both high and low Ki values of the drug for hepatocytes. These findings indicate that the alpha- and beta-adrenergic receptors in hepatocytes are predominantly alpha 1-type and beta 2-type, but that those in AH130 cells are predominantly alpha 2-type and beta 1-type, and the low adrenergic response of AH130 cells is due to the dominant appearance of alpha 2-adrenergic receptors, linked with the inhibitory guanine-nucleotide binding regulatory protein, instead of alpha 1-adrenergic receptors, and beta 1-adrenergic receptors with low affinity for the hormone.

  3. Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells

    PubMed Central

    Ullio, Chiara; Brunk, Ulf T; Urani, Chiara; Melchioretto, Pasquale; Bonelli, Gabriella; Baccino, Francesco M; Autelli, Riccardo

    2015-01-01

    Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other iron-binding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX. PMID:26566051

  4. Prognostic value of nuclear hepatoma-derived growth factor (HDGF) localization in patients with breast cancer.

    PubMed

    Chen, Xiaoyan; Yun, Jun; Fei, Fei; Yi, Jun; Tian, Ruifeng; Li, Sanzhong; Gan, Xiaoqiang

    2012-08-15

    Hepatoma-derived growth factor (HDGF) plays an important role in tumor progression. Highly expressed HDGF has been found to indicate poor prognosis in many cancers. However, no information is available regarding the prognostic value of nuclear or cytoplasmic HDGF staining level in breast cancer. In the present study, the nuclear or cytoplasmic HDGF staining level was investigated in 86 patients with primary breast cancer by immunohistochemistry; the relationship between nuclear or cytoplasmic HDGF staining level and clinicopathological parameters was examined by Two-tailed Mann-Whitney U-test or Krustal-Wallis. The prognostic value of nuclear or cytoplasmic HDGF staining level in disease-free survival and overall survival was analyzed by Kaplan-Meier methods and log-rank test. We found that the percentage of cases with strong nuclear HDGF staining level was significantly higher in the cases with high tumor grade, high stage, high proliferation index (Ki-67 index>20%), as well as in those with lymph node invasion and recurrence (p<0.05) compared to those without. No significant correlation was found between cytoplasmic HDGF expression and any clinicopathological variables. In addition, disease-free survival and overall survival were significantly lower in patients with high nuclear HDGF expression (level 2) than in those with low nuclear HDGF expression (level 0 and level 1). Further Cox multivariate analysis showed that high nuclear HDGF expression is an independent factor for indicating poor prognosis in breast cancer patients. No significant difference in disease-free survival rate and overall survival was found between different cytoplasmic HDGF staining levels. All these findings suggest that increased nuclear HDGF expression is involved in tumor progression and might be used as a new prognosticator for breast cancer. Crown Copyright © 2012. Published by Elsevier GmbH. All rights reserved.

  5. 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

  6. Genetic analysis of a transcriptional activation pathway by using hepatoma cell variants.

    PubMed Central

    Bulla, G A; Fournier, R E

    1994-01-01

    A hierarchy of liver-enriched transcription factors plays an important role in activating expression of many hepatic genes. In particular, hepatocyte nuclear factor 4 (HNF-4) is a major activator of the gene encoding HNF-1, and HNF-1 itself activates expression of more than 20 liver genes. To dissect this activation pathway genetically, we prepared somatic cell variants that were deficient in expression of the liver-specific alpha 1-antitrypsin (alpha 1AT) gene, which requires both HNF-1 and HNF-4 for high-level gene activity. This was accomplished in two steps. First, hepatoma transfectants that stably expressed two selectable markers under alpha 1AT promoter control were prepared; second, variant sublines that could no longer express either transgene were isolated by direct selection. In this report, we demonstrate that the variants contain defects in the HNF-4/HNF-1 activation pathway. These defects functioned in trans, as expression of many liver genes was affected, but the variant phenotypes were recessive to wild type in somatic cell hybrids. Three different variant classes could be discriminated by their phenotypic responses to ectopic expression of either HNF-4 or HNF-1. Two variant clones appeared specifically deficient in HNF-4 expression, as transfection with an HNF-4 expression cassette fully restored their hepatic phenotypes. Another line activated HNF-1 in response to forced HNF-4 expression, but activation of downstream genes failed to occur. One clone was unresponsive to either HNF-1 or HNF-4. Using the variants, we demonstrate further that the chromosomal genes encoding alpha 1AT, aldolase B, and alpha-fibrinogen display strict requirements for HNF-1 activation in vivo, while other liver genes were unaffected by the presence or absence of HNF-1 or HNF-4. We also provide evidence for the existence of an autoregulatory loop in which HNF-1 regulates its own expression through activation of HNF-4. Images PMID:7935424

  7. Ascorbic acid enhances iron-induced ferritin translation in human leukemia and hepatoma cells.

    PubMed

    Toth, I; Rogers, J T; McPhee, J A; Elliott, S M; Abramson, S L; Bridges, K R

    1995-02-10

    Ascorbate is an important cofactor in many cellular metabolic reactions and is intimately linked to iron homeostasis. Continuously cultured cells are ascorbate deficient due to the lability of the vitamin in solution and to the fact that daily supplementation of media with ascorbate is unusual. We found that ascorbate repletion alone did not alter ferritin synthesis. However, ascorbate-replete human hepatoma cells, Hep3B and HepG2, as well as K562 human leukemia cells achieved a substantially higher cellular ferritin content in response to a challenge with iron than did their ascorbate-deficient counterparts grown under standard culture conditions. Most of the elevation in ferritin content was due to an increase in de novo ferritin synthesis of greater than 50-fold, as shown by in vivo labeling with [35S]methionine and immunoprecipitation. RNA-blot analysis showed only minor changes in steady state levels of ferritin mRNA, suggesting that ascorbate enhances iron-induced ferritin synthesis primarily by post-transcriptional events. Transient gene expression experiments using chloramphenicol acetyltransferase reporter gene constructs showed that the ascorbate effect on ferritin translation is not mediated through the stem-loop near the translational start site that transduces ferritin synthesis in response to cytokines. The data suggest that ascorbate possibly modifies the action of the iron-responsive element on ferritin translation, although more precise structure-function studies are needed to clarify this issue. These data demonstrate a novel role of ascorbate as a signaling molecule in post-transcriptional gene regulation. The mechanism by which ascorbate modulates cellular iron metabolism is complex and requires additional detailed investigation.

  8. Hexokinase isoenzymes from the Novikoff hepatoma. Purification, kinetic and structural characterization, with emphasis on hexokinase C.

    PubMed Central

    Radojković, J; Ureta, T

    1987-01-01

    The purification to homogeneity of hexokinases B and C from the cytosol of rat Novikoff hepatoma was achieved by a protocol using an initial chromatography on Blue 2-agarose to separate the isoenzymes from each other. After that step each hexokinase was subjected to chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-300, followed by re-chromatography on hydroxyapatite. The final preparations of hexokinases B and C had specific activities of 86 and 23.5 units/mg of protein respectively, and gave single bands on electrophoresis under non-denaturing conditions or in SDS/polyacrylamide gels. Mr values of about 100,000 were found for both isoenzymes either by Sephacryl S-300 chromatography or by SDS/polyacrylamide-gel electrophoresis. Values of apparent Km for glucose and ATP of pure hexokinase B were similar to those reported for the enzyme from other sources. The apparent Km value for glucose of hexokinase C was 0.025 mM. Marked inhibition of hexokinase C by glucose concentrations above 0.2 mM was found. The effect was partially relieved by ATP concentrations above 1 mM and was independent of pH. Glucose 6-phosphate was inhibitory, but the Ki value (0.18 mM) is higher than those reported for other animal hexokinases. The amino acid composition of hexokinase C was found to be similar to those reported for hexokinases B and D. Also, an immune serum directed against hexokinase A was able, at low dilutions, to bind hexokinases B and C. An immune serum directed against hexokinase C was able, at low dilutions, to bind hexokinase B and also, but weakly, hexokinase A. Images Fig. 3. PMID:3593283

  9. 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

  10. 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.

  11. Induction of phenolsulfotransferase expression by phenolic acids in human hepatoma HepG2 cells.

    PubMed

    Yeh, Chi-Tai; Huang, Shang-Ming; Yen, Gow-Chin

    2005-06-15

    Phenolic acids are antioxidant phenolic compounds, widespread in plant foods, which contribute significant biological and pharmacological properties; some have demonstrated a remarkable ability to alter sulfate conjugation. However, the modulation mechanisms of antioxidant phenolic acids on phenolsulfotransferase activity have not yet been described. In the present study, the human hepatoma cell line, HepG2, was used as a model to investigate the effect of antioxidant phenolic acids on enzymatic activity and expression of one of the major phase II sulfate conjugation enzymes, P-form phenolsulfotransferase (PST-P). The results showed that gallic acid, gentisic acid, p-hydroxybenzoic acid, and p-coumaric acid increased PST-P activity, in a dose-dependent manner. A maximum of 4- and 5-fold induction of PST-P activity was observed for both gallic acid and gentisic acid; however, they showed an adverse effect on cell growth at higher concentrations. A 2- or 2.5-fold increase of PST-P activity was found with either p-coumaric or p-hydroxybenzoic acid treatment, whereas no significant effect was found for ferulic acid treatment. PST-P induction, by gallic acid, was further confirmed, using reverse transcription PCR and Western blotting techniques to measure mRNA expression and protein translation. A significant correlation (r = 0.74, p < 0.01) between the expressions of PST-P mRNA and the corresponding PST-P activity was observed. Thus, gallic acid increased PST-P protein expression in HepG2 cells, in a dose- and time-dependent manner. The results demonstrated that certain antioxidant phenolic acids could induce PST-P activity in HepG2 cells, by promoting PST-P mRNA and protein expression, suggesting a novel mechanism by which phenolic acids may be implicated in phase II sulfate conjugation.

  12. 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.

  13. An Occult Hepatitis B-Derived Hepatoma Cell Line Carrying Persistent Nuclear Viral DNA and Permissive for Exogenous Hepatitis B Virus Infection

    PubMed Central

    Lin, Chih-Lang; Chien, Rong-Nan; Lin, Shi-Ming; Ke, Po-Yuan; Lin, Chen-Chun; Yeh, Chau-Ting

    2013-01-01

    Occult hepatitis B virus (HBV) infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg). Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV) and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2). Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection. PMID:23734258

  14. An occult hepatitis B-derived hepatoma cell line carrying persistent nuclear viral DNA and permissive for exogenous hepatitis B virus infection.

    PubMed

    Lin, Chih-Lang; Chien, Rong-Nan; Lin, Shi-Ming; Ke, Po-Yuan; Lin, Chen-Chun; Yeh, Chau-Ting

    2013-01-01

    Occult hepatitis B virus (HBV) infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg). Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV) and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2). Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection.

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

    PubMed

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

    2004-10-01

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

  16. Differentially profiling the low-expression transcriptomes of human hepatoma using a novel SSH/microarray approach

    PubMed Central

    Pan, Yi-Shin; Lee, Yun-Shien; Lee, Yung-Lin; Lee, Wei-Chen; Hsieh, Sen-Yung

    2006-01-01

    Background The main limitation in performing genome-wide gene-expression profiling is the assay of low-expression genes. Approaches with high throughput and high sensitivity for assaying low-expression transcripts are urgently needed for functional genomic studies. Combination of the suppressive subtractive hybridization (SSH) and cDNA microarray techniques using the subtracted cDNA clones as probes printed on chips has greatly improved the efficiency for fishing out the differentially expressed clones and has been used before. However, it remains tedious and inefficient sequencing works for identifying genes including the great number of redundancy in the subtracted amplicons, and sacrifices the original advantages of high sensitivity of SSH in profiling low-expression transcriptomes. Results We modified the previous combination of SSH and microarray methods by directly using the subtracted amplicons as targets to hybridize the pre-made cDNA microarrays (named as "SSH/microarray"). mRNA prepared from three pairs of hepatoma and non-hepatoma liver tissues was subjected to the SSH/microarray assays, as well as directly to regular cDNA microarray assays for comparison. As compared to the original SSH and microarray combination assays, the modified SSH/microarray assays allowed for much easier inspection of the subtraction efficiency and identification of genes in the subtracted amplicons without tedious and inefficient sequencing work. On the other hand, 5015 of the 9376 genes originally filtered out by the regular cDNA microarray assays because of low expression became analyzable by the SSH/microarray assays. Moreover, the SSH/microarray assays detected about ten times more (701 vs. 69) HCC differentially expressed genes (at least a two-fold difference and P < 0.01), particularly for those with rare transcripts, than did the regular cDNA microarray assays. The differential expression was validated in 9 randomly selected genes in 18 pairs of hepatoma/non-hepatoma

  17. Molecular cloning of cDNA for rat argininosuccinate lyase and its expression in rat hepatoma cell lines.

    PubMed Central

    Lambert, M A; Simard, L R; Ray, P N; McInnes, R R

    1986-01-01

    Using antibody and plaque hybridization screening, we isolated rat argininosuccinate lyase (AS lyase) cDNA clones from a liver cDNA library prepared in the phage expression vector lambda gt11. Five overlapping cDNAs covering 1.7 kilobases of the estimated 2.0-kilobase AS lyase mRNA were characterized and confirmed as AS lyase sequences by hybrid selection. We examined the differential expression of AS lyase in rat liver and four rat hepatoma cell lines (7800C1, H4, HTC, and MH1C1). These cells exhibited a 60-fold range of AS lyase enzyme activity, with a direct correlation between activity, amount of AS lyase immunoreactive protein, and quantity of specific AS lyase mRNA. These observations suggest that the differences in AS lyase expression between rat liver and the hepatoma cell lines result from variations in AS lyase transcriptional activity or alterations in nuclear processing of AS lyase RNA. Images PMID:3785176

  18. Carnitine palmitoyl transferase activity in Morris Hepatoma 7777 mitochondria and its sensitivity to malonyl CoA inhibition

    SciTech Connect

    Woldegiorgis, G.; Shrago, E.

    1986-05-01

    Earlier reports in the literature have indicated no detectable Carnitine Palymitoyl Transferase (CPT) activity in homogenates prepared from Morris Hepatoma 7777. In its study CPT activity in isolated mitochondria (mito) was measured by butanol extraction of the (/sup 3/H)palmitoyl carnitine formed as outlined by Bremer et al. Contrary to the earlier work where no appreciable activity of CPT was observed the authors find significant levels of CPT (2.6 nMol/min/mg protein) in isolated mito from Morris Hepatoma 7777 (MH 7777). The level of CPT activity observed in MH 7777 mito was, however, 36% lower compared to the host liver CPT activity (4.1 nMol/min/mg protein). The enzyme in MH 7777 mito showed 83% inhibition in the presence of 10 ..mu..M malonyl CoA, in agreement with the degree of sensitivity observed with the host liver isolated mito. On freeze thawing host mito, total CPT activity increased and the sensitivity of the enzyme to malonyl CoA decreased. Frozen thawed MH 7777 mito showed a similar response to malonyl CoA but no change in the total CPT level was observed. The authors results establish for the first time the presence of a malonyl CoA sensitive CPT in MH 7777 mito, which may have slightly different properties from normal due to the membrane environment of the enzyme.

  19. Critical roles of cellular glutathione homeostasis and jnk activation in andrographolide-mediated apoptotic cell death in human hepatoma cells.

    PubMed

    Ji, Lili; Shen, Kaikai; Jiang, Ping; Morahan, Grant; Wang, Zhengtao

    2011-08-01

    Andrographolide (ANDRO), isolated from the traditional herbal medicine Andrographis paniculata, is reported to have the potential therapeutic effects for hepatocellular carcinoma (HCC) in our previous reports. Here, we investigated the mechanism of ANDRO-mediated apoptotic cell death, focusing on the involvement of cellular reduced glutathione (GSH) homeostasis and c-Jun NH(2) -Terminal kinase (JNK). Buthionine sulfoximine (BSO), an inhibitor of cellular GSH biosynthesis, significantly augmented ANDRO-induced cytotoxicity in hepatoma Hep3B and HepG2 cells. BSO depleted cellular GSH, and augmented ANDRO-induced apoptosis, inhibition of colony formation and JNK activation in Hep3B cells. All these effects could be reversed by GSH monoethyl ester (GSH.EE), whose deacetylation replenishes cellular GSH. BSO also augmented ANDRO-induced activation of apoptosis signal-regulating kinase 1 (ASK1), mitogen-activated protein kinase kinase-4 (MKK4) and c-Jun, which are all up-stream or down-stream signals of JNK. Further results showed that JNK inhibitor SP600125 and 420116 both reversed ANDRO-induced cytotoxicity, and SP600125 also decreased ANDRO-increased intracellular GSH and GCL activity. Finally, we showed that in nude mice bearing xenografted Hep3B tumors, BSO improved the inhibition of tumor growth by ANDRO. Taken together, our results suggest that there is a crosstalk between JNK activation and cellular GSH homeostasis, and ANDRO targets this to induce cytotoxicity in hepatoma cells.

  20. 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

    2015-09-18

    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.

  1. 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

  2. Degradable and biocompatible nanoparticles decorated with cyclic RGD peptide for efficient drug delivery to hepatoma cells in vitro.

    PubMed

    Loyer, Pascal; Bedhouche, Wahib; Huang, Zhi Wei; Cammas-Marion, Sandrine

    2013-10-01

    Amphiphilic derivatives of poly(benzyl malate) were synthesized and characterized with the aim of being used as degradable and biocompatible building blocks for the design of functional nanoparticles (NPs). An anti-cancer model drug, doxorubicin, has been successfully encapsulated into the prepared NPs and its release profile has been evaluated in water and in culture medium. NPs bearing biotin molecules were prepared either for site-specific drug delivery via the targeting of biotin receptors overexpressed on the surface of several cancer cells, or for grafting biotinylated cyclic RGD peptide onto their surface using the strong and highly specific interactions between biotin and the streptavidin protein. We have shown that this binding did not affect dramatically the physico-chemical properties of the corresponding NPs. Cyclic RGD grafted fluorescent NPs were more efficiently uptaken by the HepaRG hepatoma cells than biotinylated fluorescent NPs. Furthermore, the targeting of HepaRG hepatoma cells with NPs bearing cyclic RGD was very efficient and much weaker for HeLa and HT29 cell lines confirming that cyclic RGD is a suitable targeting agent for liver cells. Our results also provide a new mean for rapid screening of short hepatotropic peptides in order to design NPs showing specific liver targeting properties.

  3. Active compounds from Saussurea lappa Clarks that suppress hepatitis B virus surface antigen gene expression in human hepatoma cells.

    PubMed

    Chen, H C; Chou, C K; Lee, S D; Wang, J C; Yeh, S F

    1995-05-01

    We have examined the antiviral activity of the crude extract prepared from the root of Saussurea lappa Clarks, a Chinese medicinal herb which is widely used for many illnesses including cancer. Two active components, costunolide and dehydrocostus lactone, were identified which show strong suppressive effect on the expression of the hepatitis B surface antigen (HBsAg) in human hepatoma Hep3B cells, but have little effect on the viability of the cells. Both costunolide and dehydrocostus lactone suppress the HBsAg production by Hep3B cells in a dose-dependent manner with IC50s of 1.0 and 2.0 microM, respectively. Northern blotting analysis shows that the suppression of HBsAg gene expression by both costunolide and dehydrocostus lactone were mainly at the mRNA level. Furthermore, the suppressive effect of costunolide and dehydrocostus lactone on HBsAg and hepatitis B e antigen (HBeAg), a marker for hepatitis B viral genome replication in human liver cells, was also observed in another human hepatoma cell line HepA2 which was derived from HepG2 cells by transfecting a tandemly repeat hepatitis B virus (HBV) DNA. Similarly, the mRNA of HBsAg in HepA2 cells was also suppressed by these two compounds. Our findings suggest that costunolide and dehydrocostus lactone may have potential to develop as specific anti-HBV drugs in the future.

  4. Retroviral-mediated gene transfer of human phenylalanine hydroxylase into NIH 3T3 and hepatoma cells

    SciTech Connect

    Ledley, F.D.; Grenett, H.E.; McGinnis-Shelnutt, M.; Woo, S.L.C.

    1986-01-01

    Phenylketonuria (PKU) is caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). A full-length human PAH cDNA sequence has been inserted into pzip-neoSV(X), which is a retroviral vector containing the bacterial neo gene. The recombinant has been transfected into Psi2 cells, which provide synthesis of the retroviral capsid. Recombinant virus was detected in the culture medium of the transfected Psi2 cells, which is capable of transmitting the human PAH gene into mouse NIH 3T3 cells by infection leading to stable incorporation of the recombinant provirus. Infected cells express PAH mRNA, immunoreactive PAH protein, and exhibit pterin-dependent phenylaline hydroxylase activity. The recombinant virus is also capable of infecting a mouse hepatoma cell line that does not normal synthesize PAH. PAH activity is present in the cellular extracts and the entire hydroxylation system is reconstituted in the hepatoma cells infected with the recombinant viruses. Thus, recombinant viruses containing human PAH cDNA provide a means for introducing functional PAH into mammalian cells of hepatic origin and can potentially be introduced into whole animals as a model for somatic gene therapy for PKU.

  5. Neutral red uptake inhibition in adhered and adhering rat hepatoma-derived Fa32 cells to predict human toxicity.

    PubMed

    Dierickx, Paul J; Scheers, Ellen M

    2002-01-01

    The cytotoxicity of the MEIC (Multicentre Evaluation of In vitro Cytotoxicity) reference chemicals was investigated by measuring the neutral red uptake inhibition in adhered and adhering rat hepatoma-derived Fa32 cells. The adhered cells were seeded and then treated and the adhering cells were treated simultaneously upon seeding. Five of the 44 test chemicals were twofold more toxic in adhering cells; ethylene glycol was 28-fold more toxic and mercuric chloride was 5.2-fold more toxic than in adhered cells. The cytotoxicity of dithiothreitol was altered in the same way as that of ethylene glycol, probably by interacting with calcium. When the neutral red uptake inhibition was compared with human toxicity, the correlation coefficient for adhering cells was almost identical to that obtained previously in human hepatoma-derived Hep G2 cells and slightly higher for adhered cells. The Hep G2 assay was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. An obviously better correlation was obtained when the strong intoxicant mercuric chloride was withdrawn from the comparison, both for the adhered and the adhering cells. Altogether, the results can be integrated very well with the basal cytotoxicity concept.

  6. A study of chromosomal changes associated with amplified dihydrofolate reductase genes in rat hepatoma cells and their dedifferentiated variants

    PubMed Central

    1984-01-01

    We have examined the karyological consequences of dihydrofolate reductase gene amplification in a series of six rat hepatoma cell lines, all derived from the same clone. Cells of three of these lines express a series of liver-specific functions whereas those of three others fail to express these functions. Cells of each line have been subjected to stepwise selection for methotrexate resistance and, in most cases, resistance is associated with a 40-50-fold amplification of sequences hybridizing to a dihydrofolate reductase cDNA probe. In one line no modified chromosome is observed, whereas in two others the amplified genes are associated with an expanded chromosomal region. R- banding analysis of these karyotypes showed that few changes have occurred. These observations apply to two of the well-differentiated lines, and to a variant able to revert to the differentiated state. In contrast, in the two stably dedifferentiated hepatoma cell lines, amplified dihydrofolate reductase genes are found on large chromosomes of variable size, on ring chromosomes, and on chromosomes containing terminal, median, or multiple centromeres. We conclude that the nature of the chromosomal changes associated with dihydrofolate reductase gene amplification are the result of differences in cell lines rather than in the protocols employed for selection. PMID:6746737

  7. 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

  8. Amphiregulin impairs apoptosis-stimulating protein 2 of p53 overexpression-induced apoptosis in hepatoma cells.

    PubMed

    Liu, Kai; Lin, Dongdong; Ouyang, Yabo; Pang, Lijun; Guo, Xianghua; Wang, Shanshan; Zang, Yunjin; Chen, Dexi

    2017-03-01

    Overexpression of apoptosis-stimulating protein 2 of p53 (ASPP2) induces apoptotic cell death in hepatoma cells (e.g. HepG2 cells) by enhancing the transactivation activity of p53, but long-term ASPP2 overexpression fails to induce more apoptosis since activation of the epidermal growth factor/epidermal growth factor receptor/SOS1 pathway impairs the pro-apoptotic role of ASPP2. In this study, in recombinant adenovirus-ASPP2-infected HepG2 cells, ASPP2 overexpression induces amphiregulin expression in a p53-dependent manner. Although amphiregulin initially contributes to ASPP2-induced apoptosis, it eventually impairs the pro-apoptotic function of ASPP2 by activating the epidermal growth factor/epidermal growth factor receptor/SOS1 pathway, leading to apoptosis resistance. Moreover, blocking soluble amphiregulin with a neutralizing antibody also significantly increased apoptotic cell death of HepG2 cells due to treatment with methyl methanesulfonate, cisplatin, or a recombinant p53 adenovirus, suggesting that the function of amphiregulin involved in inhibiting apoptosis may be a common mechanism by which hepatoma cells escape from stimulus-induced apoptosis. Thus, our data elucidate an apoptosis-evasion mechanism in hepatocellular carcinoma and have potential implications for hepatocellular carcinoma therapy.

  9. Influence of BGP-15, a nicotinic amidoxime derivative, on the vascularization and growth of murine hepatoma xenografts.

    PubMed

    Kardon, Tamás; Nagy, Gábor; Csala, Miklós; Kiss, András; Schaff, Zsuzsa; Nagy, Péter Literáti; Wunderlich, Livius; Bánhegyi, Gábor; Mandl, József

    2006-01-01

    The expression of vascular endothelial growth factor (VEGF), a key regulator of angiogenesis, is controlled by the oxygen supply. Previous observations suggested that nicotinic amidoxime derivatives (i.e. BGP-15) might interfere with the induction of hypoxia-sensitive genes. Hence, the effect of BGP-15 on angiogenesis was studied in Hepa 1c1c7 tumor xenografts. Hepa 1c1c7 hepatoma cells were implanted under the dorsal skin of female CD-1-nu/nu immunodeficient mice. One group of animals was given 100 mg/kg body weight/day BGP-15 intraperitoneally during tumor development. Vascularization, apoptotic and mitotic indices were determined by the histological and immunohistochemical analysis of the tumors. VEGF and GLUT-1 expressions were measured by Northern blot. The in vivo administration of BGP-15 resulted in a decrease in tumor weight and mitotic index, while it did not affect the apoptotic rate in the xenograft. Furthermore, BGP-15 treatment depressed microvascular density and the level of VEGF mRNA by 50%, and similarly decreased GLUT-1 mRNA levels. These findings suggest that BGP-15 suppresses hepatoma development by affecting angiogenesis.

  10. Apoptosis-inducing activity of cisplatin (CDDP) against human hepatoma and oral squamous cell carcinoma cell lines.

    PubMed

    Okamura, Masahiko; Hashimoto, Ken; Shimada, Jun; Sakagami, Hiroshi

    2004-01-01

    The sensitivity of human hepatoma (HepG2) and oral squamous cell carcinoma (HSC-2) cell lines against various apoptosis-inducing agents was compared. HepG2 cells were generally more resistant to an oxidant (H2O2), antioxidants (sodium ascorbate, gallic acid, epigallocatechin gallate) and anticancer drugs (doxorubicin, methotrexate, cisplatin (CDDP), etoposide, 5-fluoro-2,4(1H,3H)-pyrimidinedione (5-FU), peplomycin sulfate) as compared to HSC-2 cells. Lower concentrations of CDDP, but not other anticancer drugs, induced comparable cytostatic effects on both HSC-2 and HepG2 cells. CDDP induced internucleosomal DNA fragmentation and activation of caspases 3, 8 and 9 in HepG2 cells. On the other hand, CDDP did not induce DNA fragmentation and activated caspase 3 only marginally in HSC-2 cells. Combination treatment with CDDP (10 microM) and 5-FU (100 microM) additively activated all three caspases in HepG2 cells, but not in HSC-2 cells. The present study demonstrated the chemotherapeutic potential of combined treatment of CDDP and 5-FU against hepatoma cells and the considerable variation of drug sensitivity between cancer cell lines.

  11. [Mechanism of regulation of hepatoma cell cycle by XPD/P44 subcomplex : an in vitro experiment].

    PubMed

    Wang, Hong-yun; Xiong, Gao-fei; Wu, Bo-lin; Zhang, Ji-xiang

    2008-07-22

    To explore the effects of xeroderma pigmentosum group D(XPD)/P44 subcomplex on the cell cycle of the hepatoma cells. Human hematoma cells of the line SMMC-7721 were cultured and transfected with human XPD gene by Lipofectamine and 2 strains with stably transfected plasmid pEGFG-N2 and stably transfected recombinant plasmid pEGFG-N2/XPD were selected. After stably transfection,the antisense oligonucleotides of P44 were added to treat the stably transfected cells. The cells were divided into 6 groups: Group (1) (control group), Group (2) transfected with the blank plasmid pEGFP-N2, Group (3) transfected with the recombinant plasmid pEGFP-N2/XPD, Group (4) transfected with ASODN complementary to the translation initiation site of pEGFP-N2/XPD, Group (5) transfected with antisense oligodeoxynucleotides (ASODN) complementary to the translation terminal site of pEGFP-N2/XPD, and Group (6) transfected with ASODN complementary to the translation exon5 site of pEGFP-N2/XPD. The expression levels of wild-type P44, XPD, cdk7, cdk2, c-myc, and cdc25A were detected by RT-PCR and Western blotting. The cell growth and the cell cycle were examined by MTT and flow cytometry (FCM). The P44 and XPD mRNA expression levels of Group (4) were significantly higher than those of Groups (1) and (2) (both P < 0.01). Western blotting indicated that the changes of P44 and XPD protein expression levels were consistent with those of their mRNAs respectively; while the mRNA and protein expression levels of cdk7, cdk2, c-myc, and cdc25A were all decreased. MTF method showed that the hepatoma cells grew slowly, FCM showed that the number of the cells arrested at the G1 stage of Group (3) were higher than those of Groups (1) and (2). After the blockage of P44 gene expression, the expression levels of XPD mRNA and protein were decreased. The XPD mRNA and protein expression levels of Groups (4), (5), and (6) were significantly higher than those of Group (3) (all P < 0.01). The mRNA and protein

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

    PubMed

    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 of

  13. Antisense oligonucleotide targeting at the initiator of hTERT arrests growth of hepatoma cells

    PubMed Central

    Liu, Su-Xia; Sun, Wen-Sheng; Cao, Ying-Lin; Ma, Chun-Hong; Han, Li-Hui; Zhang, Li-Ning; Wang, Zhen-Guang; Zhu, Fa-Liang

    2004-01-01

    AIM: To evaluate the inhibitory effect of antisense phosphorothioate oligonucleotide (asON) complementary to the initiator of human telomerase catalytic subunit (hTERT) on the growth of hepatoma cells. METHODS: The as-hTERT was synthesized by using a DNA synthesizer. HepG2.2.15 cells were treated with as-hTERT at the concentration of 10 μmol/L. After 72 h, these cells were obtained for detecting growth inhibition, telomerase activity using the methods of MTT, TRAP-PCR-ELISA, respectively. BALB/c(nu/nu) mice were injected HepG2.2.15 cells and a human-nude mice model was obtained. There were three groups for anti-tumor activity study. Once tumors were established, these animals in the first group were administered as-hTERT and saline. Apoptosis of tumor cells was detected by FCM. In the 2nd group, the animals were injected HepG2.2.15 cells together with as-hTERT. In the third group, the animals were given as-hTERT 24 hours postinjection of HepG2.2.15 cells. The anti-HBV effects were assayed with ELISA in vitro and in vivo. RESULTS: Growth inhibition was observed in cells treated with as-hTERT in vitro. A significant different in the value of A570 - A630 was found between cells treated with as-hTERT and control (P < 0.01) by MTT method. The telomerase activity of tumor cells treated with as-hTERT was reduced, the value of A450 nm was 0.42 compared to control (1.49) with TRAP-PCR-ELISA. The peak of apoptosis in tumor cells given as-hTERT was 21.12%, but not seen in saline-treated control. A prolonged period of carcinogenesis was observed in the second and third group animals. There was inhibitory effect on the expression of HBsAg and HBeAg in vivo and in vitro. CONCLUSION: As-hTERT has an anti-tumor activity, which may be useful for gene therapy of tumors. PMID:14760759

  14. Observation of DNA damage of human hepatoma cells irradiated by heavy ions using comet assay

    PubMed Central

    Qiu, Li-Mei; Li, Wen-Jian; Pang, Xin-Yue; Gao, Qing-Xiang; Feng, Yan; Zhou, Li-Bin; Zhang, Gao-Hua

    2003-01-01

    AIM: Now many countries have developed cancer therapy with heavy ions, especially in GSI (Gesellschaft fürSchwerionenforschung mbH, Darmstadt, Germany), remarkable results have obtained, but due to the complexity of particle track structure, the basic theory still needs further researching. In this paper, the genotoxic effects of heavy ions irradiation on SMMC-7721 cells were measured using the single cell gel electrophoresis (comet assay). The information about the DNA damage made by other radiations such as X-ray, γ-ray, UV and fast neutron irradiation is very plentiful, while little work have been done on the heavy ions so far. Hereby we tried to detect the reaction of liver cancer cells to heavy ion using comet assay, meanwhile to establish a database for clinic therapy of cancer with the heavy ions. METHODS: The human hepatoma cells were chosen as the test cell line irradiated by 80Mev/u 20Ne10+ on HIRFL (China), the radiation-doses were 0, 0.5, 1, 2, 4 and 8 Gy, and then comet assay was used immediately to detect the DNA damages, 100-150 cells per dose-sample (30-50 cells were randomly observed at constant depth of the gel). The tail length and the quantity of the cells with the tail were put down. EXCEL was used for statistical analysis. RESULTS: We obtained clear images by comet assay and found that SMMC-7721 cells were all damaged apparently from the dose 0.5 Gy to 8 Gy (t-test: P < 0.001, vs control). The tail length and tail moment increased as the doses increased, and the number of cells with tails increased with increasing doses. When doses were higher than 2 Gy, nearly 100% cells were damaged. Furthermore, both tail length and tail moment, showed linear equation. CONCLUSION: From the clear comet assay images, our experiment proves comet assay can be used to measure DNA damages by heavy ions. Meanwhile DNA damages have a positive correlation with the dose changes of heavy ions and SMMC-7721 cells have a great radiosensitivity to 20Ne10+. Different

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

    PubMed Central

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

    2009-01-01

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

  16. Involvement of the prostaglandin E receptor EP2 in paeoniflorin-induced human hepatoma cell apoptosis.

    PubMed

    Hu, Shanshan; Sun, Wuyi; Wei, Wei; Wang, Di; Jin, Juan; Wu, Jingjing; Chen, Jingyu; Wu, Huaxun; Wang, Qingtong

    2013-02-01

    Prostaglandin E2 (PGE2) has been shown to play an important role in tumor development and progression. PGE2 mediates its biological activity by binding any one of four prostanoid receptors (EP1 through EP4). The present study was designed to determine the role of the EP2 receptor during the proliferation and apoptosis of human HepG2 and SMMC-7721 hepatoma cell lines and the effect of paeoniflorin, a monoterpene glycoside. The proliferation of HepG2 and SMMC-7721 cells was determined by methyl thiazolyl tetrazolium after exposure to the selective EP2 receptor agonists butaprost and paeoniflorin. Apoptosis of HepG2 and SMMC-7721 cells was also quantified by flow cytometry with annexin V-fluorescein isothiocyanate and propidium iodide staining. The expression levels of Bcl-2 and Bax were quantified by western blotting and immunohistochemistry. The expression of the EP2 receptor and cysteine-aspartic acid protease (caspase)-3 was determined by western blotting. Butaprost significantly increased proliferation in HepG2 and SMMC-7721 cells. Paeoniflorin significantly inhibited the proliferation of HepG2 and SMMC-7721 cells stimulated by butaprost at multiple time points (24, 48, and 72 h). Paeoniflorin induced apoptosis in HepG2 and SMMC-7721 cells, which was quantified by annexin-V and propidium iodide staining. Our results indicate that the expression of the EP2 receptor and Bcl-2 was significantly increased, whereas that of Bax and cleaved caspase-3 was decreased in HepG2 and SMMC-7721 cells after stimulation by butaprost. Paeoniflorin significantly decreased the expression of the EP2 receptor and Bcl-2 and increased Bax and caspase-3 activation in HepG2 and SMMC-7721 cells on addition of butaprost. Our results show that the PGE2 receptor subtype EP2 may play a vital role in the survival of both HepG2 and SMMC-7721 cells. Paeoniflorin, which may be a promising agent in the treatment of liver cancer, induced apoptosis in hepatocellular carcinoma cells by downregulating

  17. Inhibitory effects of vegetable and fruit ferment liquid on tumor growth in Hepatoma-22 inoculation model.

    PubMed

    Lu, Ming; Toshima, Yasushi; Wu, Xiao-li; Zhang, Xu; Cai, Yun-qing

    2007-01-01

    The aim of this study was to determine the anti-tumor effect of vegetables and fruits ferment liquid (VFFL) in human hepatoma-22(H22)-bearing mice. Mice bearing H22 were randomly divided into four groups, that is a control group and three VFFL groups (16.7, 33.3 and 66.6 ml/kg). Inhibition rates of tumor, thymus and spleen index were observed. The apoptosis was analyzed by flow cytometry and the apoptotic body was observed under an electron microscope. A survival study was performed on the same model for the duration of 60 days. For this survival study, the mice were divided into five groups, which included a control group, three VFFL groups (16.7, 33.3 and 66.6 ml/kg) and a CP group. Tumor inhibition rates for VFFL16.7, 33.3 and 66.6ml/kg were 25.7%, 35.0 % (p<0.05) and 49.1 % (p<0.01) respectively at 30d, increasing in proportion to the concentration of VFFL given. Thymus and spleen indices of the VFFL groups were also higher than that of the control group. The apoptotic rates in VFFL 16.7, 33.3 and 66.6 ml/kg groups were 20.5%, 24.0% and 15.8% respectively, while it was only 6.82% in control group. In particular, the apoptotic body in the 66.6 ml/kg group exhibited typical apoptotic characteristics, e.g., condensation of nucleus, chromatin fragmentation, and shrinkage of cytoplasm. For the survival study, the mice in the VFFL 66.6ml/kg group exhibited significantly extended survival rates compared with the mice in the control group (p<0.05). This study concludes that VFFL possesses anti-tumor properties, which it exhibits by inducing apoptosis and prolonging life in H22 tumor-bearing mice.

  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. Mapping of the gene encoding the human hepatoma-derived growth factor (HDGF) with homology to the high-mobility group (HMG)-1 protein to Xq25

    SciTech Connect

    Wanschura, S.; Bartnitzke, S.; Bullerdiek, J.

    1996-03-01

    This report describes the localization of the human hepatoma-derived growth factor (HDGF) gene to human chromosome Xq25 using fluorescence in situ hybridization. Homology to the high-mobility group (HMG)-1 protein was also demonstrated. 13 refs., 1 fig.

  20. Sodium butyrate enhances STAT 1 expression in PLC/PRF/5 hepatoma cells and augments their responsiveness to interferon-alpha.

    PubMed

    Hung, W C; Chuang, L Y

    1999-05-01

    Although interferon-alpha (IFN-alpha) has shown great promise in the treatment of chronic viral hepatitis, the anti-tumour effect of this agent in the therapy of liver cancer is unclear. Recent studies have demonstrated that differentiation-inducing agents could modulate the responsiveness of cancer cells to IFN-alpha by regulating the expression of signal transducers and activators of transcription (STAT) proteins, a group of transcription factors which play important roles in the IFN signalling pathway. We have reported that sodium butyrate is a potent differentiation inducer for human hepatoma cells. In this study, we investigated whether this drug could regulate the expression of STAT proteins and enhance the anti-tumour effect of IFN-alpha in hepatoma cells. We found that sodium butyrate specifically activated STAT1 gene expression and enhanced IFN-alpha-induced phosphorylation and activation of STAT1 proteins. Co-treatment with these two drugs led to G1 growth arrest, accompanied by down-regulation of cyclin D1 and up-regulation of p21WAF-1, and accumulation of hypophosphorylated retinoblastoma protein in hepatoma cells. Additionally, internucleosomal DNA fragmentation, a biological hallmark of apoptosis, was detected in hepatoma cells after continuous incubation with a combination of these two drugs for 72 h. Our results show that sodium butyrate potently enhances the anti-tumour effect of IFN-alpha in vitro and suggest that a rational combination of these two drugs may be useful for the treatment of liver cancer.

  1. 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

  2. Co-culture of hepatoma cells with hepatocytic precursor (stem-like) cells inhibits tumor cell growth and invasion by downregulating Akt/NF-κB expression

    PubMed Central

    Sui, Cheng-Jun; Xu, Miao; Li, Wei-Qing; Yang, Jia-Mei; Yan, Hong-Zhu; Liu, Hui-Min; Xia, Chun-Yan; Yu, Hong-Yu

    2016-01-01

    Hepatocytic stem cells (HSCs) have inhibitory effects on hepatocarcinoma cells. The present study investigated the effects of HSC activity in hepatocarcinoma cells in vitro. A Transwell co-culture system of hepatocytic precursor (stem-like) WB-F344 cells and hepatoma CBRH-7919 cells was used to assess HSC activity in metastasized hepatoma cells in vitro. Nude mouse xenografts were used to assess HSC activity in vivo. Co-culture of hepatoma CBRH-7919 cells with WB-F344 cells suppressed the growth and colony formation, tumor cell migration and invasion capacity of CBRH-7919 cells. The nude mouse xenograft assay demonstrated that the xenograft size of CBRH-7919 cells following co-culture with WB-F344 cells was significantly smaller compared with that of control cells. Furthermore, the expression levels of the epithelial markers E-cadherin and β-catenin were downregulated, while the mesenchymal markers α-SMA and vimentin were upregulated. Co-culture of CBRH-7919 cells with WB-F344 cells downregulated NF-κB and phospho-Akt expression. In conclusion, hepatocytic precursor (stem-like) WB-F344 cells inhibited the growth, colony formation and invasion capacity of metastasized hepatoma CBRH-7919 cells in vitro and in vivo by downregulating Akt/NF-κB signaling. PMID:27895771

  3. 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

  4. 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.

  5. [Features of the immune proteasome expression in ascite Zajdela hepatoma after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis].

    PubMed

    Mel'nikova, V I; Khegaĭ, I I; Popova, N A; Lifantseva, N V; Ivanova, L N; Zaharova, L A

    2014-01-01

    The expression of the total proteasome pool, immune proteasome subunits LMP2 and LMP7, TAP1 and TAP2 transporters, as well as RT1A molecule of MHC class I was investigated in the ascite Zajdela hepatoma at the 10th day after implantation into Brattleboro rats with the hereditary defect of hypothalamic arginine-vasopressin synthesis (AVP) and into WAG rats with normal AVP expression. In Zajdela hepatoma cells implanted into Brattleboro rats the 3-fold increase of the total proteasome pool and LMP2 level and 8-fold increase of the LMP7 level was detected by Western blotting as compared to those in WAG rats. Differences in the LMP2 and LMP7 expression suggest variations in their functions, namely the important role of LMP7 in anti-tumor immunity. The growth of Zajdela hepatoma in WAG rats was accompanied by the decreased level of total proteasome pool as well as immune proteasome expression as compared to those in Brattleboro rats during the regression of tumor. The analysis of TAP1 and TAP2 revealed the pronounced expression of these peptide transporters in Zajdela hepatoma cells implanted into Brattleboro and WAG rats. The expression level of RT1A molecule of MHC class I was increased 3 times in Zajdela hepatoma cells implanted into Brattleboro rats as compared to WAG rats. Moreover, flow cytometric analysis of CD4- and CD8-lymphocytes number in the spleen of Brattleboro and WAG rats was performed at the 10th day after implantation of Zajdela hepatoma. The increased number of CD4- and CD8-lymphocytes was observed in the spleen of Brattleboro as compared to WAG. The increased subpopulations of cytotoxic T-lymphocytes and T-helpers might promote the tumor regression in Brattleboro rats. The reduced populations of CD4- and CD8-lymphocytes in the spleen of WAG rats were accompanied by the splenomegaly and tumor progression. The data obtained suggest that AVP deficiency in Brattleboro rats leads to the increase of the immune proteasome and MHC class I expression in

  6. 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.

  7. 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

  8. [Intervention of glypican-3 genetic transcription on anti-proliferative effect of hepatoma cells with high metastatic potentiality].

    PubMed

    Yao, Min; Wang, Li; Shi, Yun; Qian, Qi; Yu, Dandan; Shi, Yang; Lu, Shaolin; Yao, Dengfu

    2014-08-26

    To explore the silencing glypican-3 (GPC-3) gene transcription by specific small hairpin RNA (shRNA) on the inhibition of hepatoma cells with high metastatic potentiality and hepatoma growth. After MHCC-97H cells were transfected with higher effective GPC-3-shRNA, GPC-3 mRNA was analyzed by multiple FG-RT-PCR or protein by Western blot. Cell proliferation was detected by 5-ethynyl-2'-deoxyuridine and sulforhodamine B assay, its migratory metastasis and invasiveness by wound healing or transwell chamber system and cell apoptosis was detected by Caspase-Glo(®) 3/7 Luminescence assay. Nude mice were subcutaneously injected with stable MHCC-97H cells for observing the forming time or volume of xenograft tumors. And the expressions of GPC-3, β-catenin, p-GSK3β and CyclinD1 were analyzed by immunohistochemistry. After shRNA1 transfection with high efficiency (>80%), the expression of GPC-3 was down-regulated to 75.6% (t = 15.473, P < 0.001) at mRNA level in accordance with its protein, inhibiting cell proliferation (71.1%, t = 10.468, P < 0.001) notably, decreasing its migration (80.1%, t = 32.697, P < 0.001) and invasiveness (69.1%, t = 39.647, P < 0.001). β-catenin was down-regulated (67.7%, t = 18.4, P < 0.001) and Gli1 increased (53.5%, t = -4.824, P = 0.008) with its protein. The average forming time of subcutaneous tumors was 11.2 days (d) in the shRNA group and it was significantly longer (P < 0.01) than that in the control (5.3 d) or shRNA-neg (5.5 d) group. And the average volume (65.5 mm(3)) of tumors with decreased GPC-3, β-catenin, p-GSK3β, and cyclinD1 expressions in the shRNA group was significantly smaller (P < 0.01) than that in the shRNA-neg (365.7 mm(3)) or control (404.8 mm(3)) group, respectively. Specific shRNA might intervene effectively the GPC-3 gene transcription and inhibit invasion and tumor growth. Thus GPC-3 may become a potential molecular target for hepatoma gene therapy.

  9. Prolonged perturbation of the oscillations of hepatoma Fao cell proliferation by a single small dose of methotrexate.

    PubMed

    Guerroui, S; Deschatrette, J; Wolfrom, C

    2005-06-01

    The proliferation rate of various cell types in vitro, including hepatoma Fao cells, displays aperiodic oscillations. The frequency of these oscillations is about one every 3-5 weeks, and there are variations in cell functions and polarity. Topological analysis has showed that these oscillations in growth rate are determined, and presumably chaotic. One characteristic of complex chaotic systems is that their dynamics can be persistently modified by a small external perturbation. We show that treatment with a single small dose of the anticancer drug methotrexate causes long-term stable alteration of the oscillatory dynamics of Fao cell proliferation. The oscillations of growth rate are shifted, and their mean level decreased according to a fractal pattern.

  10. Influence of riboflavin on disturbances in trytophan metabolism and hepatoma production after a single dose of aflatoxin B1.

    PubMed

    Lemonnier, F J; Scotto, J M; Thuong-Trieu, C

    1975-11-01

    Female Wistar rats were given a single oral dose of aflatoxin B1, either alone or with a large amount of riboflavin. Biochemical and histologic studies were performed for 30 months. Nine animals of 19 in the aflatoxin-treated group and only 5 of 18 in the riboflavin-aflatoxin-treated group developed hepatomas. The number of rats was insufficient for tests of statistical significance to be fruitful. Urinary excretion of tryptophan metabolites was studied in aflatoxin- and riboflavin-treated rats after an oral administration of 10 mg tryptophan/100 g rat. Riboflavin did not affect the percentage of aflatoxin-treated animals with abnormal urinary excretion patterns, but did increase the magnitude of the disturbances in elimination of kynurenic and xanthurenic acids. The hepatic tryptophan-oxygenase activity was increased only in the two groups given riboflavin, and the levels of nucleic acids were the same in all groups.

  11. A comparison of adrenergic receptors of rat ascites hepatoma AH130 cells with those of normal rat hepatocytes.

    PubMed

    Sanae, F; Miyamoto, K; Koshiura, R

    1988-04-01

    The pharmacological specificity of adrenergic receptors in the plasma membrane of rat ascites hepatoma AH130 cells was compared with that in normal rat hepatocytes. The number of [125I]iodocyanopindolol-binding sites was much greater in AH130 cells than in the hepatocytes. We characterized the alpha-adrenergic receptor subtypes using the alpha 1-selective ligand [3H]prazosin and the alpha 2-selective ligand [3H]clonidine. AH130 cells had fewer prazosin-binding sites than the hepatocytes and about 8 times as many clonidine-binding sites of high affinity. The results showed that the adrenergic receptors in AH130 cells have pharmacological properties that are very different from those of the receptors in normal rat hepatocytes.

  12. Butein induces G(2)/M phase arrest and apoptosis in human hepatoma cancer cells through ROS generation.

    PubMed

    Moon, Dong-Oh; Kim, Mun-Ock; Choi, Yung Hyun; Hyun, Jin Won; Chang, Weon Young; Kim, Gi-Young

    2010-02-28

    We investigated the molecular effects of 3,4,2',4'-tetrahydroxychalcone (butein) treatment in two human hepatoma cancer cell lines-HepG2 and Hep3B. Butein treatment inhibited cancer cell growth by inducing G(2)/M phase arrest and apoptosis. Butein-induced G(2)/M phase arrest was associated with increased ATM, Chk1, and Chk2 phosphorylations and reduced cdc25C levels. Additionally, butein treatment enhanced inactivated phospho-Cdc2 levels, reduced Cdc2 kinase activity, and generated reactive oxygen species (ROS) that was accompanied by JNK activation. The extent of butein-induced G(2)/M phase arrest significantly decreased following pretreatment with N-acetyl-l-cysteine or glutathione and following JNK phosphorylation reduction by SP600125. Both N-acetyl-l-cysteine and glutathione also decreased butein-mediated apoptosis. Taken together, these results imply a critical role of ROS and JNK in the anticancer effects of butein.

  13. Oleoylethanolamide, a natural ligand for PPAR-alpha, inhibits insulin receptor signalling in HTC rat hepatoma cells.

    PubMed

    Martínez de Ubago, María; García-Oya, Inmaculada; Pérez-Pérez, Antonio; Canfrán-Duque, Alberto; Quintana-Portillo, Rocio; Rodríguez de Fonseca, Fernando; González-Yanes, Carmen; Sánchez-Margalet, Víctor

    2009-08-01

    Oleoylethanolamide (OEA) is a lipid mediator belonging to the fatty acid ethanolamides family. It is produced by intestine and adipose tissue. It inhibits food intake and body weight gain, and has hypolipemiant action in vivo, as well as a lipolytic effect in vitro. OEA is a PPAR-alpha agonist, and recently it has been found that OEA is an endogenous ligand of an orphan receptor. Previously, we have shown that OEA inhibits insulin-stimulated glucose uptake in isolated adipocytes, and produces glucose intolerance in rats. In the present work, we have studied another insulin target cell, the hepatocyte using a rat hepatoma cell line (HTC), and we have studied the cross-talk of OEA signalling with metabolic and mitotic signal transduction of insulin receptor. OEA dose-dependently activates JNK and p38 MAPK, and inhibits insulin receptor phosphorylation. OEA inhibits insulin receptor activation, blunting insulin signalling in the downstream PI3K pathway, decreasing phosphorylation of PKB and its target GSK-3. OEA also inhibits insulin-dependent MAPK pathway, as assessed by immunoblot of phosphorylated MEK and MAPK. These effects were reversed by blocking JNK or p38 MAPK using pharmacological inhibitors (SP 600125, and SB 203580). Since OEA is an endogenous PPAR-alpha agonist, we investigated whether a pharmacologic agonist (WY 14643) may mimic the OEA effect on insulin receptor signalling. Activation of PPAR-alpha by the pharmacological agonist WY14643 in HTC hepatoma cells is sufficient to inhibit insulin signalling and this effect is also dependent on p38 MAPK but not JNK kinase. In summary, OEA inhibits insulin metabolic and mitogenic signalling by activation of JNK and p38 MAPK via PPAR-alpha.

  14. 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.

  15. Inhibitory and immunological effects induced by the combination of photodynamic therapy and dendritic cells on mouse transplanted hepatoma.

    PubMed

    Zhang, Nan-zheng; Bai, Shuang; Cai, Xiao-jun; Li, Li-bo

    2016-03-01

    To investigate the anti-tumor and immune efficacy of photodynamic immune-therapy (PIT), the combination of photodynamic therapy and dendritic cells (DC), on murine Heps hepatoma. DCs were derived from syngeneic mouse bone marrow and then labeled with DAPI in vitro. The hepatoma model was established by subcutaneous inoculation with Heps cells in one hundred and twenty-eight mice. They were then divided into four groups at random: control group, PDT group, DC group and PIT group. Tumors in the control group were injected with normal saline. Mice in the PDT group were injected with the photosensitizer Deuteporfin 24 h before irradiation. Mice in the DC group were injected with DAPI labeled dendritic cells intratumorally. Mice in the PIT group were further given an injection of DCs after photoirradiation. Tumor growth and survival time were recorded after treatment. Fluorescence of tumor draining lymph nodes was evaluated under fluorescence microscope. Cytotoxic activity of splenocytes was tested by standard lactate dehydrogenase (lactate dehydrogenase, LDH) release assay. (1) Tumor growth was significantly slowed down in the PDT and PIT group compared to the control group (P<0.01). (2) The mean survival time was significantly prolonged in the PDT and PIT group. (3) The number of fluorescent cells in the draining lymph nodes from DC group was higher than that of the PIT group. (4) The anti-tumor activity of splenocytes in the PDT and PIT group was significantly higher than that of the DC and control groups (P<0.01, P<0.01). The present study suggests that PDT can inhibit tumor growth and induce anti-tumour immune response. The combination of PDT induced by Deuteporfiin and dendritic cell is capable of amplifying the antitumor immune response. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Pharmacological evaluation of several major ingredients of Chinese herbal medicines in human hepatoma Hep3B cells.

    PubMed

    Chou, C C; Pan, S L; Teng, C M; Guh, J H

    2003-08-01

    Long-dan-tan (Chinese name) is one of the most common herbal medicines used by Chinese people with chronic liver disease. Accumulated anecdotal evidence suggests that Long-dan-tan may show a beneficial effect in patients with hepatocellular carcinoma. Long-dan-tan is made from five plants: Gentiana root, Scutellaria root, Gardenia fruit, Alisma rhizome, and Bupleurum root. In this study, we have examined the cytotoxic effects of the five major ingredients isolated from the above plants, i.e. gentiopicroside, baicalein, geniposide, alisol B acetate and saikosaponin-d, respectively, on human hepatoma Hep3B cells. Annexin V immunofluorescence detection, DNA fragmentation assays and FACScan analysis of propidium iodide-staining cells showed that gentiopicroside, baicalein, and geniposide had little effect, whereas alisol B acetate and saikosaponin-d profoundly induced apoptosis in Hep3B cells. Alisol B acetate, but not saikosaponin-d, induced G2/M arrest of the cell cycle as well as a significant increase in caspase-3 activity. Interestingly, baicalein by itself induced an increase in H(2)O(2) generation and the subsequent NF-kappaB activation; furthermore, it effectively inhibited the transforming growth factor-beta(1) (TGF-beta(1))-induced caspase-3 activation and cell apoptosis. We suggest that alisol B acetate and saikosaponin-d induced cell apoptosis through the caspase-3-dependent and -independent pathways, respectively. Instead of inducing apoptosis, baicalein inhibits TGF-beta(1)-induced apoptosis via increase in cellular H(2)O(2) formation and NF-kappaB activation in human hepatoma Hep3B cells.

  17. Interaction of Na+ and K+ transport with aerobic energy metabolism in slices of Morris hepatoma 3924A.

    PubMed

    Galeotti, T; van Rossum, G D; Russo, M A; Palombini, G

    1976-11-01

    Addition of increasing concentrations of glucose to slices of Morris hepatoma 3924A greatly stimulated aerobic lactate production and reduced respiration by 20%. Neither the adenine nucleotide content of the slices nor the calculated rate of adenosine 5'-triphosphate synthesis was altered. Ouabain reduced the rate of O2 uptake (by 20 to 25%) and of aerobic lactate production (by 25 to 50%) without affecting adenine nucleotide contents. The reduction by ouabain of the calculated rate of adenosine 5'-triphosphate synthesis was similar whether the slices were utilizing only endogenous substrate or exogenous glucose also. Raising the medium K+ concentration (and correspondingly reducing Na+) partially overcame the inhibition of ion transport by ouabain and partially restored the rates of respiration and aerobic lactate production toward control levels. Electron microscopic observations of mitochondria within the slices incubated under different conditions showed variations in configuration between "orthodox," "condensed" and degenerating forms. Slices preincubated at 1 degrees showed mitochondria in the condensed form: they were restored to the orthodox configuration during incubation at 38 degrees in oxygenated medium. Oligomycin and glucose enhanced the transition, but ouabain reduced the number of mitochondria undergoing the change. The results suggest that in hepatoma 3924A utilization of adenosine 5'-triphosphate by ion transport exerts a simultaneous control of both respiration and aerobic glycolysis, which is presumably mediated by alterations in the availability of adenosine 5-diphosphate. The mitochondria undergo conformational transitions under conditions likely to affect local availability of adenosine 5'-diphosphate within cell compartments, but the transitions are not all externally added adenosine diphosphate on isolated mitochondria.

  18. 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.

  19. 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

  20. 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

    2003-06-04

    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.

  1. Critical role of c-Jun N-terminal protein kinase activation in troglitazone-induced apoptosis of human HepG2 hepatoma cells.

    PubMed

    Bae, Myung-Ae; Song, Byoung J

    2003-02-01

    The peroxisome proliferator-activated receptor agonist troglitazone (TRO) was used for treatment of non-insulin-dependent diabetes until its removal from the market because of its severe hepatotoxicity. However, the mechanism for its hepatotoxicity is still poorly understood. In this study, we investigated whether TRO caused cell death by altering signaling pathways associated with cell damage and survival in human hepatoma cells. Our data reveal that TRO caused time- and concentration-dependent apoptosis of HepG2 and Chang liver human hepatoma cells, as evidenced by DNA fragmentation and staining with Hoechst 33342. In contrast, 50 or 100 microM rosiglitazone, a structural analog of TRO, did not cause apoptosis in these hepatoma cells. TRO activated both c-Jun N-terminal protein kinase (JNK) and p38 kinase about 5-fold between 0.5 and 8 h before they returned to control levels at 16 h in HepG2 cells. In contrast, TRO failed to activate the extracellular signal-regulated kinase. Furthermore, TRO increased the levels of proapoptotic proteins, Bad, Bax, release of cytochrome c, and cleavage of Bid in a time-dependent manner. The antiapoptotic Bcl-2 protein level decreased in hepatoma cells treated with TRO. Pretreatment of hepatoma cells with a selective JNK inhibitor, anthra[1,9-cd]pyrazol-6(2H)-one (SP600125), significantly reduced the rate of TRO-induced cell death, whereas 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), an inhibitor of p38 kinase, had little effect on apoptosis. Pretreatment with SP600125 also prevented JNK activation and c-Jun phosphorylation. In addition, rosiglitazone, which is not as toxic to hepatoma cells as TRO, did not stimulate JNK activity. Transfection of cDNA for the dominant-negative mutant JNK-KR (Lys-->Arg) or SEK1-KR (Lys-->Arg), an immediate upstream kinase of JNK, significantly reduced TRO-induced JNK activation and cell death rate. Furthermore, SP600125 pretreatment effectively prevented the

  2. Exogenous hydrogen sulfide exerts proliferation/anti-apoptosis/angiogenesis/migration effects via amplifying the activation of NF-κB pathway in PLC/PRF/5 hepatoma cells.

    PubMed

    Zhen, Yulan; Pan, Wanying; Hu, Fen; Wu, Hongfu; Feng, Jianqiang; Zhang, Ying; Chen, Jingfu

    2015-05-01

    Hydrogen sulfide (H2S) takes part in a diverse range of intracellular pathways and hss physical and pathological properties in vitro and in vivo. However, the effects of H2S on cancer are controversial and remain unclear. The present study investigates the effects of H2S on liver cancer progression via activating NF-κB pathway in PLC/PRF/5 hepatoma cells. PLC/PRF/5 hepatoma cells were pretreated with 500 µmol/l NaHS (a donor of H2S) for 24 h. The expression levels of CSE, CBS, phosphosphorylate (p)-NF-κB p65, caspase-3, COX-2, p-IκB and MMP-2 were measured by western blot assay. Cell viability was detected by cell counter kit 8 (CCK-8). Apoptotic cells were observed by Hoechst 33258 staining assay. The production level of H2S in cell culture medium was measured by using the sulfur-sensitive electrode method. The production of vascular endothelial growth factor (VEGF) was tested by enzyme-linked immunosorbent assay (ELISA). Our results showed that the production of H2S was dramatically increased in the PLC/PRF/5 hepatoma cells, compared with human LO2 hepatocyte cells group, along with the overexpression levels of CSE and CBS. Treatment of PLC/PRF/5 hepatoma cells with 500 µmol/l NaHS (a donor of H2S) for 24 h markedly increased the expression levels of CSE, CBS, p-IκB and NF-κB activation, leading to COX-2 and MMP-2 overexpression, and decreased caspase-3 production, as well as increased cell viability and decreased number of apoptotic cells. Otherwise, the production level of H2S and VEGF were also significantly increased. Furthermore, co-treatment of PLC/PRF/5 hepatoma cells with 500 µmol/l NaHS and 200 µmol/l PDTC for 24 h significantly overturned these indexes. The findings of the present study provide evidence that the NF-κB is involved in the NaHS-induced cell proliferation, anti-apoptisis, angiogenesis, and migration in PLC/PRF/5 hepatoma cells, and that the PDTC against the NaHS-induced effects were by inhibition of the NF-κB pathway.

  3. Stimulation of NADH oxidase activity from rat liver plasma membranes by growth factors and hormones is decreased or absent with hepatoma plasma membranes.

    PubMed Central

    Bruno, M; Brightman, A O; Lawrence, J; Werderitsh, D; Morré, D M; Morre, D J

    1992-01-01

    Plasma membranes of rat liver isolated by aqueous two-phase partition exhibited basal levels of NADH oxidase activity that were increased approx. 2-fold by addition of hormones and growth factors to which liver cells were known to respond. In contrast, hepatoma plasma membranes demonstrated an intrinsically increased level of NADH oxidase, which was not stimulated further by addition of growth factors. The results suggest that the NADH oxidase of the hepatoma plasma membrane is no longer correctly coupled to hormone and growth-factor receptors. This biochemical defect may parallel the loss of growth control that is characteristic of neoplastic transformation in hepatocarcinogenesis and other transformation systems. Images Fig. 3. PMID:1622384

  4. CD18/ICAM-1-dependent oxidative NF-kappaB activation leading to nitric oxide production in rat Kupffer cells cocultured with syngeneic hepatoma cells.

    PubMed Central

    Kurose, I; Saito, H; Miura, S; Ebinuma, H; Higuchi, H; Watanabe, N; Zeki, S; Nakamura, T; Takaishi, M; Ishii, H

    1997-01-01

    Previous studies have indicated that nitric oxide (NO) released from Kupffer cells modulates biological viability of cocultured hepatoma cells. This study was designed to evaluate the mechanisms by which Kupffer cells synthesize and release NO in reponse to cocultured hepatoma cells. Kupffer cells isolated from male Wistar rats were cocultured with rat hepatoma cell line, AH70 cells. The sum of nitrite and nitrate levels increased in the culture medium of Kupffer cells with AH70 cells as compared with those of Kupffer cells or AH70 cells alone. Increased expressions of iNOS and iNOS mRNA in Kupffer cells cocultured with AH70 cells were detected by an immunofluorescence staining and a fluorescence in situ hybridization study, respectively. A fluorescence in situ DNA-protein binding assay revealed that NF-kappaB activation occurs in Kupffer cells and activated NF-kappaB moved into the nuclei preceding to an increased production of NO. Oxidative stress indicated by dichlorofluorescein fluorescence was observed in Kupffer cells cocultured with AH70 cells. An increased calcium mobilization indicated as increased fluo-3-associated fluorescence was also induced in Kupffer cells after coculture with AH70 cells. Monoclonal antibodies directed against rat CD18 and ICAM-1, as well as TMB-8, a calcium inhibitor, prevented the calcium mobilization, active oxygen production, and NF-kappaB activation in addition to the increased production of NO. Pyrrolidine dithiocarbamate, an inhibitor of oxidative NF-kappaB activation, diphenylene iodonium, an NADPH oxidase inhibitor, and quinacrine, a phospholipase A2 inhibitor, significantly attenuated the increase in dichlorofluorescein fluorescence, NF-kappaB activation, and NO production. Therefore, this study suggests that CD18/ICAM-1-dependent cell-to-cell interaction with hepatoma cells causes calcium mobilization and oxidative activation of NF-kappaB, which may lead to the increased production of NO in Kupffer cells. PMID:9062344

  5. [Analysis on anti-hepatoma effect of medicine invigorating blood circulation and eliminating blood stasis based on warm-pungent-liver efficiency network].

    PubMed

    Gu, Hao; Ma, Li; Yuan, Bin; Zhang, Yan-Ling; Wang, Yun; Qiao, Yan-Jiang

    2014-07-01

    The efficiency network is a complicated network for revealing the efficient mechanism of traditional Chinese medicines (TCMs) and relations among efficiencies. The efficiency-property relations were used to establish a warm-pungent-liver efficiency network to explain the principle of treating hepatoma with medicines invigorating blood circulation and eliminating blood stasis. Safflower, a warm-pungent medicine distributing along the live meridian, was taken for example to discuss the efficiency network' s application in the identification of active ingredients of TCMs and the combination. In the early stage of this study, combined warm-pungent-liver medicines distributed along the liver meridian and invigorating blood circulation and eliminating blood stasis were taken as the study objects to collect the pharmacological effect data of warm-pungent-liver medicines and obtain the pharmacological effect combinations with the highest blood circulation-invigorating association by the association rules and the chi-square test. The pharmacological target data recorded in the DrugBank database is used to establish the warm-pungent-liver efficiency network according to the principle line of "efficiency-property-pharmacology-target-protein interaction" under the background of the protein interaction network. The blood circulation-invigorating medicines could directly treat hepatoma by impacting protooncogene, cancer suppressor gene, cell apoptosis and anti-inflammation, and indirectly treat hepatoma by resisting coagulation and adhesion, regulating local blood circulation, preventing cancer cell metastasis and enhancing the tissues' sensitivity to the anticancer drugs. Among the active ingredients of safflower screened based on the blood circulation-invigorating network targets, carthamin yellow, quercetin and luteolin have been proved to have the anti-hepatoma effect in literatures, which indicated the reliability of this study's results and the purpose of the efficiency

  6. Emodin inhibits the growth of hepatoma cells: finding the common anti-cancer pathway using Huh7, Hep3B, and HepG2 cells.

    PubMed

    Hsu, Chin-Mu; Hsu, Yu-An; Tsai, Yuhsin; Shieh, Fa-Kuen; Huang, Su-Hua; Wan, Lei; Tsai, Fuu-Jen

    2010-02-19

    Emodin--a major component of Rheum palmatum L.-exerts antiproliferative effects in cancer cells that are regulated by different signaling pathways. Hepatocellular carcinoma has high-incidence rates and is associated with poor prognosis and high mortality rates. This study was designed to evaluate the effects of emodin on human hepatocarcinoma cell viability and investigate its mechanisms of action in Huh7, Hep3B, and HepG2 cells. To define the molecular changes associated with this process, expression profiles were compared in emodin-treated hepatoma cells by cDNA microarray hybridization, quantitative RT-PCRs, and Western blot analysis. G2/M phase arrest was observed in all 3 cell lines. Cell cycle regulatory gene analysis showed increased protein levels of cyclin A, cyclin B, Chk2, Cdk2, and P27 in hepatoma cells after time courses of emodin treatment, and Western blot analysis showed decreased protein levels of Cdc25c and P21. Microarray expression profile data and quantitative PCR revealed that 15 representative genes were associated with emodin treatment response in hepatoma cell lines. The RNA expression levels of CYP1A1, CYP1B1, GDF15, SERPINE1, SOS1, RASD1, and MRAS were upregulated and those of NR1H4, PALMD, and TXNIP were downregulated in all three hepatoma cells. Moreover, at 6h after emodin treatment, the levels of GDF15, CYP1A1, CYP1B1, and CYR61 were upregulated. Here, we show that emodin treatment caused G2/M arrest in liver cancer cells and increased the expression levels of various genes both in mRNA and protein level. It is likely that these genes act as biomarkers for hepatocellular carcinoma therapy.

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

    NASA Astrophysics Data System (ADS)

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

    2011-08-01

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

  8. Orphan receptor TR3 enhances p53 transactivation and represses DNA double-strand break repair in hepatoma cells under ionizing radiation.

    PubMed

    Zhao, Bi-xing; Chen, Hang-zi; Du, Xiao-dan; Luo, Jie; He, Jian-ping; Wang, Rong-hao; Wang, Yuan; Wu, Rong; Hou, Ru-rong; Hong, Ming; Wu, Qiao

    2011-08-01

    In response to ionizing radiation (IR)-induced DNA double-strand breaks (DSB), cells elicit an evolutionarily conserved checkpoint response that induces cell cycle arrest and either DNA repair or apoptosis, thereby maintaining genomic stability. DNA-dependent protein kinase (DNA-PK) is a central enzyme involved in DSB repair for mammalian cells that comprises a DNA-PK catalytic subunit and the Ku protein, which act as regulatory elements. DNA-PK also functions as a signaling molecule to selectively regulate p53-dependent apoptosis in response to IR. Herein, we demonstrate that the orphan nuclear receptor TR3 suppresses DSB repair by blocking Ku80 DNA-end binding activity and promoting DNA-PK-induced p53 activity in hepatoma cells. We find that TR3 interacts with Ku80 and inhibits its binding to DNA ends, which then suppresses DSB repair. Furthermore, TR3 is a phosphorylation substrate for DNA-PK and interacts with DNA-PK catalytic subunit in a Ku80-independent manner. Phosphorylated TR3, in turn, enhances DNA-PK-induced phosphorylation and p53 transcription activity, thereby enhancing IR-induced apoptosis in hepatoma cells. Together, our findings reveal novel functions for TR3, not only in DSB repair regulation but also in IR-induced hepatoma cell apoptosis, and they suggest that TR3 is a potential target for cancer radiotherapy.

  9. 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.

  10. Inhibition of bleomycin-induced [3H] thymidine 5'-triphosphate incorporation into liver and hepatoma nuclei by N-ethyl maleimide and daunomycin.

    PubMed

    Coetzee, M L; Sartiano, G P; Klein, K; Ove, P

    1977-02-01

    The addition of bleomycin to a nuclear incorporating system results in an increased incorporation of 3H-thymidine 5'-triphosphate (3H-TTP) into the DNA of liver and hepatoma nuclei. Bleomycin added to the nuclear incorporating system also produces scissions of DNA as determined by sucrose density gradient centrifugation of the extracted DNA. The action of bleomycin is dependent on the presence of sulfhydryl agents in the incubation mixture. Two compounds, N-ethyl maleimide and daunomycin, inhibit the bleomycin-induced incorporation of 3H-TTP preferentially. N-Ethyl maleimide inhibits bleomycin-induced activity in liver and hepatoma 7777 nuclei equally. Lower levels of daunomycin inhibit the bleomycin-induced activity in the hepatoma 7777 nuclei than are required to inhibit the activity in liver nuclei. The two compounds inhibit the bleomycin effect by different mechanisms. The addition of N-ethyl maleimide to bleomycin in the incubation system prevents bleomycin from causing breaks in the DNA. The addition of daunomycin, despite inhibition of bleomycin-induced 3H-TTP incorporation, does not affect the bleomycin-produced breaks in the DNA. N-Ethyl maleimide acts by binding to the DNA and by competing with a sulfhydryl agent for bleomycin-sensitive sites on the DNA. Daunomycin apparently inhibits a repair enzyme that is responsible for the increased incorporation following bleomycin treatment.

  11. 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.

  12. 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.

  13. A fast and sensitive colorimetric assay for IL-6 in hepatoma cells based on the production of a secreted form of alkaline phosphatase (SEAP).

    PubMed

    Gregory, B; Savino, R; Ciliberto, G

    1994-03-29

    Structure-function studies of cytokines require that simple, sensitive and reliable biological assays are available. A well known property of interleukin-6 (IL-6) is that of being able to induce transcription from several liver-specific promoters in human hepatoma cells. However, the available assays of IL-6 in hepatoma cells, which are either based on the detection of increased expression of endogenous acute phase response genes or on the activation of reporter genes transfected under the control of IL-6 responsive promoters, are not very sensitive and are time consuming. We have established a new assay for IL-6 in hepatoma cells which is based on the transfection of an IL-6 inducible promoter/secreted alkaline phosphatase (SEAP) gene fusion and which measures the inducible production and release of SEAP in the culture medium. SEAP activity is measured with a simple colorimetric assay that requires no cell manipulation, thus allowing a large set of samples to be analysed simultaneously. The CRP/SEAP assay can be used in studies on the structure-function relationships of human IL-6.

  14. 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.

  15. Probing the control elements of the CYP1A1 switching module in H4IIE hepatoma cells.

    PubMed

    Broccardo, Carolyn J; Billings, Ruth E; Andersen, Melvin E; Hanneman, William H

    2005-11-01

    Previous research from our laboratory has shown a switch-like response to PCB 126 mediated CYP1A1 induction in primary rat hepatocytes and in H4IIE rat hepatoma cells. On a single cell level, cells appear to be either "on" or "off" for CYP1A1 induction at a given dose; some cells never respond to PCB 126. These cells represent a non-responding population. Cells that are switched "on" by PCB 126 display varying levels of induction, much like the dimmer on a light switch. The goal of the present research is to begin to uncover the mechanism for this switch-like response to CYP1A1 induction in H4IIE rat hepatoma cells. The AhR pathway is modulated by multiple co-activators and by phosphorylation. This research focuses on the phosphorylation cascades initiated by PCB 126 and the role they play in CYP1A1 induction. Our research reveals a likely role for protein kinase C (PKC) in this switch response. Inhibition of PKC by H-7 dramatically reduced the percent of cells that express CYP1A1 in response to PCB 126 treatment, as determined by flow cytometry. The effect of H-7 was concentration dependent, decreasing the number of cells expressing CYP1A1 rather than decreasing the level of CYP1A1 in all cells. This finding provides further evidence for the switch-like behavior of CYP1A1 induction and implicates PKC in this response to PCB126. The protein kinase inhibitor, HA-1004, had only a minor effect on CYP1A1 induction. A high-throughput immunoblot screen for 40 proteins revealed the regulation of several proteins/phosphoproteins by PCB 126. Most importantly, two proteins containing phosphoserine/phoshothreonine residues were increased by PCB126 treatment. However, PKC translocation studies and activity studies failed to verify that PCB126 activates PKC. It is possible that constitutive PKC activity is sufficient to maintain phosphorylation of critical components of the AhR pathway. Immunoblotting studies showed that MAP kinases ERK and JNK are not activated by PCB 126 in H

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

    PubMed

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

    2015-10-01

    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 (IC 50 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, 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.

  17. [Effects of intratumoral injection of microspheres containing cobra venom cytotoxin on transplanted human hepatoma in nude mice].

    PubMed

    Wang, Yan; Lin, Li-wu; Chen, Zhi-kui; Xue, En-sheng; Lin, Xiao-dong; Yu, Li-Yun; Lin, Zhen-hu

    2009-09-01

    To evaluate the safety and efficacy of intratumoral injection of polylactic-co-glycolic acid (PLGA) microspheres containing cobra venom cytotoxin in nude mice with transplanted human hepatoma. Cytotoxic activity of cytotoxin from cobra venom was determined by using methyl thiazolyl tetrazolium method in vitro. Microspheres containing cobra venom cytotoxin were prepared with a double emulsion-solvent evaporation method. Forty BALB/c nude mice were inoculated subcutaneously in right flank with hepatoma BEL-7404 cells. Thirty-two mice whose tumor size reached about 1.0 cm in diameter, were randomly assigned into normal saline group, blank microsphers group, cytotoxin group and cytotoxin-PLGA group. Nude mice were intratumorally injected with normal saline, blank microspheres, cytotoxin or cytotoxin-PLGA microspheres respectively. Internal echo characteristics and blood flow of tumors were observed by high-frequency ultrasound every week after treatment. Twenty-six days after treatment, the tumors were removed to calculate the inhibition rate of tumor growth. The tumor, heart, liver and kidney tissues were obtained for histopathological examination. The cytotoxin separated and purified from crude cobra venom caused intense cytotoxic effects to the BEL-7404 cells in vitro. The diameter of PLGA microspheres containing cobra venom cytotoxin was about (34.45+/-9.85)microm. Encapsulation rate was up to (78.13+/-8.92)%, and cumulative amount of cobra venom cytotoxin released from the PLGA microspheres in vitro during 30 days was up to 84.3%. After intratumoral injection, tumor volumes and weights in the cytotoxin-PLGA group were lower than those in the normal saline group, with a tumor growth inhibition rate of 52.36%. Observed under a light microscope, most tumor tissues were necrotic. No obvious morphological change could be seen on the liver, kidney and heart tissues. The above findings indicate that intratumoral injection of cytotoxin-PLGA microspheres has strong

  18. Long term and large-scale cultivation of human hepatoma Hep G2 cells in hollow fiber bioreactor. Cultivation of human hepatoma Hep G2 in hollow fiber bioreactor.

    PubMed

    Liu, J J; Chen, B S; Tsai, T F; Wu, Y J; Pang, V F; Hsieh, A; Hsieh, J H; Chang, T H

    1991-02-01

    Long-term and large scale cultivation of an anchorage-dependent cell line using an industrial scale hollow fiber perfusion bioreactor is described. Hep G2 cells (a human hepatoma cell line) were cultivated in an Acusyst-P (Endotronic) with a total fiber surface area of 7.2 m2 6 x 1.2m2) to produce Hep G2 crude conditioned medium (CCM). Pretreatment of the cellulose acetate hollow fibers with collagen enhances the attachment of the anchorage-dependent cells. We have succeeded in growing the Hep G2 cells in an antibiotics- and serum-free IMDM medium, supplemented with 50 micrograms/ml of Hep G2 CCM protein at inoculation. The Hep G2 cells replicate and secrete CCM protein in quantities comparable to those produced in DMEM containing 10% fetal calf serum (FCS). The highest CCM protein productivity during the 80-day cultivation was 1.1 g/day with a total of 30 g of protein accumulated. Hep G2 CCM (20-40 micrograms protein/ml) was comparable to or even better than 10% FCS in supporting the growth of Molt-4 (a human T leukemia cell line) and FO (a mouse myeloma cell line) cells in vitro. The availability of this large amount of Hep G2 CCM will aid the further purification and characterization of growth factor(s) which could be used as serum substituents.

  19. DMFC (3,5-dimethyl-(7)H-furo[3,2-g]chromen-7-one) regulates Bim to trigger Bax and Bak activation to suppress drug-resistant human hepatoma.

    PubMed

    Xiang, Jun; Wang, Zhe; Liu, Qianqian; Li, Xia; Sun, Jianguo; Fung, Kwok-Pui; Liu, Feiyan

    2017-03-01

    3,5-Dimethyl-(7)H-furo[3,2-g]chromen-7-one (DMFC) is a coumarin derivative with anti-cancer activity against human hepatoma cells, but the mechanisms underlying DMFC function in cancer suppression is unknown. In this study, we aimed at elucidating the molecular mechanisms underlying DMFC anti-cancer activity and determining whether DMFC is effective in suppression of drug-resistant human hepatocellular carcinoma. We show here that DMFC effectively suppresses both the parent and the multidrug-resistant hepatoma cell growth in vitro and DMFC suppresses hepatoma cell growth at least in part through inducing tumor cell apoptosis. In the molecular level, we observed that DMFC treatment decreases Bcl-2 level by a post-transcriptional mechanism and activates Bim transcription to increase Bim mRNA and protein level in hepatoma cells. Furthermore, co-immunoprecipitation studies revealed that DMFC-induced Bim interrupts interactions between Bcl-2 and Bax and between Mcl-1 and Bak, resulting in dissociation of Bax from Bcl-2 and Bak from Mcl-1 and subsequent activation of both Bax and Bak. Activation of Bax and Bak leads to mitochondrial outer membrane permeabilization and cytochrome c release. Consistent with its potent apoptosis-inducing activity, DMFC exhibited potent activity against the multidrug-resistant hepatoma xenograft growth in vivo. Therefore, we determine that DMFC suppresses hepatoma growth through decreasing Bcl-2 and increasing Bim to induce tumor cell apoptosis and hold great promise for further development as a therapeutic agent to treat chemoresistant hepatoma.

  20. Cytotoxic effects and the mechanism of three types of magnetic nanoparticles on human hepatoma BEL-7402 cells

    NASA Astrophysics Data System (ADS)

    Kai, Wei; Xiaojun, Xu; Ximing, Pu; Zhenqing, Hou; Qiqing, Zhang

    2011-07-01

    The evaluation of the toxicity of magnetic nanoparticles (MNPs) has attracted much attention in recent years. The current study aimed to investigate the cytotoxic effects of Fe3O4, oleic acid-coated Fe3O4 (OA-Fe3O4), and carbon-coated Fe (C-Fe) nanoparticles on human hepatoma BEL-7402 cells and the mechanisms. WST-1 assay demonstrated that the cytotoxicity of three types of MNPs was in a dose-dependent manner. G1 (Fe3O4 and OA-Fe3O4) phase and G2 (C-Fe) phase cell arrests and apoptosis induced by MNPs were detected by flow cytometry analysis. The increase in apoptosis was accompanied with the Bax over-expression, mitochondrial membrane potential decrease, and the release of cytochrome C from mitochondria into cytosol. Moreover, apoptosis was further confirmed by morphological and biochemical hallmarks, such as swollen mitochondria with lysing cristae and caspase-3 activation. Our results revealed that certain concentrations of the three types of MNPs affect BEL-7402 cells viability via cell arrest and inducing apoptosis, and the MNPs-induced apoptosis is mediated through the mitochondrial-dependent pathway. The influence potency of MNPs observed in all experiments would be: C-Fe > Fe3O4 > OA-Fe3O4.

  1. Selection of rat hepatoma cells defective in hormone-regulated production of mouse mammary tumor virus RNA.

    PubMed Central

    Grove, J R; Ringold, G M

    1981-01-01

    We have been studying the mechanism of glucocorticoid hormone action by using mouse mammary tumor virus (MMTV)-infected rat hepatoma cells as a model system. J2.17, a clonal cell line that contains one MMTV provirus, induces tyrosine aminotransferase (TyrATase; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5), viral RNA, and the cell surface viral glycoprotein gp52 in response to dexamethasone. Using a fluorescence-activated cell sorter and a rabbit antiserum directed against gp52, we selected a cell population that displays a reduced hormone-mediated increase in cell surface gp52. Fourteen clones of this population were assayed for induction of viral gp52 and RNA and of cellular TyrATase. The results of these assays revealed that the clones display a variety of responses to hormone. One clone has retained wild-type responses of both TyrATase and gp52. Six clones exhibit coordinately reduced or abolished responses of both markers. Seven clones show normal induction of TyrATase but reduced or undetectable induction of gp52. These latter clones exhibit reduced production of MMTV RNA and thus may represent a unique class of variants defective in the regulation of MMTV gene expression. Images PMID:6117075

  2. Modulated expression of a nuclear-associated glycoprotein during normal rat liver development and in various hepatoma cells.

    PubMed

    Goulet, Francine; Napa, Ioana Diana; Solomon, Luc; Morin, Odette; Islam, Nazrul

    2006-01-01

    Liver plays a major role in systemic detoxification and drug metabolism. NF-164, a protein of 164 kDa predominantly localized in hepatocyte nuclei, was found to be present in increasing amounts during liver maturation. In addition, fetal rat hepatocytes had ten times, and neonatal five times less of this protein than adult hepatocytes. It was also detected in an albumin producing hepatoma cell line, but not in three other lines that have lost several differentiated functions. These data suggest that NF-164 expression is development-dependent and that it may be a marker for both normal and malignant hepatocyte differentiation. NF-164 seems to be liver-specific, since it was not detected in rat brain, spleen, kidney, lung and bovine thymus. It was purified from adult rat hepatocyte nuclei. Its estimated pI is 6.8. Its total amino acid composition and partial amino acid sequence is also being reported. Despite major differences between their respective contents in amino acids, partial sequences showed homologies with carbamyl phosphate synthetase I (CPSI). These observations may suggest that NF-164 also shares some functional features with this enzyme.

  3. Aqueous extract of Polygonum bistorta modulates proteostasis by ROS-induced ER stress in human hepatoma cells

    PubMed Central

    Liu, Yu-Huei; Weng, Yui-Ping; Lin, Hsuan-Yuan; Tang, Sai-Wen; Chen, Chao-Jung; Liang, Chi-Jung; Ku, Chung-Yu; Lin, Jung-Yaw

    2017-01-01

    Hepatocellular carcinoma (HCC) remains the leading cause of cancer mortality with limited therapeutic targets. The endoplasmic reticulum (ER) plays a pivotal role in maintaining proteostasis in normal cells. However, alterations in proteostasis are often found in cancer cells, making it a potential target for therapy. Polygonum bistorta is used in traditional Chinese medicine owing to its anticancer activities, but the molecular and pharmacological mechanisms remain unclear. Using hepatoma cells as a model system, this study demonstrated that P. bistorta aqueous extract (PB) stimulated ER stress by increasing autophagosomes but by blocking degradation, followed by the accumulation of ubiquitinated proteins and cell apoptosis. In addition, an autophagy inhibitor did not enhance ubiquitinated protein accumulation whereas a reactive oxygen species (ROS) scavenger diminished both ubiquitinated protein accumulation and ligand-stimulated epidermal growth factor receptor (EGFR) expression, suggesting that ROS generation by PB may be upstream of PB-triggered cell death. Nevertheless, PB-exerted proteostasis impairment resulted in cytoskeletal changes, impairment of cell adhesion and motility, and inhibition of cell cycle progression. Oral administration of PB delayed tumour growth in a xenograft model without significant body weight loss. These findings indicate that PB may be a potential new alternative or complementary medicine for HCC. PMID:28134285

  4. Preparation of carotenoids and chlorophylls from Gynostemma pentaphyllum (Thunb.) Makino and their antiproliferation effect on hepatoma cell.

    PubMed

    Tsai, Yu-Chian; Wu, Wen-Bin; Chen, Bing-Huei

    2010-12-01

    A preparative column chromatographic method for isolation of carotenoids and chlorophylls from Gynostemma pentaphyllum, a traditional Chinese herb, was developed to evaluate their antiproliferative effects on the hepatoma cell Hep3B. An open column containing 70 g of magnesium oxide-diatomaceous earth (1:2.5, wt/wt) was used to elute carotenoid with 2% ethanol in ethyl acetate and chlorophyll with 50% ethanol in acetone. After high-performance liquid chromatography-mass spectrometry analysis, the carotenoid fraction was composed of all-trans- and cis-isomers of lutein, α-carotene, and β-carotene as well as epoxy-containing carotenoids, while the chlorophyll fraction consisted of chlorophylls a and b and their derivatives. Both carotenoid and chlorophyll fractions as well as lutein and chlorophyll a standards at 50-100 μg/mL were effective against Hep3B cells with a dose-dependent response with the following order: carotenoid fraction > chlorophyll fraction > lutein > chlorophyll a. For all treatments, the cell cycle was arrested in the G₀/G₁ phase, with Hep3B cells undergoing necrosis or apoptosis.

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

    PubMed Central

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

    2005-01-01

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

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

    PubMed

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

    2017-09-27

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

  7. Mutation of N-linked glycosylation at Asn548 in CD133 decreases its ability to promote hepatoma cell growth.

    PubMed

    Liu, Ying; Ren, Shifang; Xie, Liqi; Cui, Chunhong; Xing, Yang; Liu, Chanjuan; Cao, Benjin; Yang, Fan; Li, Yinan; Chen, Xiaoning; Wei, Yuanyan; Lu, Haojie; Jiang, Jianhai

    2015-08-21

    The membrane glycoprotein CD133 is a popular marker for cancer stem cells and contributes to cancer initiation and invasion in a number of tumor types. CD133 promotes tumorigenesis partly through an interaction between its phosphorylated Y828 residue and the PI3K regulatory subunit p85, and the interaction with β-catenin. Although CD133 glycosylation is supposed to be associated with its function, the contribution of N-glycosylation to its functions remains unclear. Here we analyzed the exact site(s) of N-glycosylation in CD133 by mass spectrometry and found that all eight potential N-glycosylation sites of CD133 could be indeed occupied by N-glycans. Loss of individual N-glycosylation sites had no effect on the level of expression or membrane localization of CD133. However, mutation at glycosylation site Asn548 significantly decreased the ability of CD133 to promote hepatoma cell growth. Furthermore, mutation of Asn548 reduced the interaction between CD133 and β-catenin and inhibited the activation of β-catenin signaling by CD133 overexpression. Our results identified the characteristics and function of CD133 glycosylation sites. These data could potentially shed light on molecular regulation of CD133 by glycosylation and enhance our understanding of the utility of glycosylated CD133 as a target for cancer therapies.

  8. Ellagic acid modulates lipid accumulation in primary human adipocytes and human hepatoma Huh7 cells via discrete mechanisms.

    PubMed

    Okla, Meshail; Kang, Inhae; Kim, Da Mi; Gourineni, Vishnupriya; Shay, Neil; Gu, Liwei; Chung, Soonkyu

    2015-01-01

    Previously, we have reported that consumption of a muscadine grape phytochemical powder (MGP) decreased lipid accumulation in high-fat fed mice. The aim of this study was to identify the responsible polyphenolic constituents and elucidate the underlying mechanisms. In mice, MGP supplementation significantly reduced visceral fat mass as well as adipocyte size. To determine whether MGP affects adipogenesis or hypertrophic lipid accumulation, we used a human adipogenic stem cell (hASCs) model. Among the MGP, ellagic acid (EA) was identified as a potent negative regulator of adipogenesis of hASCs. In addition, EA substantially decreased the conversion of [(3)H]-acetyl CoA into fatty acids (FAs), suggesting that EA inhibits de novo synthesis of FA in mature adipocytes. Similarly, MGP supplementation significantly decreased hepatic triglyceride (TG) levels. The TG-lowering effects of EA were confirmed in human hepatoma Huh7 cells. EA reduced [(3)H]-oleic acid esterification into [(3)H]-TG as well as the de novo synthesis of FA from [(3)H]-acetyl CoA in Huh7 cells. Intriguingly, EA also increased oxygen consumption rate and β-oxidation-related gene expression. Taken together, EA attenuated new fat cell formation and FA biosynthesis in adipose tissue, while it reduced the synthesis of TG and FA and increased FA oxidation in the liver. These results suggest that EA exerts unique lipid-lowering effects both in adipose tissue and liver via discrete mechanisms. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. 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.

  10. 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. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  11. Spheroid organization kinetics of H35 rat hepatoma model cell system on elastin-like polypeptide-polyethyleneimine copolymer substrates.

    PubMed

    Turner, Paul A; Weeks, C Andrew; McMurphy, Austin J; Janorkar, Amol V

    2014-03-01

    Though two-dimensional systems have yielded some success in deriving morphological and functional markers of hepatocyte culture, they largely fail to capture the three-dimensional organization, long-term viability, and functionality of the hepatic tissue. We have engineered a system for inducing self-assembly of model H35 rat hepatoma spheroids using a copolymer comprised of biocompatible elastin-like polypeptide (ELP) chemically conjugated to positively charged polyethyleneimine (PEI). We have achieved a conjugation ratio of 30 mol %, though our studies analyzing spheroid organization kinetics indicate conjugate ratios of 5 mol % and greater to be optimal for cell culture based on least variability in spheroid sizes and minimum incidence of overgrown aggregates. Furthermore, our ELP-PEI system indicated the potential for influencing ultimate spheroid dimensions, with spheroid size inversely related to polyelectrolyte conjugation. Overall, this study provides a good starting point to investigate functional correlations between spheroid size and functional markers and their future use as an in vitro diagnostic or tissue engineering tool.

  12. Genotoxicity of marine sediments in the fish hepatoma cell line PLHC-1 as assessed by the Comet assay.

    PubMed

    Šrut, Maja; Traven, Luka; Štambuk, Anamaria; Kralj, Sonja; Žaja, Roko; Mićović, Vladimir; Klobučar, Göran I V

    2011-02-01

    The main goal of this study was to test the usefulness of the Comet assay in the PLHC-1 hepatoma fish cell line as a tool for detecting the presence of genotoxic compounds in contaminated marine sediments. The system has been tested using both model chemicals (benzo[a]pyrene (B[a]P) and ethyl methanesulfonate (EMS)) and extracts of sediment samples obtained with solvent dichloromethane/methanol. For all of the analysed sediment extracts as well as for the model chemicals a concentration dependent genotoxic effect was observed. The sediment with the highest observed genotoxic potential was additionally extracted using various solvents in order to test which class of compounds, according to their polarity, is most responsible for the observed genotoxic effect. Non-polar solvents (cyclohexane and dichloromethane) yielded stronger genotoxic effect but the highest level of DNA damage was determined after exposure to sediment extract obtained with the solvent mixture dichloromethane/methanol which extracts a wide range of contaminants. Our results indicate that the PLHC-1 cell line is a suitable in vitro model in sediment genotoxicity assessment and encourage the use of fish cell lines as versatile tools in ecogenotoxicology.

  13. Biological response of hepatomas to an extract of Fagopyrum esculentum M. (buckwheat) is not mediated by inositols or rutin.

    PubMed

    Curran, Julianne M; Stringer, Danielle M; Wright, Brenda; Taylor, Carla G; Przybylski, Roman; Zahradka, Peter

    2010-03-10

    Buckwheat contains d-chiro-inositol (D-CI) and myo-inositol (MI), possible insulin-mimetic compounds; thus, this study investigated the insulin-mimetic activities of a buckwheat concentrate (BWC), D-CI, and MI on insulin signal transduction pathways and glucose uptake with H4IIE rat hepatoma cells. BWC stimulated phosphorylation of p42/44 extracellular-related kinase (p42/44 ERK) and its downstream target, p70(S6K), on Thr(421). In contrast, D-CI, MI, rutin, or its agylcone form, quercetin, did not activate these signal transduction proteins. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), another target of insulin, was also up-regulated upon BWC treatment. The effects of BWC on glucose uptake were subsequently investigated using H4IIE cells. Insulin and D-CI stimulated glucose uptake, whereas BWC inhibited basal and insulin-stimulated glucose uptake. Although results from this work suggest that BWC has insulin-mimetic effects on select protein phosphorylation events in H4IIE cells, D-CI and MI were not the active components responsible for the observed effects. The inhibition of glucose uptake by BWC suggests that buckwheat may affect hepatic glucose metabolism, possibly by inhibiting glucose flux. Furthermore, the fact that D-CI and MI stimulated glucose uptake in H4IIE cells suggests that other compounds are responsible for inhibition of glucose uptake by BWC.

  14. Effects of N-acetyl-L-cysteine on fish hepatoma cells treated with mercury chloride and ionizing radiation.

    PubMed

    Kim, Jin Kyu; Han, Min; Nili, Mohammad

    2011-11-01

    Organisms are exposed to natural radiations from cosmic or terrestrial origins. Furthermore the combined action of radiation with various chemicals is an inevitable feature of modern life. Radiation is known to cause cell death, mainly due to its ability to produce reactive oxygen species in cells. N-acetyl-L-cysteine (NAC) is a well-known sulfhydryl-containing antioxidant whose role in radioprotection has been reported. Synergistic effects of radiation and mercury chloride on human cells was previously reported by the authors. Based on the previous report, this study was designed to assess the synergistic effects of radiation and mercury chloride on fish hepatoma cells, as well as to investigate the protective effects of NAC on the cells. The cytotoxicity of radiation was enhanced in the presence of mercury chloride. NAC in lower concentrations prevented cells from death after irradiation with lower doses (<300 Gy) while it did not prevent cells from radiation-induced death after irradiation with higher doses (300, 500 Gy). The intracellular glutathione (GSH) levels significantly decreased after irradiation while the combined treatment of NAC and radiation alleviated the decrease in the GSH levels. The investigations give a clue for the action mechanism of synergistic or protective effects of NAC on the cells. Due to their high resistance to ionizing radiation, the PLHC-1 cells can be effectively used as a screening tool for assessing the combined effects of radiation with toxic chemicals.

  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.

  16. 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.

  17. Dibenzylbutane- and butyrolactone-type lignans as apoptosis inducers in human hepatoma HuH-7 cells.

    PubMed

    Mansoor, Tayyab A; Ramalho, Rita M; Rodrigues, Cecília M P; Ferreira, Maria-José U

    2012-05-01

    Seven lignans, previously isolated from Pycnanthus angolensis or obtained by derivatization, namely the dibenzylbutane-type lignans threo-4,4'-dihydroxy-3-methoxylignan (1), 4'-hydroxy-3,3',4-trimethoxylignan (2), (-)-dihydroguaiaretic acid (3), 3,3',4,4'-tetramethoxylignan (4), 4,4'-diacetyl-3,3'-dimethoxylignan (5), heliobuphthalmin (6) and the butyrolactone lignan hinokinin (7), were evaluted for their ability as apoptosis inducers in human hepatoma HuH-7 cells. Cell viability assays, morphological evaluation of apoptosis and enzymatic analyses of caspase activity in HuH-7 cells were carried out. Using the lactate dehydrogenase lactate dehydrogenase (LDH) assay, it was demonstrated that the lignans (1-7) tested significantly reduced viability of HuH-7 cells. Morphologic evaluation of HuH-7 cells using Hoechst staining and fluorescence microscopy revealed that lignans 1-7 were strong inducers of apoptosis. In fact, HuH-7 cells developed morphological changes of apoptosis, including chromatin condensation, nuclear fragmentation and formation of apoptotic bodies. However, lignans 2 and 7 were the most promising compounds in this study, inducing 2.4- and 2.5-fold increases in apoptotic cells as compared to controls. Caspase-3-like activity assays confirmed the morphologic data. Copyright © 2011 John Wiley & Sons, Ltd.

  18. 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. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Mutation of N-linked glycosylation at Asn548 in CD133 decreases its ability to promote hepatoma cell growth

    PubMed Central

    Xie, Liqi; Cui, Chunhong; Xing, Yang; Liu, Chanjuan; Cao, Benjin; Yang, Fan; Li, Yinan; Chen, Xiaoning; Wei, Yuanyan; Lu, Haojie; Jiang, Jianhai

    2015-01-01

    The membrane glycoprotein CD133 is a popular marker for cancer stem cells and contributes to cancer initiation and invasion in a number of tumor types. CD133 promotes tumorigenesis partly through an interaction between its phosphorylated Y828 residue and the PI3K regulatory subunit p85, and the interaction with β-catenin. Although CD133 glycosylation is supposed to be associated with its function, the contribution of N-glycosylation to its functions remains unclear. Here we analyzed the exact site(s) of N-glycosylation in CD133 by mass spectrometry and found that all eight potential N-glycosylation sites of CD133 could be indeed occupied by N-glycans. Loss of individual N-glycosylation sites had no effect on the level of expression or membrane localization of CD133. However, mutation at glycosylation site Asn548 significantly decreased the ability of CD133 to promote hepatoma cell growth. Furthermore, mutation of Asn548 reduced the interaction between CD133 and β-catenin and inhibited the activation of β-catenin signaling by CD133 overexpression. Our results identified the characteristics and function of CD133 glycosylation sites. These data could potentially shed light on molecular regulation of CD133 by glycosylation and enhance our understanding of the utility of glycosylated CD133 as a target for cancer therapies. PMID:26029999

  20. Growth Characteristics and Imaging Properties of the Morris Hepatoma 3924A in ACI Rats: A Suitable Model for Transarterial Chemoembolization

    SciTech Connect

    Truebenbach, Jochen; Graepler, Florian; Pereira, Philippe L.; Ruck, Peter; Lauer, Ulrich; Gregor, Michael; Claussen, Claus-D.; Huppert, Peter E.

    2000-03-15

    Purpose: For experimental studies investigating modalities and efficacy of transarterial chemoembolization (TACE) in hepatocellular carcinoma (HCC) an animal model resembling the human situation as closely as possible would be appropriate. Specifically, reproducible tumor growth characteristics with the capability for appropriate in vivo imaging to monitor treatment efficacy are required.Methods: Morris hepatoma 3924A was implanted into the liver of 30 ACI rats. Tumor growth was followed by angiography (n = 10), ultrasound (US, n = 30), native computed tomography (CT, n = 16), and native magnetic resonance imaging (MRI, n = 30) between day 8 and day 36 after implantation. The radiological morphological characteristics were compared with the macroscopic and microscopic histological findings of the explanted tumors.Results: In all 30 animals a solitary liver tumor was found and macroscopically no signs of metastases, ascites, or peritoneal tumor were visible. On histopathological examination tumor sizes ranged between 27 {+-} 3 mm{sup 3} (day 8) and 3468 {+-} 79 mm{sup 3} (day 36). The first signs of tumor necrosis occurred at day 16. US allowed tumor visualization from day 8, MRI from day 8, angiography from day 10, and CT from day 14.Conclusions: The tumor model has the potential to be used for the visualization of tumor growth by MRI and US. The potential for monitoring therapeutic effects of TACE needs to be investigated.

  1. A Taiwanese Propolis Derivative Induces Apoptosis through Inducing Endoplasmic Reticular Stress and Activating Transcription Factor-3 in Human Hepatoma Cells

    PubMed Central

    Suk, Fat-Moon; Lien, Gi-Shih; Huang, Wei-Jan; Chen, Chia-Nan; Lu, Shao-Yu; Yan, Ming-De

    2013-01-01

    Activating transcription factor-(ATF-) 3, a stress-inducible transcription factor, is rapidly upregulated under various stress conditions and plays an important role in inducing cancer cell apoptosis. NBM-TP-007-GS-002 (GS-002) is a Taiwanese propolin G (PPG) derivative. In this study, we examined the antitumor effects of GS-002 in human hepatoma Hep3B and HepG2 cells in vitro. First, we found that GS-002 significantly inhibited cell proliferation and induced cell apoptosis in dose-dependent manners. Several main apoptotic indicators were found in GS-002-treated cells, such as the cleaved forms of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). GS-002 also induced endoplasmic reticular (ER) stress as evidenced by increases in ER stress-responsive proteins including glucose-regulated protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153), phosphorylated eukaryotic initiation factor 2α (eIF2α), phosphorylated protein endoplasmic-reticular-resident kinase (PERK), and ATF-3. The induction of ATF-3 expression was mediated by mitogen-activated protein kinase (MAPK) signaling pathways in GS-002-treated cells. Furthermore, we found that GS-002 induced more cell apoptosis in ATF-3-overexpressing cells. These results suggest that the induction of apoptosis by the propolis derivative, GS-002, is partially mediated through ER stress and ATF-3-dependent pathways, and GS-002 has the potential for development as an antitumor drug. PMID:24222778

  2. 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.

  3. 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.

  4. Cancer stem-like cells can be induced through dedifferentiation under hypoxic conditions in glioma, hepatoma and lung cancer

    PubMed Central

    Wang, Pan; Wan, Wen-wu; Xiong, Shuang-Long; Feng, Hua; Wu, Nan

    2017-01-01

    Traditional studies have shown that transcription factors, including SOX-2, OCT-4, KLF-4, Nanog and Lin-28A, contribute to the dedifferentiation and reprogramming process in normal tissues. Hypoxia is a physiological phenomenon that exists in tumors and promotes the expression of SOX-2, OCT-4, KLF-4, Nanog and Lin-28A. Therefore, an interesting question is whether hypoxia as a stimulating factor promotes the process of dedifferentiation and induces the formation of cancer stem-like cells. Studies have shown that OCT-4 and Nanog overexpression induced the formation of cancer stem cell-like cells through dedifferentiation and enhanced malignancy in lung adenocarcinoma, and reprogramming SOX-2 in pancreatic cancer cells also promoted the dedifferentiation process. Therefore, we investigated this phenomenon in glioma, lung cancer and hepatoma cells and found that the transcription factors mentioned above were highly expressed under hypoxic conditions and induced the formation of spheres, which exhibited asymmetric division and cell cycle arrest. The dedifferentiation process induced by hypoxia highlights a new pattern of cancer development and recurrence, demonstrating that all kinds of cancer cells and the hypoxic microenvironment should be taken into consideration when developing tumor therapies. PMID:28179999

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

    PubMed

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

    2016-05-01

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

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

    PubMed

    Lee, Yong Soo

    2004-12-01

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

  7. Cytotoxic effects and the mechanism of three types of magnetic nanoparticles on human hepatoma BEL-7402 cells

    PubMed Central

    2011-01-01

    The evaluation of the toxicity of magnetic nanoparticles (MNPs) has attracted much attention in recent years. The current study aimed to investigate the cytotoxic effects of Fe3O4, oleic acid-coated Fe3O4 (OA-Fe3O4), and carbon-coated Fe (C-Fe) nanoparticles on human hepatoma BEL-7402 cells and the mechanisms. WST-1 assay demonstrated that the cytotoxicity of three types of MNPs was in a dose-dependent manner. G1 (Fe3O4 and OA-Fe3O4) phase and G2 (C-Fe) phase cell arrests and apoptosis induced by MNPs were detected by flow cytometry analysis. The increase in apoptosis was accompanied with the Bax over-expression, mitochondrial membrane potential decrease, and the release of cytochrome C from mitochondria into cytosol. Moreover, apoptosis was further confirmed by morphological and biochemical hallmarks, such as swollen mitochondria with lysing cristae and caspase-3 activation. Our results revealed that certain concentrations of the three types of MNPs affect BEL-7402 cells viability via cell arrest and inducing apoptosis, and the MNPs-induced apoptosis is mediated through the mitochondrial-dependent pathway. The influence potency of MNPs observed in all experiments would be: C-Fe > Fe3O4 > OA-Fe3O4. PMID:21801413

  8. Selenium methylselenocysteine protects human hepatoma HepG2 cells against oxidative stress induced by tert-butyl hydroperoxide.

    PubMed

    Cuello, Susana; Ramos, Sonia; Mateos, Raquel; Martín, M Angeles; Madrid, Yolanda; Cámara, Carmen; Bravo, Laura; Goya, Luis

    2007-12-01

    Selenium methylselenocysteine (Se-MeSeCys) is a common selenocompound in the diet with a tested chemopreventive effect. This study investigated the potential protective effect of Se-MeSeCys against a chemical oxidative stress induced by tert-butyl hydroperoxide (t-BOOH) on human hepatoma HepG2 cells. Speciation of selenium derivatives by liquid chromatography-inductively coupled plasma mass spectrometry depicts Se-MeSeCys as the only selenocompound in the cell culture. Cell viability (lactate dehydrogenase) and markers of oxidative status--concentration of reduced glutathione (GSH) and malondialdehyde (MDA), generation of reactive oxygen species (ROS) and activity of the antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR)--were evaluated. Pretreatment of cells with Se-MeSeCys for 20 h completely prevented the enhanced cell damage, MDA concentration and GR and GPx activity and the decreased GSH induced by t-BOOH but did not prevent increased ROS generation. The results show that treatment of HepG2 cells with concentrations of Se-MeSeCys in the nanomolar to micromolar range confers a significant protection against an oxidative insult.

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

    PubMed

    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.

  10. Size-mediated cytotoxicity of nanocrystalline titanium dioxide, pure and zinc-doped hydroxyapatite nanoparticles in human hepatoma cells

    NASA Astrophysics Data System (ADS)

    Devanand Venkatasubbu, G.; Ramasamy, S.; Avadhani, G. S.; Palanikumar, L.; Kumar, J.

    2012-03-01

    Nanoparticles are highly used in biological applications including nanomedicine. In this present study, the interaction of HepG2 hepatocellular carcinoma cells (HCC) with hydroxyapatite (HAp), zinc-doped hydroxyapatite, and titanium dioxide (TiO2) nanoparticles were investigated. Hydroxyapatite, zinc-doped hydroxyapatite and titanium dioxide nanoparticles were prepared by wet precipitation method. They were subjected to isochronal annealing at different temperatures. Particle morphology and size distribution were characterized by X-ray diffraction and transmission electron microscope. The nanoparticles were co-cultured with HepG2 cells. MTT assay was employed to evaluate the proliferation of tumor cells. The DNA damaging effect of HAp, Zn-doped HAp, and TiO2 nanoparticles in human hepatoma cells (HepG2) were evaluated using DNA fragmentation studies. The results showed that in HepG2 cells, the anti-tumor activity strongly depend on the size of nanoparticles in HCC cells. Cell cycle arrest analysis for HAp, zinc-doped HAp, and TiO2 nanoparticles revealed the influence of HAp, zinc-doped HAp, and titanium dioxide nanoparticles on the apoptosis of HepG2 cells. The results imply that the novel nano nature effect plays an important role in the biomedicinal application of nanoparticles.

  11. Comparison of polysomal and nuclear poly(A)-containing RNA populations from normal rat liver and Novikoff hepatoma.

    PubMed Central

    Capetanaki, Y G; Alonso, A

    1980-01-01

    Polysomal and nuclear poly(A)-containing RNA of normal rat liver and Novikoff hepatoma cells have been compared by cDNA.RNA hybridization kinetics. Homologous hybridization reactions revealed at total kinetic complexity of about 1.6 X 10(10) and 1.38 X 10(10) daltons for liver and Novikoff mRNA respectively. The high abundance component present in liver cannot be detected in Novikoff. It was found from heterologous reactions that about 30% by weight of mRNA sequences are specific to liver. Determination of the nuclear poly(A)-containing RNA complexities revealed that about 5.5% and 4% of the haploid genome is expressed in the liver and Novikoff respectively. In a heterologous reaction, up to 30% of the liver cDNA failed to form hybrids with Novikoff nuclear RNA. Cross hybridizations have further revealed abundance shifts in both nuclear and polysomal RNA populations. Some sequences abundant in liver are less abundant in Novikoff and some rare liver sequences are relatively abundant in Novikoff. PMID:6160467

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

    PubMed

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

    2008-01-10

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

  13. S-adenosylmethionine and methylthioadenosine are antiapoptotic in cultured rat hepatocytes but proapoptotic in human hepatoma cells.

    PubMed

    Ansorena, Eduardo; García-Trevijano, Elena R; Martínez-Chantar, Maria L; Huang, Zong-Zhi; Chen, Lixin; Mato, José M; Iraburu, Maria; Lu, Shelly C; Avila, Matías A

    2002-02-01

    S-adenosylmethionine (AdoMet) is an essential compound in cellular transmethylation reactions and a precursor of polyamine and glutathione synthesis in the liver. In liver injury, the synthesis of AdoMet is impaired and its availability limited. AdoMet administration attenuates experimental liver damage, improves survival of alcoholic patients with cirrhosis, and prevents experimental hepatocarcinogenesis. Apoptosis contributes to different liver injuries, many of which are protected by AdoMet. The mechanism of AdoMet's hepatoprotective and chemopreventive effects are largely unknown. The effect of AdoMet on okadaic acid (OA)-induced apoptosis was evaluated using primary cultures of rat hepatocytes and human hepatoma cell lines. AdoMet protected rat hepatocytes from OA-induced apoptosis dose dependently. It attenuated mitochondrial cytochrome c release, caspase 3 activation, and poly(ADP-ribose) polymerase cleavage. These effects were independent from AdoMet-dependent glutathione synthesis, and mimicked by 5'-methylthioadenosine (MTA), which is derived from AdoMet. Interestingly, AdoMet and MTA did not protect HuH7 cells from OA-induced apoptosis; conversely both compounds behaved as proapoptotic agents. AdoMet's proapoptotic effect was dose dependent and observed also in HepG2 cells. In conclusion, AdoMet exerts opposing effects on apoptosis in normal versus transformed hepatocytes that could be mediated through its conversion to MTA. These effects may participate in the hepatoprotective and chemopreventive properties of this safe and well-tolerated drug.

  14. 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

  15. 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

  16. A high expression of heme oxygenase-1 in the liver of LEC rats at the stage of hepatoma: the possible implication of induction in uninvolved tissue.

    PubMed

    Matsumoto, A; Hanayama, R; Nakamura, M; Suzuki, K; Fujii, J; Tatsumi, H; Taniguchi, N

    1998-04-01

    We have examined changes in the expression of heme oxygenase-1 (HO-1), an inducible isoform and HO-2, a constitutive isoform, in the liver of Long-Evans with a Cinnamon-like color (LEC) rat, a mutant strain which spontaneously develops acute hepatitis and hepatoma. HO-1 expression was highly enhanced in the LEC rat livers with jaundice, and then decreased slightly, but overall remained at a higher level than in the Long-Evans with Agouti color (LEA) control rats, as judged by Northern blotting analysis of the whole liver extract. The high expression of HO-1 in the LEC rat liver was, however, not due to the actual cancer lesion but, rather, due to the surrounding uninvolved tissues including hepatocytes. Immunohistochemical analysis also supported this conclusion. Among normal tissues, the expression of HO-1 but not HO-2 was high in only the spleen of both LEC and LEA rats. The high expression observed in the stage of acute hepatitis and hepatoma stages in the LEC rat is probably due to the oxidative stress caused by the accumulation of free copper and free iron levels which has been reported earlier by our group (Suzuki et al., Carcinogenesis, 1993, 14, 1881-1884 and Koizumi et al., Free Radical Research, in press) as well as by free heme levels. The inflammatory cytokines produced by the surrounding tissue at the hepatoma stage would also be expected to play a role in the induction mechanism. The physiological relevance of HO-1 induction might be an adaptive response to oxidative stress and vasodilatory effect of carbon monoxide on sinusoidal circulation.

  17. Anti-tumoural effects of PlgK1-5 are directly linked to reduced ICAM expression, resulting in hepatoma cell apoptosis.

    PubMed

    Schmitz, Volker; Sauerbruch, Tilman; Raskopf, Esther

    2012-08-01

    Angiostatin and angiostatin-like molecules are known as anti-angiogenic factors, which inhibit endothelial cell functions resulting in reduced tumour growth. Recent data indicate that these molecules, especially PlgK1-5, directly affect tumour cells, which could explain the strong anti-tumoural effects of PlgK1-5. Therefore, we have analysed whether PlgK1-5 alters tumour cell functions and expression levels of cell adhesion molecules in murine and human hepatoma cells in vitro and in vivo. First, effects on tumour growth, proliferation and apoptosis were investigated in vivo in a subcutaneous tumour model. In vitro, effects of PlgK1-5 on tumour cell apoptosis, clonal expansion, migration, corresponding ICAM expression and intracellular signal transduction in murine Hepa129 and human HuH7 hepatoma cells have been analysed. In vivo, subcutaneous tumour growth was reduced by 75% in PlgK1-5-treated animals compared to the controls. This was accompanied by increased tumour cell apoptosis (up to 33%) and decreased tumour cell proliferation (by up to 21%). In vitro, PlgK1-5 induced apoptosis in hepatoma cells, corresponding to increased caspase-8 cleavage and reduced AKT phosphorylation. Migration and clonal expansion was also diminished in PlgK1-5-treated Hepa129, corresponding to decreased ICAM expression levels. Here, we show that PlgK1-5 directly affects tumour cells by decreasing cell adhesion resulting-at least partly-in apoptosis. This is mediated by altered intracellular signal transduction and by activation of the caspase cascade. These findings further underscore the potential therapeutic role of PlgK1-5 in the treatment of HCC.

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

    PubMed

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

    2017-04-01

    JS-K is a novel anticancer nitric oxide (NO) prodrug effective against a variety of cancer cells, including the inhibition of AM-1 hepatoma cell growth in rats. To further evaluate anticancer effects of JS-K, human hepatoma Hep3B cells were treated with JS-K and the compound control JS-43-126 at various concentrations (0-100μM) for 24h, and cytotoxicity was determined by the MTS assay. The compound control JS-43-126 was not cytotoxic to Hep3B cells at concentrations up to 100μM, while the LC50 for JS-K was about 10μM. To examine the molecular mechanisms of antitumor effects of JS-K, Hep3B cells were treated with 1-10μM of JS-K for 24h, and then subjected to gene expression analysis via real time RT-PCR and protein immunostain via confocal images. JS-K is a GST-α targeting NO prodrug, and decreased immunostaining for GST-α was associated with JS-K treatment. JS-K activated apoptosis pathways in Hep3B cells, including induction of caspase-3, caspase-9, Bax, TNF-α, and IL-1β, and immunostaining for caspase-3 was intensified. The expressions of thrombospondin-1 (TSP-1) and the tissue inhibitors of metalloproteinase-1 (TIMP-1) were increased by JS-K at both transcript and protein levels. JS-K treatment also increased the expression of differentiation-related genes CD14 and CD11b, and depressed the expression of c-myc in Hep3B cells. Thus, multiple molecular events appear to be associated with anticancer effects of JS-K in human hepatoma Hep3B cells, including activation of genes related to apoptosis and induction of genes involved in antiangiogenesis and tumor cell migration.

  19. Inhibition of insulin degradation by hepatoma cells after microinjection of monoclonal antibodies to a specific cytosolic protease.

    PubMed Central

    Shii, K; Roth, R A

    1986-01-01

    Four monoclonal antibodies were identified by their ability to bind to 125I-labeled insulin covalently linked to a cytosolic insulin-degrading enzyme from human erythrocytes. All four antibodies were also found to remove more than 90% of the insulin-degrading activity from erythrocyte extracts. These antibodies were shown to be directed to different sites on the enzyme by mapping studies and by their various properties. Two antibodies recognized the insulin-degrading enzyme from rat liver; one inhibited the erythrocyte enzyme directly; and two recognized the enzyme after gel electrophoresis and transfer to nitrocellulose filters. By this latter procedure and immunoprecipitation from metabolically labeled cells, the enzyme from a variety of tissues was shown to be composed of a single polypeptide chain of apparent Mr 110,000. Finally, these monoclonal antibodies were microinjected into the cytoplasm of a human hepatoma cell line to assess the contribution of this enzyme to insulin degradation in the intact cell. In five separate experiments, preloading of cells with these monoclonal antibodies resulted in an inhibition of insulin degradation of 18-54% (average 39%) and increased the amount of 125I-labeled insulin associated with the cells. In contrast, microinjection of control antibody or an extraneous monoclonal antibody had no effect on insulin degradation or on the amount of insulin associated with the cells. Moreover, the monoclonal antibodies to the insulin-degrading enzyme caused no significant inhibition of degradation of another molecule, low density lipoprotein. Thus, these results support a role for this enzyme in insulin degradation in the intact cell. Images PMID:2424018

  20. P2X4 Activation Modulates Volume-sensitive Outwardly Rectifying Chloride Channels in Rat Hepatoma Cells*

    PubMed Central

    Varela, Diego; Penna, Antonello; Simon, Felipe; Eguiguren, Ana Luisa; Leiva-Salcedo, Elías; Cerda, Oscar; Sala, Francisco; Stutzin, Andrés

    2010-01-01

    Volume-sensitive outwardly rectifying (VSOR) Cl− channels are critical for the regulatory volume decrease (RVD) response triggered upon cell swelling. Recent evidence indicates that H2O2 plays an essential role in the activation of these channels and that H2O2 per se activates the channels under isotonic isovolumic conditions. However, a significant difference in the time course for current onset between H2O2-induced and hypotonicity-mediated VSOR Cl− activation is observed. In several cell types, cell swelling induced by hypotonic challenges triggers the release of ATP to the extracellular medium, which in turn, activates purinergic receptors and modulates cell volume regulation. In this study, we have addressed the effect of purinergic receptor activation on H2O2-induced and hypotonicity-mediated VSOR Cl− current activation. Here we show that rat hepatoma cells (HTC) exposed to a 33% hypotonic solution responded by rapidly activating VSOR Cl− current and releasing ATP to the extracellular medium. In contrast, cells exposed to 200 μm H2O2 VSOR Cl− current onset was significantly slower, and ATP release was not detected. In cells exposed to either 11% hypotonicity or 200 μm H2O2, exogenous addition of ATP in the presence of extracellular Ca2+ resulted in a decrease in the half-time for VSOR Cl− current onset. Conversely, in cells that overexpress a dominant-negative mutant of the ionotropic receptor P2X4 challenged with a 33% hypotonic solution, the half-time for VSOR Cl− current onset was significantly slowed down. Our results indicate that, at high hypotonic imbalances, swelling-induced ATP release activates the purinergic receptor P2X4, which in turn modulates the time course of VSOR Cl− current onset in a extracellular Ca2+-dependent manner. PMID:20056605

  1. 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

    2006-02-05

    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. (c) 2005 Wiley Periodicals, Inc.

  2. 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.

  3. 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.

  4. 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.

  5. Human papillomavirus type 18 E6 and E7 genes integrate into human hepatoma derived cell line Hep G2.

    PubMed

    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

    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. 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. 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. 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.

  6. 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. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  7. 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.

  8. Resveratrol induces apoptotic cell death in rat H4IIE hepatoma cells but necrosis in C6 glioma cells.

    PubMed

    Michels, G; Wätjen, W; Weber, N; Niering, P; Chovolou, Y; Kampkötter, A; Proksch, P; Kahl, R

    2006-08-15

    Resveratrol (trans-3,5,4',-trihydroxystilbene) is assumed to possess cancer-preventive and cancer-therapeutic properties. The aim of this project was to analyze cellular effects of resveratrol in metabolically active H4IIE rat hepatoma cells in comparison to metabolically poorly active C6 rat glioma cells. Resveratrol is rapidly taken up by both cell types and acts as a potent intracellular antioxidant. On the other hand, resveratrol in higher concentrations is relatively toxic to both cell lines as measured by the neutral red accumulation assay. In H4IIE cells, resveratrol concentrations rapidly decline to very low levels during the first hours of incubation due to formation of resveratrol glucuronides. The first resveratrol effect found at 3h after the start of resveratrol treatment was the induction of mild DNA damage as detected by the comet assay. Cell death was caused via induction of apoptosis as detected by caspase activation, oligonucleosomal DNA fragmentation and formation of apoptotic nuclei. Following DNA damage, resveratrol led to an activation of caspases 2 and 8/10 at 6h and consequently of caspase 3 at 12h, but failed to activate caspase 9. In contrast to H4IIE cells, resveratrol is not metabolised in C6 glioma cells and accumulates to concentrations which are assumed to drive the cell into necrosis. This suggests that the mode of cell death caused by resveratrol and the usefulness of resveratrol for cancer prevention and treatment critically depends on the metabolic capacity of the tumor cell to be eradicated.

  9. 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

  10. 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.

  11. Determining oxidative and non-oxidative genotoxic effects driven by estuarine sediment contaminants on a human hepatoma cell line.

    PubMed

    Pinto, M; Costa, P M; Louro, H; Costa, M H; Lavinha, J; Caeiro, S; Silva, M J

    2014-04-15

    Estuarine sediments may be reservoirs of hydrophilic and hydrophobic pollutants, many of which are acknowledged genotoxicants, pro-mutagens and even potential carcinogens for humans. Still, studies aiming at narrowing the gap between ecological and human health risk of sediment-bound contaminant mixtures are scarce. Taking an impacted estuary as a case study (the Sado, SW Portugal), HepG2 (human hepatoma) cells were exposed in vitro for 48 h to extracts of sediments collected from two areas (urban/industrial and Triverine/agricultural), both contaminated by distinct mixtures of organic and inorganic toxicants, among which are found priority mutagens such as benzo[a]pyrene. Comparatively to a control test, extracts of sediments from both impacted areas produced deleterious effects in a dose-response manner. However, sediment extracts from the industrial area caused lower replication index plus higher cytotoxicity and genotoxicity (concerning total DNA strand breakage and clastogenesis), with emphasis on micronucleus induction. On the other hand, extracts from the rural area induced the highest oxidative damage to DNA, as revealed by the FPG (formamidopyrimidine-DNA glycosylase) enzyme in the Comet assay. Although the estuary, on its whole, has been classified as moderately contaminated, the results suggest that the sediments from the industrial area are significantly genotoxic and, furthermore, elicit permanent chromosome damage, thus potentially being more mutagenic than those from the rural area. The results are consistent with contamination by pro-mutagens like polycyclic aromatic hydrocarbons (PAHs), potentiated by metals. The sediments from the agriculture-influenced area likely owe their genotoxic effects to metals and other toxicants, probably pesticides and fertilizers, and able to induce reactive oxygen species without the formation of DNA strand breakage. The findings suggest that the mixtures of contaminants present in the assayed sediments are genotoxic

  12. Antimutagenic activity and in vitro anticancer effects of bamboo salt on HepG2 human hepatoma cells.

    PubMed

    Zhao, Xin; Ju, Jaehyun; Kim, Hyung-Min; Park, Kun-Young

    2013-01-01

    Bamboo salt is a traditional Korean baked solar salt processed by packing the solar salt in bamboo joint cases and heating it several times to high temperatures. The antimutagenic activity and in vitro anticancer effects of bamboo salt on HepG2 human hepatoma cells were investigated and compared to those of other salt samples. Although solar salt and purified salt exhibited comutagenicity with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the Salmonella typhimurium TA100 strain, bamboo salt was associated with a lower degree of comutagenicity or antimutagenic activity. Bamboo salt baked nine times (9×) showed a greater increase in antimutagenic activity than salts baked once (1×) or three times (3×). At a concentration of 1%, the growth rate of HepG2 cells treated with 9× bamboo salt determined by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MIT) assay was reduced by 65%; this rate of inhibition was higher than that achieved with 1× baked bamboo salt (40%). Purified and solar salts had relatively lower inhibitory effects on growth rate (25% and 29%, respectively). Compared to the other salt samples, 9× bamboo salt significantly (p<0.05) induced apoptosis as determined by 4,6-diamidino-2-phenylindole (DAPI) staining and flow cytometry analysis. It also upregulated the expression of Bax, caspase-9 and caspase-3; and downregulated Bcl-2 expression. The bamboo salts, especially 9× bamboo salt, also significantly (p<0.05) downregulated the expression of inflammation-related NF-κB, iNOS, and COX-2, and upregulated the gene expression of IκB-α compared to the other salt sample.

  13. Complex regulation of transcription from the hepatitis B virus major surface antigen promoter in human hepatoma cell lines.

    PubMed Central

    Raney, A K; Milich, D R; McLachlan, A

    1991-01-01

    A detailed mutational analysis of the regulatory DNA sequence elements that control expression of the hepatitis B virus major surface antigen gene was performed in the human hepatoma cell lines HepG2.1 and Huh7, using transient transfection assays. Seven regions (A to G) of the major surface antigen promoter located within 200 nucleotides of the RNA initiation site have been identified which influence the level of transcription from this promoter. The three distal regions (A to C), located between -188 and -68, appear to possess a level of redundancy in their ability to influence the transcriptional activity from the major surface antigen promoter. The simultaneous deletion of regions A, B, and C resulted in an approximately fourfold reduction in transcription from the major surface antigen promoter. Region D, located between -67 and -49, is an essential element of the major surface antigen promoter. The three proximal regions (E to G) are located within 45 nucleotides of the major transcription initiation site. Region E prevents the negative influence of region F and can compensate for the effect of mutation of region G on transcription from the major surface antigen promoter. Region G can compensate for the effect of the loss of a functional region E sequence on the transcriptional activity of the major surface antigen promoter only in the absence of a functional region F sequence. These results imply that the level of expression of the major surface antigen gene is controlled by the complex interplay between a minimum of six transcription factors which activate and one transcription factor which represses transcription from this gene. PMID:1651407

  14. 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.

  15. Comparison of primary human hepatocytes and hepatoma cell line Hepg2 with regard to their biotransformation properties.

    PubMed

    Wilkening, Stefan; Stahl, Frank; Bader, Augustinus

    2003-08-01

    Cultures of primary hepatocytes and hepatoma cell line HepG2 are frequently used in in vitro models for human biotransformation studies. In this study, we characterized and compared the capacity of these model systems to indicate the presence of different classes of promutagens. Genotoxic sensitivity, enzyme activity, and gene expression were monitored in response to treatment with food promutagens benzo[a]pyrene, dimethylnitrosamine (DMN), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). DNA damage could be detected reliably with the comet assay in primary human hepatocytes, which were maintained in sandwich culture. All three promutagens caused DNA damage in primary cells, but in HepG2 no genotoxic effects of DMN and PhIP could be detected. We supposed that the lack of specific enzymes accounts for their inability to process these promutagens. Therefore, we quantified the expression of a broad range of genes coding for drug-metabolizing enzymes with real-time reverse transcription-polymerase chain reaction. The genes code for cytochromes p450 and, in addition, for a series of important phase II enzymes. The expression level of these genes in human hepatocytes was similar to those previously reported for human liver samples. On the other hand, expression levels in HepG2 differed significantly from that in human. Activity and expression, especially of phase I enzymes, were demonstrated to be extremely low in HepG2 cells. Up-regulation of specific genes by test substances was similar in both cell types. In conclusion, human hepatocytes are the preferred model for biotransformation in human liver, whereas HepG2 cells may be useful to study regulation of drug-metabolizing enzymes.

  16. The effect of zinc and D-penicillamine in a stable human hepatoma ATP7B knockout cell line.

    PubMed

    Chandhok, Gursimran; Schmitt, Nadine; Sauer, Vanessa; Aggarwal, Annu; Bhatt, Mohit; Schmidt, Hartmut H J

    2014-01-01

    Mutations in the copper (Cu) transporter gene ATP7B, the primary cause of Wilson disease (WD), result in high liver Cu and death of hepatocytes. Cu chelators and zinc salts are the two most important drugs used in the treatment of WD patients; however, the molecular mechanisms of the drugs with regard to ATP7B expression have not been determined. A targeted knockout of ATP7B (KO) was established in the most widely used human hepatoma cell line, HepG2 for molecular studies of the pathogenesis and treatment of the disease. KO cells showed similar growth, Cu uptake, release, and gene expression as compared to parental cells. However, in the presence of Cu, morphological changes, oxidative stress, apoptosis, and loss of viability were observed. Induction of metallothionein (MT1X) after Cu exposure was significantly reduced in KO cells. Following zinc treatment, MT1X expression was strongly induced and a high percentage of KO cells could be rescued from Cu induced toxicity. D-penicillamine treatment had a minor effect on the viability of KO cells whereas the parental cell line showed a pronounced improvement. Combined treatment displayed a highly synergistic effect in KO cells. The data suggest that zinc has a previously unrecognized effect on the viability of hepatocytes that lack ATP7B due to a high induction of MT1X expression that compensates low gene expression after Cu exposure. A combination therapy that simultaneously targets at MT1X induction and Cu chelation improves the overall survival of hepatocytes for most efficient therapy of patients having WD.

  17. Exploring the potential interference of estuarine sediment contaminants with the DNA repair capacity of human hepatoma cells.

    PubMed

    Pinto, Miguel Ferreira; Louro, Henriqueta; Costa, Pedro M; Caeiro, Sandra; Silva, Maria João

    2015-01-01

    Estuaries may be reservoirs of a wide variety of pollutants, including mutagenic and carcinogenic substances that may impact on the ecosystem and human health. A previous study showed that exposure of human hepatoma (HepG2) cells to extracts from sediment samples collected in two areas (urban/industrial and riverine/agricultural) of an impacted estuary (Sado, Portugal), produced differential cytotoxic and genotoxic effects. Those effects were found to be consistent with levels and nature of sediment contamination. The present study aimed at evaluating whether the mixtures of contaminants contained in those extracts were able to modulate DNA repair capacity of HepG2 cells. The residual level of DNA damage was measured by the comet assay in cells exposed for 24 or 48 h to different extracts, after a short preexposure to a challenging concentration range of ethyl methanesulfonate (EMS), as a model alkylating agent. The results suggested that the mixture of contaminants present in the tested samples, besides a potential direct effect on the DNA molecule, may also interfere with DNA repair mechanisms in HepG2 cells, thus impairing their ability to deal with genotoxic stress and, possibly, facilitating accumulation of mutations. Humans are environmentally/occupationally exposed to mixtures rather than to single chemicals. Thus, the observation that estuarine contaminants induce direct and indirect DNA strand breakage in human cells, the latter through the impairment of DNA repair, raises additional concerns regarding potential hazards from exposure and the need to further explore these endpoints in the context of environmental risk assessment.

  18. Geranylgeranylacetone has anti-hepatitis C virus activity via activation of mTOR in human hepatoma cells.

    PubMed

    Takeshita, Shigeyuki; Ichikawa, Tatsuki; Taura, Naota; Miyaaki, Hisamitsu; Matsuzaki, Toshihisa; Otani, Masashi; Muraoka, Toru; Akiyama, Motohisa; Miuma, Satoshi; Ozawa, Eisuke; Ikeda, Masanori; Kato, Nobuyuki; Isomoto, Hajime; Takeshima, Fuminao; Nakao, Kazuhiko

    2012-02-01

    Geranylgeranylacetone (GGA), an isoprenoid compound which includes retinoids, has been used orally as an anti-ulcer drug in Japan. GGA acts as a potent inducer of anti-viral gene expression by stimulating ISGF3 formation in human hepatoma cells. This drug has few side effects and reinforces the effect of IFN when administered in combination with peg-IFN and ribavirin. This study verified the anti-HCV activity of GGA in a replicon system. In addition, mechanisms of anti-HCV activity were examined in the replicon cells. OR6 cells stably harboring the full-length genotype 1 replicon containing the Renilla luciferase gene, ORN/C-5B/KE, were used to examine the influence of the anti-HCV effect of GGA. After treatment, the cells were harvested with Renilla lysis reagent and then subjected to a luciferase assay according to the manufacturer's protocol. The results showed that GGA had anti-HCV activity. GGA induced anti-HCV replicon activity in a time- and dose-dependent manner. GGA did not activate the tyrosine 701 and serine 727 on STAT-1, and did not induce HSP-70 in OR6 cells. The anti-HCV effect depended on the GGA induced mTOR activity, not STAT-1 activity and PKR. An additive effect was observed with a combination of IFN and GGA. GGA has mTOR dependent anti-HCV activity. There is a possibility that the GGA anti-HCV activity can be complimented by IFN. It will be necessary to examine the clinical effectiveness of the combination of GGA and IFN for HCV patients in the future.

  19. Glucocorticoid-regulated localization of cell surface glycoproteins in rat hepatoma cells is mediated within the Golgi complex

    PubMed Central

    1988-01-01

    Glucocorticoid hormones regulate the post-translational maturation and sorting of cell surface and extracellular mouse mammary tumor virus (MMTV) glycoproteins in M1.54 cells, a stably infected rat hepatoma cell line. Exposure to monensin significantly reduced the proteolytic maturation and externalization of viral glycoproteins resulting in a stable cellular accumulation of a single 70,000-Mr glycosylated polyprotein (designated gp70). Cell surface- and intracellular-specific immunoprecipitations of monensin-treated cells revealed that gp70 can be localized to the cell surface only in the presence of 1 microM dexamethasone, while in uninduced cells gp70 is irreversibly sequestered in an intracellular compartment. Analysis of oligosaccharide processing kinetics demonstrated that gp70 acquired resistance to endoglycosidase H with a half-time of 65 min in the presence or absence of hormone. In contrast, gp70 was inefficiently galactosylated after a 60-min lag in uninduced cells while rapidly acquiring this carbohydrate modification in the presence of dexamethasone. Furthermore, in the absence or presence of monensin, MMTV glycoproteins failed to be galactosylated in hormone-induced CR4 cells, a complement-selected sorting variant defective in the glucocorticoid-regulated compartmentalization of viral glycoproteins to the cell surface. Since dexamethasone had no apparent global effects on organelle morphology or production of total cell surface-galactosylated species, we conclude that glucocorticoids induce the localization of cell surface MMTV glycoproteins by regulating a highly selective step within the Golgi apparatus after the acquisition of endoglycosidase H- resistant oligosaccharide side chains but before or at the site of galactose attachment. PMID:2836430

  20. Antitumor and immunomodulatory effects of low-dose 5-FU on hepatoma 22 tumor-bearing mice

    PubMed Central

    CAO, ZHIYUN; ZHANG, ZHIDENG; HUANG, ZHENGRONG; WANG, RONGPING; YANG, AILIAN; LIAO, LIANMING; DU, JIAN

    2014-01-01

    Low-dose 5-fluorouracil (5-FU), a widely used chemotherapeutic, has been reported to have immunomodulatory effects. This study aimed to evaluate the optimal dose of 5-FU that produces antitumor and immunomodulatory effects. In a hepatoma 22 tumor-bearing mouse model, 0, 10, 20 and 40 mg/kg 5-FU (i.p.) was administered for 10 days. Tumor weight and volume were measured, thymus index (TI) and spleen index (SI) were calculated, and the number of white blood cells (WBCs) and lymphocytes (LYs) were counted following treatment. The percentages of CD3+, CD4+, CD8+ and natural killer (NK) cells were measured by flow cytometry. In addition, the body weights of the mice were measured and the average diet consumption was calculated. Administration of 5-FU produced a potent antitumor effect in a dose-dependent manner (P<0.01). At 20 and 40 mg/kg, a significant reduction of body weight and food consumption was observed. TI and SI decreased in the 20- and 40-mg/kg groups (P<0.01) for 10 days. The number of WBCs significantly decreased in each group (P<0.01); however, the number of LYs only decreased in the 40-mg/kg group (P<0.01). Percentages of CD3+ and CD4+ cells were increased in the 10- and 20-mg/kg groups (P<0.01). Thus, 5-FU at 10 mg/kg inhibits tumor growth while maintaining the immune function of the mice. 5-FU may exert its antitumor effect at a low dose with low toxicity and stimulate the host immune system. Future clinical trials taking into account the immunostimulatory capacity of chemotherapeutic agents are desirable for certain patients. PMID:24660037

  1. 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.

  2. Decorin protects human hepatoma HepG2 cells against oxygen-glucose deprivation via modulating autophagy.

    PubMed

    Ju, Wenbo; Li, Shubo; Wang, Zhaohui; Liu, Yanfeng; Wang, Dawei

    2015-01-01

    This study is to investigate the effects of decorin (DCN) on human hepatoma HepG2 cells under oxygen-glucose deprivation (OGD) condition. HepG2 cells were cultured under OGD condition. CCK-8 assay was used to assess the cell survival, and flow cytometry was performed to detect the apoptosis. Protein expression levels were detected with Western blot analysis. Transfection was performed with liposome, and cells were screened with G418. The cell survival rates were significantly decreased in the OGD groups. When treated with autophagy inhibitor 3-MA, the survival rates were further declined in these cells. Moreover, flow cytometry indicated that apoptosis occurred in the HepG2 cells under OGD condition, and the apoptosis rates were significantly increased by the 3-MA treatment. Western blot analysis showed that, the expression levels of DCN were significantly elevated in OGD-preconditioned HepG2 cells. Meanwhile, the expression level of Beclin1 and the LC3BI/LC3BII ratio were significantly increased, while the expression level of P62 was significantly decreased, in HepG2 cells under OGD condition. Over-expression of DCN significantly increased the expression level of Beclin1 and the LC3BI/LC3BII ratio, while no significant changes were observed in the P62 expression level, in HepG2 cells. Under the OGD condition, the apoptosis rate was also significantly decreased in DCN-transfected HepG2 cells. DCN protects HepG2 cells against OGD-induced injury, via regulating autophagy. These results might contribute to a better understanding of the roles of DCN and autophagy in hepatocellular carcinoma, and the potential treatment for the disease.

  3. Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells.

    PubMed

    Aninat, Caroline; Piton, Amélie; Glaise, Denise; Le Charpentier, Typhen; Langouët, Sophie; Morel, Fabrice; Guguen-Guillouzo, Christiane; Guillouzo, André

    2006-01-01

    Most human hepatocyte cell lines lack a substantial set of liver-specific functions, especially major cytochrome P450 (P450)-related enzyme activities, making them unrepresentative of in vivo hepatocytes. We have used the HepaRG cells, derived from a human hepatocellular carcinoma, which exhibit a high differentiation pattern after 2 weeks at confluency to determine whether they could mimic human hepatocytes for drug metabolism and toxicity studies. We show that when passaged at low density, these cells reversed to an undifferentiated morphology, actively divided, and, after having reached confluency, formed typical hepatocyte-like colonies surrounded by biliary epithelial-like cells. By contrast, when seeded at high density, hepatocyte-like clusters retained their typical differentiated morphology. Transcripts of various nuclear receptors (aryl hydrocarbon receptor, pregnane X receptor, constitutive androstane receptor, peroxisome proliferator-activated receptor alpha), P450s (CYP1A2, 2C9, 2D6, 2E1, 3A4), phase 2 enzymes (UGT1A1, GSTA1, GSTA4, GSTM1), and other liver-specific functions were estimated by reverse transcriptase-quantitative polymerase chain reaction and were found to be expressed, for most of them, at comparable levels in both confluent differentiated and high-density differentiated HepaRG cells and in cultured primary human hepatocytes. For several transcripts, the levels were strongly increased in the presence of 2% dimethyl sulfoxide. Measurement of basal activities of several P450s and their response to prototypical inducers as well as analysis of metabolic profiles and cytotoxicity of several compounds confirmed the functional resemblance of HepaRG cells to primary cultured human hepatocytes. In conclusion, HepaRG cells constitute the first human hepatoma cell line expressing high levels of the major P450s involved in xenobiotic metabolism and represent a reliable surrogate to human hepatocytes for drug metabolism and toxicity studies.

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

    PubMed Central

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

    2009-01-01

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

  5. Downregulation of tumorogenicity and changes in the actin cytoskeleton of murine hepatoma after irradiation with polychromatic visible and IR light.

    PubMed

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

    2015-04-01

    This study evaluated the function and structural consequences of direct exposure of murine hepatoma MH-22a cells to polychromatic polarized light, to determine potential risk of malignancy following irradiation. Visible (VIS) and infrared (IR) light have been actively used for prevention and treatment of complications developed after conventional tumor therapy. However, the safety associated with this irradiation has not been determined. Polychromatic light (480-3400 and 385-750 nm), were used at different doses (4.8-38.4 J/cm(2)) to determine the viability, proliferation, and actin cytoskeleton in vitro by flow cytometry and confocal microscopy. Tumorogenic properties of cells were studied in vivo after transplantation in C3HA mice. Polychromatic light of a wide range of doses did not change the viability and proliferation of cells. After transplantation of cells irradiated with VIS-IR light (4.8 and 9.6 J/cm(2)) and VIS light (38.4 J/cm(2)) the tumor volume was lower in the treated group than in the control group in vivo. Transplantability of the irradiated cells also decreased, whereas survival of tumor-bearing mice increased. Three cell populations with different cytoskeleton structure were identified. After irradiation, the reorganized part of the actin cytoskeleton changed its localization to the submembranous area. A decrease of tumorigenicity in cells irradiated with polychromatic light used in non-damaging doses correlated with an increase in the number of cells with reorganized actin in the submembranous area. The results of the present study argue in favor of the oncological safety of polychromatic VIS-IR light (480-3400 nm).

  6. Differential expression of five protein kinase C isoenzymes in FAO and HepG2 hepatoma cell lines compared with normal rat hepatocytes.

    PubMed

    Ducher, L; Croquet, F; Gil, S; Davy, J; Féger, J; Bréhier, A

    1995-12-14

    We analyzed the expression of five protein kinase C (PKC) isoforms in cytosolic and membrane fractions from normal rat hepatocytes compared with those of two tumorigenic cell lines FAO and HepG2. Western blots with PKC-specific isoenzymes polyclonal antibodies provide evidences for the presence of the five isoforms alpha, beta II, delta, epsilon and zeta in normal rat hepatocytes. In hepatoma cells, we show differences in the level of expression, the molecular sizes and the responses to Phorbol 12-myristate 13-acetate (PMA).

  7. Alpha particle-induced bystander effect is mediated by ROS via a p53-dependent SCO2 pathway in hepatoma cells.

    PubMed

    Li, Jitao; He, Mingyuan; Shen, Bo; Yuan, Dexiao; Shao, Chunlin

    2013-12-01

    The radiation-induced bystander effect (RIBE) has important implications for the efficiency of radiotherapy but the underlying role of cellular metabolism is widely unknown. The roles of synthesis of cytochrome c oxidase 2 (SCO2), a key effector for respiratory chain, and related signaling factors in α-particle-induced bystander damage were currently investigated in a liver cell co-culture system. Human hepatoma cells of HepG2 with wild-type p53 (wtp53) and Hep3B (p53 null) were irradiated with 0.4 Gy of α-particles and co-cultured with non-irradiated normal liver cells HL-7702 for 6 h, then the incidence of micronucleus (MN) in the bystander HL-7702 cells was analyzed. The expressions of total P53, phospho-P53 (p-P53), SCO2, and reactive oxygen species (ROS) in the irradiated hepatoma cells were detected. In some experiments, the hepatoma cells were respectively treated with p53 siRNA, SCO2 siRNA, or dimethyl sulfoxide (DMSO) before irradiation. Bystander damage in HL-7702 cells was induced by α-irradiated HepG2 cells but not by α-irradiated Hep3B cells, and this bystander effect was diminished when the irradiated HepG2 cells were pretreated with p53 siRNA, SCO2 siRNA, or DMSO. Meanwhile, the expressions of p-P53 protein and SCO2 mRNA, the activity of SCO2 protein, and intracellular ROS were all increased in the irradiated HepG2 cells but not Hep3B cells and these expressions were eliminated by p53 siRNA treatment. Moreover, the radiation-enhanced expressions of SCO2 and ROS were inhibited by SCO2 siRNA. α-particle-induced bystander effect was regulated by p53 and its downstream SCO2 in the irradiated hepatoma cells, and ROS generation could be an early event for triggering this bystander response.

  8. Effect of vitamin E and human placenta cysteine peptidase inhibitor on expression of cathepsins B and L in implanted hepatoma Morris 5123 tumor model in Wistar rats

    PubMed Central

    Sebzda, Tadeusz; Hanczyc, Piotr; Saleh, Yousif; Akinpelumi, Bernice F; Siewinski, Maciej; Rudnicki, Jerzy

    2005-01-01

    AIM: To examine the effectiveness of human placental inhibitors, by injecting vitamin E to rats with transplanted Morris-5123 hepatoma, on the expression of cathepsins B and L in tumor, liver, lung and blood sera after transplantation of Morris 5123 hepatoma. METHODS: Animals were divided into 10 groups receiving three different concentrations of vitamin E and inhibitors along or in combination and compared with negative control (healthy rats) and positive control (tumor rats). Effectiveness of treatment was evaluated with regard to survival time, tumor response and determination of the activities of proteolytic enzymes and their inhibitors using flurogenic substrates. RESULTS: Cathepsins B and L activities were elevated by 16-fold in comparison with negative control tissues, and their endogenous inhibitor activity decreased by 1.2-fold before treatment. In several cases, tumors completely disappeared following vitamin E plus human placental cyteine protease inhibitor (CPI) compared with controls. The number of complete tumor responses was higher when 20 m/kg vitamin E plus 400 μg of CPI was used, i.e., 7/10 rats survived more than two mo. Cathepsins B and L were expressed significantly in tumor, liver, lung tissues and sera in parallel to the increasing of the endogenous inhibitor activity compared with the controls after treatment (P<0.0001). CONCLUSION: The data indicate formation of metastasis significantly reduced in treated rats, which might provide a therapeutic basis for anti-cancer therapy. PMID:15641152

  9. Glutamine, insulin and glucocorticoids regulate glutamine synthetase expression in C2C12 myotubes, Hep G2 hepatoma cells and 3T3 L1 adipocytes.

    PubMed

    Wang, Yanxin; Watford, Malcolm

    2007-04-01

    The cell-specific regulation of glutamine synthetase expression was studied in three cell lines. In C2C12 myotubes, glucocorticoids increased the abundance of both glutamine synthetase protein and mRNA. Culture in the absence of glutamine also resulted in very high glutamine synthetase protein abundance but mRNA levels were unchanged. Glucocorticoids also increased the abundance of glutamine synthetase mRNA in Hep G2 hepatoma cells but this was not reflected in changes in protein abundance. Culture of Hep G2 cells without glutamine resulted in very high levels of protein, again with no change in mRNA abundance. Insulin was without effect in both C2C12 and Hep G2 cells. In 3T3 L1 adipocytes glucocorticoids increased the abundance of both glutamine synthetase mRNA and protein, insulin added alone had no effect but in the presence of glucocorticoids resulted in lower mRNA levels than seen with glucocorticoids alone, although protein levels remained high under such conditions. In contrast to the other cell lines glutamine synthetase protein levels were relatively unchanged by culture in the absence of glutamine. The results support the hypothesis that in myocytes, and hepatomas, but not in adipocytes, glutamine acts to moderate glutamine synthetase induction by glucocorticoids.

  10. The Chinese medicine Bu-Zhong-Yi-Qi-Tang inhibited proliferation of hepatoma cell lines by inducing apoptosis via G0/G1 arrest.

    PubMed

    Kao, S T; Yeh, C C; Hsieh, C C; Yang, M D; Lee, M R; Liu, H S; Lin, J G

    2001-08-17

    Bu-Zhong-Yi-Qi-Tang (BZYQT), a Chinese herbal medicine, inhibited the proliferation of human hepatoma cell lines (Hep3B, HepG2 and HA22T) dose-dependently. The IC50s of BZYQT on the proliferation of Hep3B, HepG2 and HA22T were 432.5+/-31.8 microg/ml, 455.4+/-24.2 microg/ml, and 2284.3+/-77.2 microg/ml respectively on day 3. However, BZYQT did not significantly inhibit the proliferation of normal human hepatocytes (Chang liver, CCL-13) at the concentration under 5,000 microg/ml. Major compounds of BZYQT, including astragaloside IV, ginsenoside Rb1 and Rg1, saikosaponin a and c, and glycyrrhizin, have been identified. To investigate the key inhibitors of BZYQT. Hep3B cells were treated with BZYQT, individual major compounds of BZYQT, and mixture of major compounds in the same ratio as present in BZYQT. Significant inhibition of proliferation was detected in BZYQT and its major compounds mixture in a comparable level. Not any individual major compound examined could suppress the proliferation of Hep3B cells. This data indicated that there could be synergistic or additive effects of the ingredients in BZYQT. BrdU incorporation, cell cycle analysis and DNA fragmentation assay revealed that BZYQT suppressed the proliferation of hepatoma cells via G0/G1 cell cycle arrest and inhibition of DNA synthesis followed by apoptosis.

  11. Enhancement of carboplatin- and quercetin-induced cell death by roscovitine is Akt dependent and p53 independent in hepatoma cells.

    PubMed

    Sharma, Aanchal; Bhat, Manoj Kumar

    2011-12-01

    Hepatocellular carcinoma (HCC) is a common malignancy worldwide and has an annual occurrence of one million new cases. Novel therapeutic strategies of increased efficacy in the treatment of HCC-bearing patients would certainly be helpful. Hence, the authors explored the effect of combination treatment of roscovitine with chemotherapeutic drugs or quercetin (Qctn) in hepatoma cells, HepG2 and Hep3B. Cell viability was assessed by MTT assay, cell growth assay, and nuclear morphological changes by DAPI staining. The altered expression of signaling proteins and apoptotic molecules was established by Western blotting. Roscovitine pretreatment considerably enhanced the drugs and Qctn-induced cell death in HepG2 and Hep3B cells. The exploratory studies revealed that augmented cell killing in HepG2 and Hep3B was mediated via Akt pathway and was independent of p53. pAkt was found to be significantly downregulated in combination treatment of roscovitine with carboplatin or Qctn. Corresponding to reduced expression of pAkt, the downstream molecules Bcl-2 and proactive forms of caspase 9 and caspase 3 were also downregulated indicating apoptosis. The present study reports for the first time, in hepatoma cells, the potentiation of carboplatin- and Qctn-induced cell death by the cell cycle inhibitor roscovitine. Roscovitine can thus be considered as a potential therapeutic target in combination with chemotherapeutic drugs or Qctn for treatment of HCC.

  12. Arecoline inhibits the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1A1 activation in human hepatoma cells.

    PubMed

    Chang, Eddy Essen; Miao, Zhi-Feng; Lee, Wen-Jhy; Chao, How-Ran; Li, Lih-Ann; Wang, Ya-Fen; Ko, Ying-Chin; Tsai, Feng-Yuan; Yeh, Szu Ching; Tsou, Tsui-Chun

    2007-07-19

    In the present study, we investigated the effect of arecoline, a major areca nut alkaloid, on the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced activation of cytochrome P4501A1 (CYP1A1) in a human hepatoma cell line Huh-7. We treated Huh-7 cells with 10nM TCDD in the presence of different concentrations of arecoline (50-300 microM). Our results indicated that arecoline attenuated the TCDD-induced CYP1A1 enzyme activation with an inhibitory effect on cell proliferation. By using real-time RT-PCR, we demonstrated that arecoline inhibited the TCDD-induced activations of CYP1A1 and AhR repressor (AhRR) mRNA expression in a similar pattern. Our results revealed that arecoline inhibited AhR mRNA expression with no direct effect on CYP1A1 enzyme activity. Therefore, in our present study, the observed inhibitory effect of arecoline on CYP1A1 activation was not due to the up-regulation of AhRR or direct inhibitory effect on CYP1A1. Taken together, here we have demonstrated that arecoline attenuates the TCDD-induced CYP1A1 activation mainly via down-regulation of AhR expression in human hepatoma cells, suggesting the possible involvement of arecoline in the AhR-mediated metabolism of environmental toxicants in liver.

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

    PubMed Central

    2011-01-01

    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. PMID:21711951

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

    PubMed

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

    2011-06-13

    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.

  15. Role of reactive oxygen species-mediated mitochondrial dysregulation in 3-bromopyruvate induced cell death in hepatoma cells : ROS-mediated cell death by 3-BrPA.

    PubMed

    Kim, Ji Su; Ahn, Keun Jae; Kim, Jeong-Ah; Kim, Hye Mi; Lee, Jong Doo; Lee, Jae Myun; Kim, Se Jong; Park, Jeon Han

    2008-12-01

    Hexokinase type II (HK II) is the key enzyme for maintaining increased glycolysis in cancer cells where it is overexpressed. 3-bromopyruvate (3-BrPA), an inhibitor of HK II, induces cell death in cancer cells. To elucidate the molecular mechanism of 3-BrPA-induced cell death, we used the hepatoma cell lines SNU449 (low expression of HKII) and Hep3B (high expression of HKII). 3-BrPA induced ATP depletion-dependent necrosis and apoptosis in both cell lines. 3-BrPA increased intracellular reactive oxygen species (ROS) leading to mitochondrial dysregulation. NAC (N-acetyl-L: -cysteine), an antioxidant, blocked 3-BrPA-induced ROS production, loss of mitochondrial membrane potential and cell death. 3-BrPA-mediated oxidative stress not only activated poly-ADP-ribose (PAR) but also translocated AIF from the mitochondria to the nucleus. Taken together, 3-BrPA induced ATP depletion-dependent necrosis and apoptosis and mitochondrial dysregulation due to ROS production are involved in 3-BrPA-induced cell death in hepatoma cells.

  16. 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.

  17. 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

  18. 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

  19. Specific growth stimulation by linoleic acid in hepatoma cell lines transfected with the target protein of a liver carcinogen.

    PubMed Central

    Keler, T; Barker, C S; Sorof, S

    1992-01-01

    The hepatic carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) was shown previously to interact specifically with its target protein, liver fatty acid binding protein (L-FABP), early during hepatocarcinogenesis in rats. In search of the significance of the interaction, rat L-FABP cDNA in the sense and antisense orientations was transfected into a subline of the rat hepatoma HTC cell line that did not express L-FABP. After the transfections, the basal doubling times of the cells were not significantly different. However, at 10(-5)-10(-7) M, linoleic acid, which is an essential fatty acid, a ligand of L-FABP, and the precursor of many eicosanoids and related lipids, stimulated the incorporation of [3H]thymidine in three randomly isolated and stably transfected cell clones that expressed L-FABP, but virtually did not stimulate the incorporation of [3H]thymidine in three L-FABP-nonexpressing clones transfected with the antisense DNA. Linoleic acid at 10(-6) M increased cell number almost 3-fold (38% vs. 14%; P less than 0.0001) and thymidine incorporation nearly 5-fold (23.2% vs. 4.9%; P less than 0.001) in the L-FABP-expressing cells compared to that in the transfected nonexpressing cells. L-FABP acted specifically and cooperatively with linoleic acid, inasmuch as all the proteins other than L-FABP in the transfected L-FABP nonexpressing cells and four other fatty acids (gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, and palmitoleic acid) were unable to effect a significant elevation or difference in the level of DNA synthesis that was attributable to the transfection. Metabolism of the linoleic acid to oxygenated derivatives was apparently necessary, since the cyclooxygenase inhibitor indomethacin partly inhibited and the antioxidant lipoxygenase inhibitors nordihydroguariaretic acid and alpha-tocopherol completely abolished the growth stimulation. The evidence supports the idea that L-FABP, the target protein of the liver carcinogen

  20. The aryl hydrocarbon receptor mediates raloxifene-induced apoptosis in estrogen receptor-negative hepatoma and breast cancer cells.

    PubMed

    O'Donnell, E F; Koch, D C; Bisson, W H; Jang, H S; Kolluri, S K

    2014-01-30

    Identification of new molecular targets for the treatment of breast cancer is an important clinical goal, especially for triple-negative breast cancer, which is refractory to existing targeted treatments. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known primarily as the mediator of dioxin toxicity. However, the AhR can also inhibit cellular proliferation in a ligand-dependent manner and act as a tumor suppressor in mice, and thus may be a potential anticancer target. To investigate the AhR as an anticancer target, we conducted a small molecule screen to discover novel AhR ligands with anticancer properties. We identified raloxifene, a selective estrogen receptor (ER) modulator currently used in the clinic for prevention of ER-positive breast cancer and osteoporosis in post-menopausal women, as an AhR activator. Raloxifene directly bound the AhR and induced apoptosis in ER-negative mouse and human hepatoma cells in an AhR-dependent manner, indicating that the AhR is a molecular target of raloxifene and mediates raloxifene-induced apoptosis in the absence of ER. Raloxifene selectively induced apoptosis of triple-negative MDA-MB-231 breast cancer cells compared with non-transformed mammary epithelial cells via the AhR. Combined with recent data showing that raloxifene inhibits triple-negative breast cancer xenografts in vivo (Int J Oncol. 43(3):785-92, 2013), our results support the possibility of repurposing of raloxifene as an AhR-targeted therapeutic for triple-negative breast cancer patients. To this end, we also evaluated the role of AhR expression on survival of patients diagnosed with breast cancer. We found that higher expression of the AhR is significantly associated with increased overall survival and distant metastasis-free survival in both hormone-dependent (ER-positive) and hormone-independent (ER and progesterone receptor (PR)-negative) breast cancers. Together, our data strongly support the possibility of using the Ah

  1. The aryl hydrocarbon receptor mediates raloxifene-induced apoptosis in estrogen receptor-negative hepatoma and breast cancer cells

    PubMed Central

    O'Donnell, E F; Koch, D C; Bisson, W H; Jang, H S; Kolluri, S K

    2014-01-01

    Identification of new molecular targets for the treatment of breast cancer is an important clinical goal, especially for triple-negative breast cancer, which is refractory to existing targeted treatments. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known primarily as the mediator of dioxin toxicity. However, the AhR can also inhibit cellular proliferation in a ligand-dependent manner and act as a tumor suppressor in mice, and thus may be a potential anticancer target. To investigate the AhR as an anticancer target, we conducted a small molecule screen to discover novel AhR ligands with anticancer properties. We identified raloxifene, a selective estrogen receptor (ER) modulator currently used in the clinic for prevention of ER-positive breast cancer and osteoporosis in post-menopausal women, as an AhR activator. Raloxifene directly bound the AhR and induced apoptosis in ER-negative mouse and human hepatoma cells in an AhR-dependent manner, indicating that the AhR is a molecular target of raloxifene and mediates raloxifene-induced apoptosis in the absence of ER. Raloxifene selectively induced apoptosis of triple-negative MDA-MB-231 breast cancer cells compared with non-transformed mammary epithelial cells via the AhR. Combined with recent data showing that raloxifene inhibits triple-negative breast cancer xenografts in vivo (Int J Oncol. 43(3):785-92, 2013), our results support the possibility of repurposing of raloxifene as an AhR-targeted therapeutic for triple-negative breast cancer patients. To this end, we also evaluated the role of AhR expression on survival of patients diagnosed with breast cancer. We found that higher expression of the AhR is significantly associated with increased overall survival and distant metastasis-free survival in both hormone-dependent (ER-positive) and hormone-independent (ER and progesterone receptor (PR)-negative) breast cancers. Together, our data strongly support the possibility of using the Ah

  2. Internalization and degradation of recombinant human coagulation factor VIIa by the human hepatoma cell line HuH7.

    PubMed

    Chang, G T; Kisiel, W

    1995-02-01

    Previous studies demonstrated that several normal and transformed cultured human cell lines specifically bind human coagulation factors VII and VIIa via tissue factor. In the present study, we show that 125I-radiolabeled recombinant human factor VIIa (125I-rFVIIa) binds to a human hepatoma cell line (HuH7). In the presence of rabbit polyclonal anti-human tissue factor apoprotein IgG, binding of 125I-rFVIIa to the HuH7 cells was decreased approximately 60%, suggesting of tissue factor-independent binding sites for 125I-rFVIIa on these cells. The binding isotherm of 125I-rFVIIa for the HuH7 cells in the presence of anti-tissue factor IgG exhibited a hyperbolic profile and was time-, temperature- and calcium-dependent. Furthermore, binding at 4 degrees C was specific, dose-dependent and saturable. Scatchard analysis of the binding data demonstrated a single class of binding sites with a dissociation constant (Kd) of 3.2 nM and 27,000 binding sites per cell. At 4 degrees C, 125I-rFVIIa bound to, and eluted from, the cell was indistinguishable from offered 125I-rFVIIa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. The molecular properties of the tissue factor-independent binding protein were studied by using the cleavable cross-linking agent 3,3'-dithiobis(sulfosuccinimidylpropionate). A cross-linking product of 125I-rFVIIa and a cell surface protein with an apparent M(r) approximately 100,000 was observed. The cross-linking reaction was strongly inhibited by a 100-fold molar excess of unlabeled rFVIIa, but not by rabbit polyclonal anti-human tissue factor apoprotein IgG, indicating that cross-linking does not involve the extracellular domain of tissue factor.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Fuzheng Qingjie Granules Inhibit Growth of Hepatoma Cells via Inducing Mitochondria-Mediated Apoptosis and Enhancing Immune Function.

    PubMed

    Chen, Xuzheng; Cao, Zhiyun; Zhang, Youquan; Li, Jinnong; Wang, Suqing; Du, Jian; Liao, Lianming

    2017-09-01

    Fuzheng Qingjie (FZQJ) granules, a compound Chinese medicine, have been used as an adjuvant therapy for alimentary tract cancers. However, the underlying anticancer mechanisms are still not well understood. In the present study, HepG2 cells were treated with FZQJ-containing serum. Cell proliferation was evaluated using MTT assay. Apoptosis was analyzed using a flow cytometer. Cell ultrastructure was observed under a transmission electron microscope. The mitochondrial membrane potential (Δψ) was examined with JC-1 dye. In H22 tumor-bearing mice, CD4(+) T cells, CD8(+) T cells, CD3(+) T cells, and natural killer (NK) cells in peripheral blood were evaluated cytometrically. Interleukin (IL)-2 and tumor necrosis factor (TNF)-α levels were measured using radioimmunoassay.The mRNA levels of Bax and Bcl-2 were examined by reverse transcription-polymerase chain reaction. The protein levels of Bax, Bcl-2, cytochrome C, caspase 3 and 9, PARP, and CD69 were examined by Western blotting. The apoptotic cells in tissues were observed using TUNEL method. Alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (CRE) were detected by an automatic biochemical analyzer. The results showed that FZQJ-containing serum remarkably inhibited proliferation of HepG2 cells in dose- and time-dependent manners, induced HepG2 cell apoptosis and caused a decrease of Δψ. Analysis of tumor tissue showed that FZQJ-induced apoptosis was accompanied by downregulation of Bcl-2 and upregulation of Bax, release of cytochrome c, activation of caspase 3 and 9, and cleavage of PARP. In addition, FZQJ increased the percentages of CD4(+) T and NK cells, the ratio of CD4(+)/CD8(+) T cells as well as the levels of serum TNF-α. FZQJ also increased CD69 expression in tumor tissue. No hepatorenal toxicity was observed in H22 tumor-bearing mice. These results indicated that FZQJ could inhibit the growth of hepatoma cells via regulating immune function and inducing

  4. [Inhibitory effect of magnesium cantharidate on human hepatoma SMMC-7721 cell proliferation by blocking MAPK signaling pathway].

    PubMed

    Liu, Yun; Li, Xiaofei; Zou, Qianqian; Liu, Liu; Zhu, Xinting; Jia, Qi; Wang, Lingjun; Yan, Rong

    2017-03-01

    Objective To investigate the anticancer mechanism of magnesium cantharidate by observing its effect on the mitogen-activated protein kinase (MAPK) signaling pathway in human hepatoma SMMC-7721 cells. Methods The protein phosphatase 2A (PP2A) activity detection kit was used to detect the effects of magnesium cantharidate and okadaic acid (OA) on PP2A activity. After the treatment of SMMC-7721 cells with magnesium cantharidate and/or OA, mRNA levels of extracellular signal-regulated kinase 1 (ERK1), ERK2, p38MAPK, c-Jun N-terminal kinase 1 (JNK1) and JNK2 were detected by real-time quantitative PCR, and the protein expression levels and protein phosphorylation of ERK1, ERK2, p38 MAPK and JNK were determined by Western blotting. Results The effect of magnesium cantharidate on the activity of PP2A in SMMC-7721 cells was not evident at the concentration of 0.283 μmol/L, but the activity of PP2A was declined significantly at 0.567 μmol/L or higher concencentrations in a concentration-dependent manner. Likewise, OA also displayed apparent inhibitory effect on the activity of PP2A at 0.059 nmol/L. Compared with the control group, mRNA levels of ERK1 and ERK2 were not changed by magnesium cantharidate at 0.283 μmol/L, but they significantly declined at the concentrations greater than 0.567 μmol/L. In contrast, mRNA levels of ERK1 and ERK2 were significantly elevated by 0.059 nmol/L OA. mRNA levels of p38MAPK, JNK1 and JNK2 significantly increased after the treatment of 0.059 nmol/L OA or magnesium cantharidate at varying concentrations. Compared with the control group, phosphorylation levels of ERK1 and ERK2 were not changed by 0.283 μmol/L magnesium cantharidate, but decreased significantly when the concentration was 0.567 μmol/L or above. In contrast, the phosphorylation levels of ERK1 and ERK2 showed a significant increase in 0.059 nmol/L OA treated group. The phosphorylation levels of p38 MAPK, JNK1 and JNK2 were also significantly increased by 0.059 nmol/L OA or

  5. Hepatitis B Virus X Protein Driven Alpha Fetoprotein Expression to Promote Malignant Behaviors of Normal Liver Cells and Hepatoma Cells

    PubMed Central

    Zhu, Mingyue; Lu, Yan; Li, Wei; Guo, Junli; Dong, Xu; Lin, Bo; Chen, Yi; Xie, Xieju; Li, Mengsen

    2016-01-01

    Background: The infection of Hepatitis B virus (HBV) is closely associated with the development of hepatocellular carcinoma(HCC), HBV-X protein(HBx) is able to induce expression of alpha-fetoprotein(AFP) in normal liver cells, and AFP harbors a function to promote malignant transformation of normal liver cells, but the role AFP playing in malignant behaviors of HCC cells is still unclear. Methods: Fifty-six liver tissue samples were collected from the clinical patients through hepatectomy(include normal liver tissues, HBV-related hepatitis liver tissues and HBV-related HCC tissues), and diagnosis of these tissues by pathology section, expression of AFP, Ras and CXCR4 were evidenced by immunohisochemical staining and Western blotting; The proliferation of human normal liver cells line L-02 cells and human hepatoma cells line, HLE cells(non AFP-producing) were performed by MTT method; Repaired capacity of L-02 and HLE cells were compared by wound healing assay; Migration and invasion of these cells were analyzed by Transwell chamber assay; HBx expressed vectors(pcDNA3.1-HBx) were constructed and transfected into L-02 and HLE cells, effects of pcDNA3.1-HBx on the malignant behaviors were also detected by MTT, Transwell chamber assay and the expression of AFP, Ras and CXCR4 were evidenced by Western blotting. Results: we found that expression of AFP, Ras and CXCR4 in HBV-related HCC and lymph nodes metastasis tissues were significantly elevated compared with HBV-related HCC, non metastasis tissues and HBV-related hepatitis tissues; Expression of AFP, Ras and CXCR4 in HBV-related hepatitis tissues were significantly enhanced compared with normal liver tissues; The growth ratio, migratory and invasive ability, expression of AFP, Ras and CXCR4 of the cells were outstanding promoted while L-02 and HLE cells were transfected with pcDNA3.1-HBx vectors. The proliferation ratio, migration and invasion ability, and expression of Ras and CXCR4 were significantly inhibited while

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

    SciTech Connect

    Wang, Yijun; Lu, Hongjuan; Wang, Dongxu; Li, Shengrong; Sun, Kang; Wan, Xiaochun; Taylor, Ethan Will; Zhang, Jinsong

    2012-12-15

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

  7. Association of topoisomerase II with the hepatoma cell nuclear matrix: the role of intermolecular disulfide bond formation.

    PubMed

    Kaufmann, S H; Shaper, J H

    1991-02-01

    Previous studies have resulted in conflicting data regarding the recovery of the nuclear enzymes topoisomerase (topo) II and topo I in the nuclear matrix fraction. In the present study we have assessed the effect of systematically altering a single extraction procedure on the distribution of these enzymes during the subfractionation of nuclei from HTC hepatoma tissue culture cells. When nuclear monolayers (prepared by treating attached cells in situ with the neutral detergent Nonidet-P40 at 4 degrees C) were isolated in the presence of the irreversible sulfhydryl blocking reagent iodoacetamide, subsequent treatment with DNase I and RNase A followed by 1.6 M NaCl resulted in structures which were extensively depleted of intranuclear components as assessed by phase contrast microscopy and conventional transmission electron microscopy. These structures contained 12 +/- 4% of the total protein present in the original nuclear monolayers. The lamins and polypeptides with molecular weights comparable to those of actin and vimentin were the predominant polypeptides present on SDS-polyacrylamide gels. Western blotting revealed that less than 5% of the total nuclear topo II molecules were present in these structures. In contrast, when the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) was substituted for iodoacetamide, the same extraction procedure yielded structures containing components of the nucleolus and an extensive intranuclear network. These structures contained a wide variety of nonlamin, nonhistone nuclear polypeptides including 23 +/- 4% of the total nuclear topo II. SDS-polyacrylamide gel electrophoresis performed under nonreducing conditions revealed that topo II in these nuclear matrices was present as part of a large disulfide cross-linked complex. Treatment of these structures with reducing agents in 1.6 M NaCl released the topo II. In contrast, topo I did not form disulfide cross-linked oligomers and was not detectable in any of these nuclease

  8. Different Contribution of Redox-Sensitive Transient Receptor Potential Channels to Acetaminophen-Induced Death of Human Hepatoma Cell Line

    PubMed Central

    Badr, Heba; Kozai, Daisuke; Sakaguchi, Reiko; Numata, Tomohiro; Mori, Yasuo

    2016-01-01

    Acetaminophen (APAP) is a safe analgesic antipyretic drug at prescribed doses. Its overdose, however, can cause life-threatening liver damage. Though, involvement of oxidative stress is widely acknowledged in APAP-induced hepatocellular death, the mechanism of this increased oxidative stress and the associated alterations in Ca2+ homeostasis are still unclear. Among members of transient receptor potential (TRP) channels activated in response to oxidative stress, we here identify that redox-sensitive TRPV1, TRPC1, TRPM2, and TRPM7 channels underlie Ca2+ entry and downstream cellular damages induced by APAP in human hepatoma (HepG2) cells. Our data indicate that APAP treatment of HepG2 cells resulted in increased reactive oxygen species (ROS) production, glutathione (GSH) depletion, and Ca2+ entry leading to increased apoptotic cell death. These responses were significantly suppressed by pretreatment with the ROS scavengers N-acetyl-L-cysteine (NAC) and 4,5-dihydroxy-1,3-benzene disulfonic acid disodium salt monohydrate (Tiron), and also by preincubation of cells with the glutathione inducer Dimethylfumarate (DMF). TRP subtype-targeted pharmacological blockers and siRNAs strategy revealed that suppression of either TRPV1, TRPC1, TRPM2, or TRPM7 reduced APAP-induced ROS formation, Ca2+ influx, and cell death; the effects of suppression of TRPV1 or TRPC1, known to be activated by oxidative cysteine modifications, were stronger than those of TRPM2 or TRPM7. Interestingly, TRPV1 and TRPC1 were labeled by the cysteine-selective modification reagent, 5,5′-dithiobis (2-nitrobenzoic acid)-2biotin (DTNB-2Bio), and this was attenuated by pretreatment with APAP, suggesting that APAP and/or its oxidized metabolites act directly on the modification target cysteine residues of TRPV1 and TRPC1 proteins. In human liver tissue, TRPV1, TRPC1, TRPM2, and TRPM7 channels transcripts were localized mainly to hepatocytes and Kupffer cells. Our findings strongly suggest that APAP

  9. The effects of urotensin II on migration and invasion are mediated by NADPH oxidase-derived reactive oxygen species through the c-Jun N-terminal kinase pathway in human hepatoma cells.

    PubMed

    Li, Ying-Ying; Shi, Zheng-Ming; Yu, Xiao-Tong; Feng, Ping; Wang, Xue-Jiang

    2017-02-01

    Urotensin II (UII) is a vasoactive neuropeptide involved in migration and invasion in various cell types. However, the effects of UII on human hepatoma cells still remain unclear. The aim of this study was to investigate the role and mechanism of UII on migration and invasion in human hepatoma cells. Migration was measured by wound healing assays and a Transwell(®) methodology, and invasion was analyzed using Matrigel(®) invasion chambers. Reactive oxygen species (ROS) levels were detected using a 2', 7'-dichlorofluorescein diacetate probe, and flow cytometry, and protein expression levels were evaluated by western blotting. Cell proliferation and actin polymerization were examined using cell proliferation reagent WST-1 and F-actin immunohistochemistry staining. Exposure to UII promoted migration and invasion in hepatoma cells compared with that in cells without UII. UII also increased matrix metalloproteinase-2 (MMP2) expression in a time-independent manner. Furthermore, UII markedly enhanced ROS generation and NADPH oxidase subunit expression, and consequently facilitated the phosphorylation of c-Jun N-terminal kinase (JNK). The UT antagonist urantide or the antioxidant/NADPH oxidase inhibitor apocynin decreased UII-induced ROS production. JNK phosphorylation, migration, invasion, and MMP9/2 expression were also reversed by pretreatment with apocynin. Urantide and JNK inhibitor SP600125 abrogated migration, invasion, or MMP9/2 expression in response to UII. UII induced actin polymerization and fascin protein expression, and could be reversed by apocynin and SP600125. Exogenous UII induced migration and invasion in hepatoma cells that mainly involved NADPH oxidase-derived ROS through JNK activation. UT played an additional role in regulating hepatoma cells migration and invasion. Thus, our data suggested an important effect of UII in hepatocellular carcinoma metastasis. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Hepatitis B virus X protein upregulates Lin28A/Lin28B through Sp-1/c-Myc to enhance the proliferation of hepatoma cells.

    PubMed

    You, X; Liu, F; Zhang, T; Lv, N; Liu, Q; Shan, C; Du, Y; Kong, G; Wang, T; Ye, L; Zhang, X

    2014-01-23

    Hepatitis B virus X protein (HBx) plays critical roles in the pathogenesis of hepatocellular carcinoma (HCC). Here, we were interested in knowing whether the oncogene Lin28A and its homolog Lin28B are involved in the hepatocarcinogenesis mediated by HBx. We showed that the expression levels of Lin28A and Lin28B were increased in clinical HCC tissues, HepG2.2.15 cell line and liver tissues of p21-HBx transgenic mice. Interestingly, the expression levels of HBx were positively associated with those of Lin28A/Lin28B in clinical HCC tissues. Moreover, the overexpression of HBx resulted in the upregulation of Lin28A/Lin28B in hepatoma HepG2/H7402 cell lines by transient transfection, suggesting that HBx was able to upregulate Lin28A and Lin28B. Then, we examined the mechanism by which HBx upregulated Lin28A and Lin28B. We identified that the promoter region of Lin28A regulated by HBx was located at nt -235/-66 that contained Sp-1 binding element. Co-immunoprecipitation showed that HBx was able to interact with Sp-1 in HepG2-X cells. Moreover, chromatin immunoprecipitation (ChIP) demonstrated that HBx could bind to the promoter of Lin28A, which failed to work when Sp-1 was silenced. Electrophoretic mobility shift assay (EMSA) further identified that HBx was able to interact with Sp-1 element in Lin28A promoter via transcription factor Sp-1. In addition, we found that c-Myc was involved in the activation of Lin28B mediated by HBx. In function, Lin28A/Lin28B played important roles in HBx-enhanced proliferation of hepatoma cells in vitro and in vivo. In conclusion, HBx activates Lin28A/Lin28B through Sp-1/c-Myc in hepatoma cells. Lin28A/Lin28B serves as key driver genes in HBx-induced hepatocarcinogenesis.

  11. Herbal compound “Songyou Yin” attenuates hepatoma cell invasiveness and metastasis through downregulation of cytokines secreted by activated hepatic stellate cells

    PubMed Central

    2013-01-01

    Background Activated hepatic stellate cells (aHSCs) play an important role in the progression of hepatocellular carcinoma (HCC). Here, we determined if cytokines secreted in response to the herbal compound “Songyou Yin” (SYY) treatment of aHSCs could influence invasiveness and metastatic capabilities of hepatoma cells. Methods Primary rat hepatic stellate cells (HSCs) were isolated, activated, divided into SYY treated and untreated (nSYY) groups, and conditioned media (CM-SYY and CM-nSYY, respectively) were collected. The hepatoma cell line, McA-RH7777 was cultured for 4 weeks with SYY, CM-SYY, and CM-nSYY, designated McA-SYY, McA-SYYCM and McA-nSYYCM. The invasiveness and metastatic capabilities were evaluated using Matrigel invasion assay in vitro and pulmonary metastasis in vivo. Matrix metalloproteinase-2 (MMP-2), MMP-9, E-cadherin, N-cadherin, and vimentin protein levels in McA-SYYCM and McA-nSYYCM were evaluated by Western blot. Cytokine levels in conditioned media were tested using enzyme-linked immunosorbent assay (ELISA). Results Matrigel invasion assay indicated that the number of McA-SYYCM cells passing through the basement membrane was less than in McA-nSYYCM cells (P < 0.01). Similar results were also observed in vivo for lung metastasis. McA-SYYCM cells showed less pulmonary metastasis capabilities than McA-nSYYCM cells (P < 0.001). The reduced expression of MMP-2 and reversed epithelial to mesenchymal transition with E-cadherin upregulation, and N-cadherin and vimentin downregulation were also found in McA-SYYCM compared to McA-nSYYCM. Metastasis-promoting cytokines hepatocyte growth factor, interleukin-6, transforming growth factor-β1, and vascular endothelial growth factor were markedly decreased in CM-SYY compared to CM-nSYY. Conclusions SYY attenuates hepatoma cell invasiveness and metastasis capabilities through downregulating cytokines secreted by activated hepatic stellate cells. PMID:23622143

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

    SciTech Connect

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

    2012-10-15

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

  13. MicroRNA-148a suppresses the epithelial-mesenchymal transition and metastasis of hepatoma cells by targeting Met/Snail signaling.

    PubMed

    Zhang, J-P; Zeng, C; Xu, L; Gong, J; Fang, J-H; Zhuang, S-M

    2014-07-31

    Metastasis is responsible for the rapid recurrence and poor survival of malignancies. Epithelial-mesenchymal transition (EMT) has a critical role in metastasis. Increasing evidence indicates that EMT can be regulated by microRNAs (miRNAs). miR-148a is a liver-abundant miRNA. However, the role of miR-148a in the development of liver cancer remains largely unknown. In this study, we found that, compared with normal livers, miR-148a was significantly decreased in hepatocellular carcinoma (HCC) tissues, especially in those with the portal vein tumor thrombus. An in vitro transwell assay and an in vivo orthotopic liver xenograft model showed that the restoration of miR-148a expression significantly repressed the migration and pulmonary metastasis of hepatoma cells. Linear regression analysis revealed a positive correlation between the expression of miR-148a and the mRNA level of E-cadherin gene in human HCC tissues. Both gain- and loss-of-function studies disclosed that miR-148a promoted the expression of epithelial marker (E-cadherin) and reduced the levels of mesenchymal markers (N-cadherin, fibronectin or vimentin) in hepatoma cells. These data suggest that miR-148a may suppress EMT and cancer metastasis. Further mechanistic investigations showed that miR-148a directly inhibited Met expression by binding to its 3'-UTR. Moreover, the reintroduction of miR-148a attenuated the downstream signaling of Met, like activated phosphorylation of AKT-Ser473 and inhibitory phosphorylation of GSK-3β-Ser9, and consequently reduced the nuclear accumulation of Snail, a transcription factor that promotes EMT. Taken together, miR-148a may negatively regulate Met/Snail signaling and therefore inhibit the EMT and metastasis of hepatoma cells. These findings highlight the significance of miR-148a downregulation in tumor progression and implicate miR-148a as an attractive candidate for cancer therapy.

  14. Increase of O6-methylguanine-DNA-methyltransferase and N3-methyladenine glycosylase RNA transcripts in rat hepatoma cells treated with DNA-damaging agents

    SciTech Connect

    Laval, F. )

    1991-05-15

    A variety of DNA-damaging agents increase the O6-methylguanine-DNA-methyltransferase (transferase) and the N3-methyladenine (3-meAde)-DNA-glycosylase activities in a rat hepatoma cell line (H4 cells). Using two cDNA expressing either the rat 3-meAde-DNA-glycosylase or the transferase, the level of mRNA transcripts was measured by hybridization in H4 cells treated with three different inducing agents, gamma-rays, cis-dichlorodiammine platinum II or N-methyl-9-hydroxy ellipticinium. The two mRNA increased 24 hours after the cell treatments but this enhanced transcription was a transient phenomenon, as it was no longer observed after 96 hours. No significant DNA amplification was detectable in the treated cells.

  15. Detection for cross-reactive proteins in filarial worm Setaria equina, MCF-7 human breast cancer, and Huh-7 hepatoma cells.

    PubMed

    Abdel-Latif, Mahmoud; Sakran, Thabet

    2016-01-01

    This study aimed to detect the cross-reactive proteins in filarial parasite adult worm Setaria equina and two different tumor cell lines (MCF-7 human breast cancer and Huh-7 hepatoma cells). This was performed using rabbit anti-S. equina extract (SeqE) or DEC (Diethylcarbamazine citrate) polyclonal IgG antibodies by indirect ELISA and western blotting. The results indicated cross-reactive bands at 70 and 75 kDa in all extracts by anti-DEC and SeqE antibodies, respectively. In addition, the expression of 70 kDa protein was only reduced in filarial worms and Huh-7 after in vitro DEC treatment compared to the control.

  16. Monocyte-conditioned medium, interleukin-1, and tumor necrosis factor stimulate the acute phase response in human hepatoma cells in vitro

    PubMed Central

    1986-01-01

    Human hepatoma cells mimic the acute phase response after treatment with monocyte-conditioned medium. Levels of secreted fibrinogen, alpha- 1 acid glycoprotein, C-reactive protein, haptoglobin, and the third component of complement were elevated compared with control levels after 48 h of incubation with conditioned supernatant medium from an enriched fraction of normal peripheral monocytes. Albumin levels declined and alpha-1 antitrypsin remained unchanged. Levels of specific mRNA were measured by hybridization to slot blots and Northern blots and changed in correspondence with protein alterations. Interleukin-1 and tumor necrosis factor stimulated the third component of complement, but did not elevate any other member of the acute phase group and were therefore only partially active in this system. The identification of an in vitro model of the human acute phase response will permit analysis of the molecular basis for coordinate regulation of this group of facultative genes. PMID:3017995

  17. Sulphation of proteins secreted by a human hepatoma-derived cell line. Sulphation of N-linked oligosaccharides on alpha 2HS-glycoprotein.

    PubMed Central

    Hortin, G; Green, E D; Baenziger, J U; Strauss, A W

    1986-01-01

    Several human glycoproteins, including alpha 1-antitrypsin, alpha 1-acid glycoprotein, transferrin, caeruloplasmin and alpha 2HS-glycoprotein, synthesized by the hepatoma-derived cell line HepG2 were observed to contain covalently linked sulphate. These proteins were estimated to contain about 0.1 mol of sulphate/mol of protein. The most abundant of the sulphated glycoproteins, alpha 2HS-glycoprotein, was analysed in detail. All of the sulphate on this protein was attached to N-linked oligosaccharides which contained sialic acid and resisted release by endoglycosidase H. Several independent analytical approaches established that approx. 10% of the molecules of alpha 2HS-glycoprotein contained sulphate. Our results suggest that a number of human plasma proteins contain small amounts of sulphate linked to oligosaccharides. Images Fig. 1. Fig. 2. Fig. 3. PMID:3017304

  18. Long chain acyl-CoA synthetase 3-mediated phosphatidylcholine synthesis is required for assembly of very low density lipoproteins in human hepatoma Huh7 cells.

    PubMed

    Yao, Hongbing; Ye, Jin

    2008-01-11

    Hepatocytes play a crucial role in regulating lipid metabolism by exporting cholesterol and triglyceride into plasma through secretion of very low density lipoproteins (VLDL). VLDL production is also required for release of hepatitis C virus (HCV) from infected hepatocytes. Here, we show that long chain acyl-CoA synthetase 3 (ACSL3) plays a crucial role in secretion of VLDL and HCV from hepatocytes. In cultured human hepatoma Huh7 cells, ACSL3 is specifically required for incorporation of fatty acids into phosphatidylcholine. In cells receiving small interfering RNA targeting ACSL3, secretion of apolipoprotein B, the major protein component of VLDL, was inhibited and the lipoprotein was rapidly degraded. This inhibition in secretion was completely eliminated when these cells were treated with phosphatidylcholine. Treatment of cells with small interfering RNA targeting ACSL3 also inhibited secretion of HCV from Huh7-derived cells. These results identify ACSL3 as a new enzymatic target to limit VLDL secretion and HCV infection.

  19. Comparative cytotoxicity induced by bulk and nanoparticulated ZnO in the fish and human hepatoma cell lines PLHC-1 and Hep G2.

    PubMed

    Fernández-Cruz, Maria Luisa; Lammel, Tobias; Connolly, Mona; Conde, Estefania; Barrado, Ana Isabel; Derick, Sylvain; Perez, Yolanda; Fernandez, Marta; Furger, Christophe; Navas, Jose Maria

    2013-08-01

    The increasing presence of ZnO nanoparticles (NPs) in consumer products may be having a dramatic impact in aquatic environments. The evaluation of ZnO NP toxicity represents a great challenge. This study aimed at evaluating the cytotoxic effect of micro- and nanosized ZnO in a fish and a mammalian hepatoma cell line. A detailed characterisation of the particles in exposure media showed that ZnO NPs formed large aggregates. ZnO cytotoxicity was evaluated with a battery of in vitro assays including LUCS, a new approach based on DNA alteration measurements. In fish cells, ZnO NP aggregates contributed substantially to the cytotoxic effects whereas toxicity in the human cells appeared to be mainly produced by the dissolved fraction. ROS production did not contribute to the observed cytotoxicity. This work also showed that measuring concentrations of NPs is essential to understand the mechanisms underlying their toxicity.

  20. Synthesis of spirolactone-type diterpenoid derivatives from kaurene-type oridonin with improved antiproliferative effects and their apoptosis-inducing activity in human hepatoma Bel-7402 cells.

    PubMed

    Li, Dahong; Cai, Hao; Jiang, Bowen; Liu, Guyue; Wang, Yuetong; Wang, Lei; Yao, Hequan; Wu, Xiaoming; Sun, Yijun; Xu, Jinyi

    2013-01-01

    A series of novel spirolactone-type diterpenoid derivatives of oridonin (12a-j) were designed and synthesized. All the target compounds showed improved anti-proliferative activity against a panel of human cancer cell lines and the most effective compound 12j was more potent than positive control Taxol in K562 and Bel-7402 cells with IC(50) values of 0.39 μM and 1.39 μM, respectively. The cellular mechanisms showed that compound 12j induced apoptosis at low micromolar concentrations in human hepatoma Bel-7402 cells. These results demonstrate that the spirolactone-type diterpenoid derivatives of oridonin have optimized growth inhibitory activity against cancer cells and interesting apoptosis-inducing ability.

  1. Preparation of novel butyryl galactose ester-modified coix component microemulsions and evaluation on hepatoma-targeting in vitro and in vivo.

    PubMed

    Liu, Ming Jian; Qu, Ding; Chen, Yan; Liu, Cong Yan; Liu, Yu Ping; Ding, Xue Fang

    2016-11-01

    The butyryl galactose ester-modified coix component microemulsions (But-Gal-CMEs) was developed for enhanced liver tumor-specific targeting. The study was aimed to evaluate the hepatoma-targeting potential of But-Gal-CMEs in vitro and in vivo. But-Gal-CMEs with a uniform spherical shape exhibited a small particle size (56.68 ± 0.07 nm), a narrow polydispersity (PDI, 0.144 ± 0.005) and slightly negative surface charge (-0.102 ± 0.008 mV). In the cell uptake studies, But-Gal-CMEs showed a significant enhancement on the intracellular fluorescent intensity on HepG2 cells model, which was 1.93-fold higher relative to coix component microemulsions (CMEs). The IC50 of But-Gal-CMEs against HepG2 cells was 64.250 μg/mL, which was notably stronger than that of CMEs. In the cell apoptosis studies, compared with CMEs, But-Gal-CMEs (50 μg/mL) treatment resulted in a 1.34-fold rise in total apoptosis cells of HepG2. In the biodistribution studies in vivo, the intratumorous fluorescence of Cy5-loaded But-Gal-CMEs was 1.43-fold higher relative to that of Cy5-loaded CMEs, suggesting an obviously enhanced accumulation in the tumor sites. Taken as together, But-Gal could be incorporated into the coix component microemulsions as a novel ligand for realizing hepatoma-targeting drugs delivery.

  2. Differential action of 13-HPODE on PPARalpha downstream genes in rat Fao and human HepG2 hepatoma cell lines.

    PubMed

    König, Bettina; Eder, Klaus

    2006-06-01

    In rats, oxidized fats activate the peroxisome proliferator-activated receptor alpha (PPARalpha), leading to reduced triglyceride concentrations in liver, plasma and very low density lipoproteins. Oxidation products of linoleic acid constitute an important portion of oxidized dietary fats. This study was conducted to check whether the primary lipid peroxidation product of linoleic acid, 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE), might be involved in the PPARalpha-activating effect of oxidized fats. Therefore, we examined the effect of 13-HPODE on the expression of PPARalpha target genes in the rat Fao and the human HepG2 hepatoma cell lines. In Fao cells, 13-HPODE increased the mRNA concentration of the PPARalpha target genes acyl-CoA oxidase (ACO), cytochrome P450 4A1 and carnitine-palmitoyltransferase 1A (CPT1A). Furthermore, the concentration of cellular and secreted triglycerides was reduced in Fao cells treated with 13-HPODE. Because PPARalpha mRNA was not influenced, we conclude that these effects are due to an activation of PPARalpha by 13-HPODE. In contrast, HepG2 cells seemed to be resistant to PPARalpha activation by 13-HPODE because no remarkable induction of the PPARalpha target genes ACO, CPT1A, mitochondrial HMG-CoA synthase and delta9-desaturase was observed. Consequently, cellular and secreted triglyceride levels were not changed after incubation of HepG2 cells with 13-HPODE. In conclusion, this study shows that 13-HPODE activates PPARalpha in rat Fao but not in human HepG2 hepatoma cells.

  3. HAb18G/CD147 inhibits starvation-induced autophagy in human hepatoma cell SMMC7721 with an involvement of Beclin 1 down-regulation.

    PubMed

    Gou, Xingchun; Ru, Qiang; Zhang, Hongxin; Chen, Yanke; Li, Ling; Yang, Hushan; Xing, Jinliang; Chen, Zhinan

    2009-05-01

    HAb18G/CD147, a transmembrane glycoprotein highly expressed in various types of malignant cells, mainly functions as an inducer of matrix metalloproteinases to promote tumor growth, invasion and metastasis. However, whether there are other mechanisms underlying the role of HAb18G/CD147 in tumor progression remains to be elucidated. In this study, we investigated the functional effects of HAb18G/CD147 on autophagy in hepatoma cell line SMMC7721 using immunofluorescence staining, Western blot and transmission electronmicroscopy. Our data showed that specific small interference RNA (siRNA) considerably down-regulated the expression of HAb18G/CD147 in SMMC7721 cells at both messenger RNA (mRNA) and protein levels. The down-regulation of HAb18G/CD147 significantly promoted starvation-induced autophagy in a dose-dependent manner. Using trypan blue exclusion assay, we found that HAb18G/CD147 notably enhanced the survival of SMMC7721 cells through inhibiting starvation-induced autophagy. In addition, we demonstrated that HAb18G/CD147 down-regulated the expression of autophagy-regulating protein Beclin 1 in SMMC7721 cells. Furthermore, our data indicated that HAb18G siRNA-transfected SMMC7721 cells had a significantly decreased level of phosphorylated serine/threonine protein kinase B (pAkt) and the expression of Beclin 1 was inversely associated with the level of pAkt, suggesting that the Class I phosphatidylinositol 3 kinase-Akt pathway may be involved in the down-regulation of Beclin 1 by HAb18G/CD147. Overall, we provide the first experimental evidence to show that HAb18G/CD147 may play an important role in the inhibitory regulation of autophagy. Therefore, our data suggest a new molecular mechanism for HAb18G-mediated hepatoma progression.

  4. Intracellular trafficking and cellular uptake mechanism of mPEG-PLGA-PLL and mPEG-PLGA-PLL-Gal nanoparticles for targeted delivery to hepatomas.

    PubMed

    Liu, Peifeng; Sun, Yanming; Wang, Qi; Sun, Ying; Li, He; Duan, Yourong

    2014-01-01

    The lysosomal escape of nanoparticles is crucial to enhancing their delivery and therapeutic efficiency. Here, we report the cellular uptake mechanism, lysosomal escape, and organelle morphology effect of monomethoxy (polyethylene glycol)-poly (D,L-lactide-co-glycolide)-poly (L-lysine) (mPEG-PLGA-PLL, PEAL) and 4-O-beta-D-Galactopyranosyl-D-gluconic acid (Gal)-modified PEAL (PEAL-Gal) for intracellular delivery to HepG2, Huh7, and PLC hepatoma cells. These results indicate that PEAL is taken up by clathrin-mediated endocytosis of HepG2, Huh7 and PLC cells. For PEAL-Gal, sialic acid receptor-mediated endocytosis and clathrin-mediated endocytosis are the primary uptake pathways in HepG2 cells, respectively, whereas PEAL-Gal is internalized by sag vesicle- and clathrin-mediated endocytosis in Huh7 cells. In the case of PLC cells, clathrin-mediated endocytosis and sialic acid receptor play a primary role in the uptake of PEAL-Gal. TEM results verify that PEAL and PEAL-Gal lead to a different influence on organelle morphology of HepG2, Huh7 and PLC cells. In addition, the results of intracellular distribution reveal that PEAL and PEAL-Gal are less entrapped in the lysosomes of HepG2 and Huh7 cells, demonstrating that they effectively escape from lysosomes and contribute to enhance the efficiency of intracellular delivery and tumor therapy. In vivo tumor targeting image results demonstrate that PEAL-Gal specifically delivers Rhodamine B (Rb) to the tumor tissue of mice with HepG2, Huh7, and PLC hepatomas and remains at a high concentration in tumor tissue until 48 h, properties that will greatly contribute to enhanced antitumor efficiency. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  5. The role of inorganic metals and metalloporphyrins in the induction of haem oxygenase and heat-shock protein 70 in human hepatoma cells.

    PubMed Central

    Mitani, K; Fujita, H; Fukuda, Y; Kappas, A; Sassa, S

    1993-01-01

    The role of inorganic metals and metalloporphyrins in the induction of mRNAs for haem oxygenase and heat-shock protein 70 (hsp70), the two heat-shock proteins, was examined in human HepG2 and Hep3B hepatoma cells. SnCl2, but not Sn-protoporphyrin, was found to be a potent inducer of both haem oxygenase and hsp70 mRNAs. In contrast, CoCl2, ZnCl2 and FeCl2 caused little induction of haem oxygenase and hsp70 mRNAs, whereas the porphyrin complexes of these metals strongly induced haem oxygenase mRNA, without influencing the level of hsp70 mRNA. The induction process was largely transcriptional, as judged by the inhibition of induction by actinomycin D, but not by cycloheximide, and by increased transcription demonstrated by nuclear run-off analysis. Since CoCl2 is a potent inducer of haem oxygenase in vivo in animals, the possibility of the biosynthesis of Co-protoporphyrin was examined in human hepatoma cells by incubating them with CoCl2 and protoporphyrin, or delta-aminolaevulinate (ALA), the precursor of protoporphyrin. Both types of treatment led to a potent induction of haem oxygenase mRNA. Co-protoporphyrin formation was also spectrally demonstrated in cells incubated with the metal and ALA. The results of this study indicate that certain metals, e.g. SnCl2, may directly induce haem oxygenase mRNA, whereas with other elements, incorporation of the metal into the porphyrin macrocycle is necessary for induction. Therefore CoCl2, like haemin, may activate the haem oxygenase gene via a haem-responsive transcription factor, whereas SnCl2 may exert its effect via a metal-responsive transcription factor. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8384446

  6. Type I Interferon Supports Inducible Nitric Oxide Synthase in Murine Hepatoma Cells and Hepatocytes and during Experimental Acetaminophen-Induced Liver Damage.

    PubMed

    Bachmann, Malte; Waibler, Zoe; Pleli, Thomas; Pfeilschifter, Josef; Mühl, Heiko

    2017-01-01

    Cytokine regulation of high-output nitric oxide (NO) derived from inducible NO synthase (iNOS) is critically involved in inflammation biology and host defense. Herein, we set out to characterize the role of type I interferon (IFN) as potential regulator of hepatic iNOS in vitro and in vivo. In this regard, we identified in murine Hepa1-6 hepatoma cells a potent synergism between pro-inflammatory interleukin-β/tumor necrosis factor-α and immunoregulatory IFNβ as detected by analysis of iNOS expression and nitrite release. Upregulation of iNOS by IFNβ coincided with enhanced binding of signal transducer and activator of transcription-1 to a regulatory region at the murine iNOS promoter known to support target gene expression in response to this signaling pathway. Synergistic iNOS induction under the influence of IFNβ was confirmed in alternate murine Hepa56.1D hepatoma cells and primary hepatocytes. To assess iNOS regulation by type I IFN in vivo, murine acetaminophen (APAP)-induced sterile liver inflammation was investigated. In this model of acute liver injury, excessive necroinflammation drives iNOS expression in diverse liver cell types, among others hepatocytes. Herein, we demonstrate impaired iNOS expression in type I IFN receptor-deficient mice which associated with diminished APAP-induced liver damage. Data presented indicate a vital role of type I IFN within the inflamed liver for fine-tuning pathological processes such as overt iNOS expression.

  7. HSV-1 Cgal+ Infection Promotes Quaking RNA Binding Protein Production and Induces Nuclear-Cytoplasmic Shuttling of Quaking I-5 Isoform in Human Hepatoma Cells*

    PubMed Central

    Sánchez-Quiles, Virginia; Mora, María I.; Segura, Victor; Greco, Anna; Epstein, Alberto L.; Foschini, Maria Giovanna; Dayon, Loïc; Sanchez, Jean-Charles; Prieto, Jesús; Corrales, Fernando J.; Santamaría, Enrique

    2011-01-01

    Herpesvirus type 1 (HSV-1) based oncolytic vectors arise as a promising therapeutic alternative for neoplastic diseases including hepatocellular carcinoma. However, the mechanisms mediating the host cell response to such treatments are not completely known. It is well established that HSV-1 infection induces functional and structural alterations in the nucleus of the host cell. In the present work, we have used gel-based and shotgun proteomic strategies to elucidate the signaling pathways impaired in the nucleus of human hepatoma cells (Huh7) upon HSV-1 Cgal+ infection. Both approaches allowed the identification of differential proteins suggesting impairment of cell functions involved in many aspects of host-virus interaction such as transcription regulation, mRNA processing, and mRNA splicing. Based on our proteomic data and additional functional studies, cellular protein quaking content (QKI) increases 4 hours postinfection (hpi), when viral immediate-early genes such as ICP4 and ICP27 could be also detected. Depletion of QKI expression by small interfering RNA results in reduction of viral immediate-early protein levels, subsequent decrease in early and late viral protein content, and a reduction in the viral yield indicating that QKI directly interferes with viral replication. In particular, HSV-1 Cgal+ induces a transient increase in quaking I-5 isoform (QKI-5) levels, in parallel with an enhancement of p27Kip1 protein content. Moreover, immunofluorescence microscopy showed an early nuclear redistribution of QKI-5, shuttling from the nucleus to the cytosol and colocalizing with nectin-1 in cell to cell contact regions at 16–24 hpi. This evidence sheds new light on mechanisms mediating hepatoma cell response to HSV-1 vectors highlighting QKI as a central molecular mediator. PMID:21467216

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

    PubMed

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

    2017-12-01

    Orange Jessamine [Murraya paniculata L. (Rutaceae)] has been used worldwide in folk medicine as an anti-inflammatory, antibiotic and analgesic. The objective of this study is to investigate the in vitro antioxidant, cytotoxic, antibacterial and antifungal activity and the time-kill curve studies of orange jessamine essential oil and β-caryophyllene, as well as the chemical composition of the essential oil. The cytotoxic activity of M. paniculata and β-caryophyllene (7.8-500 μg/mL) was evaluated using the MTT assay on normal fibroblasts and hepatoma cells. The minimal inhibitory concentration and time-kill curves (24 h) were evaluated against those of Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Enterococcus faecallis, Aspergillus (niger, fumigates and parasiticum) and F. solani by the broth microdilution method. The antioxidant activity was measured by the DPPH and ABTS assays. Chemical composition was evaluated by GC/MS analyses. GC/MS analyses identified 13 compounds, with β-caryophyllene as the major compound. The oil exhibited moderate antibacterial activity (MIC <1.0 mg/mL) and strong antifungal activity. Time-kill curve studies showed that either the essential oil or β-caryophyllene presented rapid bacterial killing (4 h for S. aureus) and fungicidal effect (2-4 h for F. solani); however, both displayed weak free radical scavenger capacity. The cytotoxic activity exhibited a prominent selective effect against hepatoma cancer cells (IC50 value =63.7 μg/mL) compared with normal fibroblasts (IC50 value =195.0 μg/mL), whereas the β-caryophyllene showed low cytotoxicity. The experimental data suggest that the activities of M. paniculata essential oil are due to the synergistic action among its components.

  9. Insulin-like growth factor-I stimulates H{sub 4}II rat hepatoma cell proliferation: Dominant role of PI-3'K/Akt signaling

    SciTech Connect

    Alexia, Catherine; Fourmatgeat, Pascal; Delautier, Daniele; Groyer, Andre . E-mail: groyer@bichat.inserm.fr

    2006-04-15

    Although hepatocytes are the primary source of endocrine IGF-I and -II in mammals, their autocrine/paracrine role in the dysregulation of proliferation and apoptosis during hepatocarcinogenesis and in hepatocarcinomas (HCC) remains to be elucidated. Indeed, IGF-II and type-I IGF receptors are overexpressed in HCC cells, and IGF-I is synthesized in adjacent non-tumoral liver tissue. In the present study, we have investigated the effects of type-I IGF receptor signaling on H{sub 4}II rat hepatoma cell proliferation, as estimated by {sup 3}H-thymidine incorporation into DNA. IGF-I stimulated the rate of DNA synthesis of serum-deprived H{sub 4}II cells, stimulation being maximal 3 h after the onset of IGF-I treatment and remaining elevated until at least 6 h. The IGF-I-induced increase in DNA replication was abolished by LY294002 and only partially inhibited by PD98059, suggesting that phosphoinositol-3' kinase (PI-3'K) and to a lesser extent MEK/Erk signaling were involved. Furthermore, the 3- to 19-fold activation of the Erks in the presence of LY294002 suggested a down-regulation of the MEK/Erk cascade by PI-3'K signaling. Finally, the effect of IGF-I on DNA replication was almost completely abolished in clones of H{sub 4}II cells expressing a dominant-negative form of Akt but was unaltered by rapamycin treatment of wild-type H{sub 4}II cells. Altogether, these data support the notion that the stimulation of H{sub 4}II rat hepatoma cell proliferation by IGF-I is especially dependent on Akt activation but independent on the Akt/mTOR signal0009i.

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

    PubMed Central

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

    2016-01-01

    Although it has showed that icaritin can apparently suppress growth of HCC by reducing the level of AFP, the intrinsic mechanism remains unclear. In this study, we explored the possible mechanism of miRNAs on post-transcriptional regulation of AFP gene, as well as the effects of HBV infection and icaritin in hepatoma cells. The results showed that miR-620, miR-1236 and miR-1270 could bind target sites in the range of 9–18 nt and 131–151 nt downstream of the stop codon in the AFP mRNA 3′-UTR to suppress the expression of AFP. Mutation of these target sites could reverse the effects of these miRNAs. Icaritin (10 μM) might reduce the stability and translational activity of AFP mRNA by increasing the expression levels of these mentioned miRNAs. HBV infection resulted in apparent decreases of these miRNAs and, consequently, increased AFP expression. The results indicated that miR-620, miR-1236 and miR-1270 are critical factors in the post-transcriptional regulation of AFP. Icaritin can counteract the effect of HBV. These findings will contribute to full understanding of the regulatory mechanism of AFP expression in hepatoma cells. And also it revealed a synergistic mechanism of HBV infection and elevation of AFP in the pathogenesis of HCC, as well as the potential clinical significance of icaritin on the therapy of HCC induced by HBV. PMID:27835879

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

    PubMed

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

    2017-10-01

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

  12. Claudin-1, miR-122 and apolipoprotein E transductions improve the permissivity of SNU-182, SNU-398 and SNU-449 hepatoma cells to hepatitis C virus.

    PubMed

    Fournier, C; Hoffmann, T W; Morel, V; Descamps, V; Dubuisson, J; Brochot, E; Francois, C; Duverlie, G; Castelain, S; Helle, F

    2017-08-03

    Hepatitis C virus (HCV) is a human hepatotropic virus, but many hepatoma cell lines are not permissive to this virus. In a previous study, we observed that SNU-182, SNU-398 and SNU-449 hepatoma cell lines were nonpermissive to HCV. To understand the nonpermissivity, we evaluated the ability of each cell line to support the different steps of HCV life cycle (entry, replication and production of infectious particles). Using retroviral pseudoparticles pseudotyped with HCV envelope proteins and recombinant HCV produced in cell culture, we observed that low level or absence of claudin-1 (CLDN1) expression limited the viral entry process in SNU-182 and SNU-398 cells, respectively. Our results also showed that supplementation of the three cell lines with miR-122 partly restored the replication of a JFH1 HCV replicon. Finally, we observed that expression of apolipoprotein E (ApoE) was very low or undetectable in the three cell lines and that its ectopic expression permits the production of infectious viral particles in SNU-182 and SNU-398 cells but not in SNU-449 cells. Nevertheless, the supplementation of SNU-182, SNU-398 and SNU-449 cells with CLDN1, miR-122 and ApoE was not sufficient to render these cells as permissive as HuH-7 cells. Thus, these cell lines could serve as cell culture models for functional studies on the role of CLDN1, miR-122 and ApoE in HCV life cycle but also for the identification of new restriction and/or dependency host factors essential for HCV infection. © 2017 John Wiley & Sons Ltd.

  13. Trace element profiles of the oxyanions of selenium in the development of murine hepatoma and the effect of Se supplementation on the essential trace element concentrations

    SciTech Connect

    Tariq, M.A.

    1992-01-01

    To determine trace quantities of selenium in tissue samples a simplified pre-concentration procedure is developed based upon sample digestion and separation of selenium from the bulk by solvent extraction and its determination by radioisotope induced x-ray fluorescence (RIXRF) spectrometry. Signal to background ratio is improved and interference from Br and As peaks is significantly reduced. Submicrogram quantities of selenium in animal tissues can be analyzed. The distribution and retention of selenate and selenite in C57L/J mice was investigated using intra-ocular injection of [sup 75]Se. The distribution and retention of species were studied during the progression of BBW756 murine hepatoma. Comparison is made with the normal distribution of selenium studied by RIXRF method. The distribution of the two oxidation states measured as activity of [sup 75]Se was almost identical but differed from the normal trace elemental profile, TEP. The excretion rates of the two selenium species in the initial phase are different with more selenate being excreted than selenite in the first two days. The whole body excretion rates followed a pure first order pattern after day two. This was not true for the individual organs of the animals. Selenium has shown both inhibiting and enhancing effects on the development of various cancer types. The effects of selenium supplementation on the development of BW7756 murine hepatoma have been monitored. Various non-lethal levels of Se are administrated in either drinking water or by intraperitoneal injection. The inhibitory effects are not specific to any stage of tumor development and the decrease in tumor growth is related to the general toxicity of the element. Supplementation of selenium at 2 [mu]g/ml level in the drinking water of the mice strongly influenced the concentration levels of Fe, Cu, Zn, Br, and Rb. The effect of selenite supplementation on the tissue levels of these elements is significantly different than that of selenate.

  14. Effects of simultaneous dietary fish oil ingestion and sulfur amino acid supplementation on the lipid metabolism in hepatoma-bearing rats with hyperlipidemia.

    PubMed

    Kawasaki, Masashi; Miura, Yutaka; Funabiki, Ryuhei; Yagasaki, Kazumi

    2010-01-01

    The effects of simultaneous dietary fish oil ingestion and sulfur amino acid (L-methionine and L-cystine) supplementation on serum lipid concentrations and various parameters related to the lipid metabolism were studied in Donryu rats subcutaneously implanted with an ascites hepatoma cell line, AH109A. A diet containing 10% fish oil was found to reduce serum triglyceride, total cholesterol, (very-low-density lipoprotein plus low-density lipoprotein)-cholesterol, phospholipid and nonesterified fatty acid (NEFA) concentrations in these animals, and dietary supplementation of 1.2% L-methionine and L-cystine also suppressed these serum lipid concentrations. Hepatic fatty acid synthesis and the availability of serum NEFA were decreased, and epididymal adipose tissue lipoprotein lipase (LPL) activity was elevated by dietary fish oil, while LPL activity in various tissues and hepatic fatty acid oxidation were increased by dietary sulfur amino acids, resulting in a reduction in the serum triglyceride concentration by dietary fish oil and sulfur amino acids, respectively. Dietary fish oil suppressed the hepatoma-induced increase in cholesterogenesis in the host liver, and dietary methionine and cystine enhanced bile acid excretion into feces, which were the causes of the hypocholesterolemic effect. In these serum lipid concentrations, there were significant effects of fish oil ingestion and sulfur amino acid supplementation, but no significant interaction between these two factors was seen. These results indicate that dietary fish oil and sulfur amino acid, L-methionine and L-cystine, have hypolipidemic effects in cancer-related hyperlipidemia, and that the effects of these two factors on the decrease in these serum lipid concentrations are additive; these two factors may affect the lipid metabolism via different pathways and mechanisms.

  15. Melatonin Represses Oxidative Stress-Induced Activation of the MAP Kinase and mTOR Signaling Pathways in H4IIE Hepatoma Cells Through Inhibition of Ras

    PubMed Central

    Kimball, Scot R.; Abbas, Ahmed; Jefferson, Leonard S.

    2009-01-01

    SUMMARY Reactive oxygen species (ROS) have been implicated in the pathogenesis of a variety of diseases, and antioxidant treatment is currently being investigated as a potential therapy to attenuate the detrimental effects of ROS-mediated oxidative stress. Melatonin is a potent naturally produced antioxidant, which acts through various mechanisms to ameliorate the toxic effects of ROS. However, little is known about the mechanisms or signaling pathways through which melatonin acts to reverse the effects of ROS. In the present study, the effect of melatonin treatment on the hydrogen peroxide (H2O2)-induced activation of the mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling pathways was assessed in H4IIE hepatoma cells. It was found that melatonin strongly attenuated H2O2-induced activation of the ERK1/2 and p38 MAP kinases, as well as several of their downstream targets. Melatonin also attenuated the H2O2-induced phosphorylation of Akt and the Akt substrate mTOR, as well as a downstream target of mTOR action, 4E-BP1. Upregulation of ERK1/2, p38, and Akt signaling by H2O2 was accompanied by activation of Ras, an effect that was blocked by melatonin. Overall, the results suggest that melatonin acts to prevent many of the H2O2-induced alterations in the MAPK and mTOR signaling pathways through inhibition of Ras, at least in H4IIE hepatoma cells. PMID:18410586

  16. Antcin K, an active triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea, induces mitochondria and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells

    PubMed Central

    Lai, Chiao-I.; Chu, Yung-Lin; Ho, Chi-Tang; Su, Yu-Cheng; Kuo, Yueh-Hsiung; Sheen, Lee-Yan

    2015-01-01

    Liver cancer is the second leading cause of cancer deaths in Taiwan as per the 2011 statistics and ranks fourth in cancer-related mortality in the world. Recent researches have shown that Antrodia cinnamomea, a Taiwan-specific medicinal mushroom, has biological activities, including hepatoprotection, anti-inflammation, antihepatitis B virus activity, and anticancer activity. In the present study, the antiproliferative activity and molecular mechanisms of antcin K, the most abundant ergostane triterpenoid from the fruiting bodies of basswood cultivated A. cinnamomea, were investigated using human hepatoma Hep 3B cells. The results showed that antcin K effectively reduced Hep 3B cells viability within 48 hours. Antcin K induced phosphatidylserine exposure, chromatin condensation, and DNA damage, but did not significantly increase autophagosome content or cause cell expansion and cell lysis. Thus, the principal mode of Hep 3B cells death induced by antcin K was apoptosis, rather than autophagy or necrosis. In-depth investigation of the molecular mechanisms revealed that antcin K first promoted reactive oxygen species generation and adenosine triphosphate depletion, leading to endoplasmic reticulum stress and resulting in mitochondrial membrane permeability changes. After losing the mitochondrial membrane potential, caspase-independent and caspase-dependent apoptosis-related proteins were released, including HtrA2, apoptotic-induced factor, endonuclease G, and cytochrome c. Cytochrome c activated caspase-9 and caspase-3, and cut downstream protein PARP, ultimately leading to cell apoptosis. These results suggested that antcin K induced mitochondrial and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells. Coupled with these findings, antcin K has a potential to be a complementary agent in liver cancer therapy. PMID:26870680

  17. Gypenosides induce apoptosis by ca2+ overload mediated by endoplasmic-reticulum and store-operated ca2+ channels in human hepatoma cells.

    PubMed

    Sun, Da-Peng; Li, Xiao-Xi; Liu, Xin-Li; Zhao, Dan; Qiu, Feng-Qi; Li, Yan; Ma, Ping

    2013-05-01

    Gypenosides (Gyps) are triterpenoid saponins contained in an extract from Gynostemma pentaphyllum Makino and reported to induce apoptosis in human hepatoma cells through Ca(2+)-implicated endoplasmic reticulum (ER) stress and mitochondria-dependent pathways. The mechanism underlying the Gyp-increased intracellular Ca(2+) concentration ([Ca(2+)]i) is unclear. Here, we examined Gyp-induced necrosis and apoptosis in human hepatoma HepG2 cells. Gyp-induced apoptotic cell death was accompanied by a sustained increase in [Ca(2+)]i level. Gyp-increased [Ca(2+)]i level was partly inhibited by removal of extracellular Ca(2+) by Ca(2+) chelator EGTA, store-operated Ca(2+) channel (SOC) inhibitor 2- aminoethoxydiphenyl borate (2-APB), and ER Ca(2+)-release-antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8). The strongest inhibitory effect was observed with TMB-8. EGTA, 2-APB, and TMB-8 also protected against Gyp-induced apoptosis in HepG2 cells. The combination of 2-APB and TMB-8 almost completely abolished the Gyp-induced Ca(2+) response and apoptosis. In contrast, the sarco/endoplasmic-reticulum-Ca(2+)-ATPase (SERCA) inhibitor thapsigargin slightly elevated Gyp-induced [Ca(2+)]i increase and apoptosis in HepG2 cells. Exposure to 300 μg/mL Gyp for 24 hours upregulated protein levels of inositol 1,4,5-trisphosphate receptor and SOC and downregulated that of SERCA for at least 72 hours. Thus, Gyp-induced increase in [Ca(2+)]i level and consequent apoptosis in HepG2 cells may be mainly due to enhanced Ca(2+) release from ER stores and increased store-operated Ca(2+) entry.

  18. Enhancement of the cytotoxicity and selectivity of doxorubicin to hepatoma cells by synergistic combination of galactose-decorated γ-poly(glutamic acid) nanoparticles and low-intensity ultrasound.

    PubMed

    Tsai, Wei-Bor; Lai, Hsin-Yu; Lee, Jyun-Lin; Lo, Chia-Wen; Chen, Wen-Shiang

    2014-05-20

    Specific drug delivery to solid tumors remains one of the challenges in cancer therapy. The aim of this study was to combine three drug-targeting strategies, polymer-drug conjugate, ligand presentation and ultrasound treatment, to enhance the efficacy and selectivity of doxorubicin (DXR) to hepatoma cells. The conjugation of DXR to γ-poly(glutamic acids) (γ-PGA) decreased the cytotoxicity of DXR, while the conjugation of galactosamine (Gal) to the γ-PGA-DXR conjugate restored the cytotoxic efficacy of DXR on hepatoma cells due to increased uptake of DXR. Furthermore, low-intensity ultrasound treatment increased the cell-killing ability of γ-PGA-DXR conjugates by 20%. The in vitro results showed the potential of the γ-PGA-DXR-Gal conjugate for future clinical applications.

  19. Interferon α–Stimulated Natural Killer Cells From Patients With Acute Hepatitis C Virus (HCV) Infection Recognize HCV-Infected and Uninfected Hepatoma Cells via DNAX accessory molecule-1

    PubMed Central

    Stegmann, Kerstin A.; Björkström, Niklas K.; Ciesek, Sandra; Lunemann, Sebastian; Jaroszewicz, Jerzy; Wiegand, Johannes; Malinski, Phillipp; Dustin, Lynn B.; Rice, Charles M.; Manns, Michael P.; Pietschmann, Thomas; Cornberg, Markus; Ljunggren, Hans-Gustaf

    2012-01-01

    Background. Natural killer (NK) cells are an important component of the innate immune defense against viruses, including hepatitis C virus (HCV). The cell culture system using HCV-permissive Huh-7.5 cells make studies on interaction of NK cells and HCV-infected target cells possible. We used this system to characterize interactions of HCV-infected Huh-7.5 cells and NK cells from healthy controls and patients with acute HCV infection. Methods. IFNα- and IL-2 stimulated NK cells were cultured with HCV-infected hepatoma cells and subsequently analyzed (for degranulation and cytokine production) via multicolour flow cytometry. Luciferase assyas have been used to study inhibition of HCV replication. Further, PBMC from patients with acute hepatitis C as well as HCV-infected Huh7.5 cells have been analyzed via flow cytometry for expression of NK cell receptors and ligands, respectively. Results. After interferon (IFN) α stimulation, NK cells from healthy controls and patients with acute hepatitis C efficiently recognized both HCV-infected and uninfected hepatoma cells. Subsequent dissection of receptor-ligand interaction revealed a dominant role for DNAM-1 and a complementary contribution of NKG2D for NK cell activation in this setting. Furthermore, IFN-α–stimulated NK cells effectively inhibited HCV replication in a DNAM-1–dependent manner. Conclusions. Human NK cells recognize HCV-infected hepatoma cells after IFN-α stimulation in a DNAM-1–dependent manner. Furthermore, interaction of IFN-α–stimulated NK cells with HCV-infected hepatoma cells efficiently reduced HCV replication. This study opens up future studies of NK cell interaction with HCV-infected hepatocytes to gain further insight into the pathogenesis of human HCV infection and the therapeutic effects of IFN-α. PMID:22457290

  20. Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. II. Temporal and spatial kinetics

    PubMed Central

    1982-01-01

    The activation of cyclic AMP-dependent protein kinase has been found to be the predominant mode by which cyclic AMP (cAMP) leads to alterations of a large variety of cellular functions. The activation of the kinase results in the release of the catalytic subunit which as the free enzyme possesses phosphotransferase activity for a variety of specific protein substrates. Using a sensitive and specific cytofluorometric technique we monitored the appearance of free catalytic subunit in Reuber H35 hepatoma cells in culture after incubation with N6-1'-O- dibutyryl-cyclic AMP (DBcAMP), 8-bromoadenosine-3':5'-cyclic monophosphate (8-BrcAMP), and glucagon. The cytochemical method employs the heat-stable inhibitor of the free catalytic subunit which has been conjugated to fluorescein isothiocyanate (F:PKI) and was validated as described in the companion paper (Fletcher and Byus. 1982. J. Cell Biol. 93:719-726). Here we studied the temporal and spatial kinetics of the free catalytic subunit following activation of cAMP-dependent protein kinase by increasing concentrations of DBcAMP,8-BrcAMP, and glucagon. Under similar conditions protein kinase activation was also assessed biochemically in H35 cell supernatants by assaying the protein kinase activity ratio. Incubation of the hepatoma cells with DBcAMP (0.1 mM) led to an increase in the activity ratio from 0.2 in control cultures to a value of nearly 1.0 within a 1- to 2-h period. During this same period using the F:PKI probe, a significant increase in cytoplasmic and nucleolar fluorescence indicative of the release of the free catalytic subunit was coincidentally observed. In contrast to the rapid appearance of catalytic subunit in the cytoplasm and nucleolus of the cell within 5-15 min of the addition of DBcAMP, discernible nucleoplasmic fluorescence did not occur until after 1 h. H35 cell cultures incubated with 8-BrcAMP (0.01-1.0 mM) exhibited a more rapid activation of the protein kinase measured cytochemically compared

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

    SciTech Connect

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

    2009-05-15

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

  2. Development and characterization of P-glycoprotein 1 (Pgp1, ABCB1)-mediated doxorubicin-resistant PLHC-1 hepatoma fish cell line

    SciTech Connect

    Zaja, Roko; Caminada, Daniel; Loncar, Jovica; Fent, Karl; Smital, Tvrtko

    2008-03-01

    The development of the multidrug resistance (MDR) phenotype in mammals is often mediated by the overexpression of the P-glycoprotein1 (Pgp, ABCB1) or multidrug resistance-associated protein (MRP)-like ABC transport proteins. A similar phenomenon has also been observed and considered as an important part of the multixenobiotic resistance (MXR) defence system in aquatic organisms. We have recently demonstrated the presence of ABC transporters in the widely used in vitro fish model, the PLHC-1 hepatoma cell line. In the present study we were able to select a highly resistant PLHC-1 sub-clone (PLHC-1/dox) by culturing the wild-type cells in the presence of 1 {mu}M doxorubicin. Using quantitative PCR a 42-fold higher expression of ABCB1 gene was determined in the PLHC-1/dox cells compared to non-selected wild-type cells (PLHC-1/wt). The efflux rates of model fluorescent Pgp1 substrates rhodamine 123 and calcein-AM were 3- to 4-fold higher in the PLHC-1/dox in comparison to the PLHC-1/wt cells. PLHC-1/dox were 45-fold more resistant to doxorubicin cytotoxicity than PLHC-1/wt. Similarly to mammalian cell lines, typical cross-resistance to cytotoxicity of other chemotherapeutics such as daunorubicin, vincristine, vinblastine, etoposide and colchicine, occurred. Furthermore, cyclosporine A, verapamil and PSC833, specific inhibitors of Pgp1 transport activity, completely reversed resistance of PLHC-1/dox cells to all tested drugs, resulting in EC50 values similar to the EC50 values found for PLHC-1/wt. In contrast, MK571, a specific inhibitor of MRP type of efflux transporters, sensitized PLHC-1/dox cells, neither to doxorubicin, nor to any other of the chemotherapeutics used in the study. These data demonstrate for the first time that a specific Pgp1-mediated doxorubicin resistance mechanism is present in the PLHC-1 fish hepatoma cell line. In addition, the fact that low micromolar concentrations of specific inhibitors may completely reverse a highly expressed doxorubicin

  3. MiR-520b suppresses proliferation of hepatoma cells through targeting ten-eleven translocation 1 (TET1) mRNA

    SciTech Connect

    Zhang, Weiying; Lu, Zhanping; Gao, Yuen; Ye, Lihong; Song, Tianqiang; Zhang, Xiaodong

    2015-05-08

    Accumulating evidence indicates that microRNAs are able to act as oncogenes or tumor suppressor genes in human cancer. We previously reported that miR-520b was down-regulated in hepatocellular carcinoma (HCC) and its deregulation was involved in hepatocarcinogenesis. In the present study, we report that miR-520b suppresses cell proliferation in HCC through targeting the ten-eleven translocation 1 (TET1) mRNA. Notably, we identified that miR-520b was able to target 3′-untranslated region (3′UTR) of TET1 mRNA by luciferase reporter gene assays. Then, we revealed that miR-520b was able to reduce the expression of TET1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blotting analysis. In terms of function, 5-ethynyl-2-deoxyuridine (EdU) incorporation and colony formation assays demonstrated that the forced miR-520b expression remarkably inhibited proliferation of hepatoma cells, but TET1 overexpression could rescue the inhibition of cell proliferation mediated by miR-520b. Furthermore, anti-miR-520b enhanced proliferation of hepatoma cells, whereas silencing of TET1 abolished anti-miR-520b-induced acceleration of cell proliferation. Then, we validated that the expression levels of miR-520b were negatively related to those of TET1 mRNA in clinical HCC tissues. Thus, we conclude that miR-520b depresses proliferation of liver cancer cells through targeting 3′UTR of TET1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • TET1 is a novel target gene of miR-520b. • TET1 is upregulated in clinical HCC tissues. • MiR-520b is negatively correlated with TET1 in clinical HCC tissues. • MiR-520b depresses the proliferation of HCC cells through targeting TET1 mRNA.

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

    SciTech Connect

    Feng, Guoxing; Shi, Hui; Li, Jiong; Yang, Zhe; Fang, Runping; Ye, Lihong; Zhang, Weiying; Zhang, Xiaodong

    2016-04-08

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

  5. Selective expression and induction of cytochrome P450PB and P450MC during the development of hereditary hepatitis and hepatoma of LEC rats.

    PubMed

    Sugiyama, T; Suzuki, K; Ookawara, T; Kurosawa, T; Taniguchi, N

    1989-11-01

    The Long-Evans rat with a cinnamon-like coat color (LEC rat) is a mutant strain displaying hereditary hepatitis with severe jaundice. The age related difference in microsomal dealkylation of pentoxyresorufin and ethoxyresorufin was examined. The enzyme activity levels of pentoxyresorufin O-depentylase in LEC rats were decreased to 25% of the levels in control [Long-Evans rats with an agouti coat color (LEA rats)]. In contrast, ethoxyresorufin O-deethylase exhibited a much less marked difference between the strains. In parallel with these strain differences in enzyme activities, a decrease in phenobarbital (PB) inducible P450 isozymes, mainly P450b and P450e, was observed by Western blot analysis. The level of P450PB in LEC rats was more markedly depressed than in the LEA strain. On the other hand, microsomes from uninduced LEC rat liver had more 3-methylcholanthrene (MC) inducible P450MC, mainly P450c and P450d, than microsomes from LEA rat liver and these isozymes in the LEC were markedly induced by 3-methylcholanthrene treatment. The great difference in cytochrome P450PB content of the liver microsomes between LEC and LEA rats and the maintained constitutive levels of hepatic cytochrome P450MC in the LEC rats suggest a possible role of these cytochrome isozymes in the onset of spontaneous hepatitis and hepatoma.

  6. FoxO3a mediates transforming growth factor-beta1-induced apoptosis in FaO rat hepatoma cells.

    PubMed

    Kim, Byung-Chul

    2008-10-31

    FoxO3a is a member of the forkhead box class O (FoxO) transcription factor family and an important regulator of apoptosis. This work aimed to elucidate the involvement of FoxO3a in transforming growth factor-beta1 (TGF-beta1)-induced apoptosis in FaO rat hepatoma cells. TGF-beta1 caused a time-dependent activation of FoxO3a and a subsequent increase in FoxO response-element-containing luciferase reporter activity, which was Akt-sensitive. The FaO cells stably transfected with a wild type FoxO3a were more susceptible to the formation of apoptotic bodies, populations of sub-G1 apoptotic cells, and collapse of the mitochondrial-membrane potential triggered by TGF-beta1. In contrast, transfection with small-interfering RNA (siRNA) oligonucleotide specific for FoxO3a significantly inhibited caspase activation in FaO cells treated with TGF-beta1. It thus appears that FoxO3a plays a crucial mediatory role in the TGF-beta1 signaling pathway leading to apoptosis.

  7. Separation and quantitation of hepatoma-associated gamma-glutamyltransferase by affinity chromatography with Affi-Gel blue and Con A-Sepharose.

    PubMed

    Izumi, M; Taketa, K

    1983-01-01

    Isozymes of serum gamma-glutamyltransferase (GGT) in patients with hepatocellular carcinoma (HCC) and other liver diseases were separated into two groups by double-affinity column chromatography with Affi-Gel blue and Con A-Sepharose, one recovered in the unbound fraction and the other in the bound fraction. Upon electrophoresis with polyacrylamide gradient gel slabs, the unbound fraction gave a GGTI1 band and a faint II1 band and the bound fraction gave a GGT I band and faint bands of GGT I", II' and X, when the original serum contained hepatoma-associated GGT (I1, I" and II') and high-molecular-weight lipid-protein complex, GGT(X). GGT I was present in all cases as a common isozyme. Other lipoprotein-associated GGT isozymes, III-IX, were removed by passing through Affi-Gel blue. GGT activities of unbound fraction in patients with HCC were generally higher than those in patients with non-HCC liver diseases, although the difference was not significant. When the percent of GGT activity of unbound (unbound + bound) was taken, 54% of patients with HCC had a ratio greater than 22%, whereas none of the healthy subjects or patients with other liver diseases gave values greater than this. The present technique may prove to be a useful clinical test for the diagnosis of HCC.

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

    SciTech Connect

    Yun, Hong Shik; Hong, Eun-Hee; Lee, Su-Jae; Baek, Jeong-Hwa; Lee, Chang-Woo; Yim, Ji-Hye; Um, Hong-Duck; Hwang, Sang-Gu

    2013-09-27

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

  9. Microarray data and pathway analyses for primary human activated hepatic stellate cells compared to HepG2 human hepatoma cells.

    PubMed

    Hetherington, Alexandra M; Sawyez, Cynthia G; Borradaile, Nica M

    2017-02-01

    As nonalcoholic fatty liver disease progresses to end-stage diseases, including fibrosis, cirrhosis and hepatocellular carcinoma, fibrotic activated hepatic stellate cells and cancerous epithelial cells can become abundant, changing the cellular composition of this organ. Despite potentially residing within the same diseased tissue, direct comparisons of global gene expression between activated hepatic stellate cells and hepatocellular carcinoma cells are lacking. Here we provide data collected using Affymetrix GeneChip microarrays to identify differential gene expression in cultured primary human activated hepatic stellate cells compared to HepG2 human hepatoma cells. The dataset includes many genes involved in intermediary metabolism which were investigated in greater depth in our associated article (A.M. Hetherington, C.G. Sawyez, E. Zilberman, A.M. Stoianov, D.L. Robson, J.M. Hughes-Large, et al., 2016) [1]. Pathway analyses of known protein coding genes down-regulated or up-regulated by greater than 2.0-fold are also provided.

  10. Radiosensitivity of hepatoma cell lines and human normal liver cell lines exposed in vitro to carbon ions and argon ions at the HIRFL

    NASA Astrophysics Data System (ADS)

    Jing, Xigang; Li, Wenjian; Wang, Zhuanzi; Wei, Wei; Guo, Chuanling; Lu, Dong; Yang, Jianshe

    2009-05-01

    Human hepatoma (SMMC-7721) and normal liver (L02) cells were irradiated with γ-rays, 12C 6+ and 36Ar 18+ ion beams at the Heavy Ion Research Facility in Lanzhou (HIRFL). By using the Calyculin-A induced premature chromosome condensation technique, chromatid-type breaks and isochromatid-type breaks were scored separately. Tumor cells irradiated with heavy ions produced a majority of isochromatid break, while chromatid breaks were dominant when cells were exposed to γ-rays. The relative biological effectiveness (RBE) for irradiation-induced chromatid breaks were 3.6 for L02 and 3.5 for SMMC-7721 cell lines at the LET peak of 96 keVμm -112C 6+ ions, and 2.9 for both of the two cell lines of 512 keVμm -136Ar 18+ ions. It suggested that the RBE of isochromatid-type breaks was pretty high when high-LET radiations were induced. Thus we concluded that the high production of isochromatid-type breaks, induced by the densely ionizing track structure, could be regarded as a signature of high-LET radiation exposure.

  11. Enhancement of 1,3-bis(2-chloroethyl)-1-nitrosourea resistance by gamma-irradiation or drug pretreatment in rat hepatoma cells

    SciTech Connect

    Habraken, Y.; Laval, F. )

    1991-02-15

    Treatment of rat hepatoma cells (H4 cells) with various DNA-damaging agents increases the number of O6-methylguanine-DNA-methyltransferase (transferase) molecules per cell. Because the cellular resistance to chloroethylnitrosoureas depends on the number of transferase molecules, we studied the influence of pretreatment with gamma-irradiation, cis-dichlorodiammineplatinum(II), or 2-methyl-9-hydroxyellipticinium on the sensitivity of H4 cells to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The BCNU resistance depends on the gamma-ray dose and increases with time after irradiation: it is maximum when the drug is added 48 h after irradiation, which corresponds to the maximum enhancement of the transferase activity in the cells. Pretreatment with a single dose of cis-dichlorodiammineplatinum(II) or 2-methyl-9-hydroxyellipticinium also increases the cellular resistance to BCNU. This resistance is not due to a modification of the alkylation of the cellular DNA in the pretreated cells but is related to the increased transferase activity, as it is no longer observed when this activity is depleted by incubating the pretreated cells with the free base O6-methylguanine before BCNU treatment. These results suggest that tumor cells surviving after gamma-irradiation or drug treatment may become resistant to chemotherapy with chloroethylnitrosoureas.

  12. Profiling of promoter occupancy by the SND1 transcriptional coactivator identifies downstream glycerolipid metabolic genes involved in TNFα response in human hepatoma cells

    PubMed Central

    Arretxe, Enara; Armengol, Sandra; Mula, Sarai; Chico, Yolanda; Ochoa, Begoña; Martínez, María José

    2015-01-01

    The NF-κB-inducible Staphylococcal nuclease and tudor domain-containing 1 gene (SND1) encodes a coactivator involved in inflammatory responses and tumorigenesis. While SND1 is known to interact with certain transcription factors and activate client gene expression, no comprehensive mapping of SND1 target genes has been reported. Here, we have approached this question by performing ChIP-chip assays on human hepatoma HepG2 cells and analyzing SND1 binding modulation by proinflammatory TNFα. We show that SND1 binds 645 gene promoters in control cells and 281 additional genes in TNFα-treated cells. Transcription factor binding site analysis of bound probes identified motifs for established partners and for novel transcription factors including HSF, ATF, STAT3, MEIS1/AHOXA9, E2F and p300/CREB. Major target genes were involved in gene expression and RNA metabolism regulation, as well as development and cellular metabolism. We confirmed SND1 binding to 21 previously unrecognized genes, including a set of glycerolipid genes. Knocking-down experiments revealed that SND1 deficiency compromises the glycerolipid gene reprogramming and lipid phenotypic responses to TNFα. Overall, our findings uncover an unexpected large set of potential SND1 target genes and partners and reveal SND1 to be a determinant downstream effector of TNFα that contributes to support glycerophospholipid homeostasis in human hepatocellular carcinoma during inflammation. PMID:26323317

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

    PubMed

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

    2016-04-15

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2012-01-01

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

  16. The p53-inducible gene 3 involved in flavonoid-induced cytotoxicity through the reactive oxygen species-mediated mitochondrial apoptotic pathway in human hepatoma cells.

    PubMed

    Zhang, Qiang; Cheng, Guangdong; Qiu, Hongbin; Zhu, Liling; Ren, Zhongjuan; Zhao, Wei; Zhang, Tao; Liu, Lei

    2015-05-01

    Flavonoids have been reported to exhibit prooxidant cytotoxicity against cancer cells, but the underlying mechanism is still poorly understood. Here we investigated the potential mechanism that p53-inducible gene 3 (PIG3), a NADPH:quinone oxidoreductase, mediated the prooxidant cytotoxicity of flavonoids on human hepatoma HepG2 cells. The results showed that flavonoids (apigenin, luteolin, kaempferol, and quercetin) inhibited the growth of HepG2 cells in a dosage- and time-dependent manner, and induced the morphological changes characteristic of apoptosis in HepG2 cells. We also found that expression of PIG3 was increased markedly in HepG2 cells treated with flavonoids at both mRNA and protein levels, which was accompanied by increased intracellular ROS production and a decreased mitochondrial membrane potential (ΔΨm). All these effects were largely reversed through knockdown of the PIG3 gene in HepG2 cells. Western blotting indicated that flavonoids increased cytochrome c release, upregulated the ratio of Bax/Bcl-2, and activated the caspases-9 and -3. Moreover, knockdown of PIG3 could reverse the changes of these apoptotic-related proteins. These results suggest that PIG3 plays an important role in regulating the prooxidant activity and apoptosis-inducing action of flavonoids on HepG2 cells though the ROS-triggered mitochondrial apoptotic pathway.

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

    PubMed

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

    2014-07-01

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

  18. Effect of coffee melanoidin on human hepatoma HepG2 cells. Protection against oxidative stress induced by tert-butylhydroperoxide.

    PubMed

    Goya, Luis; Delgado-Andrade, Cristina; Rufián-Henares, José A; Bravo, Laura; Morales, Francisco J

    2007-05-01

    Soluble high-molecular weight fraction (named melanoidin) from coffee brew was isolated by ultrafiltration, subsequently digested by simulating a gastric plus pancreatic digestive condition and partly characterized by CZE, gel-filtration and browning. The objective of the present study was to investigate the potential protective effect of the coffee melanoidin submitted to gastrointestinal digestion on cell viability (lactate dehydrogenase leakage) and redox status of cultured human hepatoma HepG2 cells submitted to oxidative stress induced by tert-butylhydroperoxide (t-BOOH). Concentration of reduced glutathione (GSH) and malondialdehyde (MDA), generation of reactive oxygen species (ROS) and activity of antioxidant enzymes glutathione peroxidase (GPx) and reductase (GR) were used as markers of cellular oxidative status. Pretreatment of cultured HepG2 cells with 0.5-10 microg/mL digested coffee melanoidin (DCM) for 2 or 20 h completely prevented the increase in cell damage and GR and partly prevented the decrease of GSH and the increase of MDA and GPx evoked by t-BOOH in HepG2 cells. In contrast, increased ROS generation induced by t-BOOH was not prevented when cells were pretreated with DCM. The results show that treatment of HepG2 cells with concentrations of DCM within the expected physiological range confers the cells a significant protection against an oxidative insult.

  19. Molecular mechanism of extinction of liver-specific functions in mouse hepatoma x rat fibroblast hybrids: extinction of the albumin gene

    SciTech Connect

    Papaconstantinou, J.; Wong, E.; Ratrie, H.; Szpirer, C.; Szpirer, J.

    1982-01-01

    Hybrids formed by the fusion of mouse hepatoma (BWTG3) and rat fibroblast (JF1) cells exhibit the extinction of mouse albumin and ..cap alpha..-fetoprotein synthesis. Karyotype analyses suggest that all parental chromosomes are present in the hybrids. The extinction, therefore, of mouse hepatocyte genes is attributed to the inhibitory action of the rat genome. In these studies, we show that these hybrids possess and express the mouse ..beta..-glucyronidase gene (which is encoded on the same chromosome as the mouse albumin and ..cap alpha..-fetoprotein gene), and we present data of Southern blot analysis which demonstrate that such hybrids have indeed retained both mouse and rat albumin DNA sequences. In addition, using mouse albumin cDNA, we have shown by cDNA-RNA reassociation kinetics that albumin mRNA is virtually absent in these hybrids. We conclude from these studies that the extinction of albumin synthesis involves a mechanism which results in the loss of cytoplasmic albumin mRNA.

  20. Intratumoural GM-CSF microspheres and CTLA-4 blockade enhance the antitumour immunity induced by thermal ablation in a subcutaneous murine hepatoma model.

    PubMed

    Chen, Zubing; Shen, Shiqiang; Peng, Baogang; Tao, Jianpin

    2009-08-01

    We evaluated the effect of a new antitumour immunity regimen that included microwave ablation, intratumoural microspheres encapsulating granulocyte-macrophage colony stimulating factor (GM-CSF), and blockade of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). C57BL6 mice with an established subcutaneous Hepa 1-6 hepatoma underwent microwave ablation, followed by intratumoural injection of GM-CSF microspheres, and intraperitoneal injection of anti-CTLA-4 antibodies. The therapeutic effects were evaluated by tumour growth, survival analysis, and cytotoxicity of T lymphocytes against Hepa 1-6. The co-administration of microwave thermal ablation, GM-CSF microspheres, and anti-CTLA-4 rejected tumour rechallenge in 90% of treated mice in a subcutaneous murine Hepa 1-6 model, and cured established distant tumour in 50% of the treated mice. This antitumour immune response was tumour-specific and mediated by natural killer (NK), CD4+, and CD8+ T cells. Microwave ablation, followed by intratumoural GM-CSF microspheres, and anti-CTLA-4 antibodies results in the local eradication of tumours, rejection of tumours following rechallenge, and cures established distant tumours, suggesting that this is a promising regimen and one that is readily applicable in the clinic.

  1. Hepatitis C virus E2 protein promotes human hepatoma cell proliferation through the MAPK/ERK signaling pathway via cellular receptors

    SciTech Connect

    Zhao Lanjuan; Wang Lu; Ren Hao; Cao Jie; Li Li; Ke Jinshan; Qi Zhongtian . E-mail: qizt53@hotmail.com

    2005-04-15

    Dysregulation of mitogen-activated protein kinase (MAPK) signaling pathways by various viruses has been shown to be responsible for viral pathogenicity. The molecular mechanism by which hepatitis C virus (HCV) infection caused human liver diseases has been investigated on the basis of abnormal intracellular signal events. Current data are very limited involved in transmembrane signal transduction triggered by HCV E2 protein. Here we explored regulation of the MAPK/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway by E2 expressed in Chinese hamster oval cells. In human hepatoma Huh-7 cells, E2 specifically activated the MAPK/ERK pathway including downstream transcription factor ATF-2 and greatly promoted cell proliferation. CD81 and low density lipoprotein receptor (LDLR) on the cell surface mediated binding of E2 to Huh-7 cells. The MAPK/ERK activation and cell proliferation driven by E2 were suppressed by blockage of CD81 as well as LDLR. Furthermore, pretreatment with an upstream kinase MEK1/2 inhibitor U0126 also impaired the MAPK/ERK activation and cell proliferation induced by E2. Our results suggest that the MAPK/ERK signaling pathway triggered by HCV E2 via its receptors maintains survival and growth of target cells.

  2. CYP3A4 inhibition by Psoralea corylifolia and its major components in human recombinant enzyme, differentiated human hepatoma HuH-7 and HepaRG cells.

    PubMed

    Liu, Yitong; Flynn, Thomas J

    2015-01-01

    Psoralea corylifolia (P. corylifolia) is a medicinal plant used primarily in herbal dietary supplements to treat skin diseases, such as vitiligo and psoriasis. Case reports of liver toxicity have recently emerged from its use, which often includes co-administration with other herbal products. In this study, CYP3A4 inhibition and hepatotoxicity of P. corylifolia and its major components were evaluated in human recombinant CYP3A4 enzyme, differentiated human hepatoma HuH-7 and HepaRG cells. LC/MS-TOF was used to identify the major components of P. corylifolia fruit methanol-water extract. P. corylifolia and its major bioactive components psoralen and isopsoralen were then incubated with human recombinant CYP3A4 (10 min) or differentiated HuH-7 and HepaRG cells (24 h) prior to CYP3A4 activity and cytotoxicity assays. P. corylifolia extract, psoralen, and isopsoralen concentration dependently inhibited CYP3A4 activity with different potency in the three in vitro systems. No cytotoxicity was observed at any concentration tested. In vitro CYP3A4 inhibition by P. corylifolia and its major components suggests potential drug-dietary supplement interactions that warrant further investigations in vivo.

  3. In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride.

    PubMed

    Fotakis, George; Timbrell, John A

    2006-01-05

    The aim of this study was to compare four in vitro cytotoxicity assays and determine their ability to detect early cytotoxic events. Two hepatoma cell lines, namely HTC and HepG2 cells, were exposed to cadmium chloride (0-300 microM) for 3, 5 and 8 h. Following exposure to the toxic metal cytotoxicity was determined with the lactate dehydrogenase leakage assay (LDH), a protein assay, the neutral red assay and the methyl tetrazolium (MTT) assay. In HTC cells no toxicity was observed for any incubation period when the LDH leakage, the MTT and the protein assay were employed whereas the neutral red assay revealed early cytotoxicity starting after incubation of HTC cells with CdCl(2) for 3 h. In the case of HepG2 cells the MTT assay reveals cytotoxicity due to CdCl(2) exposure after 3 h whereas no such effect is seen with the other three assays. Following 5 h exposure of HepG2 cells to CdCl(2), toxicity is observed with the MTT assay at lower concentrations compared to the ones required for detection of toxicity with the LDH leakage and the neutral red assay. In conclusion different sensitivity was observed for each assay with the neutral red and the MTT assay being the most sensitive in detecting cytotoxic events compared to the LDH leakage and the protein assay.

  4. Anti-Inflammatory Activity of Cyanobacterial Serine Protease Inhibitors Aeruginosin 828A and Cyanopeptolin 1020 in Human Hepatoma Cell Line Huh7 and Effects in Zebrafish (Danio rerio)

    PubMed Central

    Faltermann, Susanne; Hutter, Simon; Christen, Verena; Hettich, Timm; Fent, Karl

    2016-01-01

    Intensive growth of cyanobacteria in freshwater promoted by eutrophication can lead to release of toxic secondary metabolites that may harm aquatic organisms and humans. The serine protease inhibitor aeruginosin 828A was isolated from a microcystin-deficient Planktothrix strain. We assessed potential molecular effects of aeruginosin 828A in comparison to another cyanobacterial serine protease inhibitor, cyanopeptolin 1020, in human hepatoma cell line Huh7, in zebrafish embryos and liver organ cultures. Aeruginosin 828A and cyanopeptolin 1020 promoted anti-inflammatory activity, as indicated by transcriptional down-regulation of interleukin 8 and tumor necrosis factor α in stimulated cells at concentrations of 50 and 100 µmol·L−1 aeruginosin 828A, and 100 µmol·L−1 cyanopeptolin 1020. Aeruginosin 828A induced the expression of CYP1A in Huh7 cells but did not affect enzyme activity. Furthermore, hatched zebrafish embryos and zebrafish liver organ cultures were exposed to aeruginosin 828A. The transcriptional responses were compared to those of cyanopeptolin 1020 and microcystin-LR. Aeruginosin 828A had only minimal effects on endoplasmic reticulum stress. In comparison to cyanopeptolin 1020 our data indicate that transcriptional effects of aeruginosin 828A in zebrafish are very minor. The data further demonstrate that pathways that are influenced by microcystin-LR are not affected by aeruginosin 828A. PMID:27428998

  5. Novel Pd(II)-salen complexes showing high in vitro anti-proliferative effects against human hepatoma cancer by modulating specific regulatory genes.

    PubMed

    Azam, Mohammad; Hussain, Zahid; Warad, Ismail; Al-Resayes, Saud I; Khan, Mohd Shahnawaz; Shakir, Mohammad; Trzesowska-Kruszynska, Agata; Kruszynski, Rafal

    2012-09-21

    We have reported the synthesis of a novel salen ligand and its mononuclear Pd-salen complexes derived from 2-{[2-hydroxy-3-{[(E)-(2-hydroxyphenyl)methylidene]amino}propyl)imino]methyl}phenol. The newly synthesized and isolated Pd(II) complexes have been identified and fully characterized by various physico-chemical studies viz., elemental analyses, IR, UV-Vis, (1)H, (13)C NMR spectroscopy, electron spray ionization mass spectrometry (ESI-MS) and TGA/DTA studies. The molecular structure of the salen ligand has been ascertained by single-crystal XRD and it is coordinated to Pd(II) ion through two nitrogen and two oxygen atoms. The UV-Vis data clearly suggest a square-planar environment around both the Pd(II) ions. The DNA binding studies of the synthesized compounds has been investigated by electron spectroscopy and fluorescence measurements. The results suggest that Pd(II) complexes bind to DNA strongly as compared to the free ligand. The free salen ligand and its Pd(II) complexes have also been tested against human hepatoma cancer cell line (Huh7) and results manifested exceptional anti-proliferative effects of the Pd(II) complexes. The anti-proliferative activity of Pd(II) complexes has been modulated by specific regulatory genes.

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

    PubMed

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

    2008-01-01

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

  7. Comparative effects of food-derived polyphenols on the viability and apoptosis of a human hepatoma cell line (HepG2).

    PubMed

    Ramos, Sonia; Alía, Mario; Bravo, Laura; Goya, Luis

    2005-02-23

    Consumption of fruits and vegetables, which are rich in polyphenols, has been associated with a reduced risk of chronic diseases such as cancer. Dietary polyphenols have antioxidant and antiproliferative properties that might explain their beneficial effect on cancer prevention. The aim of this study was to investigate the effects of different pure polyphenols [quercetin, chlorogenic acid, and (-)-epicatechin] and natural fruit extracts (strawberry and plum) on viability or apoptosis of human hepatoma HepG2 cells. The treatment of cells for 18 h with quercetin and fruit extracts reduced cell viability in a dose-dependent manner; however, chlorogenic acid and (-)-epicatechin had no prominent effects on the cell death rate. Similarly, quercetin and strawberry and plum extracts, rather than chlorogenic acid and (-)-epicatechin, induced apoptosis in HepG2 cells. Moreover, quercetin and fruit extracts arrested the G1 phase in the cell cycle progression prior to apoptosis. Quercetin and strawberry and plum extracts may induce apoptosis and contribute to a reduced cell viability in HepG2 cells.

  8. Molecular mechanisms of (-)-epicatechin and chlorogenic acid on the regulation of the apoptotic and survival/proliferation pathways in a human hepatoma cell line.

    PubMed

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

    2007-03-07

    Dietary polyphenols have been associated with reduced risk of chronic diseases, but the precise molecular mechanisms of protection remain unclear. This work was aimed at studying the effect of (-)-epicatechin (EC) and chlorogenic acid (CGA) on the regulation of apoptotic and survival/proliferation pathways in a human hepatoma cell line (HepG2). EC or CGA treatment for 18 h had a slight effect on cell viability and decreased reactive oxygen species formation, and EC alone promoted cell proliferation, whereas CGA increased glutathione levels. Phenols neither induced the caspase cascade for apoptosis nor affected expression levels of Bcl-xL or Bax. A sustained activation of the major survival signals AKT/PI-3-kinase and ERK was shown in EC-treated cells, rather than in CGA-exposed cells. These data suggest that EC and CGA have no effect on apoptosis and enhance the intrinsic cellular tolerance against oxidative insults either by activating survival/proliferation pathways or by increasing antioxidant potential in HepG2.

  9. Antiproliferative activity of Humulus lupulus extracts on human hepatoma (Hep3B), colon (HT-29) cancer cells and proteases, tyrosinase, β-lactamase enzyme inhibition studies.

    PubMed

    Cömert Önder, Ferah; Ay, Mehmet; Aydoğan Türkoğlu, Sümeyye; Tura Köçkar, Feray; Çelik, Ayhan

    2016-01-01

    The aims of this study were to examine the antiproliferation of Humulus lupulus extracts on human hepatoma carcinoma (Hep3B) and human colon carcinoma (HT-29) cell lines along with enzyme inhibitory effects of the crude extracts. Potential cell cytotoxicity of six different H. lupulus extracts were assayed on various cancer cells using MTT assay at 24, 48 and 72 h intervals. Methanol-1 extract has inhibited the cell proliferation with doses of 0.6-1 mg/mL in a time dependent (48 and 72 hours) manner in Hep3B cells with 70% inhibition, while inhibitory effect was not seen in colon cancer cells. Acetone extract has increased the cell proliferation at low doses of 0.1 mg/mL for 72 h in Hep3B cells and 0.1-0.2 mg/mL for 48 and 72 h in HT29 cells. The inhibitory effects of the extracts were compared by relative maximum activity values (V(max)) using proteases such as α-chymotrypsin, trypsin and papain, tyrosinase and β-lactamase (penicillinase).

  10. Proteolysis of integrin alpha5 and beta1 subunits involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells.

    PubMed

    Hsu, S L; Cheng, C C; Shi, Y R; Chiang, C W

    2001-06-26

    Our previous report demonstrated that all-trans-retinoic acid (ATRA) induces detachment and death under serum starvation in several human tumor cell lines. In this study, we examined the influence of cell-extracellular matrix interaction on the ability of ATRA to induce apoptosis. Plating of human hepatoma Hep3B cells onto poly-hydroxyethylmethacrylate-coated plates in the absence of serum resulted in the acceleration of ATRA-induced apoptosis. In contrast, ATRA-induced apoptosis was significantly suppressed by plating cells onto Matrigel-coated plates but not suppressed by culturing onto collagen-, laminin-, vitronectin-, or fibronectin-coated plates. Exogenously added soluble collagen, laminin, fibronectin, vitronectin or Matrigel failed to suppress ATRA-induced apoptosis. Results from the adhesion assay indicated that the cell attachment to fibronectin was significantly inhibited by ATRA. Treatment with perturbing antibody against integrin alpha5 or beta1 subunits resulted in promotion of ATRA-induced apoptosis. Moreover, the proteolytic cleavage of alpha5beta1 integrin and focal adhesion kinase (FAK) proteins is linked to the early phase of the ATRA-induced apoptotic process. Furthermore, ATRA-induced detachment, death, and cleavage of alpha5beta1 integrin and FAK were drastically suppressed by plating cells onto Matrigel-coated plates. These findings provide evidence that abrogation of cell adhesion, through proteolysis of alpha5beta1 integrin and FAK, is closely linked to ATRA-induced apoptosis in Hep3B cells.

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

    PubMed

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

    2016-11-01

    Hepatoma-derived growth factor (HDGF) is a heparin-binding growth factor, which has previously been shown to be expressed in a variety of cancers. HDGF overexpression has also previously been correlated with a poor prognosis in several cancers. The significance of HDGF in prostate cancer, however, has not been investigated. Here, we show that HDGF is overexpressed in both androgen-sensitive LNCaP cells and androgen-insensitive DU145, 22RV1, and PC-3 cells. Forced overexpression enhanced cell viability of RWPE-1 cells, whereas HDGF knockdown reduced cell proliferation in human prostate cancer cells. We also show that HDGF may serve as a survival-related protein as ectopic overexpression of HDGF in RWPE cells up-regulated the expression of antiapoptosis proteins cyclin E and BCL-2, whereas simultaneously down-regulating proapoptotic protein BAX. Western blot analysis also showed that HDGF overexpression modulated the activity of phospho-AKT as well as NF-kB, and these results correlated with in vitro migration and invasion assays. We next assessed the therapeutic potential of HDGF inhibition with a HDGF monoclonal antibody and vitamin k2, showing reduced cell proliferation as well as inhibition of NF-kB expression in HDGF overexpressed RWPE cells treated with a HDGF monoclonal antibody and vitamin K2. Collectively, our results suggest that HDGF is a relevant protein in prostate oncogenesis and may serve as a potential therapeutic target in prostate cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Inversely repeating integrated hepatitis B virus DNA and cellular flanking sequences in the human hepatoma-derived cell line huSP.

    PubMed Central

    Mizusawa, H; Taira, M; Yaginuma, K; Kobayashi, M; Yoshida, E; Koike, K

    1985-01-01

    Among recombinant phages carrying integrated hepatitis B virus (HBV) DNA sequences cloned from the human hepatoma-derived cell line huSP, one clone, lambda hu-489, revealed some unusual features. The 2.25-kilobase Eco D fragment from the insert of this clone hybridized to the HBV DNA probe only and its nucleotide sequence was determined. The viral sequence, as well as a cellular flanking sequence, showed extensive rearrangement accompanied by inverted repetition. The Eco D fragment contained HBV DNA from the 5'-end region of gene S to the middle of gene X, followed by a long cellular flanking sequence. Moreover, a part of gene X was found inversely repeated at the head of the same gene S in a head-to-head configuration truncated by the same cellular sequence. Therefore, the same junction sequence of viral DNA and the cellular sequence was found at two different sites in the Eco D fragment in opposite polarities. Images PMID:2982143

  13. Bis(hydroxyphenyl)methane-bisphenol F-metabolism by the HepG2 human hepatoma cell line and cryopreserved human hepatocytes.

    PubMed

    Dumont, Coralie; Perdu, Elisabeth; de Sousa, Georges; Debrauwer, Laurent; Rahmani, Roger; Cravedi, Jean-Pierre; Chagnon, Marie-Christine

    2011-10-01

    Bisphenol F (BPF) is present in the environment and as a contaminant of food. Humans may, therefore, be exposed to BPF, and an assessment of this risk is required. BPF has been shown to have genotoxic and endocrine-disruptor properties in a human hepatoma cell line (HepG2), which is a model system for studies of xenobiotic toxicity. In this study, we investigated the ability of HepG2 cells to biotransform BPF, because metabolism may affect the observed effects of BPF, and we compared this metabolic capacity with that of human hepatocytes. Cells were incubated for 24 hours with [(3)H]-BPF. The culture medium was then concentrated and its metabolites were isolated by high-performance liquid chromatography and identified by mass spectrometry. BPF was largely metabolized into the corresponding sulfate by the HepG2 cell line. BPF was metabolized into both sulfate and glucuronide by human hepatocytes, but with differences between individuals. The metabolism of BPF in both HepG2 cells and human hepatocytes suggests the existence of a detoxification pathway. Thus, these two cell models differ in metabolic capacity. It is, therefore, very important, when assessing the toxic effects of substances in vitro, to determine, in parallel, the biotransformation capacities of the model used to extrapolate in vivo.

  14. Double-Stranded Linear Duck Hepatitis B Virus (DHBV) Stably Integrates at a Higher Frequency than Wild-Type DHBV in LMH Chicken Hepatoma Cells

    PubMed Central

    Gong, Shih S.; Jensen, Anne D.; Chang, C. J.; Rogler, Charles E.

    1999-01-01

    Integration of hepadnavirus DNAs into host chromosomes can have oncogenic consequences. Analysis of host-viral DNA junctions of DHBV identified the terminally duplicated r region of the viral genome as a hotspot for integration. Since the r region is present on the 5′ and 3′ ends of double-stranded linear (DSL) hepadnavirus DNAs, these molecules have been implicated as integration precursors. We have produced a LMH chicken hepatoma cell line (LMH 66-1 DSL) which replicates exclusively DSL duck hepatitis B virus (DHBV) DNA. To test whether linear DHBV DNAs integrate more frequently than the wild type open circular DHBV DNAs, we have characterized the integration frequency in LMH 66-1 DSL cells by using a subcloning approach. This approach revealed that 83% of the LMH 66-1 DSL subclones contained new integrations, compared to only 16% of subclones from LMH-D2 cells replicating wild-type open circular DHBV DNA. Also, a higher percentage of the LMH 66-1 DSL subclones contained two or more new integrations. Mathematical analysis suggests that the DSL DHBV DNAs integrated stably once every three generations during subcloning whereas wild-type DHBV integrated only once every four to five generations. Cloning and sequencing of new integrations confirmed the r region as a preferred integration site for linear DHBV DNA molecules. One DHBV integrant was associated with a small deletion of chromosomal DNA, and another DHBV integrant occurred in a telomeric repeat sequence. PMID:9882355

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

    PubMed Central

    Manganiello, Vincent; Vaughan, Martha

    1972-01-01

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

  16. Inhibition of hypoxia-inducible factor via upregulation of von Hippel-Lindau protein induces “angiogenic switch off” in a hepatoma mouse model

    PubMed Central

    Iwamoto, Hideki; Nakamura, Toru; Koga, Hironori; Izaguirre-Carbonell, Jesus; Kamisuki, Shinji; Sugawara, Fumio; Abe, Mitsuhiko; Iwabata, Kazuki; Ikezono, Yu; Sakaue, Takahiko; Masuda, Atsutaka; Yano, Hirohisa; Ohta, Keisuke; Nakano, Masahito; Shimose, Shigeo; Shirono, Tomotake; Torimura, Takuji

    2015-01-01

    “Angiogenic switch off” is one of the ideal therapeutic concepts in the treatment of cancer. However, the specific molecules which can induce “angiogenic switch off” in tumor have not been identified yet. In this study, we focused on von Hippel-Lindau protein (pVHL) in hepatocellular carcinoma (HCC) and investigated the effects of sulfoquinovosyl-acylpropanediol (SQAP), a novel synthetic sulfoglycolipid, for HCC. We examined mutation ratio of VHL gene in HCC using 30 HCC samples and we treated the HCC-implanted mice with SQAP. Thirty clinical samples showed no VHL genetic mutation in HCC. SQAP significantly inhibited tumor growth by inhibiting angiogenesis in a hepatoma mouse model. SQAP induced tumor “angiogenic switch off” by decreasing hypoxia-inducible factor (HIF)-1, 2α protein via pVHL upregulation. pVHL upregulation decreased HIFα protein levels through different multiple mechanisms: (i) increasing pVHL-dependent HIFα protein degradation; (ii) decreasing HIFα synthesis with decrease of NF-κB expression; and (iii) decrease of tumor hypoxia by vascular normalization. We confirmed these antitumor effects of SQAP by the loss-of-function experiments. We found that SQAP directly bound to and inhibited transglutaminase 2. This study provides evidence that upregulation of tumor pVHL is a promising target, which can induce “angiogenic switch off” in HCC. PMID:27119112

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

    SciTech Connect

    Lee, H.-J.; Wang, C.-J.; Kuo, H.-C.; Chou, F.-P.; Jean, L.-F.; Tseng, T.-H. . E-mail: tht@csmu.edu.tw

    2005-03-01

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

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

    PubMed Central

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

    2016-01-01

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

  19. Genotoxic potential of montmorillonite clay mineral and alteration in the expression of genes involved in toxicity mechanisms in the human hepatoma cell line HepG2.

    PubMed

    Maisanaba, Sara; Hercog, Klara; Filipic, Metka; Jos, Ángeles; Zegura, Bojana

    2016-03-05

    Montmorillonite, also known as Cloisite(®)Na(+) (CNa(+)), is a natural clay with a wide range of well-documented and novel applications, such as pharmaceutical products or food packaging. Although considered a low toxic product, the expected increased exposure to CNa(+) arises concern on the potential consequences on human and environmental health especially as its genotoxicity has scarcely been investigated so far. Thus, we investigated, for the first time, the influence of non-cytotoxic concentrations of CNa(+) (15.65, 31.25 and 62.5 μg/mL) on genomic instability of human hepatoma cell line (HepG2) by determining the formation of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) with the Cytokinesis block micronucleus cytome assay. Further on we studied the influence of CNa(+) on the expression of several genes involved in toxicity mechanisms using the real-time quantitative PCR. The results showed that CNa(+) increased the number of MNi, while the numbers of NBUDs and NPBs were not affected. In addition it deregulated genes in all the groups studied, mainly after longer time of exposure. These findings provide the evidence that CNa(+) is potentially genotoxic. Therefore further studies that will elucidate the molecular mechanisms involved in toxic activity of CNa(+) are needed for hazard identification and human safety assessment.

  20. Perturbations of triglycerides but not of cholesterol metabolism are prevented by anti-tumour necrosis factor treatment in rats bearing an ascites hepatoma (Yoshida AH-130).

    PubMed Central

    Dessì, S.; Batetta, B.; Spano, O.; Bagby, G. J.; Tessitore, L.; Costelli, P.; Baccino, F. M.; Pani, P.; Argilès, J. M.

    1995-01-01

    Rats transplanted with the ascites hepatoma Yoshida AH-130 developed a severely progressive cachexia, characterised by marked alterations in protein and lipid metabolism. In particular, high levels of serum triglycerides and free fatty acids were associated with altered levels and distribution of plasma cholesterol, with increased total and very low-density lipoprotein-low-density lipoprotein (VLDL-LDL) cholesterol and reduced high-density lipoprotein (HDL) cholesterol. The tumour cells showed high rates of cholesterol synthesis and elevated content of free and esterified cholesterol, whereas total cholesterol synthesis was reduced in the host liver. To determine whether these perturbations could be related to the elevation of tumour necrosis factor alpha (TNF-alpha) previously shown in the AH-130 bearers (Tessitore L, Costelli P, Baccino FM 1993, Br J Cancer, 67, 15-23), either anti-TNF polyclonal antibodies or non-immune IgGs were injected daily after tumour transplantation. The anti-TNF treatment neither affected tumour growth nor prevented the serum cholesterol changes, while attenuating the hypertriglyceridaemia and the elevated serum free fatty acid levels. These data indicate that TNF does not appear to be directly involved in the altered cholesterol metabolism in AH-130 hosts, thus supporting the view that cholesterol metabolism and lipid metabolism are regulated differently during tumour growth. PMID:7577459

  1. Single-channel currents of the permeability transition pore from the inner mitochondrial membrane of rat liver and of a human hepatoma cell line.

    PubMed

    Loupatatzis, Christos; Seitz, Gordon; Schönfeld, Peter; Lang, Florian; Siemen, Detlef

    2002-01-01

    Single-channel currents were recorded from inner mitochondrial membranes of HepG2 hepatoma cells and of normal rat liver cells by means of patch-clamp techniques. The current events showed variable amplitudes of up to 1.1 +/- 0.2 nS (n = 35) at room temperature (24 degrees C) and of up to 1.5 +/- 0.2 nS (n = 10) at 34 degrees C including large numbers of subconductance states. Voltages of -40 mV and below closed the channels usually with a delay of about 2 min. Increasing Ca(2+) concentrations activated the channels, whereas cyclosporin A (100 nM) blocked. The concentration-response relation for the Ca(2+)-effect could be fitted best using an EC(50) of 10 microM and a Hill coefficient of 1.5. Taken together the results indicate that we recorded from the permeability transition pore (PTP). As PTP activity may be related to apoptosis we tested if lysate from differently treated T-lymphocytes (Jurkat cells) was able to induce PTP activity in HepG2 cells. Lysate of untreated cells completely abolished the activity at a Ca(2+) concentration of 18 nM (buffered by EGTA), i.e. three orders of magnitude below the EC(50). Under these conditions the lysate is not likely to contain stable factors that could open the PTP. Preliminary experiments show PTP activity in CD95-activated lysate.

  2. Effect of polyphenols on 3-hydroxy-3-methylglutaryl-coenzyme A lyase activity in human hepatoma HepG2 cell extracts.

    PubMed

    Nakagawa, Saori; Kojima, Yuko; Sekino, Koichi; Yamato, Susumu

    2013-01-01

    When carbohydrate metabolism is impaired, fatty acid metabolism is activated. Excess acetyl-coenzyme A (CoA) is generated from fatty acids by β-oxidation and is used for the formation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) and subsequently for acetoacetate. High levels of secreted ketone bodies (acetoacetate and 3β-hydroxybutyrate) lower the pH of blood and urine, resulting in ketoacidosis. HMG-CoA lyase in hepatic cells is a rate-limiting enzyme catalyzing the cleavage of HMG-CoA to acetoacetate, and thus inhibition of this enzyme results in reduced acetoacetate production, in other words, impaired ketoacidosis. Inhibition of HMG-CoA lyase activity possibly prevents ketoacidosis and should be the therapeutic target. Polyphenols are common and abundant dietary constituents with beneficial effects on human health. We examined the inhibitory effects of dietary polyphenols on HMG-CoA lyase activity in cellular extracts of human hepatoma HepG2 cells. Of the nine representative dietary polyphenols tested, (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), and gallic acid (GA) effectively inhibited HMG-CoA lyase activity. Lineweaver-Burk analysis revealed that EGC and EGCG are likely to be mixed-type noncompetitive inhibitors. Pyrogallol with the gallyl structure also inhibited HMG-CoA lyase activity, suggesting that the gallyl moiety of polyphenols is important for the inhibition of HMG-CoA lyase activity.

  3. A new triterpene and protective effect of Periploca somaliensis Browicz fruits against CCl₄-induced injury on human hepatoma cell line (Huh7).

    PubMed

    Abdel-Monem, Azza R; Kandil, Zeinab A; Abdel-Naim, Ashraf B; Abdel-Sattar, Essam

    2015-01-01

    The potential hepatoprotective effect of the methanolic extract of Periploca somaliensis Browicz fruits, its different fractions (n-hexane, chloroform and n-butanol) and the major isolated compound ursolic acid was evaluated using the human hepatoma cell line (Huh7) based on the changes in the activity of aspartate aminotransferase, alanine transaminase, glutathione and superoxide dismutase. Each sample was tested at three different concentrations (1000, 100 and 10 μg/mL). All tested samples exhibited a potent concentration-independent cytoprotective effect relative to silymarin as a reference standard. Chromatographic fractionation of the chloroform-soluble fraction of the methanol extract of P. somaliensis Browicz fruits afforded two known triterpenes, namely ursolic acid, and 11α,12α-epoxy-3β-hydroxy-olean-13β,28-olide, and a newly discovered one, namely 3β-hydroxy-urs-11-en-13β,28-olide. The structures of the isolated compounds were elucidated by the analysis of 1D and 2D NMR spectral data.

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

    PubMed

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

    2017-03-01

    2,4,6-Trinitrotoluene (TNT) has been widely used as an explosive substance and its toxicity is still of interest as it persisted in polluted areas. TNT is metabolized in hepatocytes which are prone to its toxicity. Since analysis of the human liver or hepatocytes is restricted due to ethical reasons, we investigated the effects of TNT on cell viability, reactive oxygen species (ROS) production, peroxisome proliferation, and antioxidative enzymes in human (HepG2), mouse (Hepa 1-6), and rat (H4IIEC3) hepatoma cell lines. Under control conditions, hepatoma cells of all three species were highly comparable exhibiting identical proliferation rates and distribution of their cell cycle phases. However, we found strong differences in TNT toxicity with the lowest IC50 values (highest cell death rate) for rat cells, whereas human and mouse cells were three to sevenfold less sensitive. Moreover, a strong decrease in cellular dehydrogenase activity (MTT assay) and increased ROS levels were noted. TNT caused peroxisome proliferation with rat hepatoma cells being most responsive followed by those from mouse and human. Under control conditions, rat cells contained fivefold higher peroxisomal catalase and mitochondrial SOD2 activities and a twofold higher capacity to reduce MTT than human and mouse cells. TNT treatment caused an increase in catalase and SOD2 mRNA and protein levels in human and mouse, but not in rat cells. Similarly, human and mouse cells upregulated SOD2 activity, whereas rat cells failed therein. We conclude that TNT induced oxidative stress, peroxisome proliferation and mitochondrial damage which are highest in rat cells rendering them most susceptible toward TNT. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 989-1006, 2017. © 2016 Wiley Periodicals, Inc.

  5. Urokinase-type plasminogen activator (uPA) stimulates triglyceride synthesis in Huh7 hepatoma cells via p38-dependent upregulation of DGAT2.

    PubMed

    Paland, Nicole; Gamliel-Lazarovich, Aviva; Coleman, Raymond; Fuhrman, Bianca

    2014-11-01

    The liver is the central organ of fatty acid and triglyceride metabolism. Oxidation and synthesis of fatty acids and triglycerides is under the control of peroxisome-proliferator-activated receptors (PPAR) α. Impairment of these receptors' function contributes to the accumulation of triglycerides in the liver resulting in non-alcoholic fatty liver disease. Urokinase-type plasminogen activator (uPA) was shown to regulate gene expression in the liver involving PPARγ transcriptional activity. In this study we questioned whether uPA modulates triglyceride metabolism in the liver, and investigated the mechanisms involved in the observed processes. Huh7 hepatoma cells were incubated with increasing concentrations of uPA for 24 h uPA dose-dependently increased the cellular triglyceride mass, and this effect resulted from increased de novo triglyceride synthesis mediated by the enzyme diglyceride acyltransferase 2 (DGAT2). Also, the amount of free fatty acids was highly up regulated by uPA through activation of the transcription factor SREBP-1. Chemical activation of PPARα further increased uPA-stimulated triglyceride synthesis, whereas inhibition of p38, an upstream activator of PPARα, completely abolished the stimulatory effect of uPA on both triglyceride synthesis and DGAT2 upregulation. The effect of uPA on triglyceride synthesis in Huh7 cells was mediated via binding to its receptor, the uPAR. In vivo studies in uPAR(-/-) mice demonstrated that no lipid droplets were observed in their livers compared to C57BL/6 mice and the triglyceride levels were significantly lower. This study presents a new biological function of the uPA/uPAR system in the metabolism of triglycerides and might present a new target for an early therapeutic intervention for NAFLD. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Non-receptor-mediated actions are responsible for the lipid-lowering effects of iodothyronines in FaO rat hepatoma cells.

    PubMed

    Grasselli, Elena; Voci, Adriana; Canesi, Laura; Goglia, Fernando; Ravera, Silvia; Panfoli, Isabella; Gallo, Gabriella; Vergani, Laura

    2011-07-01

    Iodothyronines influence lipid metabolism and energy homeostasis. Previous studies demonstrated that 3,5-l-diiodothyronine (T(2)), as well as 3,3',5-L-triiodothyronine (T(3)), was able to both prevent and reverse hepatic steatosis in rats fed a high-fat diet, and this effect depends on a direct action of iodothyronines on the hepatocyte. However, the involvement of thyroid hormone receptors (TRs) in mediating the lipid-lowering effect of iodothyronines was not elucidated. In this study, we investigated the ability of T(2) and T(3) to reduce the lipid overloading using the rat hepatoma FaO cells defective for functional TRs. The absence of constitutive mRNA expression of both TRα1 and TRβ1 in FaO cells was verified by RT-qPCR. To mimic the fatty liver condition, FaO cells were treated with a fatty acid mixture and then exposed to pharmacological doses of T(2) or T(3) for 24 h. Lipid accumulation, mRNA expression of the peroxisome proliferator-activated receptors (PPAR-α, -γ, -δ) the acyl-CoA oxidase (AOX), and the stearoyl CoA desaturase (SCD1), as well as fuel-stimulated O(2) consumption in intact cells, were evaluated. Lipid accumulation was associated with an increase in triacylglycerol content, PPARγ mRNA expression, and a decrease in PPARδ and SCD1 mRNA expression. The addition of T(2) or T(3) to lipid-overloaded cells resulted in i) reduction in lipid content; ii) downregulation of PPARα, PPARγ, and AOX expression; iii) increase in PPARδ expression; and iv) stimulation of mitochondrial uncoupling. These data demonstrate, for the first time, that in the hepatocyte, the lipid-lowering actions of both T(2) and T(3) are not mediated by TRs.

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

  8. Green and black tea extracts inhibit HMG-CoA reductase and activate AMP-kinase to decrease cholesterol synthesis in hepatoma cells

    PubMed Central

    Singh, Dev K.; Banerjee, Subhashis; Porter, Todd D.

    2009-01-01

    Recent studies have demonstrated that green and black tea consumption can lower serum cholesterol in animals and in man, and suppression of hepatic cholesterol synthesis is suggested to contribute to this effect. To evaluate this hypothesis, we measured cholesterol synthesis in cultured rat hepatoma cells in the presence of green and black tea extracts and selected components. Green and black tea decreased cholesterol synthesis by up to 55% and 78%, respectively, as measured by a 3-h incorporation of radiolabeled acetate. Inhibition was much less evident when radiolabeled mevalonate was used, suggesting that the inhibition was mediated largely at or above the level of HMG-CoA reductase. Both extracts directly inhibited HMG-CoA reductase when added to microsomal preparations, although the extent of inhibition was considerably less than the decrease in cholesterol synthesis observed in whole cells. As HMG-CoA reductase activity also can be decreased by enzyme phosphorylation by AMP-kinase, the phosphorylation state of HMG-CoA reductase and AMP-kinase, which is activated by phosphorylation, was determined in lysates from cells treated with tea extracts. Both extracts increased AMP-kinase phosphorylation and HMG-CoA reductase phosphorylation by 2.5- to 4-fold, but with different time-courses: maximal phosphorylation with green tea was evident within 30 min of treatment, whereas with black tea phosphorylation was slower to develop, with maximal phosphorylation occurring 3 or more h after treatment. These results suggest that both green and black tea decrease cholesterol synthesis in whole cells by directly inhibiting HMG-CoA reductase and by promoting its inactivation by AMP-kinase. PMID:18926682

  9. Differential Cytotoxicity of Acetaminophen in Mouse Macrophage J774.2 and Human Hepatoma HepG2 Cells: Protection by Diallyl Sulfide

    PubMed Central

    Raza, Haider; John, Annie

    2015-01-01

    Non-steroidal anti-inflammatory drugs (NSAIDs), including acetaminophen (APAP), have been reported to induce cytotoxicity in cancer and non-cancerous cells. Overdose of acetaminophen (APAP) causes liver injury in humans and animals. Hepatic glutathione (GSH) depletion followed by oxidative stress and mitochondrial dysfunction are believed to be the main causes of APAP toxicity. The precise molecular mechanism of APAP toxicity in different cellular systems is, however, not clearly understood. Our previous studies on mouse macrophage J774.2 cells treated with APAP strongly suggest induction of apoptosis associated with mitochondrial dysfunction and oxidative stress. In the present study, using human hepatoma HepG2 cells, we have further demonstrated that macrophages are a more sensitive target for APAP—induced toxicity than HepG2 cells. Using similar dose- and time-point studies, a marked increase in apoptosis and DNA fragmentation were seen in macrophages compared to HepG2 cells. Differential effects of APAP on mitochondrial respiratory functions and oxidative stress were observed in the two cell lines which are presumably dependent on the varying degree of drug metabolism by the different cytochrome P450s and detoxification by glutathione S-transferase enzyme systems. Our results demonstrate a marked increase in the activity and expression of glutathione transferase (GST) and multidrug resistance (MDR1) proteins in APAP-treated HepG2 cells compared to macrophages. This may explain the apparent resistance of HepG2 cells to APAP toxicity. However, treatment of these cells with diallyl sulfide (DAS, 200 μM), a known chemopreventive agent from garlic extract, 24 h prior to APAP (10 μmol/ml for 18h) exhibited comparable cytoprotective effects in the two cell lines. These results may help in better understanding the mechanism of cytotoxicity caused by APAP and cytoprotection by chemopreventive agents in cancer and non-cancerous cellular systems. PMID:26714183

  10. HBV polymerase overexpression due to large core gene deletion enhances hepatoma cell growth by binding inhibition of microRNA-100

    PubMed Central

    Huang, Ya-Hui; Tseng, Ying-Hsin; Lin, Wey-Ran; Hung, George; Chen, Tse-Ching; Wang, Tong-Hong; Lee, Wei-Chen; Yeh, Chau-Ting

    2016-01-01

    Different types of hepatitis B virus (HBV) core gene deletion mutants were identified in chronic hepatitis B patients. However, their clinical roles in different stages of natural chronic HBV infection remained unclear. To address this issue, HBV core genes were sequenced in three gender- and age-matched patient groups diagnosed as chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC), respectively. Functional analysis of the identified mutants was performed. A novel type of large-fragment core gene deletion (LFCD) was identified exclusively in HCC patients and significantly associated with unfavorable postoperative survival. The presence of LFCDs resulted in generation of precore-polymerase fusion protein or brought the polymerase reading frame under direct control of HBV precore/core promoter, leading to its over-expression. Enhanced cell proliferation and increased tumorigenicity in nude mice were found in hepatoma cells expressing LFCDs. Because of the epsilon-binding ability of HBV polymerase, we hypothesized that the over-expressed polymerase carrying aberrant amino-terminal sequence could bind to cellular microRNAs. Screening of a panel of microRNAs revealed physical association of a precore-polymerase fusion protein with microRNA-100. A binding inhibition effect on microRNA-100 by the precore-polymerase fusion protein with up-regulation of its target, polo-like kinase 1 (PLK1), was discovered. The binding inhibition and growth promoting effects could be reversed by overexpressing microRNA-100. Together, HCC patients carrying hepatitis B large-fragment core gene deletion mutants had an unfavorable postoperative prognosis. The growth promoting effect was partly due to polymerase overexpression, leading to binding inhibition of microRNA-100 and up-regulation of PLK1. PMID:26824500

  11. Effects of oolong tea on gene expression of gluconeogenic enzymes in the mouse liver and in rat hepatoma H4IIE cells.

    PubMed

    Yasui, Kensuke; Miyoshi, Noriyuki; Tababe, Hiroki; Ishigami, Yoko; Fukutomi, Ryuuta; Imai, Shinjiro; Isemura, Mamoru

    2011-09-01

    Tea has many beneficial effects. We have previously reported that green tea and a catechin-rich green tea beverage modulated the gene expression of the gluconeogenic enzymes glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in the normal murine liver. In the present study, we examined the effects of oral administration of oolong tea on the hepatic expression of gluconeogenesis-related genes in the mouse. The intake of oolong tea for 4 weeks reduced the hepatic expression of G6Pase and PEPCK together with that of the transcription factor hepatocyte nuclear factor (HNF) 4α. When rat hepatoma H4IIE cells were incubated in the presence of oolong tea, the expression of these genes was repressed in accordance with the findings in vivo. The reduced protein expression of PEPCK and HNF4α was also demonstrated. We then fractionated oolong tea by sequential extraction with three organic solvents to give three fractions and the residual fraction (Fraction IV). In addition to organic fractions, Fraction IV, which was devoid of low-molecular-weight catechins such as (-)-epigallocatechin gallate (EGCG), had effects similar to those of oolong tea on H4IIE cells. Fraction IV repressed the gene expression of insulin-like growth factor binding protein 1, as insulin did. This activity was different from that of EGCG. The present findings suggest that drinking oolong tea may help to prevent diabetes and that oolong tea contains a component or components with insulin-like activity distinguishable from EGCG. Identification of such component(s) may open the way to developing a new drug for diabetes.

  12. Anorexia‐cachexia syndrome in hepatoma tumour‐bearing rats requires the area postrema but not vagal afferents and is paralleled by increased MIC‐1/GDF15

    PubMed Central

    Borner, Tito; Arnold, Myrtha; Ruud, Johan; Breit, Samuel N.; Langhans, Wolfgang; Lutz, Thomas A.; Blomqvist, Anders

    2016-01-01

    Abstract Background The cancer‐anorexia‐cachexia syndrome (CACS) negatively affects survival and therapy success in cancer patients. Inflammatory mediators and tumour‐derived factors are thought to play an important role in the aetiology of CACS. However, the central and peripheral mechanisms contributing to CACS are insufficiently understood. The area postrema (AP) and the nucleus tractus solitarii are two important brainstem centres for the control of eating during acute sickness conditions. Recently, the tumour‐derived macrophage inhibitory cytokine‐1 (MIC‐1) emerged as a possible mediator of cancer anorexia because lesions of these brainstem areas attenuated the anorectic effect of exogenous MIC‐1 in mice. Methods Using a rat hepatoma tumour model, we examined the roles of the AP and of vagal afferents in the mediation of CACS. Specifically, we investigated whether a lesion of the AP (APX) or subdiaphragmatic vagal deafferentation (SDA) attenuate anorexia, body weight, muscle, and fat loss. Moreover, we analysed MIC‐1 levels in this tumour model and their correlation with tumour size and the severity of the anorectic response. Results In tumour‐bearing sham‐operated animals mean daily food intake significantly decreased. The anorectic response was paralleled by a significant loss of body weight and muscle mass. APX rats were protected against anorexia, body weight loss, and muscle atrophy after tumour induction. In contrast, subdiaphragmatic vagal deafferentation did not attenuate cancer‐induced anorexia or body weight loss. Tumour‐bearing rats had substantially increased MIC‐1 levels, which positively correlated with tumour size and cancer progression and negatively correlated with food intake. Conclusions These findings demonstrate the importance of the AP in the mediation of cancer‐dependent anorexia and body weight loss and support a pathological role of MIC‐1 as a tumour‐derived factor mediating CACS, possibly via an AP

  13. Carboxyl ester lipase overexpression in rat hepatoma cells and CEL deficiency in mice have no impact on hepatic uptake or metabolism of chylomicron-retinyl ester.

    PubMed

    van Bennekum, A M; Li, L; Piantedosi, R; Shamir, R; Vogel, S; Fisher, E A; Blaner, W S; Harrison, E H

    1999-03-30

    To study the role of carboxyl ester lipase (CEL) in hepatic retinoid (vitamin A) metabolism, we investigated uptake and hydrolysis of chylomicron (CM)-retinyl esters (RE) by rat hepatoma (McArdle-RH7777) cells stably transfected with a rat CEL cDNA. We also studied tissue uptake of CM-RE in CEL-deficient mice generated by targeted disruption of the CEL gene. CEL-transfected cells secreted active enzyme into the medium. However, both control and CEL-transfected cells accumulated exogenously added CM-RE or CM remnant (CMR)-derived RE in equal amounts. Serum clearance of intravenously injected CM-RE and cholesteryl ester were not different between wild-type and CEL-deficient mice. Also, the uptake of the two compounds by the liver and other tissues did not differ. These data indicate that the lack of CEL expression does not affect the uptake of dietary CM-RE by the liver or other tissues. Moreover, the percentage of retinol formed in the liver after CM-RE uptake, the levels of retinol and retinol-binding protein in serum, and retinoid levels in various tissues did not differ, indicating that CEL deficiency does not affect hepatic retinoid metabolism and retinoid distribution throughout the body. Surprisingly, in both pancreas and liver of wild-type, heterozygous, and homozygous CEL-deficient mice, the levels of bile salt-dependent retinyl ester hydrolase (REH) activity were similar. This indicates that in the mouse pancreas and liver an REH enzyme activity, active in the presence of bile salt and distinct from CEL, is present, compatible with the results from our accompanying paper that the intestinal processing and absorption of RE were unimpaired in CEL-deficient mice.

  14. KNOCKDOWN OF GCN5 HISTONE ACETYLTRANSFERASE by siRNA DECREASES ETHANOL INDUCED HISTONE ACETYLATION AND AFFECTS DIFFERENTIAL EXPRESSION OF GENES IN HUMAN HEPATOMA CELLS

    PubMed Central

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

    2011-01-01

    We have investigated whether Gcn5, a histone acetyltransferase (HAT), is involved in ethanol induced acetylation of histone H3 at lysine 9 (H3AcK9) and has any effect on the gene expression. Human hepatoma HepG2 cells transfected with ethanol metabolizing enzyme alcohol dehydrogenase 1 (VA 13 cells) were used. Knockdown of Gcn5 by siRNA silencing decreased mRNA and protein levels of GCN5, HAT activity, and also attenuated ethanol induced H3AcK9 in VA13 cells. Illumina gene microarray analysis using total RNA showed 940 transcripts affected by GCN5 silencing or ethanol. Silencing caused differential expression of 891 transcripts (≥ 1.5 fold up- or down- regulated). Among these, 492 transcripts were up- and 399 were down- regulated compared to their respective controls. Using a more stringent threshold (≥ 2.5 fold) the array data from GCN5 silenced samples showed 57 genes differentially expressed (39 up-regulated and 18 down-regulated). Likewise, ethanol caused differential regulation of 57 transcripts with ≥ 1.5 fold change (35 gene up-regulated and 22 down-regulated). Further analysis showed that eight genes were differentially regulated that were common for both ethanol treatment and GCN5 silencing. Among these, SLC44A2 (a putative choline transporter) was strikingly up-regulated by ethanol (3 fold), and GCN5 silencing down regulated it (1.5 fold). The quantitative RT-PCR profile corroborated the array findings. This report demonstrates for the first time that (a) GCN5 differentially affects expression of multiple genes, (b) ethanol induced histone H3-lysine 9 acetylation is mediated via GCN5 and (c) that GCN5 is involved in ethanol induced expression of the putative choline transporter SLC44A2. PMID:21367571

  15. Different apoptotic effects of triterpenoid saponin-rich Gypsophila oldhamiana root extract on human hepatoma SMMC-7721 and normal human hepatic L02 cells.

    PubMed

    Zhang, Wei; Luo, Jian-Guang; Zhang, Chao; Kong, Ling-Yi

    2013-01-01

    The roots of Gypsophila oldhamiana are rich in triterpenoid saponins with antitumor properties. Although previous reports have revealed the anticancer potency of some Gypsophila species, the underlying molecular mechanisms of this activity have not been studied in detail. The purpose of the present study was to prepare a triterpenoid saponin-rich G. oldhamiana root extract (TGOE) determined by LC-electrospray ionization (ESI)-MS(n) for biological studies and to evaluate the different anti-proliferative activities and apoptotic effects of TGOE on human hepatoma SMMC-7721 and normal human hepatic L02 cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that TGOE selectively inhibited the proliferation of SMMC-7721 cells in a dose-dependent manner with IC50 value of 19.50±3.63 µg/mL, while the cytotoxic effects of TGOE on L02 cells were much lower with IC50 value of 40.48±3.74 µg/mL. Analysis of apoptotic morphological changes and flow cytometry indicated that TGOE might preferentially induce apoptosis in SMMC-7721 cells, while exhibited much lower effects on L02 cells. Western blot analysis showed that the different apoptotic effects of TGOE on SMMC-7721 and L02 cells were due to different protein regulation of caspase-3 and mitogen activated protein kinases (MAPKs). TGOE significantly activated caspase-3 and increased the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), while decreased the phosphorylation of p38 in SMMC-7721 cells. However, the expression of these proteins was not statistically changed in L02 cells, except for the up-regulation of p38 phosphorylation. These results suggest that TGOE may have potential beneficial effects against hepatocellular carcinoma.

  16. Octanoyl galactose ester-modified microemulsion system self-assembled by coix seed components to enhance tumor targeting and hepatoma therapy

    PubMed Central

    Qu, Ding; Liu, Mingjian; Huang, Mengmeng; Wang, Lixiang; Chen, Yan; Liu, Congyan; Liu, Yuping

    2017-01-01

    A nanosized drug delivery platform with a combination of rational components and tumor targeting is significant for enhancement of anticancer therapy and reduction of side effects. In this study, we developed a octanoyl galactose ester-modified microemulsion system self-assembled by coix seed components (Gal(oct)-C-MEs), which improved the tumor accumulation through asialoglycoprotein receptor-mediated endocytosis and promoted the antitumor efficacy through multicomponent-mediated synergistic effect. Octanoyl galactose ester (Gal(oct)) with a yield of 82.3% was synthesized through a green enzymatic reaction and multidimensional characterization. Gal(oct)-C-MEs with a spherical shape had a small and uniform particle size (58.49±1.03 nm), narrow polydispersity index (0.09±0.01) and neutral surface charge (−5.82±0.57 mV). In the cellular uptake studies, the internalized Gal(oct)-C-ME was 2.28-fold higher relative to that of coix seed component-based microemulsions (C-MEs). The half-maximal inhibitory concentration of Gal(oct)-C-MEs against HepG2 cells was 46.5±2.4 μg/mL, which was notably higher than that of C-MEs. Importantly, the intratumor fluorescence of HepG2 xenograft-bearing nude mice treated with Cy5/Gal(oct)-C-MEs was 1.9-fold higher relative to treatment with Cy5/C-MEs. In the study of antitumor efficacy in vivo, HepG2 xenograft-bearing nude mice intragastrically administered Gal(oct)-C-MEs for 14 days exhibited the strongest inhibition of tumor growth and the lowest toxicity against liver and kidney among all the treatments. In summary, Gal(oct)-C-ME, as a highly effective and safe anticancer drug delivery system, showed promising potential for hepatoma therapy. PMID:28352174

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

    PubMed

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

    2011-06-01

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

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

    PubMed Central

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

    2011-01-01

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

  19. Choline metabolism and membrane formation in rat hepatoma cells grown in suspension culture. II. Phosphatidylcholine synthesis during growth cycle and fluctuation of mitochondrial density.

    PubMed

    Plagemann, P G

    1969-09-01

    The incorporation of methyl-labeled choline into phosphorylcholine and phosphatidylcholine of cellular membranes by Novikoff rat hepatoma cells (line N1S1-67) during growth in suspension culture was investigated. Upon initiation of a fresh culture at 10(5) cells/ml, the rate of synthesis of phosphorylcholine by the cells was four to five times greater than that of the synthesis of phosphatidylcholine. While the rate of synthesis of the latter remained relatively constant, the rate of phosphorylation of choline decreased progressively during the course of the growth cycle of the culture to 10-20% of the initial rate when the culture reached stationary phase at 3 x 10(6) cells/ml. The decrease in phosphorylcholine synthesis during the growth cycle was not due to depletion of choline in the medium or a decrease in its concentration, but was correlated with a decrease in choline kinase activity of the cells as measured in cell-free extracts. Newly synthesized phosphatidylcholine was detectable in cells only as an integral part of cellular membranes. Its distribution among various cytoplasmic membrane structures separated by isopycnic centrifugation in sucrose density gradients remained relatively constant during the growth cycle. About 50% was associated with the mitochondria, and the remainder with plasma membrane fragments and other membranous structures with mean densities of about 1.15 and 1.13 g/cm(3), respectively. However, the density of the mitochondria increased from about 1.167 g/cm(3) in early exponential phase cells to about 1.190 g/cm(3) in stationary phase cells. The finding that the density of the entire propulation of mitochondria changed simultaneously and progressively is in agreement with the view that mitochondria grow by addition of phospholipids and structural proteins and increase in number by division.

  20. Effects of fucoidan on proliferation, AMP-activated protein kinase, and downstream metabolism- and cell cycle-associated molecules in poorly differentiated human hepatoma HLF cells.

    PubMed

    Kawaguchi, Takumi; Hayakawa, Masako; Koga, Hironori; Torimura, Takuji

    2015-05-01

    Survival rates are low in patients with poorly differentiated hepatocellular carcinoma (HCC). Fucoidan, a sulfated polysaccharide derived from brown seaweed, has anticancer activity; however, the effects of fucoidan on poorly differentiated HCC remain unclear. In this study, we investigated the effects of fucoidan on AMP-activated protein kinase (AMPK), a proliferation regulator, and its downstream metabolism- and cell cycle-related molecules in a poorly differentiated human hepatoma HLF cell line. HLF cells were treated with fucoidan (10, 50, or 100 µg/ml; n=4) or phosphate buffered saline (control; n=4) for 96 h. Proliferation was evaluated by counting cells every 24 h. AMPK, TSC2, mTOR, GSK3β, acetyl-CoA carboxylase (ACC), ATP-citrate lyase, p53, cyclin D1, cyclin-dependent kinase (CDK) 4, and CDK6 expression and/or phosphorylation were examined by immunoblotting 24 h after treatment with 100 µg/ml fucoidan. Cell cycle progression was analyzed by fluorescence-activated cell sorter 48 h after treatment. Treatment with 50 or 100 µg/ml fucoidan significantly and dose- and time-dependently suppressed HLF cell proliferation (P<0.0001). Fucoidan induced AMPK phosphorylation on Ser172 24 h after treatment. Although no differences were seen in expression and phosphorylation levels of TSC2, mTOR, GSK3β, ATP-citrate lyase, and p53 between the control and fucoidan-treated HLF cells, fucoidan induced ACC phosphorylation on Ser79. Moreover, fucoidan decreased cyclin D1, CDK4 and CDK6 expression 24 h after treatment. Furthermore, HLF cells were arrested in the G1/S phase 48 h after fucoidan treatment. We demonstrated that fucoidan suppressed HLF cell proliferation with AMPK phosphorylation. We showed that fucoidan phosphorylated ACC and downregulated cyclin D1, CDK4 and CDK6 expression. Our findings suggest that fucoidan inhibits proliferation through AMPK-associated suppression of fatty acid synthesis and G1/S transition in HLF cells.

  1. Ganglioside GM3 inhibits hepatoma cell motility via down-regulating activity of EGFR and PI3K/AKT signaling pathway.

    PubMed

    Huang, X; Li, Y; Zhang, J; Xu, Y; Tian, Y; Ma, K

    2013-07-01

    Two related sublines derived from murine ascites hepatoma cell lines Hca-F25, which were selected for their markedly different metastatic potential to lymph nodes, were found to be distinct in their ganglioside patterns. The low metastatic cell line (HcaP) contained a major ganglioside GM3, whereas the high metastatic cell line (HcaF) contained a major ganglioside GM2. Suppression of GM3 by P4 enhanced the mobility and migration of the low metastatic HcaP cells in vitro. Increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 inhibited the mobility and migration. These results suggested that the differences in lymphatic metastasis potential between these two cell lines could be attributed to the differences in their ganglioside compositions, and GM3 could suppress the motility and migration of these cells. Further, we investigated the mechanism by which GM3 suppressed the cell mobility and migration. The results showed that suppression of GM3 synthesis by P4 in low metastatic HcaP cells promoted PKB/Akt phosphorylation at Ser473 and Thr308, and phosphorylation of EGFR at the Tyr1173. In contrast, increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 into the culture medium suppressed phosphorylation of PKB/Akt and EGFR at the same residues. Taken together, these results suggested that the mechanism of GM3-suppressed cell motility and migration may involve the inhibition of phosphorylation of EGFR and the activity of PI3K/AKT signaling pathway. Copyright © 2013 Wiley Periodicals, Inc.

  2. Identification of Replication-competent HSV-1 Cgal+ Strain Signaling Targets in Human Hepatoma Cells by Functional Organelle Proteomics*S⃞

    PubMed Central

    Santamaría, Enrique; Mora, María I.; Potel, Corinne; Fernández-Irigoyen, Joaquín; Carro-Roldán, Elvira; Hernández-Alcoceba, Rubén; Prieto, Jesús; Epstein, Alberto L.; Corrales, Fernando J.

    2009-01-01

    In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal+) infection. Using a combination of differential in-gel electrophoresis and nano liquid chromatography/tandem mass spectrometry, 18 spots corresponding to 16 unique deregulated cellular proteins were unambiguously identified, which were involved in the regulation of essential processes such as apoptosis, mRNA processing, cellular structure and integrity, signal transduction, and endoplasmic-reticulum-associated degradation pathway. Based on our proteomic data and additional functional studies target proteins were identified indicating a late activation of apoptotic pathways in Huh7 cells upon HSV-1 Cgal+ infection. Additionally to changes on RuvB-like 2 and Bif-1, down-regulation of Erlin-2 suggests stimulation of Ca2+-dependent apoptosis. Moreover, activation of the mitochondrial apoptotic pathway results from a time-dependent multi-factorial impairment as inferred from the stepwise characterization of constitutive pro- and anti-apoptotic factors. Activation of serine-threonine protein phosphatase 2A (PP2A) was also found in Huh7 cells upon HSV-1 Cgal+ infection. In addition, PP2A activation paralleled dephosphorylation and inactivation of downstream mitogen-activated protein (MAP) kinase pathway (MEK½, ERK½) critical to cell survival and activation of proapoptotic Bad by dephosphorylation of Ser-112. Taken together, our results provide novel molecular information that contributes to define in detail the apoptotic mechanisms triggered by HSV-1 Cgal+ in the host cell and lead to the implication of PP2A in the transduction of cell death signals and cell survival pathway arrest. PMID:19098277

  3. Oligomeric TTR V30M aggregates compromise cell viability, erythropoietin gene expression and promoter activity in the human hepatoma cell line Hep3B.

    PubMed

    Moreira, Luciana; Beirão, João Melo; Beirão, Idalina; Pinho e Costa, Paulo

    2015-01-01

    Familial amyloidotic polyneuropathy, ATTRV30M (p. TTRV50M) amyloidosis, is a neurodegenerative disease characterized by systemic extracellular amyloid deposition of a mutant transthyretin, TTR V30M. Anemia, with low erythropoietin (EPO) levels and spared kidney function, affects about 25% of symptomatic patients, suggesting a blockage of EPO-producing cells. Early non-fibrillar TTR aggregates are highly cytotoxic, inducing oxidative stress, the expression of apoptosis-related molecules and secretion of pro-inflammatory cytokines, factors capable of inhibiting EPO production. Low EPO levels in these patients are not related to renal amyloid deposition or the presence of circulating TTR V30M. However, the role of early non-fibrillar TTR aggregates remains unexplored. We used the EPO producing Hep3B human hepatoma cell line to study the effect of TTR oligomeric aggregates on EPO expression. Hep3B cells were incubated with soluble and oligomeric TTR V30M, and cell proliferation as well as caspase 3/7 activation was evaluated. Relative quantification of EPO mRNA transcripts was performed by real-time PCR. Significant reductions in cell viability (13 ± 7.3%) and activation of caspases 3/7 were seen after 24 h in the presence of oligomeric TTR V30M. Also, EPO expression was significantly reduced (50 ± 2.8%), in normoxic conditions. A reporter assay was constructed with a PCR fragment of the EPO promoter linked to the luciferase gene to evaluate the role of transcription factors targeting the promoter. A significant reduction of EPO promoter activity (53 ± 6.5%) was observed in transfected cells exposed to TTR oligomers. Our results show that oligomeric TTR V30M reduces EPO expression, at least in part through inhibition of promoter activity.

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

    PubMed Central

    Tiwary, Bipransh Kumar; Kumar, Anoop

    2014-01-01

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

  5. HBx induced AFP receptor expressed to activate PI3K/AKT signal to promote expression of Src in liver cells and hepatoma cells.

    PubMed

    Zhu, Mingyue; Guo, Junli; Li, Wei; Xia, Hua; Lu, Yan; Dong, Xu; Chen, Yi; Xie, Xieju; Fu, Shigan; Li, Mengsen

    2015-05-06

    Hepatitis B virus (HBV)-X protein(HBx) is a transactivator of host several cellular genes including alpha-fetoprotein(AFP) and AFP receptor(AFPR) which contributes to HBV-associated tumor development. The expression of AFP/AFPR are correlated with hepatocellular carcinoma(HCC)-initial cells. But the role of AFP and AFPR in promoting occurrence of HBV-related HCC were still unclear. A total of 71 clinical patients' liver specimens, normal human liver cells L-02 and HCC cell lines, PLC/PRF/5 were selected for analyzing the effects of HBx on expression of AFP, AFPR and Src. The expression of goal proteins were detected by Immunohistochemical stained and Western blotting; HBx-expressed vectors were constructed and transfected into L-02 cells, laser confocal microscopy was applied to observe expression and location of AFP, AFPR and Src in the normal liver cells and HCC cells, soft agar colony formation assay was used to observe colonies formed of the cells. We confirmed HBx gives preference to promote the expression of AFP and AFPR; HBx priors to up-regulate the expression of AFPR and AFP in L-02 cells and in normal liver specimens; AFPR signal been able to stimulate Src expression. The results also indicated that phosphatidylinositol 3-kinase(PI3K) inhibitors Ly294002 and GDC0941 effectively suppress AFPR mediated up-regulation expression of Src in AFPR positive HCC lines. HBx priors to drive the expression of AFP and AFPR to promote expression of Src in normal liver cells and hepatoma cells; AFP and AFPR maybe play pivotal role in HBV-related hepatocarcinogenesis; Targeting AFPR is an available therapeutic strategy of HCC.

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

    PubMed

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

    2016-07-01

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

  7. The role of protein glycosylation in the compartmentalization and processing of mouse mammary tumor virus glycoproteins in mouse mammary tumor virus-infected rat hepatoma cells.

    PubMed

    Firestone, G L

    1983-05-25

    The relationship of protein glycosylation to compartmentalization and processing of mouse mammary tumor virus (MTV) glycoproteins has been examined in M1.54, a cloned line of MTV-infected rat hepatoma tissue culture cells. Previous work established that full maturation of MTV glycoproteins in this cell line requires dexamethasone, a synthetic glucocorticoid (Firestone, G. L., Payvar, F., and Yamamoto, K. R. (1982) Nature (Lond.) 300, 221-225). The ability to regulate production of the full complement of five mature membrane-associated and secreted viral glycoproteins from one initially synthesized precursor has been used to advantage in the present work. At concentrations of tunicamycin that specifically inhibit N-linked protein glycosylation, incorporation of [35S]methionine into total cellular and secreted protein is not detectably affected, MTV-specific mRNAs are produced normally, and the nonglycosylated form of the glycosylated viral precursor polyprotein accumulates within the cells. However, tunicamycin inhibits the site-specific cleavage of the glycosylated polyprotein and distribution of MTV polypeptides to the cell surface and extracellular fractions. Thus, when tunicamycin-treated cultures of M1.54 are exposed to dexamethasone and [35S]methionine, no labeled viral antigens are detected in the culture medium. Similarly, tunicamycin prevents the appearance of membrane-associated viral antigens that can be labeled externally by lactoperoxidase-mediated iodination and it protects the cells against the cytolytic effects of MTV-specific antiserum and complement. Taken together, these results are consistent with the view that while glycosylation of some proteins may be unessential for their compartmentalization and processing, it does appear to be correlated with proper maturation of others. The hormone-dependent maturation of MTV glycoproteins in M1.54 may be particularly useful for study of this latter class since glycosylation is stringently associated with

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

    PubMed

    Hossain, Zakir; Sugawara, Tatsuya; Hirata, Takashi

    2013-03-01

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

  9. The apoptotic effect of brucine from the seed of Strychnos nux-vomica on human hepatoma cells is mediated via Bcl-2 and Ca2+ involved mitochondrial pathway.

    PubMed

    Deng, Xukun; Yin, Fangzhou; Lu, Xiaoyu; Cai, Baochang; Yin, Wu

    2006-05-01

    In an attempt to dissect the mechanism of Strychnos nux-vomica, a commonly used Chinese folk medicine in the therapy of liver cancer, the cytotoxic effects of four alkaloids in Strychnos nux-vomica, brucine, brucine N-oxide, strychnine, and isostrychnine, on human hepatoma cells (HepG2) were screened by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrasolium bromide (MTT) assay. Brucine, among the four alkaloids, exhibited the strongest toxic effect, the mechanism of which was found to cause HepG2 cell apoptosis, since brucine caused HepG2 cell shrinkage, the formation of apoptotic bodies, DNA fragmentation, cell cycle arrest, as well as phosphatidylserine externalization, all of which are typical characteristics of apoptotic programmed cell death. Brucine-induced HepG2 cell apoptosis was caspase dependent, with caspase-3 activated by caspase-9. Brucine also caused the proteolytic processing of caspase-9. In addition, brucine caused depolarization of the mitochondrial membrane of HepG2 cells, the inhibition of which by cyclosporine A completely abrogated the activation of casapses and release of cytochrome c in brucine-treated HepG2 cells. These findings suggested a pivotal role of mitochondrial membrane depolarization in HepG2 cell apoptosis elicited by brucine. Furthermore, brucine induced a rapid and sustained elevation of intracellular [Ca2+], which compromised the mitochondrial membrane potential and triggered the process of HepG2 cell apoptosis. Finally, Bcl-2 was found to predominately control the whole event of cell apoptosis induced by brucine. The elevation of [Ca2+]i caused by brucine was also suppressed by overexpression of Bcl-2 protein in HepG2 cells. From the facts given above, Ca2+ and Bcl-2 mediated mitochondrial pathway were found to be involved in brucine-induced HepG2 cell apoptosis.

  10. Involvement of the p38 MAPK signaling pathway in S-phase cell-cycle arrest induced by Furazolidone in human hepatoma G2 cells.

    PubMed

    Sun, Yu; Tang, Shusheng; Jin, Xi; Zhang, Chaoming; Zhao, Wenxia; Xiao, Xilong

    2013-12-01

    Given the previously described essential role for the p38 mitogen-activation protein kinase (p38 MAPK) signaling pathway in human hepatoma G2 cells (HepG2), we undertook the present study to investigate the role of the p38 MAPK signaling pathway in cell-cycle arrest induced by Furazolidone (FZD). The aim of this study was to determine the effects of FZD on HepG2 cells by activating and inhibiting the p38 MAPK signaling pathway. The cell cycle and proliferation of HepG2 cells treated with FZD were detected by flow cytometry and MTT assay in the presence or absence of p38 MAPK inhibitors (SB203580), respectively. Cyclin D1, cyclin D3 and CDK6 were detected by quantitative real-time PCR and western blot analysis. Our data showed that p38 MAPK became phosphorylated after stimulation with FZD. Activation of p38 MAPK could arise S-phase cell-cycle arrest and suppress cell proliferation. Simultaneously, inhibition of the p38 MAPK signaling pathway significantly prevented S-phase cell-cycle arrest, increased the percentage of cell viability and decreased the expression of cyclin D1, cyclin D3 and CDK6. These results demonstrated that FZD arose S-phase cell-cycle arrest via activating the p38 MAPK signaling pathway in HepG2 cells. Cyclin D1, cyclin D3 and CDK6 are target genes functioning at the downstream of p38 MAPK in HepG2 cells induced by FZD.

  11. Polarization of Tissue-Resident TFH-Like Cells in Human Hepatoma Bridges Innate Monocyte Inflammation and M2b Macrophage Polarization.

    PubMed

    Chen, Min-Min; Xiao, Xiao; Lao, Xiang-Ming; Wei, Yuan; Liu, Rui-Xian; Zeng, Qiu-Hui; Wang, Jun-Cheng; Ouyang, Fang-Zhu; Chen, Dong-Ping; Chan, Ka-Wo; Shi, Dai-Chao; Zheng, Limin; Kuang, Dong-Ming

    2016-10-01

    The existence, regulation, and functions of IL21(+) immune cells are poorly defined in human cancers. Here, we identified a subset of protumorigenic IL21(+) TFH-like cells in human hepatocellular carcinoma. These cells were the major source of IL21 in tumors and represented about 10% of the CD4(+) T-cell population at levels comparable with the TFH cells present in lymph nodes. However, these TFH-like cells displayed a unique CXCR5(-)PD-1(lo/-)BTLA(-)CD69(hi) tissue-resident phenotype with substantial IFNγ production, which differed from the phenotype of TFH cells. Toll-like receptor 4 (TLR4)-elicited innate monocyte inflammation was important for IL21(+) TFH-like cell induction in tumors, and activation of STAT1 and STAT3 was critical for TFH-like cell polarization in this process. Importantly, the TFH-like cells operated in IL21-IFNγ-dependent pathways to induce plasma cell differentiation and thereby create conditions for protumorigenic M2b macrophage polarization and cancer progression. Thus, induction of TFH-like cells links innate inflammation to immune privilege in tumors. We identified a novel protumorigenic IL21(+) TFH-like cell subset with a CXCR5(-)PD-1(-) BTLA(-)CD69(hi) tissue-resident phenotype in hepatoma. TLR4-mediated monocyte inflammation and subsequent T-cell STAT1 and STAT3 activation are critical for TFH-like cell induction. TFH-like cells operate via IL21-IFNγ pathways to induce plasma cells and create conditions for M2b macrophage polarization. Cancer Discov; 6(10); 1182-95. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1069. 2016 American Association for Cancer Research.

  12. Measuring and modeling of binary mixture effects of pharmaceuticals and nickel on cell viability/cytotoxicity in the human