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Sample records for hepatoma hepg2 cells

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. Intermedilysin is essential for the invasion of hepatoma HepG2 cells by Streptococcus intermedius.

    PubMed

    Sukeno, Akiko; Nagamune, Hideaki; Whiley, Robert A; Jafar, Syed I; Aduse-Opoku, Joseph; Ohkura, Kazuto; Maeda, Takuya; Hirota, Katsuhiko; Miyake, Yoichiro; Kourai, Hiroki

    2005-01-01

    Streptococcus intermedius causes endogenous infections leading to abscesses. This species produces intermedilysin (ILY), a human-specific cytolysin. Because of the significant correlation between higher ILY production levels by S. intermedius and deep-seated abscesses, we constructed ily knockout mutant UNS38 B3 and complementation strain UNS38 B3R1 in order to investigate the role of ILY in deep-seated infections. Strain UNS38 reduced the viability of human liver cell line HepG2 at infection but not of rat liver cell line BRL3A. Isogenic mutant strain UNS38 B3 was not cytotoxic in either cell line. Quantification of S. intermedius revealed that in infected HepG2 cells UNS38 but not UNS38 B3 increased intracellularly concomitantly with increasing cell damage. This difference between UNS38 and UNS38 B3 was not observed with UNS38 B3R1. Invasion and proliferation in BRL3A cells was not observed. Masking UNS38 or UNS38 B3R1 with ILY antibody drastically decreased adherence and invasion of HepG2. Moreover, coating strain UNS38 B3 with ILY partially restored adherence to HepG2 but without subsequent bacterial growth. At 1 day post-infection, many intact UNS38 were detected in the damaged phagosomes of HepG2 with bacterial proliferation observed in the cytoplasm of dead HepG2 after an additional 2 day incubation. These results indicate that surface-bound ILY on S. intermedius is an important factor for invasion of human cells by this bacterium and that secretion of ILY within host cells is essential for subsequent host cell death. These data strongly implicate ILY as an important factor in the pathogenesis of abscesses in vivo by this streptococcus.

  9. 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.

  10. 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.

  11. 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

  12. DNA damage and metallothionein synthesis in human hepatoma cells (HepG2) exposed to cadmium.

    PubMed

    Fatur, T; Tusek, M; Falnoga, I; Scancar, J; Lah, T T; Filipic, M

    2002-08-01

    Cadmium is an important heavy metal environmental toxicant, which is classified as a human carcinogen. The comet assay was used to evaluate the levels of DNA damage in a metabolically competent HepG2 cell line after treatment with low, non-cytotoxic and physiologically relevant concentrations of cadmium, alone and in combination with the dietary mutagen 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and with the environmental mutagen benzo[a]pyrene (B(a)P). After exposure of the cells to 10, 100 and 1000 nM CdCl(2), a dose- and time-dependent increase of DNA damage was detected. Maximal damage was found after 12 h of treatment, but declined with further incubation with CdCl(2). The increased synthesis of metallothioneins on exposure to CdCl(2) up to 12 h suggests that they are responsible for the adaptation of HepG2 cells to the DNA damaging effects of CdCl(2). Co-treatment of the cells with CdCl(2) (10-1000 nM) and IQ (300 microM) induced a dose-dependent increase of DNA damage compared to cells treated with IQ alone. Co-genotoxic activity was also observed by increased formation of micronuclei in cells exposed to IQ and 1000 nM CdCl(2); at this concentration, CdCl(2) alone also induced micronuclei in HepG2 cells. Our results support the hypothesis that direct and indirect mechanisms are involved in cadmium-induced DNA damage.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  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. 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.

  20. 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

  1. Differential Cytotoxicity of Acetaminophen in Mouse Macrophage J774.2 and Human Hepatoma HepG2 Cells: Protection by Diallyl Sulfide.

    PubMed

    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.

  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. 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).

  4. Cytoskeleton interruption in human hepatoma HepG2 cells induced by ketamine occurs possibly through suppression of calcium mobilization and mitochondrial function.

    PubMed

    Chang, Huai-Chia; Chen, Ta-Liang; Chen, Ruei-Ming

    2009-01-01

    Ketamine is an intravenous anesthetic agent often used for inducing and maintaining anesthesia. Cytoskeletons contribute to the regulation of hepatocyte activity of drug biotransformation. In this study, we attempted to evaluate the effects of ketamine on F-actin and microtubular cytoskeletons in human hepatoma HepG2 cells and its possible molecular mechanisms. Exposure of HepG2 cells to ketamine at cell viability. Meanwhile, administration of therapeutic concentrations of ketamine obviously interrupted F-actin and microtubular cytoskeletons. In parallel, levels of intracellular calcium concentration- and time-dependently decreased after ketamine administration. Analysis by confocal microscopy further revealed that ketamine suppressed calcium mobilization from an extracellular buffer into HepG2 cells. Exposure to ketamine decreased cellular ATP levels. The mitochondrial membrane potential and complex I NADH dehydrogenase activity were both reduced after ketamine administration. Ketamine did not change the production of actin or microtubulin mRNA in HepG2 cells. Consequently, ketamine-caused cytoskeletal interruption led to suppression of CYP3A4 expression and its metabolizing activity. Therefore, this study shows that therapeutic concentrations of ketamine can disrupt F-actin and microtubular cytoskeletons possibly through suppression of intracellular calcium mobilization and cellular ATP synthesis due to down-regulation of the mitochondrial membrane potential and complex I enzyme activity. Such disruption of the cytoskeleton may lead to reductions in CYP3A4 activity in HepG2 cells.

  5. 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.

  6. A polysaccharide from Andrographis paniculata induces mitochondrial-mediated apoptosis in human hepatoma cell line (HepG2).

    PubMed

    Zou, Yanmei; Xiong, Hua; Xiong, Huihua; Lu, Tao; Zhu, Feng; Luo, Zhiyong; Yuan, Xianglin; Wang, Yihua

    2015-07-01

    In the present study, we investigated the effects and action mechanisms of a purified polysaccharide (APWP) from Andrographis paniculata, on human hepatocellular carcinoma (HCC) HepG2 cells. The results showed that APWP was able to suppress the proliferation of HepG2 cells via inducing apoptosis. Western blot analysis revealed that dose-dependent increase in proapoptotic Bax protein and no change in antiapoptotic Bcl-2 protein in APWP-treated cells. Furthermore, exposure of tumor cells to APWP resulted in a loss of mitochondrial membrane potential (MMP) and the release of cytochrome c from the mitochondria to the cytosol. Besides, caspase-9 and caspase-3 were activated while caspase-8 was not affected in HepG2 cells followed by APWP treatment. All these results point clearly to the involvement of mitochondria-mediated signaling pathway in APWP-induced apoptosis and strongly suggest that APWP seems to be safe and effective in the prevention and treatment of HCC.

  7. Peganum harmala L. differentially modulates cytochrome P450 gene expression in human hepatoma HepG2 cells.

    PubMed

    El Gendy, Mohamed A M; El-Kadi, Ayman O S

    2009-12-01

    Peganum harmala L. (Zygophyllaceae) is a common plant in Middle East and it is still used traditionally to treat several diseases. The effect of P. harmala extract on the expression of different cytochrome P450's (CYP) involved in drug metabolism was examined in human HepG2 cells. Therefore, HepG2 cells were incubated with increasing concentrations of plant extract and the CYP gene expression was determined by real-time PCR. Our results showed that P. harmala extract significantly increased the expression of CYP1A2, 2C19, and 3A4 whereas; CYP 2B6, 2D6 and 2E1 was significantly decreased. We concluded that care should be taken when P. harmala is co-administered with other drugs.

  8. 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.

  9. 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

  10. Rosemary Extracts Upregulate Nrf2, Sestrin2, and MRP2 Protein Level in Human Hepatoma HepG2 Cells

    PubMed Central

    Tong, Xiao-pei; Ma, Yan-xia; Quan, Dan-ni; Zhang, Ling

    2017-01-01

    In the past few decades, the incidence of liver cancer has been rapidly rising across the world. Rosemary is known to possess antioxidant activity and is used as natural antioxidant food preservative. It is proposed to have anticancer activity in treating different tumor models. In this study, we try to explore the impact of rosemary extracts on upregulating the level of Nrf2 and Nrf2-regulatory proteins, Sestrin2 and MRP2 in HepG2 cells, and to speculate its potential mechanism. The anticancer activity of rosemary extract, including its polyphenolic diterpenes carnosic acid and carnosol, was evaluated to understand the potential effect on HepG2 cells. Rosemary extract, carnosic acid, and carnosol induced the expression of Sestrin2 and MRP2 associate with enhancement of Nrf2 protein level in HepG2 cells, in which carnosic acid showed most obvious effect. Although the activation pathway of Nrf2/ARE was not exactly assessed, it can be assumed that the enhancement of expression of Sestrin2 and MRP2 may result from upregulation of Nrf2. PMID:28286534

  11. Rosemary Extracts Upregulate Nrf2, Sestrin2, and MRP2 Protein Level in Human Hepatoma HepG2 Cells.

    PubMed

    Tong, Xiao-Pei; Ma, Yan-Xia; Quan, Dan-Ni; Zhang, Ling; Yan, Miao; Fan, Xin-Rong

    2017-01-01

    In the past few decades, the incidence of liver cancer has been rapidly rising across the world. Rosemary is known to possess antioxidant activity and is used as natural antioxidant food preservative. It is proposed to have anticancer activity in treating different tumor models. In this study, we try to explore the impact of rosemary extracts on upregulating the level of Nrf2 and Nrf2-regulatory proteins, Sestrin2 and MRP2 in HepG2 cells, and to speculate its potential mechanism. The anticancer activity of rosemary extract, including its polyphenolic diterpenes carnosic acid and carnosol, was evaluated to understand the potential effect on HepG2 cells. Rosemary extract, carnosic acid, and carnosol induced the expression of Sestrin2 and MRP2 associate with enhancement of Nrf2 protein level in HepG2 cells, in which carnosic acid showed most obvious effect. Although the activation pathway of Nrf2/ARE was not exactly assessed, it can be assumed that the enhancement of expression of Sestrin2 and MRP2 may result from upregulation of Nrf2.

  12. 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

  13. 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.

  14. 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.

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

    PubMed Central

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

    2012-01-01

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

  16. Augmentation of 3-methylcholanthrene-induced bioactivation in the human hepatoma cell line HepG2 by the calcium channel blocker nicardipine.

    PubMed

    Hosaka, Takuomi; Sekimoto, Masashi; Nemoto, Kiyomitsu; Degawa, Masakuni

    2010-03-01

    The abilities of the dihydropyridine calcium channel blocker nicardipine (Nic) to induce cytochrome P450 1 family enzymes (CYP1s) and to enhance the 3-methylcholanthrene (MC)-mediated induction of CYP1s and formation of MC-DNA adduct were examined in the human hepatoma cell line HepG2. The results from real time RT-PCR analysis demonstrated that Nic could induce CYP1 mRNAs and enhance the MC-mediated induction of the CYP1 mRNAs. The luciferase-reporter gene assay using the HepG2-A10 cell line, which has been previously established for the screening of aryl hydrocarbon receptor (AhR) activators, also indicated the augmentation of MC-mediated activation of AhR (induction of luciferase) by Nic, although Nic showed limited capacity for the activation of AhR. Furthermore, the results from the Western blot analysis of CYP1s, the enzyme activity assay, and the assay for MC-DNA adduct formation indicated that Nic could enhance the MC-mediated induction of CYP1s, especially CYP1A1. Furthermore, the intracellular accumulation level of [(3)H]MC after treatment of HepG2 cells with [(3)H]MC significantly increased in the presence of Nic. The present findings demonstrate that Nic can enhance the MC-mediated induction of CYP1s, especially CYP1A1, and the formation of MC-DNA adduct in HepG2 cells. Furthermore, the augmentation of the MC-mediated bioactivation by Nic is demonstrated to occur mainly through an increase in intracellular accumulation of MC.

  17. Epigallocatechin gallate (EGCG) attenuates high glucose-induced insulin signaling blockade in human hepG2 hepatoma cells.

    PubMed

    Lin, Chih-Li; Lin, Jen-Kun

    2008-08-01

    Insulin resistance is the primary characteristic of type 2 diabetes which as a result of insulin signaling defects. It has been suggested that the tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) displays some antidiabetic effects, but the mechanism for EGCG insulin-enhancing effects is incompletely understood. In the present study, the investigations of EGCG on insulin signaling are performed in insulin-responsive human HepG2 cells cotreated with high glucose. We found that the high glucose condition causes significant increasing Ser307 phosphorylation of insulin receptor substrate-1 (IRS-1), leading to reduce insulin-stimulated phosphorylation of Akt. As the results, the insulin metabolic effects of glycogen synthesis and glucose uptake are inhibited by high glucose. However, the treatment of EGCG improves insulin-stimulated downsignaling by reducing IRS-1 Ser307 phosphorylation. Furthermore, we also demonstrated these EGCG effects are essential depends on the 5'-AMP-activated protein kinase (AMPK) activation. Together, our data suggest a putative link between high glucose and insulin resistance in HepG2 cells, and the EGCG treatment attenuates insulin signaling blockade by reducing IRS-1 Ser307 phosphorylation through the AMPK activation pathway.

  18. Synergistic anticancer effect of the extracts from Polyalthia evecta caused apoptosis in human hepatoma (HepG2) cells

    PubMed Central

    Machana, Sasipawan; Weerapreeyakul, Natthida; Barusrux, Sahapat; Thumanu, Kanjana; Tanthanuch, Waraporn

    2012-01-01

    Objective To evaluate the anticancer activity of the extract fraction of Polyalthia evecta (P. evecta) (Pierre) Finet & Gagnep and the synergistic anticancer effect of the extracts from P. evecta by using the ATR/FT-IR spectroscopy. Methods The 50% ethanol-water crude leaf extract of P. evecta (EW-L) was prepared and was further fractionated to isolate various fractions. The anticancer activity was investigated from cytotoxicity against HepG2 using a neutral red assay and apoptosis induction by evaluation of nuclei morphological changes after DAPI staining. Synergistic anticancer effects of the extracts from P. evecta were performed using the ATR/FT-IR spectroscopy. Results The result showed that the EW-L showed higher cytotoxicity and apoptosis induction in HepG2 cells than its fractionated extracts. The hexane extract exhibited higher cytotoxicity and apoptosis induction than the water extracts, but less than the EW-L. The combined water and hexane extracts apparently increased cytotoxicity and apoptosis induction. The %apoptotic cells induced by the extract mixture were increased about 2-fold compared to the single hexane extract. Conclusions The polar extract fraction is necessary for the anticancer activity of the non-polar extract fraction. The ATR/FT-IR spectra illustrates the physical interaction among the constituents in the extract mixture and reveals the presence of polyphenolic constituents in the EW-L, which might play a role for the synergistic anticancer effect. PMID:23569977

  19. Ionic mechanisms of regulatory volume increase (RVI) in the human hepatoma cell-line HepG2.

    PubMed

    Wehner, Frank; Lawonn, Peter; Tinel, Hanna

    2002-03-01

    We studied the effects of hypertonic stress on ion transport and cell volume regulation (regulatory volume increase; RVI) in the human tumor cell-line HepG2. Ion conductances were monitored in intracellular current-clamp measurements with rapid ion-substitutions and in whole-cell patch-clamp recordings; intracellular pH buffering capacity and activation of Na(+)/H(+) antiport were determined fluorometrically; the rates of Na(+)-K(+)-2Cl(-) symport and Na(+)/K(+)-ATPase were quantified on the basis of time-dependent and furosemide- or ouabain-sensitive (86)Rb(+) uptake, respectively; changes in cell volume were recorded by means of confocal laser-scanning microscopy. It was found that hypertonic conditions led to the activation of a cation conductance that was inhibited by Gd(3+), flufenamate as well as amiloride, but not by benzamil or ethyl-isopropyl-amiloride (EIPA). Most likely, this cation conductance was non-selective for Na(+) over K(+). Hypertonic stress did not change K(+) conductance, whereas possible changes in Cl(-) conductance remain ambiguous. The contribution of Na(+)/H(+)antiport to the RVI process appeared to be minor. Under hypertonic conditions an approximately 3.5-fold stimulation of Na(+)-K(+)-2Cl(-)symport was observed but this transporter did not significantly contribute to the overall RVI process. Hypertonic stress did not increase the activity of Na(+)/K(+)-ATPase, which even under isotonic conditions appeared to be working at its limit. It is concluded that the main mechanism in the RVI of HepG2 cells is the activation of a novel non-selective cation conductance. In contrast, there is little if any contribution of K(+) conductance, Na(+)/H(+) antiport, Na(+)-K(+)-2Cl(-) symport, and Na(+)/K(+)-ATPase to this process.

  20. 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.

  1. 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

  2. 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.

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

    SciTech Connect

    Rudzok, S.; Schlink, U.; Herbarth, O.; Bauer, M.

    2010-05-01

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

  4. 3-Nitrobenzanthrone (3-NBA) induced micronucleus formation and DNA damage in human hepatoma (HepG2) cells.

    PubMed

    Lamy, Evelyn; Kassie, Fekadu; Gminski, Richard; Schmeiser, Heinz H; Mersch-Sundermann, Volker

    2004-01-15

    3-Nitrobenzanthrone (3-NBA), identified in diesel exhaust and in airborne particulate matter, is a potent mutagen in Salmonella, induces micronuclei formation in mice and in human cells and DNA adducts in rats. In the present study, we investigated the genotoxic potency of 3-NBA in human HepG2 cells using the micronucleus (MN) assay and the single cell gel electrophoresis (SCGE). 3-NBA caused a genotoxic effect at concentrations > or =12 nM in both assays. In the micronucleus assay, we found 98.7+/-10.3 MN/1000 BNC at a concentration of 100 nM 3-NBA in comparison to 27.3+/-0.6 MN/1000 BNC with the negative control. At the same concentration, the DNA-migration (SCGE) showed an Olive tail moment (OTM) of 2.7+/-0.45 and %DNA in the tail of 8.28+/-0.76; OTM and %DNA in the tail of cells treated with the negative control were 0.73+/-0.08 and 2.81+/-0.30, respectively. The results are discussed under consideration of former studies.

  5. 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.

  6. Vitamin B-6 restriction impairs fatty acid synthesis in cultured human hepatoma (HepG2) cells

    PubMed Central

    Zhao, Mei; Ralat, Maria A.; da Silva, Vanessa; Garrett, Timothy J.; Melnyk, Stephan; James, S. Jill

    2013-01-01

    Vitamin B-6 deficiency has been reported to alter n-6 and n-3 fatty acid profiles in plasma and tissue lipids; however, the mechanisms underlying such metabolic changes remain unclear. The objective of this study was to determine the effects of vitamin B-6 restriction on fatty acid profiles and fatty acid synthesis in HepG2 cells. Cells were cultured for 6 wk in media with four different vitamin B-6 concentrations (10, 20, 50, and 2,000 nM added pyridoxal, representing deficient, marginal, adequate, and supraphysiological conditions) that induced a range of steady-state cellular concentrations of pyridoxal phosphate. Total cellular lipid content was greatest in the deficient (10 nM pyridoxal) medium. The percentage of arachidonic acid and the ratio of arachidonic acid to linoleic acid in the total lipid fraction were ∼15% lower in vitamin B-6-restricted cells, which suggests that vitamin B-6 restriction affects n-6 fatty acid interconversions. Metabolic flux studies indicated significantly lower fractional synthesis rate of oleic acid and arachidonic acid at 10, 20, and 50 nM pyridoxal, whereas that of eicosapentaenoic acid was lower in the cells cultured in 10 nM pyridoxal. Additionally, relative mRNA expressions of Δ5 and Δ6 desaturases were 40–50% lower in vitamin B-6-restricted cells. Overall, these findings suggest that vitamin B-6 restriction alters unsaturated fatty acid synthesis, particularly n-6 and n-3 polyunsaturated fatty acid synthesis. These results and observations of changes in human plasma fatty acid profiles caused by vitamin B-6 restriction suggest a mechanism by which vitamin B-6 inadequacy influences the cardiovascular risk. PMID:23211517

  7. Altered gene expression in human hepatoma HepG2 cells exposed to low-level 2,4-dichlorophenoxyacetic acid and potassium nitrate.

    PubMed

    Bharadwaj, Lalita; Dhami, Karan; Schneberger, David; Stevens, Marianne; Renaud, Condé; Ali, Adnan

    2005-08-01

    2,4-dichlorophenoxyacetic acid (2,4-D) and nitrate are agricultural contaminants found in rural ground water. It is not known whether levels found in groundwater pose a human or environmental health risk, nor is the mechanism of toxicity at the molecular/cellular level understood. This study focused on determining whether 2,4-D or nitrate at environmentally realistic levels elicit gene expression changes in exposed cells. cDNA microarray technology was used to determine the impact of 2,4-D and nitrate in an in vitro model of exposure. Human hepatoma HepG2 cells were incubated with 2,4-D or nitrate alone for 24 h. Cell viability (neutral red assay) and proliferation (BrdU incorporation) were assessed following exposure. Total RNA from treated and control cells were isolated, reverse transcribed and reciprocal labelled with Cy3 or Cy5 dyes, and hybridized to a human cDNA microarray. The hybridized microarray chips were scanned, quantified and analyzed to identify genes affected by 2,4-D or nitrate exposure based on a two-fold increase or decrease in gene expression and reproducibility (affected in three or more treatments). Following filtering, normalization and hierarchical clustering initial data indicate that numerous genes were found to be commonly expressed in at least three or more treatments of 2,4-D or nitrate tested. The affected genes indicate that HepG2 cells respond to environmental, low-level exposure and produce a cellular response that is associated with alterations in the expression of many genes. The affected genes were characterized as stress response, cell cycle control, immunological and DNA repair genes. These findings serve to highlight new pathway(s) in which to further probe the effects of environmental levels of 2,4-D and nitrate.

  8. 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.

  9. 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.

  10. 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.

  11. Bystander effect in human hepatoma HepG2 cells caused by medium transfers at different times after high-LET carbon ion irradiation

    NASA Astrophysics Data System (ADS)

    Wu, Qingfeng; Li, Qiang; Jin, Xiaodong; Liu, Xinguo; Dai, Zhongying

    2011-01-01

    Although radiation-induced bystander effects have been well documented in a variety of biological systems, whether irradiated cells have the ability to generate bystander signaling persistently is still unclear and the clinical relevance of bystander effects in radiotherapy remains to be elucidated. This study examines tumor cellular bystander response to autologous medium from cell culture irradiated with high-linear energy transfer (LET) heavy ions at a therapeutically relevant dose in terms of clonogenic cell survival. In vitro experiments were performed using human hepatoma HepG2 cell line exposed to 100 keV/μm carbon ions at a dose of 2 Gy. Two different periods (2 and 12 h) after irradiation, irradiated cell conditioned medium (ICCM) and replenished fresh medium were harvested and then transferred to unirradiated bystander cells. Cellular bystander responses were measured with the different medium transfer protocols. Significant higher survival fractions of unirradiated cells receiving the media from the irradiated cultures at the different times post-irradiation than those of the control were observed. Even replenishing fresh medium for unirradiated cells which had been exposed to the ICCM for 12 h could not prevent the bystander cells from the increased survival fraction. These results suggest that the irradiated cells could release unidentified signal factor(s), which induced the increase in survival fraction for the unirradiated bystander cells, into the media sustainedly and the carbon ions triggered a cascade of signaling events in the irradiated cells rather than secreting the soluble signal factor(s) just at a short period after irradiation. Based on the observations in this study, the importance of bystander effect in clinical radiotherapy was discussed and incorporating the bystander effect into the current radiobiological models, which are applicable to heavy ion radiotherapy, is needed urgently.

  12. Induction of intercellular adhesion molecule-1 (CD54) on human hepatoma cell line HepG2: influence of cytokines and hepatitis B virus-DNA transfection.

    PubMed Central

    Volpes, R; van den Oord, J J; Desmet, V J; Yap, S H

    1992-01-01

    Human hepatocyte expression of intercellular adhesion molecule-1 (ICAM-1) (CD54) was studied in vitro by exposing the well differentiated human hepatoblastoma cell line HepG2 to various cytokines. In addition, hepatitis B virus (HBV)-DNA transfected HepG2 cells were also analysed. Expression of ICAM-1 on HepG2 cells was then revealed with an immunohistochemical procedure. Untreated HepG2 cells were unreactive, but showed strong cytoplasmic ICAM-1 immunoreactivity after treatment with interferon-gamma (IFN-gamma). This induction was completely inhibited by addition of a neutralizing antibody directed to IFN-gamma. IL-1, IL-6, tumour necrosis factor-alpha (TNF-alpha) and IFN-alpha, used alone or in combination, did not induce ICAM-1 expression, neither did they inhibit the IFN-gamma-induced expression of this adhesion molecule on HepG2 cells. Untreated hepatitis B virus-DNA transfected HepG2 cells expressed membranous ICAM-1. These results indicate that IFN-gamma is the main cytokine trigger for ICAM-1 expression on HepG2 cells, suggesting that in areas of liver inflammation this adhesion molecule is up-regulated on hepatocytes by locally released IFN-gamma. In addition, expression of ICAM-1 by hepatitis B virus-DNA transfected HepG2 cells suggests other, still unknown, triggering mechanisms in the induction of such adhesion molecules, for instance gene activation by viral genome, or autocrine virus-induced hepatocellular cytokine production. Images Fig. 1 Fig. 2 PMID:1346374

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

    PubMed

    Pezdirc, Marko; Žegura, Bojana; Filipič, Metka

    2013-09-01

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

  14. Proteomic profiling revealed the functional networks associated with mitotic catastrophe of HepG2 hepatoma cells induced by 6-bromine-5-hydroxy-4-methoxybenzaldehyde

    SciTech Connect

    Zhang Bo; Huang Bo; Guan Hua; Zhang Shimeng; Xu Qinzhi; He Xingpeng; Liu Xiaodan; Wang Yu; Shang Zengfu; Zhou Pingkun

    2011-05-01

    Mitotic catastrophe, a form of cell death resulting from abnormal mitosis, is a cytotoxic death pathway as well as an appealing mechanistic strategy for the development of anti-cancer drugs. In this study, 6-bromine-5-hydroxy-4-methoxybenzaldehyde was demonstrated to induce DNA double-strand break, multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human hepatoma HepG2 cells. We used proteomic profiling to identify the differentially expressed proteins underlying mitotic catastrophe. A total of 137 differentially expressed proteins (76 upregulated and 61 downregulated proteins) were identified. Some of the changed proteins have previously been associated with mitotic catastrophe, such as DNA-PKcs, FoxM1, RCC1, cyclin E, PLK1-pT210, 14-3-3{sigma} and HSP70. Multiple isoforms of 14-3-3, heat-shock proteins and tubulin were upregulated. Analysis of functional significance revealed that the 14-3-3-mediated signaling network was the most significantly enriched for the differentially expressed proteins. The modulated proteins were found to be involved in macromolecule complex assembly, cell death, cell cycle, chromatin remodeling and DNA repair, tubulin and cytoskeletal organization. These findings revealed the overall molecular events and functional signaling networks associated with spindle disruption and mitotic catastrophe. - Graphical abstract: Display Omitted Research highlights: > 6-bromoisovanillin induced spindle disruption and sustained mitotic arrest, consequently resulted in mitotic catastrophe. > Proteomic profiling identified 137 differentially expressed proteins associated mitotic catastrophe. > The 14-3-3-mediated signaling network was the most significantly enriched for the altered proteins. > The macromolecule complex assembly, cell cycle, chromatin remodeling and DNA repair, tubulin organization were also shown involved in mitotic catastrophe.

  15. Involvement of p38 MAPK and Nrf2 in phenolic acid-induced P-form phenol sulfotransferase expression in human hepatoma HepG2 cells.

    PubMed

    Yeh, Chi-Tai; Yen, Gow-Chin

    2006-05-01

    Phenolic acids have significant biological and pharmacological properties and some have demonstrated remarkable ability to alter sulfate conjugation. However, the modulation mechanisms of phenolic acids on phenol sulfotransferase expression have not been described. In the present study, we investigated the effects of phenolic acids on the expression of the Phase II P-form of phenol sulfotransferase (PST-P) in human hepatoma HepG2 cells. RT-PCR and western blot data revealed that gallic acid induced increase in PST-P expression at the mRNA and protein levels, respectively. This induction was also marked by an increase in PST-P activity. Actinomycin D and cycloheximide inhibited gallic acid-responsive PST-P mRNA expression, indicating that gallic acid is a requirement for transcription and de novo protein synthesis. Transient transfection of HepG2 cells with a reporter plasmid of the upstream region of the human PST gene caused a significant increase in reporter gene activity after gallic acid exposure. Moreover, gallic acid increased the nuclear levels of Nrf2, a transcription factor governing antioxidant response element (ARE). Electrophoretic mobility shift assay showed increased binding of nuclear proteins to ARE consensus sequence after treatment with gallic acid. While investigating the signaling pathways responsible for PST-P induction, we observed that gallic acid activated the p38 mitogen-activated protein kinase (MAPK) pathway. SB203580, a specific inhibitor of p38 MAPK, abolished gallic acid-induced PST-P protein expression. Similarly, gallic acid also caused an accumulation of Nrf2. Moreover, the protective effects of gallic acid on tert-butyl hydroperoxide-induced toxicity was partially blocked by p38 MAPK and PST-P inhibitors, further demonstrating that gallic acid attenuates oxidative stress through a pathway that involves p38 MAPK and PST-P. These results indicate that gallic acid is a potent inducer of PST-P and that PST-P induction is responsible

  16. Targeting the Na(+)/K(+)-ATPase alpha1 subunit of hepatoma HepG2 cell line to induce apoptosis and cell cycle arresting.

    PubMed

    Xu, Zhong-Wei; Wang, Feng-Mei; Gao, Mo-Jie; Chen, Xiao-Yi; Hu, Wen-Liang; Xu, Rui-Cheng

    2010-01-01

    Recent research has shown that the Na(+)/K(+)-ATPase alpha1 subunit is a novel anti-cancer target, which plays pivotal roles in malignant cell ion transport, metabolism, migration and signal transduction. The purpose of the present study was to investigate the anti-cancer effects of ouabain and Na(+)/K(+)-ATPase alpha1 small interfering ribonucleic acid (siRNA) on HepG2 cell proliferation, apoptosis and cell cycle, and to explore the molecular mechanisms. The expression of Na(+)/K(+)-ATPase alpha1 subunit in human hepatocellular carcinoma (HCC), normal liver tissues and human HCC line (HepG2, SMMC-7721 and Bel-7402) has been investigated. Using the ouabain and Na(+)/K(+)-ATPase alpha1 subunit siRNA, which target the Na(+)/K(+)-ATPase, we have evaluated the effects of inhibiting Na(+)/K(+)-ATPase alpha1 in human HepG2 cells with respect to cell proliferation, morphology, cell cycle, impact on intracellular Ca2++, reactive oxygen species (ROS) concentration, and correlated gene expression level on messenger ribonucleic acid (mRNA) and protein. Our data showed that the expression Na(+)/K(+)-ATPase alpha1 subunit in HCC tissues is higher than that in normal liver tissues. Ouabain and Na(+)/K(+)-ATPase alpha1 siRNA could inhibit HepG2 cell proliferation. Ouabain could induce HepG2 cell apoptosis and generate S phase arrest, and siRNA could enhance the anti-cancer effect of ouabain that induced HepG2 cells apoptosis via an intracellular Ca(2+) and ROS increase-mediated, and generated cell cycle S phase arresting by decreasing the CyclinA1/cyclin-dependent kinase 2 (CDK2)/proliferating cell nuclear antigen (PCNA) complex product and increasing the expression of cyclin-dependent kinase inhibitor 1A (P21(CIP1)). We believe that targeting of the Na(+)/K(+)-ATPase alpha1 subunit in human HCC cells could provide new sight into the treatment of HCC.

  17. Effects of phenyl saligenin phosphate on cell viability and transglutaminase activity in N2a neuroblastoma and HepG2 hepatoma cell lines.

    PubMed

    Harris, W; Muñoz, D; Bonner, P L R; Hargreaves, A J

    2009-12-01

    The main aim of this study was to determine whether sub-lethal concentrations of the organophosphate compound phenyl saligenin phosphate (PSP) could disrupt the activity of the Ca(2+)-activated enzyme tissue transglutaminase (TGase 2) from cultured cell lines of neuronal (N2a) and hepatic (HepG2) origin. The results indicated that PSP added directly to cytosol extracts from healthy cells was able to inhibit TGase 2 activity by 40-60% of control levels at sub-lethal concentrations (0.1 microM) that were approximately 100-fold lower than their IC(50) values in cytotoxicity assays. Following 24h exposure of N2a cells to 0.3 and 3 microM PSP in situ, a similar reduction in activity was observed in subsequent assays of TGase 2 activity. However, significantly increased activity was observed following in situ exposure of HepG2 cells to PSP (ca. 4-fold at 3 microM). Western blotting analysis indicated slightly reduced levels of TGase 2 in N2a cells compared to the control, whereas an increase was observed in the level of TGase 2 in HepG2 cells. We suggest that TGase 2 represents a potential target of organophosphate toxicity and that its response may vary in different cellular environments, possibly affected by its expression pattern.

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

    PubMed

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

    2016-05-01

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

  19. Apelin-13 induces autophagy in hepatoma HepG2 cells through ERK1/2 signaling pathway-dependent upregulation of Beclin1.

    PubMed

    Huang, Qiulin; Liu, Xuan; Cao, Chao; Lei, Junyue; Han, Dong; Chen, Guodong; Yu, Jia; Chen, Linxi; Lv, Deguan; Li, Zhongyu

    2016-02-01

    The aim of the present study was to investigate the effect of Apelin-13 on autophagy in hepatocellular carcinoma HepG2 cells and the underlying mechanism of the effect. The HepG2 cells were treated with Apelin-13 at a final concentration of 0.0001, 0.001, 0.01 and 0.1 µmol/l for 24 h. Cells were also treated with 10 µmol/l PD98059 for 24 h. The expression of the extracellular signal-regulated kinase (ERK)1/2, phosphorylated ERK1/2 (pERK1/2) and Beclin1 proteins were detected by western blot analysis. Beclin1 mRNA expression was also detected by reverse transcription-polymerase chain reaction. Autophagy was observed using fluorescence microscopy subsequent to monodansylcadaverine (MDC) staining. Following treatment with the various concentrations of Apelin-13, the expression of the ERK1/2 protein remained at a similar level, whereas the expression of pERK1/2 increased in a dose-dependent manner. Compared with the control group, the increase was significant (P<0.05). Similarly, Beclin1 expression was upregulated at the protein and mRNA levels by Apelin-13 treatment in a dose-dependent manner and was significantly increased compared with the control group. However, following treatment with the Apelin-13 inhibitor PD98059, the expression of pERK1/2, Beclin1 protein and Beclin1 mRNA were significantly decreased (P<0.05). In addition, Apelin-13 induced the autophagy of HepG2 cells in a dose-dependent manner, as revealed by MDC staining. PD98059 inhibited autophagy of HepG2 cells induced by Apelin-13. Therefore, Apelin-13 may promote autophagy in HepG2 cells by inducing the phosphorylation of ERK1/2 and upregulating the expression of Beclin1.

  20. Induction of apoptosis by pistachio (Pistacia vera L.) hull extract and its molecular mechanisms of action in human hepatoma cell line HepG2.

    PubMed

    Fathalizadeh, J; Bagheri, V; Khorramdelazad, H; Kazemi Arababadi, M; Jafarzadeh, A; Mirzaei, M R; Shamsizadeh, A; Hajizadeh, M R

    2015-11-30

    Several important Pistacia species such as P. vera have been traditionally used for treating a wide range of diseases (for instance, liver-related disorders). There is a relative lack of research into pharmacological aspects of pistachio hull. Hence, this study was aimed at investigating whether pistachio rosy hull (PRH) extract exerts apoptotic impacts on HepG2 liver cancer cell line. In order to evaluate cell viability and apoptosis in response to treatment with the extract, MTT assay and Annexin-V-fluorescein/propidium iodide (PI) double staining were performed, respectively. Moreover, molecular mechanism of apoptosis induced by the extract was determined using human apoptosis PCR array. Our findings showed that PRH extract treatment reduced cell viability (IC50 ~ 0.3 mg/ml) in a dose-dependent manner. Flow cytometric analysis revealed that the extract significantly induced apoptosis in HepG2 cells. In addition, quantitative PCR array results demonstrated the regulation of a considerable number of apoptosis-related genes belonging to the TNF, BCL2, IAP, TRAF, and caspase families. We observed altered expression of both pro-apoptotic and anti-apoptotic genes associated with the extrinsic and intrinsic apoptosis signaling pathways. These results suggest that the aqueous extract of PRH possesses apoptotic activity through cytotoxic and apoptosis-inducing effects on HepG2 cells.

  1. Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2).

    PubMed

    Alía, Mario; Ramos, Sonia; Mateos, Raquel; Bravo, Laura; Goya, Luis

    2005-01-01

    The aim of this work was to investigate the response of the antioxidant defense system to two oxidative stressors, hydrogen peroxide and tert-butyl hydroperoxide, in HepG2 cells in culture. The parameters evaluated included enzyme activity and gene expression of superoxide dismutase, catalase, glutathione peroxidase, and activity of glutathione reductase. Besides, markers of the cell damage and oxidative stress evoked by the stressors such as cell viability, intracellular reactive oxygen species generation, malondialdehyde levels, and reduced glutathione concentration were evaluated. Both stressors, hydrogen peroxide and tert-butyl hydroperoxide, enhanced cell damage and reactive oxygen species generation at doses above 50 microM. The concentration of reduced glutathione decreased, and levels of malondialdehyde and activity of the antioxidant enzymes consistently increased only when HepG2 cells were treated with tert-butyl hydroperoxide but not when hydrogen peroxide was used. A slight increase in the gene expression of Cu/Zn superoxide dismutase and catalase with 500 microM tert-butyl hydroperoxide and of catalase with 200 microM hydrogen peroxide was observed. The response of the components of the antioxidant defense system evaluated in this study indicates that tert-butyl hydroperoxide evokes a consistent cellular stress in HepG2.

  2. Pharmacological induction of leukotriene B4-12-hydroxydehydrogenase suppresses the oncogenic transformation of human hepatoma HepG2 cells.

    PubMed

    Wei, Lai; Liu, Jie; Le, X Chris; Han, Yifan; Tong, Yao; Lau, Allan S Y; Rong, Jianhui

    2011-09-01

    Leukotriene B4-12-hydroxydehydrogenase (LTB4DH) is characterized as a chemopreventive and tumor suppressor gene. The aim of this study was to investigate the pharmaco-logical induction of LTB4DH and potential anticancer activity. Using HepG2 cells as a cellular detector, we successfully isolated the active compounds from the herbs Radix Astragali and Radix Paeoniae Rubra through a bioactivity-guided fractionation procedure. Using various analytical techniques including electronic spray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR), gallic acid (GA) was identified as the active compound from Radix Paeoniae Rubra whereas the active compound from Radix Astragali, designated as RA-C, was also purified to the extent that it is now suitable for further identifi-cation. We found that the active compounds from these two different herbs synergistically induced LTB4DH expression in a dose- and time-dependent manner. A key finding was that commercial GA in combination with purified RA-C attenuated the focus formation and anchorage-independent growth, two indexes of in vitro oncogenic transformation, of HepG2 cells via the induction of LTB4DH expression. Moreover, the combination of GA and purified RA-C significantly induced G2/M cell cycle arrest in HepG2 cells. Our results demon-strated for the first time that GA and purified RA-C suppress the in vitro oncogenic transformation of HepG2 cells via the induction of LTB4DH expression. Importantly, pharmaco-logical induction of LTB4DH represents a potential alternative strategy for the therapy of hepatocellular carcinoma.

  3. Quercetin induces apoptosis via caspase activation, regulation of Bcl-2, and inhibition of PI-3-kinase/Akt and ERK pathways in a human hepatoma cell line (HepG2).

    PubMed

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

    2006-11-01

    Dietary polyphenols have been associated with the reduced risk of chronic diseases such as cancer, but the precise underlying mechanism of protection remains unclear. The aim of this study was to investigate the effect of quercetin on the activation of the apoptotic pathway in a human hepatoma cell line (HepG2). Treatment of cells for 18 h with quercetin induced cell death in a dose-dependent manner; however, a shorter treatment (4 h) had no effect on cell viability. Incubation of HepG2 cells with quercetin for 18 h induced apoptosis by the activation of caspase-3 and -9, but not caspase-8. Moreover, this flavonoid decreased the Bcl-xL:Bcl-xS ratio and increased translocation of Bax to the mitochondrial membrane. A sustained inhibition of the major survival signals, Akt and extracellular regulated kinase (ERK), also occurred in quercetin-treated cells. These data suggest that quercetin may induce apoptosis by direct activation of caspase cascade (mitochondrial pathway) and by inhibiting survival signaling in HepG2.

  4. Long chain acyl-CoA synthetase-3 is a molecular target for peroxisome proliferator-activated receptor delta in HepG2 hepatoma cells.

    PubMed

    Cao, Aiqin; Li, Hai; Zhou, Yue; Wu, Minhao; Liu, Jingwen

    2010-05-28

    ACSL3 is a member of the long chain acyl-CoA synthetase (ACSL) family that plays key roles in fatty acid metabolism in various tissues in an isozyme-specific manner. Our previous studies showed that ACSL3 was transcriptionally up-regulated by the cytokine oncostatin M (OSM) in HepG2 cells, accompanied by reduced cellular triglyceride content and enhanced beta-oxidation. In this study, we investigated the molecular mechanism underlying the OSM-induced activation of ACSL3 gene transcription in HepG2 cells. We showed that OSM treatment resulted in a coordinated elevation of mRNA levels of ACSL3 and peroxisome proliferator-activated receptor delta (PPARdelta). The effect of OSM on ACSL3 mRNA expression was inhibited by cellular depletion of PPARdelta. By utilizing a PPARdelta agonist, L165041, we demonstrated that activation of PPARdelta led to increases in ACSL3 promoter activity, mRNA level, and protein level in HepG2 cells. Analysis of the ACSL3 promoter sequence identified two imperfect PPAR-responsive elements (PPRE) located in the ACSL3 promoter region -944 to -915, relative to the transcription start site. The up-regulation of ACSL3 promoter activity by PPARdelta was abolished by deletion of this PPRE-containing region or mutation to disrupt the binding sites. Direct interactions of PPARdelta with ACSL3-PPRE sequences were demonstrated by gel mobility shift and chromatin immunoprecipitation assays. Finally, we provided in vivo evidence showing that activation of PPARdelta by L165041 in hamsters increased ACSL3 mRNA and protein levels in the liver. These new findings define ACSL3 as a novel molecular target of PPARdelta in HepG2 cells and provide a regulatory mechanism for ACSL3 transcription in liver tissue.

  5. Phenolic-containing organic extracts of mulberry (Morus alba L.) leaves inhibit HepG2 hepatoma cells through G2/M phase arrest, induction of apoptosis, and inhibition of topoisomerase IIα activity.

    PubMed

    Naowaratwattana, Wanlaya; De-Eknamkul, Wanchai; De Mejia, Elvira Gonzalez

    2010-10-01

    The entire plant of Morus alba L. (Family Moraceae), or mulberry, possesses medical benefits, including anticancer properties. In this study, we investigated the effect of mulberry leaf extracts on the human hepatoma HepG2 cell line, which is related to hepatocellular carcinoma. Mulberry leaf extracts were prepared using four solvents, each with different polarities: 100% methanol (MeOH), 50% aqueous MeOH, 1-butanol (BuOH), and hot water (W). The phenolic profile, total polyphenol content, antioxidant capacity, and effect on human hepatoma HepG2 cells of the leaf extracts were analyzed by examining cytotoxicity, cell cycle progression, apoptosis, expression of topoisomerase IIα, and proteins involved in cell cycle progression. High-performance liquid chromatography-mass spectrometry analysis revealed that 100% MeOH, 50% MeOH, and BuOH extracts contained rutin, isoquercetin, and various derivatives of kaempferol and quercetin glycosides as their major constituents; the W extract contained primarily chlorogenic acid and caffeoylquinic acid derivatives. Total phenolic content based on rutin equivalents was 17.1%, 9.6%, 8.3%, and 6.5% of dry 100% MeOH, 50% MeOH, BuOH, and W extracts, respectively. 2,2-Diphenyl-1-picrylhydrazyl radical scavenging activities were 70.0%, 45.8%, 41.0%, and 33.6%, and 50% inhibitory concentration values were 33.1, 79.4, 35.6, and 204.2 μg/mL for HepG2 cell proliferation inhibition for 100% MeOH, 50% MeOH, BuOH, and W extracts, respectively. MeOH extracts caused cell cycle G2/M arrest and induced the caspase cascade and apoptosis, but the W extract had very little effect on cell cycle progression. MeOH extracts reduced the level of topoisomerase IIα but increased the level of p27(Kip1), with no significant effect on p21(Cip1/waf1). Therefore, we concluded that phenolic-containing organic extracts of mulberry leaves inhibit the growth of HepG2 hepatoma cells through coordinated actions of inducing cell cycle arrest in the G2/M phase (with

  6. Uridine uptake inhibition as a cytotoxicity test for a human hepatoma cell line (HepG2 cells): comparison with the neutral red assay.

    PubMed

    Valentin, I; Philippe, M; Lhuguenot, J; Chagnon, M

    2001-02-14

    This study describes a sensitive microassay for measuring cytotoxicity based on the degree of inhibition of RNA synthesis in HepG2 cells. RNA synthesis is measured by the kinetic uptake of radiolabeled uridine. A large number of compounds were tested in a wide range of concentrations. The concentration required to induce 50% inhibition of HepG2 uridine uptake rates (IC(50)) was determined for each compound and used to rank its potency. These IC(50)s were compared with IC(50)s measured with the neutral red assay. 2-acetylaminofluorene, benzo[a]pyrene and methylnitrosourea were not cytotoxic in the neutral red assay. Uridine uptake was always inhibited at lower concentrations than those required in the neutral red assay, suggesting that the uridine uptake assay is a more sensitive indicator of toxic action than the neutral red inclusion. Uridine uptake assay provides a rapid and quantitative method for assessing toxicity in a human cell line. Application of this method to bottled spring waters are described. Due to its high sensitivity and reproducibility, this method provides a suitable tool for screening a great number of samples and will be a helpful test for evaluating food safety and controlling the recycling process of wrapping materials.

  7. The growth inhibitory effect of conjugated linoleic acid on a human hepatoma cell line, HepG2, is induced by a change in fatty acid metabolism, but not the facilitation of lipid peroxidation in the cells.

    PubMed

    Igarashi, M; Miyazawa, T

    2001-02-26

    We investigated the growth inhibitory effect of conjugated linoleic acid (CLA) on HepG2 (human hepatoma cell line), exploring whether the inhibitory action occurs via lipid peroxidation in the cells. When the cells were incubated up to 72 h with 5-40 microM of CLA (a mixture of 9c,11t-18:2 and 10t,12c-18:2), cell proliferation was clearly inhibited in a dose and time dependent manner but such an inhibition was not confirmed with linoleic acid (LA). In order to evaluate the possible contribution of lipid peroxidation exerted by CLA to cell growth inhibition, alpha-tocopherol (5-20 microM) and BHT (1-10 microM) as potent antioxidants were added to the medium with CLA (20 microM), which did not restore cell growth at all. Furthermore, after 72 h incubation, the membranous phospholipid hydroperoxide formation in the CLA-supplemented cells was suppressed respectively to 25% and 50% of that in LA-supplemented cells and control cells. No difference was observed by a conventional lipid peroxide assay, the TBA test, between CLA-supplemented cells and LA-supplemented cells. Although the cellular lipid peroxidation was not stimulated, lipid contents (triacylglycerol, total cholesterol and free cholesterol) and fatty acid contents (palmitic acid, palmitoleic acid and stearic acid) markedly increased in CLA-supplemented cells compared with LA-supplemented and control cells. Moreover, supplementation with 20 microM LA and 20 microM arachidonic acid profoundly interfered with the inhibitory effect of CLA in HepG2. These results suggest that the growth inhibitory effect of CLA on HepG2 is due to changes in fatty acid metabolism but not to lipid peroxidation.

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

    SciTech Connect

    Subramaniam, Sudhakar R.; Ellis, Elizabeth M.

    2011-01-15

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

  9. On-line comprehensive two-dimensional HepG2 cell membrane chromatographic analysis system for charactering anti-hepatoma components from rat serum after oral administration of Radix scutellariae: A strategy for rapid screening active compounds in vivo.

    PubMed

    Jia, Dan; Chen, Xiaofei; Cao, Yan; Wu, Xunxun; Ding, Xuan; Zhang, Hai; Zhang, Chuan; Chai, Yifeng; Zhu, Zhenyu

    2016-01-25

    Cell membrane chromatography (CMC) is a bioaffinity chromatography technique for characterizing interactions between drugs and membrane receptors and has been widely used to screen active components from complex samples such as herbal medicines (HMs). However, it has never been applied in vivo due to its relatively high limit of detection (LOD) and the matrix interferences. In this study, a novel on-line comprehensive two-dimensional HepG2/CMC/enrich columns/high performance liquid chromatography/time-of-flight mass spectrometry system was developed to rapidly screen potential anti-hepatoma components from drug-containing serum of rats after oral administration of Radix scutellariae. A matrix interference deduction method with a home-written program in MATLAB was developed, which could successfully eliminate the interference of endogenous substances in serum. Baicalein, wogonin, chrysin, oroxylin A, neobaicalein and rivularin from Radix scutellariae extraction were significantly retained in the HepG2/CMC column. Three potential active components, wogonin, oroxylin A and neobaicalein were firstly screened from the drug-containing serum as well. The cell counting kit-8 assay demonstrated that wogonin, oroxylin A and chrysin showed high inhibitory activities in a dose-dependent manner on HepG2 cells at the concentration of 12.5-200 μM (p<0.05) and the IC50 values were 69.83, 16.66 and 51.6 μM, respectively. Wogonin and oroxylin A, which were screened both from Radix scutellariae extraction and the drug-containing serum, could be selected as lead compounds to obtain good anti-hepatoma effects. The proposed comprehensive 2D CMC system and matrix interference elimination strategy have significant advantages for in vivo screening of active components from complex biological samples and could be applied to other biochromatography models.

  10. Interleukin-18 Down-Regulates Multidrug Resistance-Associated Protein 2 Expression through Farnesoid X Receptor Associated with Nuclear Factor Kappa B and Yin Yang 1 in Human Hepatoma HepG2 Cells.

    PubMed

    Liu, Xiao-cong; Lian, Wei; Zhang, Liang-jun; Feng, Xin-chan; Gao, Yu; Li, Shao-xue; Liu, Chang; Cheng, Ying; Yang, Long; Wang, Xiao-Juan; Chen, Lei; Wang, Rong-quan; Chai, Jin; Chen, Wen-sheng

    2015-01-01

    Multidrug resistance-associated protein 2 (MRP2) plays an important role in bile acid metabolism by transporting toxic organic anion conjugates, including conjugated bilirubin, glutathione, sulfate, and multifarious drugs. MRP2 expression is reduced in cholestatic patients and rodents. However, the molecular mechanism of MRP2 down-regulation remains elusive. In this report, we treated human hepatoma HepG2 cells with interleukin-18 (IL-18) and measured the expression of MRP2, nuclear factor kappa B (NF-κB), farnesoid X receptor (FXR), and the transcription factor Yin Yang 1 (YY1) by quantitative real-time quantitative polymerase chain reaction (PCR) and western blotting. We found that expression of MRP2 was repressed by IL-18 at both the mRNA and protein levels in a dose- and time-dependent manner. Furthermore, the activated NF-κB pathway increased YY1 and reduced FXR. These changes were all attenuated in HepG2 cells with knockdown of the NF-κB subunit, p65. The reduced expression of FXR and MRP2 in HepG2 cells that had been caused by IL-18 treatment was also attenuated by YY1 knockdown. We further observed significantly elevated IL-18, NF-κB, and YY1 expression and decreased FXR and MRP2 expression in bile duct-ligated Sprague Dawley rat livers. Chromatin immunoprecipitation assays also showed that FXR bound to the promoter region in MRP2 was less abundant in liver extracts from bile duct-ligated rats than sham-operated rats. Our findings indicate that IL-18 down-regulates MRP2 expression through the nuclear receptor FXR in HepG2 cells, and may be mediated by NF-κB and YY1.

  11. Induction of cytochromes P450 1A1 and 1A2 by tanshinones in human HepG2 hepatoma cell line

    SciTech Connect

    Zhang Rong; Sun Jianguo; Ma Liping; Wu Xiaolan; Pan Guoyu; Hao Haiping; Zhou Fang; Jiye, A; Liu Changhui; Ai Hua; Shang Lili; Gao Haiyan; Peng Ying; Wan Ping; Wu Hui; Wang Guangji

    2011-04-01

    Diterpenoid tanshinones including tanshinone IIA (TIIA), cryptotanshinone (CTS), tanshinone I (TI) and dihydrotanshinone I (DHTI) are the major bioactive components from Danshen. The major aim of our present study was to investigate the induction potential of these four main components of tanshinones (TIIA, CTS, TI, and DHTI) on the expression of CYP1A1 and CYP1A2 in HepG2 cells. Our results showed that all of these four tanshinones caused a significant time- and concentration-dependent increase in the amount of CYP1A1/2 expression in HepG2 cells. These induction effects were further characterized through transcriptional regulation: the induction of CYP1A1/2 mRNA level by tanshinones was completely blocked by the transcription inhibitor actinomycin D; the expression of CYP1A1/2 heterogeneous nuclear RNA was induced by tanshinone treatment; and CYP1A1 mRNA stability was not influenced by these tanshinones. Interestingly, tanshinones plus B[a]P produced additive/synergistic effect on CYP1A1/2 induction. In addition, the tanshinone-induced CYP1A1/2 expression was abolished by the aryl hydrocarbon receptor (AhR) antagonist resveratrol, suggesting an AhR dependent transcription mechanism. In the reporter gene assay, while TI and DHTI significantly induced AhR-dependent luciferase activity, TIIA and CTS failed to induce this activity. Collectively, the tanshinones could induce CYP1A1 and CYP1A2 expression through transcriptional activation mechanism and exert differential effects on activating AhR in HepG2 cells. Our findings suggest that rational administration of tanshinones should be considered with respect to their effect on AhR and CYP1A1/2 expression.

  12. Hexachlorobenzene induces cell proliferation, and aryl hydrocarbon receptor expression (AhR) in rat liver preneoplastic foci, and in the human hepatoma cell line HepG2. AhR is a mediator of ERK1/2 signaling, and cell cycle regulation in HCB-treated HepG2 cells.

    PubMed

    de Tomaso Portaz, Ana Clara; Caimi, Giselle Romero; Sánchez, Marcela; Chiappini, Florencia; Randi, Andrea S; Kleiman de Pisarev, Diana L; Alvarez, Laura

    2015-10-02

    Hexachlorobenzene (HCB) is a widespread environmental pollutant, and a liver tumor promoter in rodents. Depending on the particular cell lines studied, exposure to these compounds may lead to cell proliferation, terminal differentiation, or apoptosis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is involved in drug and xenobiotic metabolism. AhR can also modulate a variety of cellular and physiological processes that can affect cell proliferation and cell fate determination. The mechanisms by which AhR ligands, both exogenous and endogenous, affect these processes involve multiple interactions between AhR and other signaling pathways. In the present study, we examined the effect of HCB on cell proliferation and AhR expression, using an initiation-promotion hepatocarcinogenesis protocol in rat liver and in the human-derived hepatoma cell line, HepG2. Female Wistar rats were initiated with a single dose of 100 mg/kg of diethylnitrosamine (DEN) at the start of the experiment. Two weeks later, daily dosing of 100 mg/kg HCB was maintained for 10 weeks. Partial hepatectomy was performed 3 weeks after initiation. The number and area of glutathione S-transferase-P (GST-P)-positive foci, in the rat liver were used as biomarkers of liver precancerous lesions. Immunohistochemical staining showed an increase in proliferating cell nuclear antigen (PCNA)-positive cells, along with enhanced AhR protein expression in hepatocytes within GST-P-positive foci of (DEN HCB) group, when compared to DEN. In a similar manner, Western blot analysis demonstrated that HCB induced PCNA and AhR protein expression in HepG2 cells. Flow cytometry assay indicated that the cells were accumulated at S and G2/M phases of the cell cycle. HCB increased cyclin D1 protein levels and ERK1/2 phosphorylation in a dose-dependent manner. Treatment of cells with a selective MEK1 inhibitor, prevented HCB-stimulatory effect on PCNA and cyclinD1, indicating that these effects

  13. The activation of the Nrf2/ARE pathway in HepG2 hepatoma cells by phytochemicals and subsequent modulation of phase II and antioxidant enzyme expression.

    PubMed

    Krajka-Kuźniak, Violetta; Paluszczak, Jarosław; Szaefer, Hanna; Baer-Dubowska, Wanda

    2015-06-01

    Previous studies have shown that naturally occurring phytochemicals, indole-3-carbinol, phenethyl isothiocyanate, protocatechuic acid, and tannic acid increased the activity and protein level of hepatic phase II enzymes in animal models. In order to further explore the mechanism of this activity, we investigated the effect of these compounds on the activation of nuclear factor erythroid-2-related factor 2 (Nrf2)-regulated transcription in human hepatocellular carcinoma HepG2 cells. Treatment with all the tested compounds resulted in the translocation from the cytosol and nuclear accumulation of active phosphorylated Nrf2. Furthermore, phenethyl isothiocyanate and indole-3-carbinol increased the transcript and protein levels of GSTA, GSTP, GSTM, GSTT, and NQO1. On the other hand, protocatechuic and tannic acids enhanced only the expression of GSTA, GSTM, and GSTT. The expression of genes encoding antioxidant enzymes CAT, SOD, GR, and GPx was increased after the treatment with all the tested phytochemicals. These results indicate that isothiocyanates/indoles and protocatechuic and tannic acids induce phase II and antioxidant gene expression in HepG2 cells through the Nrf2-Keap1-ARE signaling pathway. Moreover, the results of this study confirmed that the degradation products of glucosinolates are more effective inducers of phase II and antioxidant enzymes than protocatechuic and tannic acids.

  14. The interleukin-6-type cytokine oncostatin M induces aryl hydrocarbon receptor expression in a STAT3-dependent manner in human HepG2 hepatoma cells.

    PubMed

    Stobbe-Maicherski, Natalie; Wolff, Sandra; Wolff, Christian; Abel, Josef; Sydlik, Ulrich; Frauenstein, Katrin; Haarmann-Stemmann, Thomas

    2013-12-01

    The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates the toxicity of dioxins, polycyclic aromatic hydrocarbons and related environmental pollutants. Besides drug metabolism, several studies have provided evidence that the AHR and its downstream targets trigger important developmental, physiological and pathophysiological processes. However, in contrast to the molecular mechanisms of AHR-dependent signaling pathways, the transcriptional regulation of the AHR gene itself is as yet only marginally understood. We found that the pleiotropic interleukin (IL)-6-type cytokine oncostatin M (OSM) is an inducer of AHR mRNA and protein expression in human HepG2 hepatocarcinoma cells. Analyses of the human AHR promoter revealed the existence of a putative signal transducer and activator of transcription (STAT)-binding element 5'-upstream of the transcription start site. By means of site-directed mutagenesis, inhibitor experiments and electrophoretic mobility shift assays, we demonstrated that this STAT motif is recognized by STAT3 to regulate basal and cytokine-inducible AHR expression in HepG2 cells. The identification of the AHR as a downstream target of IL-6-type cytokine-stimulated STAT3 signaling may contribute to a better understanding of the multiple facets of AHR during development, physiology and disease. © 2013 FEBS.

  15. 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.

  16. Insulin-Mediated Downregulation of Apolipoprotein A-I Gene in Human Hepatoma Cell Line HepG2: The Role of Interaction Between FOXO1 and LXRβ Transcription Factors.

    PubMed

    Shavva, Vladimir S; Bogomolova, Alexandra M; Nikitin, Artemy A; Dizhe, Ella B; Tanyanskiy, Dmitry A; Efremov, Alexander M; Oleinikova, Galina N; Perevozchikov, Andrej P; Orlov, Sergey V

    2017-02-01

    Apolipoprotein A-I (ApoA-I) is a key component of high density lipoproteins which possess anti-atherosclerotic and anti-inflammatory properties. Insulin is a crucial mediator of the glucose and lipid metabolism that has been implicated in atherosclerotic and inflammatory processes. Important mediators of insulin signaling such as Liver X Receptors (LXRs) and Forkhead Box A2 (FOXA2) are known to regulate apoA-I expression in liver. Forkhead Box O1 (FOXO1) is a well-known target of insulin signaling and a key mediator of oxidative stress response. Low doses of insulin were shown to activate apoA-I expression in human hepatoma HepG2 cells. However, the detailed mechanisms for these processes are still unknown. We studied the possible involvement of FOXO1, FOXA2, LXRα, and LXRβ transcription factors in the insulin-mediated regulation of apoA-I expression. Treatment of HepG2 cells with high doses of insulin (48 h, 100 nM) suppresses apoA-I gene expression. siRNAs against FOXO1, FOXA2, LXRβ, or LXRα abrogated this effect. FOXO1 forms a complex with LXRβ and insulin treatment impairs FOXO1/LXRβ complex binding to hepatic enhancer and triggers its nuclear export. Insulin as well as LXR ligand TO901317 enhance the interaction between FOXA2, LXRα, and hepatic enhancer. These data suggest that high doses of insulin downregulate apoA-I gene expression in HepG2 cells through redistribution of FOXO1/LXRβ complex, FOXA2, and LXRα on hepatic enhancer of apoA-I gene. J. Cell. Biochem. 118: 382-396, 2017. © 2016 Wiley Periodicals, Inc.

  17. Human hepatoblastoma cells (HepG2) and rat hepatoma cells are defective in important enzyme activities in the oxidation of the C27 steroid side chain in bile acid formation.

    PubMed

    Farrants, A K; Nilsson, A; Pedersen, J I

    1993-12-01

    We have examined the ability of HepG2 human hepatoblastoma cells and 7800 C1 Morris rat hepatoma cells to convert 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) to cholic acid and chenodeoxycholic acid, respectively. Cell extracts from both these cell lines could neither form cholic acid from THCA nor from the activated form, THCA-CoA. This suggests that both cell lines are defective in two enzyme activities involved in the pathway, the microsomal THCA-CoA ligase and the peroxisomal THCA-CoA oxidase. Furthermore, we show that the subsequent enzymes are active in the conversion to bile acids, because the product of the THCA-CoA oxidase, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-24-enoyl-coenzyme A (delta 24-THCA-CoA) or delta 24-THCA in the presence of THCA-CoA ligase, are converted to cholic acid by both cell lines. HepG2 cells were able to slowly form chenodeoxycholic acid and cholic acid from 5 beta-cholestane-3 alpha, 7 alpha-diol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, respectively, in 24- and 96-h incubations. The rate of cholic acid formation was lower than the rate for chenodeoxycholic acid and there was a clear accumulation of THCA. 7800 C1 Morris cells had no ability to form cholic acid or chenodeoxycholic acid after 96 h incubation. We conclude that these two cell lines have defects in two enzyme activities involved in the peroxisomal oxidation in bile acid formation, the microsomal THCA-CoA ligase and the peroxisomal THCA-CoA oxidase.

  18. Pu-erh tea supplementation suppresses fatty acid synthase expression in the rat liver through downregulating Akt and JNK signalings as demonstrated in human hepatoma HepG2 cells.

    PubMed

    Chiang, Chun-Te; Weng, Meng-Shih; Lin-Shiau, Shoei-Yn; Kuo, Kuan-Li; Tsai, Yao-Jen; Lin, Jen-Kun

    2005-01-01

    Fatty acid synthase (FAS) is a key enzyme of lipogenesis. Overexpression of FAS is dominant in cancer cells and proliferative tissues. The expression of FAS in the livers of rats fed pu-erh tea leaves was significantly suppressed. The gains in body weight, levels of triacylglycerol, and total cholesterol were also suppressed in the tea-treated rats. FAS expression in hepatoma HepG2 cells was suppressed by the extracts of pu-erh tea at both the protein and mRNA levels. FAS expression in HepG2 cells was strongly inhibited by PI3K inhibitor LY294002 and JNK inhibitor II and slightly inhibited by p38 inhibitor SB203580 and MEK inhibitor PD98059, separately. Based on these findings, we suggest that the suppression of FAS in the livers of rats fed pu-erh tea leaves may occur through downregulation of the PI3K/AKt and JNK signaling pathways. The major components of tea that have been demonstrated to be responsible for the antiobesity and hypolipidemic effects are catechins, caffeine, and theanine. The compositions of catechins, caffeine, and theanine varied dramatically in pu-erh, black, oolong, and green teas. The active principles and molecular mechanisms that exerted these biological effects in pu-erh tea deserve future exploration.

  19. The effect of WSEWS pentapeptide and WSEWS-specific monoclonal antibodies on constitutive and IL-6 induced acute-phase protein production by a human hepatoma cell line, HEPG-2.

    PubMed

    Biró, J; Bösze, S; Hudecz, F; Nagy, Z; Rajnavölgyi, E; Schmidt, B; Rákász, E; Falus, A

    1995-05-01

    Interleukin-6 receptor (IL-6R) is a member of the cytokine receptor superfamily characterised by the obligatory presence of WSXWS (Trp-Ser-X-Trp-Ser) sequence motif near the transmembrane domain. To more clearly understand the role of this motif, we treated the HepG2 hepatoma cell line with synthetic WSEWS peptide (E is glutamic acid) and checked the spontaneous and IL-6-induced production of acute-phase protein fibrinogen and C1-inhibitor (C1-INH). The peptide revealed a definitely stimulatory effect both on the constitutive synthesis of C1-INH and on the IL-6-induced fibrinogen synthesis of HepG2 cells. Monoclonal antibody specific for WSEWS pentapeptide was stimulatory for the spontaneous secretion of both fibrinogen and C1-INH. However, the IL-6-induced elevations of these acute-phase proteins were oppositely regulated, since the anti-WSEWS monoclonal antibody was inhibitory on the production of fibrinogen induced by IL-6 but strongly augmented the IL-6 induced production of C1-INH. Our study indicates that the WSEWS motif is critical in the effect of IL-6 on the acute-phase protein production influencing either the ligand binding by the WSEWS-containing receptor molecule or the signal transduction.

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

    PubMed

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

    2012-01-24

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

  1. Effects of short-chain chlorinated paraffins exposure on the viability and metabolism of human hepatoma HepG2 cells.

    PubMed

    Geng, Ningbo; Zhang, Haijun; Zhang, Baoqin; Wu, Ping; Wang, Feidi; Yu, Zhengkun; Chen, Jiping

    2015-03-03

    Short-chain chlorinated paraffins (SCCPs) have attracted considerable attention for their characteristic of persistent organic pollutants. However, very limited information is available for their toxic effects at environmentally relevant doses, limiting the evaluation of their health risks. In this study, cell viability assay and targeted metabolomic approach was used to evaluate the environmental dose (<100 μg/L) effect of SCCPs on HepG2 cells. Cell viability was found to be decreased with increases in exposure dose of SCCPs. Exposure for 48 h to C10-CPs resulted in a significant reduction in cell viability compared with 24 h, even at 1 μg/L. SCCPs exposure altered the intracellular redox status and caused significant metabolic disruptions. As a kind of peroxisome proliferator, SCCPs specifically stimulated the β-oxidation of unsaturated fatty acids and long-chain fatty acids. Meanwhile, SCCPs exposure disturbed glycolysis and amino acid metabolism, and led to the up-regulation of glutamate metabolism and urea cycle. The toxic effects of SCCPs might mainly involve the perturbation of energy production, protein biosynthesis, fatty acid metabolism, and ammonia recycling.

  2. mRNA levels of peroxisome proliferator-activated receptors and their coactivators are affected by glucose deprivation and oleate in human hepatoma hepG2 cells.

    PubMed

    Bogdanova, Katerina; Uherkova, Lenka; Poczatkova, Hana; Rypka, Miroslav; Vesely, Jaroslav

    2007-12-01

    Very modest changes in mRNA stability can affect critical points in cellular energy pathways. The aim of this study was to investigate the impact of energy abundant substrates on peroxisome proliferator-activated receptors (PPARs) and PPAR-gamma coactivators (PGCs) mRNA's steady-state levels. Quantitative RT-PCR study was performed to assess the effect of zero or normal (5 mmol/l) glucose and/or oleic acid (0.3 mmol/l) on mRNA levels of (PPARs) (PGCs) in HepG2 cells. PGC-1alpha mRNA was significantly upregulated in glucose deprived cells (123 % of the control level; p < 0.05), while PGC-1beta mRNA was significantly enhanced in oleate-fed cells (134 % and 160 % of control levels for zero glucose plus oleate and normal glucose plus oleate, respectively; p < 0.05) during the 0.5 h incubation. Upon the 4 h incubation, PPAR-gamma1 and PGC-1alpha mRNAs were significantly elevated in cells lacking glucose (142 % and 163 % of control levels, respectively; p < 0.05). Oleate significantly suppressed PPAR-alpha and PGC-1beta mRNA levels in glucose-deprived cells (58 % and 49 % of control levels, respectively; p < 0.05). PPAR-gamma1 and -gamma2 mRNAs were significantly superinduced when the cells were treated with cycloheximide, whereas PPAR-alpha and PGC-1alpha and-1beta mRNAs were destabilized. Upon actinomycin D treatment, glucose shortage significantly stabilized PPAR-alpha mRNA, while PGC-1alpha mRNA was destabilized by oleate in glucose-deprived cells. Our findings provide evidence that transcriptional processes that are under the control of energetic substrates are interconnected with concurrent translational processes that can change stability of mRNAs.

  3. Metabolism of a representative oxygenated polycyclic aromatic hydrocarbon (PAH) phenanthrene-9,10-quinone in human hepatoma (HepG2) cells.

    PubMed

    Huang, Meng; Zhang, Li; Mesaros, Clementina; Zhang, Suhong; Blaha, Michael A; Blair, Ian A; Penning, Trevor M

    2014-05-19

    Exposure to polycyclic aromatic hydrocarbons (PAHs) in the food chain is the major human health hazard associated with the Deepwater Horizon oil spill. Phenanthrene is a representative PAH present in crude oil, and it undergoes biological transformation, photooxidation, and chemical oxidation to produce its signature oxygenated derivative, phenanthrene-9,10-quinone. We report the downstream metabolic fate of phenanthrene-9,10-quinone in HepG2 cells. The structures of the metabolites were identified by HPLC-UV-fluorescence detection and LC-MS/MS. O-mono-Glucuronosyl-phenanthrene-9,10-catechol was identified, as reported previously. A novel bis-conjugate, O-mono-methyl-O-mono-sulfonated-phenanthrene-9,10-catechol, was discovered for the first time, and evidence for both of its precursor mono conjugates was obtained. The identities of these four metabolites were unequivocally validated by comparison to authentic enzymatically synthesized standards. Evidence was also obtained for a minor metabolic pathway of phenanthrene-9,10-quinone involving bis-hydroxylation followed by O-mono-sulfonation. The identification of 9,10-catechol conjugates supports metabolic detoxification of phenanthrene-9,10-quinone through interception of redox cycling by UGT, COMT, and SULT isozymes and indicates the possible use of phenanthrene-9,10-catechol conjugates as biomarkers of human exposure to oxygenated PAH.

  4. Metabolism of a Representative Oxygenated Polycyclic Aromatic Hydrocarbon (PAH) Phenanthrene-9,10-quinone in Human Hepatoma (HepG2) Cells

    PubMed Central

    2014-01-01

    Exposure to polycyclic aromatic hydrocarbons (PAHs) in the food chain is the major human health hazard associated with the Deepwater Horizon oil spill. Phenanthrene is a representative PAH present in crude oil, and it undergoes biological transformation, photooxidation, and chemical oxidation to produce its signature oxygenated derivative, phenanthrene-9,10-quinone. We report the downstream metabolic fate of phenanthrene-9,10-quinone in HepG2 cells. The structures of the metabolites were identified by HPLC–UV–fluorescence detection and LC–MS/MS. O-mono-Glucuronosyl-phenanthrene-9,10-catechol was identified, as reported previously. A novel bis-conjugate, O-mono-methyl-O-mono-sulfonated-phenanthrene-9,10-catechol, was discovered for the first time, and evidence for both of its precursor mono conjugates was obtained. The identities of these four metabolites were unequivocally validated by comparison to authentic enzymatically synthesized standards. Evidence was also obtained for a minor metabolic pathway of phenanthrene-9,10-quinone involving bis-hydroxylation followed by O-mono-sulfonation. The identification of 9,10-catechol conjugates supports metabolic detoxification of phenanthrene-9,10-quinone through interception of redox cycling by UGT, COMT, and SULT isozymes and indicates the possible use of phenanthrene-9,10-catechol conjugates as biomarkers of human exposure to oxygenated PAH. PMID:24646012

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-10-19

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

  7. Dehydroepiandrosterone effects on the mRNA levels of peroxisome proliferator-activated receptors and their coactivators in human hepatoma HepG2 cells.

    PubMed

    Poczatková, H; Bogdanová, K; Uherková, L; Cervenková, K; Riegrová, D; Rypka, M; Veselý, J

    2007-12-01

    To investigate the effect of dehydroepiandrosterone (DHEA) on intracellular mRNA levels of peroxisome proliferator-activated receptors (PPARs) and PPAR-gamma coactivators (PGCs), we conducted a quantitative real-time RT-PCR study using HepG2 cells. Treatment with 100 micromol/l DHEA for 2-20 h caused a time-dependent elevation of mRNA levels in the cells. Upon 20 h, PPAR-alpha, -gamma1, and -gamma2 mRNAs and PGC-1alpha and -1beta mRNAs increased to 157, 161, 155, 656, and 475% of control levels, respectively (p < 0.05 each). Treatment with actinomycin D for 2.5-8 h revealed a significant stabilization effect of DHEA on PPAR-gamma1 and PGC-1alpha mRNAs at both 2.5 and 8 h incubation periods and a mild but significant stabilization effect on PGC-1beta mRNA at the 8 h incubation period suggesting that DHEA can modulate turnover of these mRNA transcripts. Basal mRNA levels of PPAR-alpha and PGC-1alpha were significantly suppressed upon 20 h treatment with cycloheximide, while those of PPAR-gamma1, -gamma2, and PGC-1beta were elevated. Cycloheximide also significantly reduced DHEA-induced accumulation of PPAR-alpha, -gamma1, -gamma2, and PGC-1alpha mRNAs, demonstrating the dependence of the DHEA action on de novo protein synthesis. The findings demonstrate that a supraphysiological concentration of DHEA can substantially influence gene expression of the PPAR signalling machinery at both transcriptional and posttranscriptional levels.

  8. Beta-glucan extracted from the medicinal mushroom Agaricus blazei prevents the genotoxic effects of benzo[a]pyrene in the human hepatoma cell line HepG2.

    PubMed

    Angeli, José Pedro Friedmann; Ribeiro, Lúcia Regina; Bellini, Marilanda Ferreira; Mantovani, Mário Sérgio

    2009-01-01

    The mushroom Agaricus blazei is studied for its nutraceutical potential and as a medicinal supplement. The aim of the present study was to investigate the chemoprotective effect of beta-glucan extracted from the mushroom A. blazei against DNA damage induced by benzo[a]pyrene (B[a]P), using the comet assay (genotoxicity) and micronucleus assay with cytokinesis block (mutagenicity) in a human hepatoma cell line (HepG2). To elucidate the possible beta-glucan mechanism of action, desmutagenesis or bioantimutagenesis types, three treatment protocols were tested: simultaneous, pre-treatment, and presimultaneous. The results showed that beta-glucan does not exert genotoxic or mutagenic effect, but that it does protect against DNA damage caused by B[a]P in every protocol tested. The data suggest that beta-glucan acts through binding to B[a]P or the capture of free radicals produced during its activation. On the other hand, the pre-treatment results also suggest the possibility that beta-glucan modulates cell metabolism.

  9. Potentiation of LPS-Induced Apoptotic Cell Death in Human Hepatoma HepG2 Cells by Aspirin via ROS and Mitochondrial Dysfunction: Protection by N-Acetyl Cysteine

    PubMed Central

    Raza, Haider; John, Annie; Shafarin, Jasmin

    2016-01-01

    Cytotoxicity and inflammation-associated toxic responses have been observed to be induced by bacterial lipopolysaccharides (LPS) in vitro and in vivo respectively. Use of nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, has been reported to be beneficial in inflammation-associated diseases like cancer, diabetes and cardiovascular disorders. Their precise molecular mechanisms, however, are not clearly understood. Our previous studies on aspirin treated HepG2 cells strongly suggest cell cycle arrest and induction of apoptosis associated with mitochondrial dysfunction. In the present study, we have further demonstrated that HepG2 cells treated with LPS alone or in combination with aspirin induces subcellular toxic responses which are accompanied by increase in reactive oxygen species (ROS) production, oxidative stress, mitochondrial respiratory dysfunction and apoptosis. The LPS/Aspirin induced toxicity was attenuated by pre-treatment of cells with N-acetyl cysteine (NAC). Alterations in oxidative stress and glutathione-dependent redox-homeostasis were more pronounced in mitochondria compared to extra- mitochondrial cellular compartments. Pre-treatment of HepG2 cells with NAC exhibited a selective protection in redox homeostasis and mitochondrial dysfunction. Our results suggest that the altered redox metabolism, oxidative stress and mitochondrial function in HepG2 cells play a critical role in LPS/aspirin-induced cytotoxicity. These results may help in better understanding the pharmacological, toxicological and therapeutic properties of NSAIDs in cancer cells exposed to bacterial endotoxins. PMID:27441638

  10. SC-III3, a novel scopoletin derivative, induces autophagy of human hepatoma HepG2 cells through AMPK/mTOR signaling pathway by acting on mitochondria.

    PubMed

    Zhao, Peng; Dou, Yannong; Chen, Li; Li, Linhu; Wei, Zhifeng; Yu, Juntao; Wu, Xin; Dai, Yue; Xia, Yufeng

    2015-07-01

    (E)-3-(4-chlorophenyl)-N-(7-hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl) acrylamide (SC-III3), a newly synthesized derivative of scopoletin, was previously shown to reduce the viability of HepG2 cells and tumor growth of HepG2 xenograft mouse model. It induces the death of HepG2 cells by a way irrelevant to apoptosis and necrosis. To shed light on the cytotoxic mechanisms of SC-III3, the present study addresses whether and how it can induce autophagic cell death. When HepG2 cells were incubated with various concentrations of SC-III3, autophagic vacuoles could be observed by transmission electron microscopy and monodansylcadaverine staining. Increased expressions of LC3-II to LC3-I and Beclin-1, required for autophagosome formation, were accompanied. These characteristics integrally indicated that SC-III3 could initiate autophagy in HepG2 cells. N-acetyl-l-cysteine (NAC), a ROS scavenger, could reverse SC-III3-caused ROS accumulation, but it did not affect SC-III3-induced autophagy, suggesting that ROS was not involved in SC-III3-mediated autophagy in HepG2 cells. SC-III3 significantly depressed mitochondrial function, as evidenced by disruption of mitochondrial transmembrane potential and loss of the mitochondrial cristae structure, as well as decrease of Cox-I, Cox-III, Cox-IV, and ATP levels. The autophagy and activation of AMPK-TSC2-mTOR-p70s6k pathways induced by SC-III3 in HepG2 cells could be efficiently blocked by pre-treatments of compound C (an inhibitor of AMPK). Moreover, addition of extracellular ATP to the cell culture media could reverse SC-III3-caused activation of AMPK-TSC2-mTOR-p70s6k pathway, autophagy and cell viability decrease in HepG2 cells. Collectively, SC-III3 leads to autophagy through inducing mitochondrial dysfunction, depleting ATP, and activating AMPK-mTOR pathway, which thus reflects the cytotoxic effect of SC-III3 in HepG2 cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. AP-1 Inhibition by SR 11302 Protects Human Hepatoma HepG2 Cells from Bile Acid-Induced Cytotoxicity by Restoring the NOS-3 Expression

    PubMed Central

    González-Rubio, Sandra; Linares, Clara I.; Aguilar-Melero, Patricia; Rodríguez-Perálvarez, Manuel; Montero-Álvarez, José L.

    2016-01-01

    The harmful effects of bile acid accumulation occurring during cholestatic liver diseases have been associated with oxidative stress increase and endothelial nitric oxide synthase (NOS-3) expression decrease in liver cells. We have previously reported that glycochenodeoxycholic acid (GCDCA) down-regulates gene expression by increasing SP1 binding to the NOS-3 promoter in an oxidative stress dependent manner. In the present study, we aimed to investigate the role of transcription factor (TF) AP-1 on the NOS-3 deregulation during GCDCA-induced cholestasis. The cytotoxic response to GCDCA was characterized by 1) the increased expression and activation of TFs cJun and c-Fos; 2) a higher binding capability of these at position -666 of the NOS-3 promoter; 3) a decrease of the transcriptional activity of the promoter and the expression and activity of NOS-3; and 4) the expression increase of cyclin D1. Specific inhibition of AP-1 by the retinoid SR 11302 counteracted the cytotoxic effects induced by GCDCA while promoting NOS-3 expression recovery and cyclin D1 reduction. NOS activity inhibition by L-NAME inhibited the protective effect of SR 11302. Inducible NOS isoform was no detected in this experimental model of cholestasis. Our data provide direct evidence for the involvement of AP-1 in the NOS-3 expression regulation during cholestasis and define a critical role for NOS-3 in regulating the expression of cyclin D1 during the cell damage induced by bile acids. AP-1 appears as a potential therapeutic target in cholestatic liver diseases given its role as a transcriptional repressor of NOS-3. PMID:27490694

  12. Cytostatic and genotoxic effect of temephos in human lymphocytes and HepG2 cells.

    PubMed

    Benitez-Trinidad, A B; Herrera-Moreno, J F; Vázquez-Estrada, G; Verdín-Betancourt, F A; Sordo, M; Ostrosky-Wegman, P; Bernal-Hernández, Y Y; Medina-Díaz, I M; Barrón-Vivanco, B S; Robledo-Marenco, M L; Salazar, A M; Rojas-García, A E

    2015-06-01

    Temephos is an organophosphorus pesticide that is used in control campaigns against Aedes aegypti mosquitoes, which transmit dengue. In spite of the widespread use of temephos, few studies have examined its genotoxic potential. The aim of this study was to evaluate the cytotoxic, cytostatic and genotoxic effects of temephos in human lymphocytes and hepatoma cells (HepG2). The cytotoxicity was evaluated with simultaneous staining (FDA/EtBr). The cytostatic and genotoxic effects were evaluated using comet assays and the micronucleus technique. We found that temephos was not cytotoxic in either lymphocytes or HepG2 cells. Regarding the cytostatic effect in human lymphocytes, temephos (10 μM) caused a significant decrease in the percentage of binucleated cells and in the nuclear division index as well as an increase in the apoptotic cell frequency, which was not the case for HepG2 cells. The comet assay showed that temephos increased the DNA damage levels in human lymphocytes, but it did not increase the MN frequency. In contrast, in HepG2 cells, temephos increased the tail length, tail moment and MN frequency in HepG2 cells compared to control cells. In conclusion, temephos causes stable DNA damage in HepG2 cells but not in human lymphocytes. These findings suggest the importance of temephos biotransformation in its genotoxic effect. Copyright © 2015. Published by Elsevier Ltd.

  13. Xanthorrhizol induced DNA fragmentation in HepG2 cells involving Bcl-2 family proteins

    SciTech Connect

    Tee, Thiam-Tsui; Cheah, Yew-Hoong; Meenakshii, Nallappan; Mohd Sharom, Mohd Yusof; Azimahtol Hawariah, Lope Pihie

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer We isolated xanthorrhizol, a sesquiterpenoid compound from Curcuma xanthorrhiza. Black-Right-Pointing-Pointer Xanthorrhizol induced apoptosis in HepG2 cells as observed using SEM. Black-Right-Pointing-Pointer Apoptosis in xanthorrhizol-treated HepG2 cells involved Bcl-2 family proteins. Black-Right-Pointing-Pointer DNA fragmentation was observed in xanthorrhizol-treated HepG2 cells. Black-Right-Pointing-Pointer DNA fragmentation maybe due to cleavage of PARP and DFF45/ICAD proteins. -- Abstract: Xanthorrhizol is a plant-derived pharmacologically active sesquiterpenoid compound isolated from Curcuma xanthorrhiza. Previously, we have reported that xanthorrhizol inhibited the proliferation of HepG2 human hepatoma cells by inducing apoptotic cell death via caspase activation. Here, we attempt to further elucidate the mode of action of xanthorrhizol. Apoptosis in xanthorrhizol-treated HepG2 cells as observed by scanning electron microscopy was accompanied by truncation of BID; reduction of both anti-apoptotic Bcl-2 and Bcl-X{sub L} expression; cleavage of PARP and DFF45/ICAD proteins and DNA fragmentation. Taken together, these results suggest xanthorrhizol as a potent antiproliferative agent on HepG2 cells by inducing apoptosis via Bcl-2 family members. Hence we proposed that xanthorrhizol could be used as an anti-liver cancer drug for future studies.

  14. Developmental Stage-Specific Embryonic Induction of HepG2 Cell Differentiation.

    PubMed

    Li, Yanning; Zong, Yanhong; Xiao, Zhigang; Zhu, Mengxuan; Xiao, Hui; Qi, Jinsheng; Liu, Kun; Wang, Hui

    2016-04-01

    Although hepatocellular carcinoma cells can sometimes undergo differentiation in an embryonic microenvironment, the mechanism is poorly understood. The developmental stage-specific embryonic induction of tumor cell differentiation was investigated. Both chick and mouse liver extracts and hepatoblast-enriched cells at different developmental stages were used to treat human hepatoma HepG2 cells, and the effects on the induction of differentiation were evaluated. The nuclear factors controlling differentiation, hepatocyte nuclear factor (HNF)-4α, HNF-1α, HNF-6 and upstream stimulatory factor-1 (USF-1), and the oncogene Myc and alpha-fetoprotein (AFP) were measured. HNF-4α RNA interference was used to verify the role of HNF-4α. Embryonic induction effects were further tested in vivo by injecting HepG2 tumor cells into immunodeficient nude mice. The 9-11-days chick liver extracts and 13.5-14.5-days mouse hepatoblast-enriched cells could inhibit proliferation and induce differentiation of HepG2 cells, leading to either death or maturation to hepatocytes. The maturation of surviving HepG2 cells was confirmed by increases in the expressions of HNF-4α, HNF-1α, HNF-6, and USF-1, and decreases in Myc and AFP. The embryonic induction of HepG2 cell maturation could be attenuated by HNF-4α RNA interference. Furthermore, the 13.5-days mouse hepatoblast culture completely eliminated HepG2 tumors with inhibited Myc and induced HNF-4α, confirming this embryonic induction effect in vivo. This study demonstrated that developmental stage-specific embryonic induction of HepG2 cell differentiation might help in understanding embryonic differentiation and oncogenesis.

  15. Differential expression of several drug transporter genes in HepG2 and Huh-7 cell lines.

    PubMed

    Louisa, Melva; Suyatna, Frans D; Wanandi, Septelia Inawati; Asih, Puji Budi Setia; Syafruddin, Din

    2016-01-01

    Cell culture techniques have many advantages for investigation of drug transport to target organ like liver. HepG2 and Huh-7 are two cell lines available from hepatoma that can be used as a model for hepatic drug transport. The present study is aimed to analyze the expression level of several drug transporter genes in two hepatoma cell lines, HepG2 and Huh-7 and their response to inhibitors. This is an in vitro study using HepG2 and Huh-7 cells. The expression level of the following drug transporter genes was quantified: P-glycoprotein/multidrug resistance protein 1, Organic Anionic Transporter Protein 1B1 (OATP1B1) and Organic Cationic Transporter-1 (OCT1). Ribonucleic acid was extracted from the cells using Tripure isolation reagent, then gene expression level of the transporters is quantified using Applied Biosystems quantitative reverse transcriptase polymerase chain reaction. Verapamil (P-glycoprotein inhibitor), nelfinavir (OATP1B1 inhibitor), quinidine (OCT1 inhibitor) were used to differentiate the inhibitory properties of these agents to the transporter expressions in HepG2 and Huh-7 cells. Huh-7 shows a higher level of P-glycoprotein, OATP1B1 and OCT1 expressions compared with those of HepG2. Verapamil reduces the expressions of P-glycoprotein in HepG2 and Huh-7; nelfinavir reduces the expression of OATP1B1 in HepG2 and Huh-7; while quinidine reduces the OCT1 gene expressions in HepG2, but not in Huh-7 cells. This study indicates that HepG2 might be a more suitable in vitro model than Huh-7 to study drug transport in hepatocytes involving drug transporters.

  16. Phosphoramidate protides of five flavones and their antiproliferative activity against HepG2 and L-O2 cell lines.

    PubMed

    Li, Yue-Qing; Yang, Fei; Wang, Liu; Cao, Zhi; Han, Tian-Jiao; Duan, Zhe-Ang; Li, Zhen; Zhao, Wei-Jie

    2016-04-13

    A series of flavone-7-phosphoramidate derivatives were synthesized and tested for their antiproliferative activity in vitro against human hepatoma cell line HepG2 and human normal hepatic cell line L-O2. Compound 8d, 16d and 17d, incorporating the amino acid alanine, exhibited high inhibitory activity on HepG2 cell line with IC50 values of 9.0 μmol/L, 5.5 μmol/L and 6.6 μmol/L. The introduction of acyl groups played a pivotal role in the selective inhibition toward human hepatoma HepG2 cells, except for compound 8a, 9a and 16b. Compound 8d, 16d and 17d could significantly induce G2/M arrest in HepG2 cells. Specially, Compound 16d could lead early apoptosis in HepG2 cells. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  17. [Study on transient absorption spectrum of tungsten nanoparticle with HepG2 tumor cell].

    PubMed

    Cao, Lin; Shu, Xiao-Ning; Liang, Dong; Wang, Cong

    2014-07-01

    Significance of this study lies in tungsten nano materials can be used as a preliminary innovative medicines applied basic research. This paper investigated the inhibition of tungsten nanoparticles which effected on human hepatoma HepG2 cells by MTT. The authors use transient absorption spectroscopy (TAS) technology absorption and emission spectra characterization of charge transfer between nanoparticles and tumor cell. The authors discussed the role of the tungsten nanoparticles in the tumor early detection of the disease and its anti-tumor properties. In the HepG2 experiments system, 100-150 microg x mL(-1) is the best drug concentration of anti-tumor activity which recact violently within 6 hours and basically completed in 24 hours. The results showed that transient absorption spectroscopy can be used as tumor detection methods and characterization of charge transfer between nano-biosensors and tumor cells. Tungsten nanoparticles have potential applications as anticancer drugs.

  18. Effects of sargentgloryvine stem extracts on HepG-2 cells in vitro and in vivo.

    PubMed

    Wang, Ming-Hua; Long, Min; Zhu, Bao-Yi; Yang, Shu-Hui; Ren, Ji-Hong; Zhang, Hui-Zhong

    2011-06-21

    To observe the effects of sargentgloryvine stem extracts (SSE) on the hepatoma cell line HepG-2 in vitro and in vivo and determine its mechanisms of action. Cultured HepG-2 cells treated with SSE were analysed by 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-Diphenyltetrazolium bromide and clone formation assay. The cell cycle and apoptosis analysis were conducted by flow cytometric, TdT-Mediated dUTP Nick End Labeling and acridine orange/ethidium bromide staining methods, and protein expression was examined by both reverse transcriptase-polymerase chain reaction and Western blotting. The pathological changes of the tumor cells were observed by haematoxylin and eosin staining. Tumor growth inhibition and side effects were determined in a xenograft mouse model. SSE treatment could not only inhibit HepG-2 cell proliferation in a dose- and time-dependent manner but also induce apoptosis and cell cycle arrest at the S phase. The number of colonies formed by SSE-treated tumor cells was fewer than that of the controls (P < 0.05). SSE induced caspase-dependent apoptosis accompanied by a significant decrease in Bcl-xl and Mcl-1 and elevation of Bak expression (P < 0.05). Tumor necrosis factor α in the xenograft tumor tissue and the liver functions of SSE-treated mice showed no significant changes at week 8 compared with the control group (P > 0.05). Systemic administration of SSE could inhibit the HepG-2 xenograft tumor growth with no obvious toxic side effects on normal tissues. SSE can induce apoptosis of HepG-2 cells in vitro and in vivo through decreasing expression of Bcl-xl and Mcl-1 and increasing expression of Bax.

  19. 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.

  20. Potential Metabolic Activation of a Representative C2-Alkylated Polycyclic Aromatic Hydrocarbon 6-Ethylchrysene Associated with the Deepwater Horizon Oil Spill in Human Hepatoma (HepG2) Cells

    PubMed Central

    2016-01-01

    Exposure to polycyclic aromatic hydrocarbons (PAHs) is the major human health hazard associated with the Deepwater Horizon oil spill. C2-Chrysenes are representative PAHs present in crude oil and could contaminate the food chain. We describe the metabolism of a C2-chrysene regioisomer, 6-ethylchrysene (6-EC), in human HepG2 cells. The structures of the metabolites were identified by HPLC-UV-fluorescence detection and LC-MS/MS. 6-EC-tetraol isomers were identified as signature metabolites of the diol-epoxide pathway. O-Monomethyl-O-monosulfonated-6-EC-catechol, its monohydroxy products, and N-acetyl-l-cysteine(NAC)-6-EC-ortho-quinone were discovered as signature metabolites of the ortho-quinone pathway. Potential dual metabolic activation of 6-EC involving the formation of bis-electrophiles, i.e., a mono-diol-epoxide and a mono-ortho-quinone within the same structure, bis-diol-epoxides, and bis-ortho-quinones was observed as well. The identification of 6-EC-tetraol, O-monomethyl-O-monosulfonated-6-EC-catechol, its monohydroxy products, and NAC-6-EC-ortho-quinone supports potential metabolic activation of 6-EC by P450 and AKR enzymes followed by metabolic detoxification of the ortho-quinone through interception of its redox cycling capability by catechol-O-methyltransferase and sulfotransferase enzymes. The tetraols and catechol conjugates could be used as biomarkers of human exposure to 6-EC resulting from oil spills. PMID:27054409

  1. RNA-Seq gene expression profiling of HepG2 cells: the influence of experimental factors and comparison with liver tissue.

    PubMed

    Tyakht, Alexander V; Ilina, Elena N; Alexeev, Dmitry G; Ischenko, Dmitry S; Gorbachev, Alexey Y; Semashko, Tatiana A; Larin, Andrei K; Selezneva, Oksana V; Kostryukova, Elena S; Karalkin, Pavel A; Vakhrushev, Igor V; Kurbatov, Leonid K; Archakov, Alexander I; Govorun, Vadim M

    2014-12-15

    Human hepatoma HepG2 cells are used as an in vitro model of the human liver. High-throughput transcriptomic sequencing is an advanced approach for assessing the functional state of a tissue or cell type. However, the influence of experimental factors, such as the sample preparation method and inter-laboratory variation, on the transcriptomic profile has not been evaluated. The whole-transcriptome sequencing of HepG2 cells was performed using the SOLiD platform and validated using droplet digital PCR. The gene expression profile was compared to the results obtained with the same sequencing method in another laboratory and using another sample preparation method. We also compared the transcriptomic profile HepG2 cells with that of liver tissue. Comparison of the gene expression profiles between the HepG2 cell line and liver tissue revealed the highest variation, followed by HepG2 cells submitted to two different sample preparation protocols. The lowest variation was observed between HepG2 cells prepared by two different laboratories using the same protocol. The enrichment analysis of the genes that were differentially expressed between HepG2 cells and liver tissue mainly revealed the cancer-associated gene signature of HepG2 cells and the activation of the response to chemical stimuli in the liver tissue. The HepG2 transcriptome obtained with the SOLiD platform was highly correlated with the published transcriptome obtained with the Illumina and Helicos platforms, with moderate correspondence to microarrays. In the present study, we assessed the influence of experimental factors on the HepG2 transcriptome and identified differences in gene expression between the HepG2 cell line and liver cells. These findings will facilitate robust experimental design in the fields of pharmacology and toxicology. Our results were supported by a comparative analysis with previous HepG2 gene expression studies.

  2. Protective effects of xanthohumol against the genotoxicity of benzo(a)pyrene (BaP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and tert-butyl hydroperoxide (t-BOOH) in HepG2 human hepatoma cells.

    PubMed

    Plazar, Janja; Zegura, Bojana; Lah, Tamara T; Filipic, Metka

    2007-08-15

    Xanthohumol is the major prenylated flavonoid present in the hop plant Humulus lupulus L. (Cannabinaceae) and a common ingredient of beer. Recently, xanthohumol has gained considerable interest due to its potential cancer chemo-preventive effect. The aim of this study was to reveal the possible anti-genotoxic activity of xanthohumol in metabolically competent human hepatoma HepG2 cells, by use of the comet assay. Xanthohumol by itself was neither cytotoxic nor genotoxic to the cells at concentrations below 10microM. However, a significant protective effect against the pro-carcinogens benzo(a)pyrene (BaP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was observed at concentrations as low as 0.01microM. In cells treated with xanthohumol in combination with tert-butyl hydroperoxide (t-BOOH) - an inducer of reactive oxygen species (ROS) - no protective effect was observed and xanthohumol also showed no significant scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. On the other hand, HepG2 cells pre-treated with xanthohumol showed significantly reduced levels of t-BOOH-induced DNA strand breaks, indicating that its protective effect is mediated by induction of cellular defence mechanisms against oxidative stress. As xanthohumol is known to be an effective inhibitor of cytochrome P450 enzymes and an inducer of NAD(P)H: quinone reductase (QR), our findings can be explained by an inhibition of metabolic activation of pro-carcinogens and/or by induction of carcinogen-detoxifying and anti-oxidative enzymes by xanthohumol. These results provide evidence that xanthohumol displays anti-genotoxic activity in metabolically competent human cells.

  3. Quercetin protects human hepatoma HepG2 against oxidative stress induced by tert-butyl hydroperoxide

    SciTech Connect

    Alia, Mario . E-mail: luisgoya@if.csic.es

    2006-04-15

    Flavonols such as quercetin, have been reported to exhibit a wide range of biological activities related to their antioxidant capacity. The objective of the present study was to investigate the protective effect of quercetin on cell viability and redox status of cultured HepG2 cells submitted to oxidative stress induced by tert-butyl hydroperoxide. Concentrations of reduced glutathione and malondialdehyde, generation of reactive oxygen species and activity and gene expression of antioxidant enzymes were used as markers of cellular oxidative status. Pretreatment of HepG2 with 10 {mu}M quercetin completely prevented lactate dehydrogenase leakage from the cells. Pretreatment for 2 or 20 h with all doses of quercetin (0.1-10 {mu}M) prevented the decrease of reduced glutathione and the increase of malondialdehyde evoked by tert-butyl hydroperoxide in HepG2 cells. Reactive oxygen species generation induced by tert-butyl hydroperoxide was significantly reduced when cells were pretreated for 2 or 20 h with 10 {mu}M and for 20 h with 5 {mu}M quercetin. Finally, some of the quercetin treatments prevented the significant increase of glutathione peroxidase, superoxide dismutase, glutathione reductase and catalase activities induced by tert-butyl hydroperoxide. Gene expression of antioxidant enzymes was also affected by the treatment with the polyphenol. The results of the biomarkers analyzed clearly show that treatment of HepG2 cells in culture with the natural dietary antioxidant quercetin strongly protects the cells against an oxidative insult.

  4. Quercetin protects human hepatoma HepG2 against oxidative stress induced by tert-butyl hydroperoxide.

    PubMed

    Alía, Mario; Ramos, Sonia; Mateos, Raquel; Granado-Serrano, Ana Belén; Bravo, Laura; Goya, Luis

    2006-04-15

    Flavonols such as quercetin, have been reported to exhibit a wide range of biological activities related to their antioxidant capacity. The objective of the present study was to investigate the protective effect of quercetin on cell viability and redox status of cultured HepG2 cells submitted to oxidative stress induced by tert-butyl hydroperoxide. Concentrations of reduced glutathione and malondialdehyde, generation of reactive oxygen species and activity and gene expression of antioxidant enzymes were used as markers of cellular oxidative status. Pretreatment of HepG2 with 10 microM quercetin completely prevented lactate dehydrogenase leakage from the cells. Pretreatment for 2 or 20 h with all doses of quercetin (0.1-10 microM) prevented the decrease of reduced glutathione and the increase of malondialdehyde evoked by tert-butyl hydroperoxide in HepG2 cells. Reactive oxygen species generation induced by tert-butyl hydroperoxide was significantly reduced when cells were pretreated for 2 or 20 h with 10 microM and for 20 h with 5 microM quercetin. Finally, some of the quercetin treatments prevented the significant increase of glutathione peroxidase, superoxide dismutase, glutathione reductase and catalase activities induced by tert-butyl hydroperoxide. Gene expression of antioxidant enzymes was also affected by the treatment with the polyphenol. The results of the biomarkers analyzed clearly show that treatment of HepG2 cells in culture with the natural dietary antioxidant quercetin strongly protects the cells against an oxidative insult.

  5. Cacao polyphenols influence the regulation of apolipoprotein in HepG2 and Caco2 cells.

    PubMed

    Yasuda, Akiko; Natsume, Midori; Osakabe, Naomi; Kawahata, Keiko; Koga, Jinichiro

    2011-02-23

    Cocoa powder is rich in polyphenols, such as catechins and procyanidins, and has been shown to inhibit low-density lipoprotein (LDL) oxidation and atherogenesis in a variety of models. Human studies have also shown daily intake of cocoa increases plasma high-density lipoprotein (HDL) and decreases LDL levels. However, the mechanisms responsible for these effects of cocoa on cholesterol metabolism have yet to be fully elucidated. The present study investigated the effects of cacao polyphenols on the production of apolipoproteins A1 and B in human hepatoma HepG2 and intestinal Caco2 cell lines. The cultured HepG2 cells or Caco2 cells were incubated for 24 h in the presence of cacao polyphenols such as (-)-epicatechin, (+)-catechin, procyanidin B2, procyanidin C1, and cinnamtannin A2. The concentration of apolipoproteins in the cell culture media was quantified using an enzyme-linked immunoassay, and the mRNA expression was quantified by RT-PCR. Cacao polyphenols increased apolipoprotein A1 protein levels and mRNA expression, even though apolipoprotein B protein and the mRNA expression were slightly decreased in both HepG2 cells and Caco2 cells. In addition, cacao polyphenols increased sterol regulatory element binding proteins (SREBPs) and activated LDL receptors in HepG2 cells. These results suggest that cacao polyphenols may increase the production of mature form SREBPs and LDL receptor activity, thereby increasing ApoA1 and decreasing ApoB levels. These results elucidate a novel mechanism by which HDL cholesterol levels become elevated with daily cocoa intake.

  6. Upgrading HepG2 cells with adenoviral vectors that encode drug-metabolizing enzymes: application for drug hepatotoxicity testing.

    PubMed

    Gómez-Lechón, M José; Tolosa, Laia; Donato, M Teresa

    2017-02-01

    Drug attrition rates due to hepatotoxicity are an important safety issue considered in drug development. The HepG2 hepatoma cell line is currently being used for drug-induced hepatotoxicity evaluations, but its expression of drug-metabolizing enzymes is poor compared with hepatocytes. Different approaches have been proposed to upgrade HepG2 cells for more reliable drug-induced liver injury predictions. Areas covered: We describe the advantages and limitations of HepG2 cells transduced with adenoviral vectors that encode drug-metabolizing enzymes for safety risk assessments of bioactivable compounds. Adenoviral transduction facilitates efficient and controlled delivery of multiple drug-metabolizing activities to HepG2 cells at comparable levels to primary human hepatocytes by generating an 'artificial hepatocyte'. Furthermore, adenoviral transduction enables the design of tailored cells expressing particular metabolic capacities. Expert opinion: Upgraded HepG2 cells that recreate known inter-individual variations in hepatic CYP and conjugating activities due to both genetic (e.g., polymorphisms) or environmental (e.g., induction, inhibition) factors seems a suitable model to identify bioactivable drug and conduct hepatotoxicity risk assessments. This strategy should enable the generation of customized cells by reproducing human pheno- and genotypic CYP variability to represent a valuable human hepatic cell model to develop new safer drugs and to improve existing predictive toxicity assays.

  7. Protective Effects of Vitamin C and NAC on the Toxicity of Rifampin on Hepg2 Cells.

    PubMed

    Vahdati-Mashhadian, Nasser; Jafari, Mahmoud Reza; Sharghi, Nasim; Sanati, Toktam

    2013-01-01

    Rifampin, an antibiotic widely used for the treatment of mycobacterial infections, produces hepatic, renal and bone marrow toxicity in human and animals. In this study, the protective effects of vitamin C and n-acetylcysteine (NAC) on the toxicity of rifampin on HepG2 cells were investigated. Human hepatoma cells (HepG2) were cultured in 96-well M of rifampin in the presence of microplate and exposed to 10, 20, 50 and 100 vitamin C (0.1 mg/mL) and NAC (0.2 mg/mL). Protective effect of the two drugs against rifampin toxicity was assessed by MTT assay. Results show that both vitamin C and NAC significantly inhibited HepG2 cellular damage due to rifampin, and vitamin C was relatively more potent than NAC. Rifampin is metabolized by the liver and its toxic metabolites are responsible for the drug›s hepatic toxicity. Based on our results, it seems that reactive metabolites are the main agents responsible for rifampin hepatotoxicity. The importance of this finding is that if vitamin C or NAC do not affect the antibacterial activity of rifampin, they could be used as preventive agents in rifampin users.

  8. Protective Effects of Vitamin C and NAC on the Toxicity of Rifampin on Hepg2 Cells

    PubMed Central

    Vahdati-Mashhadian, Nasser; Jafari, Mahmoud Reza; Sharghi, Nasim; Sanati, Toktam

    2013-01-01

    Rifampin, an antibiotic widely used for the treatment of mycobacterial infections, produces hepatic, renal and bone marrow toxicity in human and animals. In this study, the protective effects of vitamin C and n-acetylcysteine (NAC) on the toxicity of rifampin on HepG2 cells were investigated. Human hepatoma cells (HepG2) were cultured in 96-well M of rifampin in the presence of microplate and exposed to 10, 20, 50 and 100 vitamin C (0.1 mg/mL) and NAC (0.2 mg/mL). Protective effect of the two drugs against rifampin toxicity was assessed by MTT assay. Results show that both vitamin C and NAC significantly inhibited HepG2 cellular damage due to rifampin, and vitamin C was relatively more potent than NAC. Rifampin is metabolized by the liver and its toxic metabolites are responsible for the drug›s hepatic toxicity. Based on our results, it seems that reactive metabolites are the main agents responsible for rifampin hepatotoxicity. The importance of this finding is that if vitamin C or NAC do not affect the antibacterial activity of rifampin, they could be used as preventive agents in rifampin users. PMID:24250582

  9. Amitriptyline induces mitophagy that precedes apoptosis in human HepG2 cells.

    PubMed

    Villanueva-Paz, Marina; Cordero, Mario D; Pavón, Ana Delgado; Vega, Beatriz Castejón; Cotán, David; De la Mata, Mario; Oropesa-Ávila, Manuel; Alcocer-Gomez, Elizabet; de Lavera, Isabel; Garrido-Maraver, Juan; Carrascosa, José; Zaderenko, Ana Paula; Muntané, Jordi; de Miguel, Manuel; Sánchez-Alcázar, José Antonio

    2016-07-01

    Systemic treatments for hepatocellular carcinoma (HCC) have been largely unsuccessful. This study investigated the antitumoral activity of Amitriptyline, a tricyclic antidepressant, in hepatoma cells. Amitriptyline-induced toxicity involved early mitophagy activation that subsequently switched to apoptosis. Amitriptyline induced mitochondria dysfunction and oxidative stress in HepG2 cells. Amitriptyline specifically inhibited mitochondrial complex III activity that is associated with decreased mitochondrial membrane potential (∆Ψm) and increased reactive oxygen species (ROS) production. Transmission electron microscopy (TEM) studies revealed structurally abnormal mitochondria that were engulfed by double-membrane structures resembling autophagosomes. Consistent with mitophagy activation, fluorescence microscopy analysis showed mitochondrial Parkin recruitment and colocalization of mitochondria with autophagosome protein markers. Pharmacological or genetic inhibition of autophagy exacerbated the deleterious effects of Amitriptyline on hepatoma cells and led to increased apoptosis. These results suggest that mitophagy acts as an initial adaptive mechanism of cell survival. However persistent mitochondrial damage induced extensive and lethal mitophagy, autophagy stress and autophagolysome permeabilization leading eventually to cell death by apoptosis. Amitriptyline also induced cell death in hepatoma cells lines with mutated p53 and non-sense p53 mutation. Our results support the hypothesis that Amitriptyline-induced mitochondrial dysfunction can be a useful therapeutic strategy for HCC treatment, especially in tumors showing p53 mutations and/or resistant to genotoxic treatments.

  10. Amitriptyline induces mitophagy that precedes apoptosis in human HepG2 cells

    PubMed Central

    Villanueva-Paz, Marina; Cordero, Mario D.; Pavón, Ana Delgado; Vega, Beatriz Castejón; Cotán, David; De la Mata, Mario; Oropesa-Ávila, Manuel; Alcocer-Gomez, Elizabet; de Lavera, Isabel; Garrido-Maraver, Juan; Carrascosa, José; Zaderenko, Ana Paula; Muntané, Jordi; de Miguel, Manuel; Sánchez-Alcázar, José Antonio

    2016-01-01

    Systemic treatments for hepatocellular carcinoma (HCC) have been largely unsuccessful. This study investigated the antitumoral activity of Amitriptyline, a tricyclic antidepressant, in hepatoma cells. Amitriptyline-induced toxicity involved early mitophagy activation that subsequently switched to apoptosis. Amitriptyline induced mitochondria dysfunction and oxidative stress in HepG2 cells. Amitriptyline specifically inhibited mitochondrial complex III activity that is associated with decreased mitochondrial membrane potential (∆Ψm) and increased reactive oxygen species (ROS) production. Transmission electron microscopy (TEM) studies revealed structurally abnormal mitochondria that were engulfed by double-membrane structures resembling autophagosomes. Consistent with mitophagy activation, fluorescence microscopy analysis showed mitochondrial Parkin recruitment and colocalization of mitochondria with autophagosome protein markers. Pharmacological or genetic inhibition of autophagy exacerbated the deleterious effects of Amitriptyline on hepatoma cells and led to increased apoptosis. These results suggest that mitophagy acts as an initial adaptive mechanism of cell survival. However persistent mitochondrial damage induced extensive and lethal mitophagy, autophagy stress and autophagolysome permeabilization leading eventually to cell death by apoptosis. Amitriptyline also induced cell death in hepatoma cells lines with mutated p53 and non-sense p53 mutation. Our results support the hypothesis that Amitriptyline-induced mitochondrial dysfunction can be a useful therapeutic strategy for HCC treatment, especially in tumors showing p53 mutations and/or resistant to genotoxic treatments. PMID:27738496

  11. Effects of PRELI in Oxidative-Stressed HepG2 Cells.

    PubMed

    Kim, Bo Yong; Cho, Min Ho; Kim, Kyung Joo; Cho, Kyung Jin; Kim, Suhng Wook; Kim, Hyun Sook; Jung, Woon-Won; Lee, Boo Hyung; Lee, Bong Hee; Lee, Seung Gwan

    2015-01-01

    Protein of relevant evolutionary and lymphoid interest (PRELI) is known for preventing apoptosis by mediating intramitochondrial transport of phosphatidic acid. However, the role of PRELI remains unclear. This study has demonstrated functions of PRELI through PRELI-knockdown in hepatocellular carcinoma (HepG2) cells exposed to oxidative stress by hydrogen peroxide. Results show that PRELI has three functions in HepG2 cells with regard to oxidative stress. First, PRELI affects expressional regulation of SOD-1 and caspase-3 genes in HepG2 cells. PRELI knockdown HepG2 cells have shown up-regulation of caspase-3 and down-regulation of SOD-1. Second, PRELI suppresses mitochondrial apoptosis in HepG2 cells. Fluorescence intensity related to mitochondrial apoptosis in PRELI-knockdown HepG2 cells increased more than two-fold compared to normal HepG2 cells. Third, PRELI suppresses senescence of HepG2 cells with oxidative stress. PRELI knockdown HepG2 cells showed higher levels of senescence than normal HepG2 cells. These results suggest that PRELI is a crucial protein in the suppression of apoptosis in HepG2 cells in response to oxidative stress. © 2015 by the Association of Clinical Scientists, Inc.

  12. Inferring Toxicological Responses of HepG2 Cells from ...

    EPA Pesticide Factsheets

    Understanding the dynamic perturbation of cell states by chemicals can aid in for predicting their adverse effects. High-content imaging (HCI) was used to measure the state of HepG2 cells over three time points (1, 24, and 72 h) in response to 976 ToxCast chemicals for 10 different concentrations (0.39-200µM). Cell state was characterized by p53 activation (p53), c-Jun activation (SK), phospho-Histone H2A.x (OS), phospho-Histone H3 (MA), alpha tubulin (Mt), mitochondrial membrane potential (MMP), mitochondrial mass (MM), cell cycle arrest (CCA), nuclear size (NS) and cell number (CN). Dynamic cell state perturbations due to each chemical concentration were utilized to infer coarse-grained dependencies between cellular functions as Boolean networks (BNs). BNs were inferred from data in two steps. First, the data for each state variable were discretized into changed/active (> 1 standard deviation), and unchanged/inactive values. Second, the discretized data were used to learn Boolean relationships between variables. In our case, a BN is a wiring diagram between nodes that represent 10 previously described observable phenotypes. Functional relationships between nodes were represented as Boolean functions. We found that inferred BN show that HepG2 cell response is chemical and concentration specific. We observed presence of both point and cycle BN attractors. In addition, there are instances where Boolean functions were not found. We believe that this may be either

  13. Effects of AFP gene silencing on Survivin mRNA expression inhibition in HepG2 cells.

    PubMed

    Fang, Z L; Fang, N; Han, X N; Huang, G; Fu, X J; Xie, G S; Wang, N R; Xiong, J P

    2015-04-10

    We investigated the effects of alpha-fetoprotein (AFP) gene silencing on Survivin expression in HepG2 cells. Small interfering RNA technology was used to downregulate AFP expression in HepG2 cells. An enzyme-linked immunosorbent assay was used to measure AFP concentration in the supernatant before and after transfection. An MTT assay was used to detect cell proliferation activity before and after transfection. We performed flow cytometric analysis to detect the cell apoptosis rate, and reverse transcription-polymerase chain reaction to detect Survivin mRNA levels before and after transfection. Forty-eight hours after transfection, AFP concentration in the supernatant of the experimental group significantly decreased, hepatocellular carcinoma cell growth was inhibited by 43.1%, and the apoptosis rate increased by 24.3%. Survivin mRNA expression was reduced by 78.0% in HepG2 cells. These indicators in the control group and in the blank group did not change significantly. Silencing of AFP expression in HepG2 cells can effectively inhibit the growth of hepatoma cells and promote apoptosis, which may be useful for reducing intracellular Survivin mRNA levels.

  14. Stable overexpression of pregnane X receptor in HepG2 cells increases its potential for bioartificial liver application.

    PubMed

    Nibourg, Geert A A; Huisman, Maarten T; van der Hoeven, Tessa V; van Gulik, Thomas M; Chamuleau, Robert A F M; Hoekstra, Ruurdtje

    2010-09-01

    To bridge patients with acute liver failure to transplantation or liver regeneration, a bioartificial liver (BAL) is urgently needed. A BAL consists of an extracorporeal bioreactor loaded with a bioactive mass that would preferably be of human origin and display high hepatic functionality, including detoxification. The human hepatoma cell line HepG2 exhibits many hepatic functions, but its detoxification function is low. In this study, we investigated whether stable overexpression of pregnane X receptor (PXR), a master regulator of diverse detoxification functions in the liver [eg, cytochrome P450 3A (CYP3A) activity], would increase the potential of HepG2 for BAL application. Stable overexpression was achieved by lentiviral expression of the human PXR gene, which yielded cell line cBAL119. In monolayer cultures of cBAL119 cells, PXR transcript levels increased 29-fold versus HepG2 cells. Upon activation of PXR by rifampicin, the messenger RNA levels of CYP3A4, CYP3A5, and CYP3A7 increased 49- to 213-fold versus HepG2 cells. According to reporter gene assays with different inducers, the highest increase in CYP3A4 promoter activity (131-fold) was observed upon induction with rifampicin. Inside BALs, the proliferation rates, as measured by the DNA content, were comparable between the 2 cell lines. The rate of testosterone 6beta-hydroxylation, a measure of CYP3A function inside BALs, increased 4-fold in cBAL119 BALs versus HepG2 BALs. Other functions, such as apolipoprotein A1 synthesis, urea synthesis, glucose consumption, and lactate production, remained unchanged or increased. Thus, stable PXR overexpression markedly increases the potential of HepG2 for BAL application. (c) 2010 AASLD.

  15. 40 GHz RF biosensor based on microwave coplanar waveguide transmission line for cancer cells (HepG2) dielectric characterization.

    PubMed

    Chen, Yu-Fu; Wu, Hung-Wei; Hong, Yong-Han; Lee, Hsin-Ying

    2014-11-15

    This paper presents a 40-GHz RF biosensor that involves using a microwave coplanar waveguide (CPW) transmission line for the dielectric characterization of cancer cells (Hepatoma G2, HepG2). In the past, conventional resonator-based biosensors were designed to operate at a specific resonant peak; however, the dielectric sensitivity of the cells was restricted to a narrow bandwidth. To provide a very wide bandwidth (1-40 GHz), biosensors were based on a microwave CPW transmission line. The proposed biosensor can rapidly measure two frequency-dependent cell-based dielectric parameters of HepG2 cells, microwave attenuation (α(f)cell) and the dielectric constant (εr(f)cell), while removing the microwave parasitic effects (including the cultured medium and substrate materials). The proposed biosensor can be applied in postoperative cancer diagnosis. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. [Observation of radiobiological characteristics in a HepG2 cell line with mitochondrial DNA deletion].

    PubMed

    Sun, Hengwen; Pan, Yi; Zeng, Zijun; Fang, Liangyi; Zhang, Hongdan; Xie, Songxi; Li, Weixiong; Xu, Jiabin

    2015-06-01

    To study the radiobiological characteristics of a HepG2 cell line with mitochondrial DNA (mtDNA) deletion. HepG2 cells were cultured in a medium containing ethidium bromide, acetylformic acid and uracil. The HepG2 cell line with mtDNA deletion (ρ(0)HepG2 cells) were acquired after 30 subcultures by limited dilution cloning. The cell survival was then observed in the absence of acetylformic acid and uracil, and the total mtDNA deletion in the cells was confirmed by PCR. The radiosensitivity of HepG2 and ρ(0)HepG2 cells was evaluated by exposure to gradient doses of 6 MV X ray irradiation. The cell apoptosis was assessed following a 2 Gy X-ray exposure with Hochest33342 staining, and the invasiveness of ρ(0)HepG2 cells was measured by Transwell assay. HepG2 cells could survive 30 subcultures in the presence of ethidium bromide, and massive cell death occurred after removal of acetylformic acid and uracil from the medium. PCR confirmed total mtDNA deletion from ρ(0)HepG2 cells, whose α/β value was significantly lower than that of HepG2 cells. ρ(0)Hep-G2 cells showed an obviously lowered cell apoptosis rate following X-ray exposure with enhanced cell invasiveness. HepG2 cells can be induced by ethidium bromide into ρ(0)HepG2 cells with an increased radiation resistance, anti-apoptosis ability and cell invasiveness.

  17. Role of metabolism by the human intestinal microflora in arbutin-induced cytotoxicity in HepG2 cell cultures.

    PubMed

    Khanal, Tilak; Kim, Hyung Gyun; Hwang, Yong Pil; Kong, Min Jeong; Kang, Mi Jeong; Yeo, Hee Kyung; Kim, Dong Hyun; Jeong, Tae Cheon; Jeong, Hye Gwang

    2011-09-23

    A possible role for metabolism by the human intestinal microflora in arbutin-induced cytotoxicity was investigated using human hepatoma HepG2 cells. When the cytotoxic effects of arbutin and hydroquinone (HQ), a deglycosylated metabolite of arbutin, were compared, HQ was more toxic than arbutin. Incubation of arbutin with a human fecal preparation could produce HQ. Following incubation of arbutin with a human fecal preparation for metabolic activation, the reaction mixture was filter-sterilized to test its toxic effects on HepG2 cells. The mixture induced cytotoxicity in HepG2 cells in a concentration-dependent manner. In addition, the mixture considerably inhibited expression of Bcl-2 together with an increase in Bax expression. Likewise, activation stimulated cleavage of caspase-3 and production of reactive oxygen species in HepG2 cell cultures. Furthermore, induction of apoptosis by the intestinal microflora reaction mixture was confirmed by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling assay. Taken together, these findings suggest that the human intestinal microflora is capable of metabolizing arbutin to HQ, which can induce apoptosis in mammalian cells. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Sodium cantharidinate induces HepG2 cell apoptosis through LC3 autophagy pathway.

    PubMed

    Tao, Ran; Sun, Wen-Yi; Yu, De-Hai; Qiu, Wei; Yan, Wei-Qun; Ding, Yan-Hua; Wang, Guang-Yi; Li, Hai-Jun

    2017-08-01

    The function of sodium cantharidinate on inducing hepatocellular carcinoma cell apoptosis was investigated for the first time. Sodium cantharidinate inhibits HepG2 cell growth mainly by LC3 autophagy pathway. MTT results show that sodium cantharidinate effectively inhibits the proliferation of HepG2 cells in a dose- and time-dependent manner and induce cell apoptosis by caspase-3 activity. The further western blotting and FACS detection show that sodium cantharidinate initiates HepG2 cell autophagy program by LC3 pathway. Autophagy-specific inhibitor 3-MA reduce sodium cantharidinate-induced caspase-3 activity and HepG2 cell apoptosis. Silence of the LC3 gene in HepG2 cell lines also reduce sodium cantharidinate-induced cell apoptosis. Collectively, our data indicate that sodium cantharidinate induces HepG2 cell apoptosis through LC3 autophagy pathway. Sodium cantharidinate has potential for development as a new drug for treatment of human HCC.

  19. Decorin inhibits the proliferation of HepG2 cells by elevating the expression of transforming growth factor-β receptor II.

    PubMed

    Liu, Yanfeng; Wang, Xuesong; Wang, Zhaohui; Ju, Wenbo; Wang, Dawei

    2016-10-01

    The aim of the present study was to investigate the effects of decorin (DCN) on the proliferation of human hepatoma HepG2 cells and the involvement of transforming growth factor-β (TGF-β) signaling pathway. A vector containing DCN was transfected into HepG2 cells with the use of Lipofectamine 2000. Cell proliferation was assessed with an MTT assay, and western blot analysis was used to detect the protein expression of TGF-β receptor I (TGF-βRI), phosphorylated TGF-βRI, p15 and TGF-βRII. In addition, small interfering RNA (siRNA) silencing was performed to knock down the target gene. The results indicated that, compared with the control group, cell proliferation was significantly decreased in HepG2 cells transfected with DCN. In addition, DCN transfection significantly increased the phosphorylation level of TGF-βRI in HepG2 cells. The expression of the downstream factor p15 was also significantly elevated in the DCN-transfected HepG2 cells. Furthermore, DCN transfection significantly elevated the expression level of TGF-βRII in HepG2 cells. By contrast, the silencing of TGF-βRII significantly decreased the phosphorylation of TGF-βRI in DCN-transfected HepG2 cells. In addition, TGF-βRII silencing abolished the effects of DCN on the proliferation of HepG2 cells. In conclusion, DCN elevated the expression level of TGF-βRII, increased the phosphorylation level of TGF-βRI, enhanced the expression of p15, and finally inhibited the proliferation of HepG2 cells. These findings may contribute to the understanding of the role of DCN in the pathogenesis of hepatic carcinoma and assist in the disease treatment.

  20. Synthesis of Functionalized Fluorescent Silver Nanoparticles and their toxicological effect in aquatic environments (Goldfish) and HEPG2 cells.

    NASA Astrophysics Data System (ADS)

    Santos, Hugo; Oliveira, Elisabete; Garcia-Pardo, Javier; Diniz, Mário; Lorenzo, Julia; Rodriguez-González, Benito; Capelo, José Luis; Lodeiro, Carlos

    2013-12-01

    Silver nanoparticles, AgNPs, are widely used in our daily life, mostly due to their antibacterial, antiviral and antifungal properties. However, their potential toxicity remains unclear. In order to unravel this issue, emissive AgNPs were first synthetized using an inexpensive photochemical method, and then their permeation was assessed in vivo in goldfish and in vitro in human hepatoma cells (HepG2). In addition, the oxidative stress caused by AgNPs was assessed in enzymes such as glutathione-S-transferase (GST), catalase (CAT) and in lipid peroxidation (LPO). This study demonstrates that the smallest sized AgNPs@3 promote the largest changes in gold fish livers, whereas AgNPs@1 were found to be toxic in HEPG2 cells depending on both the size and functionalized/stabilizer ligand.

  1. The effects of garlic-derived sulfur compounds on cell proliferation, caspase 3 activity, thiol levels and anaerobic sulfur metabolism in human hepatoblastoma HepG2 cells.

    PubMed

    Iciek, Małgorzata; Kwiecień, Inga; Chwatko, Grażyna; Sokołowska-Jeżewicz, Maria; Kowalczyk-Pachel, Danuta; Rokita, Hanna

    2012-04-01

    The aim of the present studies was to determine whether the mechanism of biological action of garlic-derived sulfur compounds in human hepatoma (HepG2) cells can be dependent on the presence of labile sulfane sulfur in their molecules. We investigated the effect of allyl sulfides from garlic: monosulfide, disulfide and trisulfide on cell proliferation and viability, caspase 3 activity and hydrogen peroxide (H(2)O(2)) production in HepG2 cells. In parallel, we also examined the influence of the previously mentioned compounds on the levels of thiols, glutathione, cysteine and cysteinyl-glycine, and on the level of sulfane sulfur and the activity of its metabolic enzymes: rhodanese, 3-mercaptopyruvate sulfurtransferase and cystathionase. Among the compounds under study, diallyl trisulfide (DATS), a sulfane sulfur-containing compound, showed the highest biological activity in HepG2 cells. This compound increased the H(2)O(2) formation, lowered the thiol level and produced the strongest inhibition of cell proliferation and the greatest induction of caspase 3 activity in HepG2 cells. DATS did not affect the activity of sulfurtransferases and lowered sulfane sulfur level in HepG2 cells. It appears that sulfane sulfur containing DATS can be bioreduced in cancer cells to hydroperthiol that leads to H(2)O(2) generation, thereby influencing transmission of signals regulating cell proliferation and apoptosis. Copyright © 2011 John Wiley & Sons, Ltd.

  2. Nile Red binding to HepG2 cells: an improved assay for in vitro studies of hepatosteatosis.

    PubMed

    McMillian, M K; Grant, E R; Zhong, Z; Parker, J B; Li, L; Zivin, R A; Burczynski, M E; Johnson, M D

    2001-01-01

    Nile Red is a fluorescent dye used extensively to study fat accumulation in many types of cells; unfortunately protocols that work well for most cells are not effective for studying drug-induced lipid accumulation in cultured liver cells and hepatocyte-derived cell lines. Using human hepatoma (HepG2) cells, we have developed a simple Nile Red binding assay as a screen for steatosis-inducing compounds. Increases in Nile Red binding in response to known hepatotoxic compounds were observed after incubating treated cells with 1 microM Nile Red for several hours, washing away free Nile Red, and then allowing redistribution, and/or clearance of the lipid-indicator dye. Several compounds known to cause hepatic fat accumulation in vivo were examined and most robustly increased Nile Red binding in HepG2 cells. These include estrogen and other steroids, ethionine, cyclosporin A, and valproic acid. Required concentrations for increased Nile Red binding were generally three-fold or more lower than the cytotoxic concentration determined by a resazurin reduction assay in the same cells. Qualitatively similar Nile Red binding results were obtained when primary canine or rat hepatocytes were used. Morphological differences in Nile Red staining were observed by confocal fluorescence microscopy in HepG2 cells after treatment with different compounds and likely reflect distinct toxicological mechanisms.

  3. Oroxylin A induced apoptosis of human hepatocellular carcinoma cell line HepG2 was involved in its antitumor activity

    SciTech Connect

    Hu Yang; Yang Yong; You Qidong . E-mail: qdyou@cpu.edu.cn; Liu Wei; Gu Hongyan; Zhao Li; Zhang Kun; Wang Wei; Wang Xiaotang; Guo Qinglong . E-mail: qinglongguo@hotmail.com

    2006-12-15

    We previously reported that wogonin, a flavonoid compound, was a potent apoptosis inducer of human hepatoma SMMC-7721 cells and murine sarcoma S180 cells. In the present study, the effect of oroxylin A, one wogonin structurally related flavonoid isolated from Scutellariae radix, on human hepatocellular carcinoma cell line HepG2 was examined and molecular mechanisms were also investigated. Oroxylin A inhibited HepG2 cell proliferation in a concentration- and time-dependent manner measured by MTT-assay. Treatment with an apoptosis-inducing concentration of oroxylin A caused typical morphological changes and apoptotic blebbing in HepG2 cells. DNA fragmentation assay was used to examine later apoptosis induced by oroxylin A. FACScan analysis revealed a dramatic increase in the number of apoptotic and G{sub 2}/M phase arrest cells after oroxylin A treatment. The pro-apoptotic activity of oroxylin A was attributed to its ability to modulate the concerted expression of Bcl-2, Bax, and pro-caspase-3 proteins. The expression of Bcl-2 protein and pro-caspase-3 protein was dramatically decreased after treatment with oroxylin A. These results demonstrated that oroxylin A could effectively induce programmed cell death and suggested that it could be a promising antitumor drug.

  4. Selectivity of biopolymer membranes using HepG2 cells.

    PubMed

    Lü, Dongyuan; Gao, Yuxin; Luo, Chunhua; Lü, Shouqian; Wang, Qian; Xu, Xianghong; Sun, Shujin; Wang, Chengzhi; Long, Mian

    2015-03-01

    Bioartificial liver (BAL) system has emerged as an alternative treatment to bridge acute liver failure to either liver transplantation or liver regeneration. One of the main reasons that the efficacy of the current BAL systems was not convincing in clinical trials is attributed to the lack of friendly interface between the membrane and the hepatocytes in liver bioreactor, the core unit of BAL system. Here, we systematically compared the biological responses of hepatosarcoma HepG2 cells seeded on eight, commercially available biocompatible membranes made of acetyl cellulose-nitrocellulose mixed cellulose (CA-NC), acetyl cellulose (CA), nylon (JN), polypropylene (PP), nitrocellulose (NC), polyvinylidene fluoride (PVDF), polycarbonate (PC) and polytetrafluoroethylene (PTFE). Physicochemical analysis and mechanical tests indicated that CA, JN and PP membranes yield high adhesivity and reasonable compressive and/or tensile features with friendly surface topography for cell seeding. Cells prefer to adhere on CA, JN, PP or PTFE membranes with high proliferation rate in spheriod-like shape. Actin, albumin and cytokeratin 18 expressions are favorable for cells on CA or PP membrane, whereas protein filtration is consistent among all the eight membranes. These results further the understandings of cell growth, morphology and spreading, as well as protein filtration on distinct membranes in designing a liver bioreactor.

  5. Effect of Fanbaicao (Herba Potentillae Discoloris) oil on the expression of p21 and CDK4 in HepG2 cells.

    PubMed

    Liu, Lei; Chen, Guang; Wang, Baixin; Chen, Liping; Wang, Shuqiu; Liu, Zhixin; Ma, Xiaoru; Wang, Fangfang; Liang, Yanfeng; Wu, Jiamei; Yang, Zhiwei

    2016-08-01

    To research the anti-cancer mechanism of the Traditional Chinese Medicine Fanbaicao (Herba Potentillae Discoloris) oil in the human hepatoma cell line HepG2. Gas chromatography was used to analyze the components of Fanbaicao (Herba Potentillae Discoloris). We tested the inhibitory effect of Fanbaicao (Herba Potentillae Discoloris) oil on the human hepatoma cell line HepG2 in vitro using 3-(4, 5-Dimet hylt hiazol-2-yl)-2,5-dip henyltetrazolium bromide assays. Fluorescence activating cell sorter analysis was used to examine the levels of apoptosis, and western blot and immunofluorescence were used to detect the expression of p21, p-p21 and CDK4 proteins. Fanbaicao (Herba Potentillae Discoloris) oil contains 45 ingredients, and L-ascorbic acid 2, 6-bispalmitate was the main component and accounted for 44.96% of total drive-off peak area. Other components included (Z)-14-met hyl-8-exadecenal- acetal (8.56%), phytol (7.74%) and lauric acid (6.31% ). Fanbaicao (Herba Potentillae Discoloris) oil treatment reduced the proliferation of HepG2 cells and the half growth inhibition concentration (IC50) was 2.03 mg/mL. Furthermore, we also observed significantly increased HepG2 cell apoptosis in a dose-dependent manner (P < 0.05). Fanbaicao (Herba Potentillae Discoloris) oil significantly increased the expression of p21 and p-p21 and significantly decreased the expression of CDK4 in HepG2 cells compared with controls (P < 0.01). Our results showed that Fanbaicao (Herba Potentillae Discoloris) oil has anti-cancer activities in HepG2 cells, which is probably related to the upregulation of p21 and p-p21 and downregulation of CDK4 expression.

  6. PLTP secreted by HepG2 cells resembles the high-activity PLTP form in human plasma.

    PubMed

    Siggins, Sarah; Jauhiainen, Matti; Olkkonen, Vesa M; Tenhunen, Jukka; Ehnholm, Christian

    2003-09-01

    Plasma phospholipid transfer protein (PLTP) is an important regulator of plasma HDL levels and HDL particle distribution. PLTP is present in plasma in two forms, one with high and the other with low phospholipid transfer activity. We have used the human hepatoma cell line, HepG2, as a model to study PLTP secreted from hepatic cells. PLTP activity was secreted by the cells into serum-free culture medium as a function of time. However, modification of a previously established ELISA assay to include a denaturing sample pretreatment with the anionic detergent sodium dodecyl sulphate was required for the detection of the secreted PLTP protein. The HepG2 PLTP could be enriched by Heparin-Sepharose affinity chromatography and eluted in size-exclusion chromatography at a position corresponding to the size of 160 kDa. PLTP coeluted with apolipoprotein E (apoE) but not with apoB-100 or apoA-I. A portion of PLTP was retained by an anti-apoE immunoaffinity column together with apoE, suggesting an interaction between these two proteins. Furthermore, antibodies against apoE but not those against apoB-100 or apoA-I were capable of inhibiting PLTP activity. These results show that the HepG2-derived PLTP resembles in several aspects the high-activity form of PLTP found in human plasma.

  7. Macelignan protects HepG2 cells against tert-butylhydroperoxide-induced oxidative damage.

    PubMed

    Sohn, Jong Hee; Han, Kyu Lee; Choo, Jeong Han; Hwang, Jae-Kwan

    2007-01-01

    In this study, we investigated the protective effect of macelignan, isolated from Myristica fragrans Houtt. (nutmeg) against tert-butylhydroperoxide (t-BHP)-induced cytotoxicity in a human hepatoma cell line, HepG2. The tetrazolium dye colorimetric test (MTT test) and lactate dehydrogenase (LDH) assay were used to monitor cell viability and necrosis, respectively. Lipid peroxidation [malondialdehyde (MDA) formation] was estimated by the fluorometric method. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA), and DNA damage was detected using single cell gel electrophoresis (comet assay). The results showed that macelignan significantly reduced the cell growth inhibition and necrosis caused by t-BHP. Furthermore, macelignan ameliorated lipid peroxidation as demonstrated by a reduction in MDA formation in a dose-dependent manner. It was also found that macelignan reduced intracellular ROS formation and DNA damaging effect caused by t-BHP. These results strongly suggest that macelignan has significant protective ability against oxidative damage caused by reactive intermediates.

  8. Cytotoxicity and induction of protective mechanisms in HepG2 cells exposed to cadmium.

    PubMed

    Urani, C; Melchioretto, P; Canevali, C; Crosta, G F

    2005-10-01

    Cadmium is a widespread industrial pollutant. The primary route of exposure occurs via contaminated drinking water or food supplies, and tobacco. Its chronic introduction and ingestion lead to bio-magnification in target organs, as the liver. The aim of this paper is to determine Cd cytotoxic concentrations in the human hepatoma cell line HepG2. Further aims are the study of the activation and involvement of protection mechanisms against Cd hepatotoxicity. Cd was accumulated within the cells, as measured by ICP-AES. Metallothioneins (MT-1 and -2), a family of metal-binding proteins, were induced in a dose-dependent way after treatment with concentrations below the IC(50) value (mean value 22 microM). The over-expression of MT by Zn pre-treatment was able to defend against Cd cytotoxicity. Heat shock protein 70 kDa (hsp70) was induced at high non-cytotoxic concentrations (5, 10 microM) probably as a consequence of proteotoxicity, but its over-expression by a sub-lethal heat shock was not able to protect the cells from Cd cytotoxic concentrations (20, 50, 100 microM).

  9. TMEM2 inhibits hepatitis B virus infection in HepG2 and HepG2.2.15 cells by activating the JAK–STAT signaling pathway

    PubMed Central

    Zhu, X; Xie, C; Li, Y-m; Huang, Z-l; Zhao, Q-y; Hu, Z-x; Wang, P-p; Gu, Y-r; Gao, Z-l; Peng, L

    2016-01-01

    We have previously observed the downregulation of TMEM2 in the liver tissue of patients with chronic hepatitis B virus (HBV) infection and in HepG2.2.15 cells with HBV genomic DNA. In the present study, we investigated the role and mechanism of TMEM2 in HepG2 and HepG2.2.15 during HBV infection HepG2 and HepG2.2.15. HepG2 shTMEM2 cells with stable TMEM2 knockdown and HepG2 TMEM2 and HepG2.2.15 TMEM2 cells with stable TMEM2 overexpression were established using lentivirus vectors. We observed reduced expression of TMEM2 in HBV-infected liver tissues and HepG2.2.15 cells. HBsAg, HBcAg, HBV DNA, and HBV cccDNA levels were significantly increased in HepG2 shTMEM2 cells but decreased in HepG2 TMEM2 and HepG2.2.15 TMEM2 cells compared with naive HepG2 cells. On the basis of the western blotting results, the JAK–STAT signaling pathway was inhibited in HepG2 shTMEM2 cells but activated in HepG2 TMEM2 and HepG2.2.15 TMEM2 cells. In addition, reduced and increased expression of the antiviral proteins MxA and OAS1 was observed in TMEM2-silenced cells (HepG2 shTMEM2 cells) and TMEM2-overexpressing cells (HepG2 TMEM2 and HepG2.2.15 TMEM2 cells), respectively. The expression of Interferon regulatory factor 9 (IRF9) was not affected by TMEM2. However, we found that overexpression and knockdown of TMEM2, respectively, promoted and inhibited importation of IRF9 into nuclei. The luciferase reporter assay showed that IRF9 nuclear translocation affected interferon-stimulated response element activities. In addition, the inhibitory effects of TMEM2 on HBV infection in HepG2 shTMEM2 cells was significantly enhanced by pre-treatment with interferon but significantly inhibited in HepG2.2.15 TMEM2 cells by pre-treatment with JAK1 inhibitor. TMEM2 inhibits HBV infection in HepG2 and HepG2.2.15 by activating the JAK–STAT signaling pathway. PMID:27253403

  10. TMEM2 inhibits hepatitis B virus infection in HepG2 and HepG2.2.15 cells by activating the JAK-STAT signaling pathway.

    PubMed

    Zhu, X; Xie, C; Li, Y-M; Huang, Z-L; Zhao, Q-Y; Hu, Z-X; Wang, P-P; Gu, Y-R; Gao, Z-L; Peng, L

    2016-06-02

    We have previously observed the downregulation of TMEM2 in the liver tissue of patients with chronic hepatitis B virus (HBV) infection and in HepG2.2.15 cells with HBV genomic DNA. In the present study, we investigated the role and mechanism of TMEM2 in HepG2 and HepG2.2.15 during HBV infection HepG2 and HepG2.2.15. HepG2 shTMEM2 cells with stable TMEM2 knockdown and HepG2 TMEM2 and HepG2.2.15 TMEM2 cells with stable TMEM2 overexpression were established using lentivirus vectors. We observed reduced expression of TMEM2 in HBV-infected liver tissues and HepG2.2.15 cells. HBsAg, HBcAg, HBV DNA, and HBV cccDNA levels were significantly increased in HepG2 shTMEM2 cells but decreased in HepG2 TMEM2 and HepG2.2.15 TMEM2 cells compared with naive HepG2 cells. On the basis of the western blotting results, the JAK-STAT signaling pathway was inhibited in HepG2 shTMEM2 cells but activated in HepG2 TMEM2 and HepG2.2.15 TMEM2 cells. In addition, reduced and increased expression of the antiviral proteins MxA and OAS1 was observed in TMEM2-silenced cells (HepG2 shTMEM2 cells) and TMEM2-overexpressing cells (HepG2 TMEM2 and HepG2.2.15 TMEM2 cells), respectively. The expression of Interferon regulatory factor 9 (IRF9) was not affected by TMEM2. However, we found that overexpression and knockdown of TMEM2, respectively, promoted and inhibited importation of IRF9 into nuclei. The luciferase reporter assay showed that IRF9 nuclear translocation affected interferon-stimulated response element activities. In addition, the inhibitory effects of TMEM2 on HBV infection in HepG2 shTMEM2 cells was significantly enhanced by pre-treatment with interferon but significantly inhibited in HepG2.2.15 TMEM2 cells by pre-treatment with JAK1 inhibitor. TMEM2 inhibits HBV infection in HepG2 and HepG2.2.15 by activating the JAK-STAT signaling pathway.

  11. C-Phycocyanin inhibits cell proliferation and may induce apoptosis in human HepG2 cells.

    PubMed

    Basha, Osama M; Hafez, Raghda A; El-Ayouty, Yassin M; Mahrous, Karima F; Bareedy, Mohammed H; Salama, Ahmed M

    2008-01-01

    C-Phycocyanin (C-Pc) is one of the major biliprotein pigments of unicellular cyanbacterium of Spirulina platenesis, it has nutritional, medicinal, and hepatoprotectant application. The growth and multiplication of human hepatoma cell lines (HepG2) under the effect of different concentrations of C-PC (0.8, 1.75, 3.5 and 7.0 microg/ml) against untreated cells as control for 24h were investigated. The results showed that the proliferating cells in presence of C-PC reached 70, 51, 44, and 39%, respectively. The results revealed that the greatest reduction in proliferation of cells was recorded at 7.0 microg/ml and LC50 at 1.75 microg/ml of C-PC. In parallel, to the previous results HCl-denatured MG-P revealed that in mass of cells there is a pattern of apoptosis because the expanded cytoplasmic area (bluish-green) reduced and appeared faintly red as C-PC concentration increased. Moreover, the cells lost all the nuclear entities then, become fragmented and having no nuclear remnants. The C-PC may be a new potential anti-cancer drug for therapy of human hepatoma cells.

  12. XPD Functions as a Tumor Suppressor and Dysregulates Autophagy in Cultured HepG2 Cells.

    PubMed

    Zheng, Jian-Feng; Li, Lin-Lin; Lu, Juan; Yan, Kun; Guo, Wu-Hua; Zhang, Ji-Xiang

    2015-05-29

    Recent clinical studies have linked polymorphisms in the xeroderma pigmentosum group D (XPD) gene, a key repair gene involved in nucleotide excision repair, to increased risk of hepatocellular carcinoma (HCC). However, the cellular effects of XPD expression in cultured HCC cells remain largely uncharacterized. Therefore, the aim of this study was to characterize the in vitro cellular effects of XPD expression on the HCC cell line HepG2. HepG2 cells were transfected as follows to create four experimental groups: pEGFP-N2/XPD plasmid (XPD) group, EGFP-N2 plasmid (N2) control group, lipofectamine™ 2000 (lipid) control group, and non-transfected (CON) control group. An MTT cell proliferation assay, Annexin V-APC apoptosis assay, colony formation assay, scratch wound migration assay, Transwell migration assay, and Western blotting of the autophagic proteins LC3 and p62 were conducted. XPD expression significantly inhibited HepG2 cell proliferation (p<0.05), significantly promoted HepG2 cell apoptosis (p<0.05), significantly inhibited HepG2 colony formation (p<0.05), significantly decreased HepG2 cells' migratory ability (p<0.05), and significantly lowered HepG2 cells' invasive capacity (p<0.05). Western blotting showed that XPD expression significantly increased LC3 expression (p<0.05) and significantly reduced p62 expression (p<0.05). XPD expression serves as a tumor suppressor and dysregulates autophagic protein degradation in HepG2 cells in vitro. Further in vivo pre-clinical studies and clinical trials are needed to validate XPD's potential as a tumor-suppressive gene therapy.

  13. VCC-1 over-expression inhibits cisplatin-induced apoptosis in HepG2 cells

    SciTech Connect

    Zhou, Zhitao; Lu, Xiao; Zhu, Ping; Zhu, Wei; Mu, Xia; Qu, Rongmei; Li, Ming

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer VCC-1 is hypothesized to be associated with carcinogenesis. Black-Right-Pointing-Pointer Levels of VCC-1 are increased significantly in HCC. Black-Right-Pointing-Pointer Over-expression of VCC-1 could promotes cellular proliferation rate. Black-Right-Pointing-Pointer Over-expression of VCC-1 inhibit the cisplatin-provoked apoptosis in HepG2 cells. Black-Right-Pointing-Pointer VCC-1 plays an important role in control the tumor growth and apoptosis. -- Abstract: Vascular endothelial growth factor-correlated chemokine 1 (VCC-1), a recently described chemokine, is hypothesized to be associated with carcinogenesis. However, the molecular mechanisms by which aberrant VCC-1 expression determines poor outcomes of cancers are unknown. In this study, we found that VCC-1 was highly expressed in hepatocellular carcinoma (HCC) tissue. It was also associated with proliferation of HepG2 cells, and inhibition of cisplatin-induced apoptosis of HepG2 cells. Conversely, down-regulation of VCC-1 in HepG2 cells increased cisplatin-induced apoptosis of HepG2 cells. In summary, these results suggest that VCC-1 is involved in cisplatin-induced apoptosis of HepG2 cells, and also provides some evidence for VCC-1 as a potential cellular target for chemotherapy.

  14. Polyphyllin I (PPI) increased the sensitivity of hepatocellular carcinoma HepG2 cells to chemotherapy

    PubMed Central

    Han, Wenhao; Hou, Guoxin; Liu, Lei

    2015-01-01

    In this study the antitumor effects of polyphyllin I (PPI) were investigated in hepatocellular carcinoma HepG2 cells. Our data showed that PPI treatment exerted dose-dependent cytotoxicity on HepG2 cells as previously reported. Furthermore, PPI could sensitize HepG2 cells to cisplastin treatment in concentration-dependent manner. The molecular mechanisms of PPI actions involved nuclear factor-κB (NF-κB) and its downstream gene products. PPI treatment dose-dependently could decrease the constitutive phosphorylation of NF-κB subunit p65 protein and its downstream target genes expression, such as Bcl-2, c-Myc and VEGF. PPI could also inhibit cisplatin-evoked increase of p65 protein phosphorylation and its downstream genes expression, which could be further decreased by combination with NF-κB specific inhibitor, PDTC. The cytotoxicity and chemosensitization effects of PPI on HepG2 cells were greatly potentiated by concomitant treatment with PDTC. Taken together, our data confirmed the cytotoxicity of PPI on hepatocellular carcinoma HepG2 cells and provided new findings about PPI sensitizing HepG2 cells to chemotherapy. Moreover, our data also indicated the involvement of NF-κB signaling pathway in PPIactions for the first time. PMID:26884988

  15. Validation of in vitro cell models used in drug metabolism and transport studies; genotyping of cytochrome P450, phase II enzymes and drug transporter polymorphisms in the human hepatoma (HepG2), ovarian carcinoma (IGROV-1) and colon carcinoma (CaCo-2, LS180) cell lines

    SciTech Connect

    Brandon, Esther F.A.; Bosch, Tessa M.; Deenen, Maarten J.; Levink, Rianne; Wal, Everdina van der; Meerveld, Joyce B.M. van; Bijl, Monique; Beijnen, Jos H. |; Schellens, Jan H.M. |; Meijerman, Irma . E-mail: I.Meijerman@pharm.uu.nl

    2006-02-15

    Human cell lines are often used for in vitro biotransformation and transport studies of drugs. In vivo, genetic polymorphisms have been identified in drug-metabolizing enzymes and ABC-drug transporters leading to altered enzyme activity, or a change in the inducibility of these enzymes. These genetic polymorphisms could also influence the outcome of studies using human cell lines. Therefore, the aim of our study was to pharmacogenotype four cell lines frequently used in drug metabolism and transport studies, HepG2, IGROV-1, CaCo-2 and LS180, for genetic polymorphisms in biotransformation enzymes and drug transporters. The results indicate that, despite the presence of some genetic polymorphisms, no real effects influencing the activity of metabolizing enzymes or drug transporters in the investigated cell lines are expected. However, this characterization will be an aid in the interpretation of the results of biotransformation and transport studies using these in vitro cell models.

  16. Effects of 3-methylcholanthrene and aspirin co-administration on ALDH3A1 in HepG2 cells.

    PubMed

    Sotiropoulou, M; Pappas, P; Marselos, M

    2001-01-30

    The effects of two different protocols of 3-methylcholanthrene (3MC) and aspirin co-administration were studied in a well-established human hepatoma cell line (HepG2). During this work, we have performed toxicity tests for cell viability/cell proliferation as well as studies on the expression of ALDH3A1 after exposure of HepG2 cells to 3MC or/and aspirin. For the evaluation of toxic concentrations of 3MC and aspirin, the WST-1 test was used. WST-1 is a reliable cytotoxicity test which is based on the cleavage of the tetrazolium salt WST-1 to formazan by mitochondrial enzymes of living cells. A broad range of drug concentrations for either 3MC (0.25-50.0 microM) or aspirin (0.05-10.0 mM) were used for cell exposure, in several periods of time. The expression of ALDH3A1 in HepG2 cells showed typical time- and dose-response curves of induction after application of 3MC (1-5 days, 1.5-5.0 microM, respectively). When cells were firstly exposed to 3MC (2.5 and 5.0 microM) and then to aspirin (0.25 mM), the induced ALDH3A1 activity was further enhanced in a statistically significant way (P<0.05). On the contrary, when aspirin application was preceded 3MC exposuring a statistically significant decrease in ALDH3A1 inducibility was observed, as compared with the application of 3MC alone.

  17. Antitumor effects of polysaccharide from Sargassum fusiforme against human hepatocellular carcinoma HepG2 cells.

    PubMed

    Fan, Sairong; Zhang, Junfeng; Nie, Wenjian; Zhou, Wenyuan; Jin, Liqin; Chen, Xiaoming; Lu, Jianxin

    2017-04-01

    Sargassum fusiforme (Harv.) Setchel, a kind of brown algae, has been applied as a therapeutic for thousands of years. This study was designed to investigate the antitumor effects of the polysaccharide (SFPS) from S. fusiform in liver cancer. The mice inoculated with HepG2 cells were orally administrated with SFPS at the doses of 100, 200 and 400 mg/kg body weight for 28 days. The products from peritoneal macrophages and serum in HepG2-bearing mice were measured. The effect of SFPS-induced cell apoptosis was measured by flow cytometry. Meanwhile, the expression levels of Bax and Bcl-2 were detected. Furthermore, the cytotoxicity of SFPS was evaluated by CCK-8 assay. Results showed that SFPS significantly inhibited growth of human HepG2 cell-transplanted tumor in nude mice, and remarkably increased serum TNF-α, IL-1, NO and IgM levels in HepG2-bearing mice. SFPS also promoted the cytokines (IL-1 and TNF-α) secreted by peritoneal macrophages in HepG2-bearing mice. SFPS exerted a stimulatory effect on apoptosis of HepG2 cells, increased the expression of Bax, and decreased the expression of Bcl-2. The results indicated that SFPS has anti-tumor and immunomodulatory activities at the high concentration, and it could be used as a potential chemopreventative and/or adjuvant chemotherapeutic agent in liver cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Role of ALA sensitivity in HepG2 cell in the presence of diode laser

    NASA Astrophysics Data System (ADS)

    Fakhar-E-Alam, M.; Atif, M.; Alsalhi, M. S.; Siddique, M.; Kishwar, S.; Qadir, M. I.; Willander, M.

    2011-05-01

    5-aminolevulinic acid (ALA) being an amazing second generation photosensitizer was studied as photodamaging drug on hepatocellular carcinoma (HepG2) cells. The mentioned photosensitizer is converted to PpIX in HepG2 cells in vitro, inducing haem in the cell causing generation of singlet oxygen leading to cell apoptosis. Cell uptake of 5-ALA was evaluated with different concentrations (ranging from 0-800 μg/ml) for 0-49 h incubation period. ALA administered in HepG2 cells is converted into Protoporphyrin IX (PpIX) which has a short half life and constitute a good hematoporphyrin derivative (HPD). Cytotoxicity of ALA in dark and cellular viability without ALA in the presence of light was studied, showing minimal toxic effects in dark with no photodamaging effect on mentioned cells in absence of ALA were observed. The optimal uptake of photosensitizer (5-ALA) in HepG2 cells was investigated by means of spectrophotometeric measurements, cellular viability was determined by means of neutral red assay (NRA). It was observed that with different concentrations (0-800 μg/ml) of ALA or light doses (0-160 J/cm2), there were no significant effect on cellular viability when studied independently. The novel of photocytotoxic study indicates that light dose of 120 J/cm2 produces convincing Photodynamic therapy (PDT) results for HepG2 cells incubated with 262 μg/ml of 5-ALA deducting that HepG2 cell line is sensitive to ALA mediated PDT. Finally morphological changes in HePG2 cells were determined before and after ALA-mediated PDT by confocal microscopy.

  19. Differential effect of manool--a diterpene from Salvia officinalis, on genotoxicity induced by methyl methanesulfonate in V79 and HepG2 cells.

    PubMed

    Nicolella, Heloiza Diniz; de Oliveira, Pollyanna Francielli; Munari, Carla Carolina; Costa, Gizela Faleiros Dias; Moreira, Monique Rodrigues; Veneziani, Rodrigo Cassio Sola; Tavares, Denise Crispim

    2014-10-01

    Salvia officinalis (sage) is a perennial woody subshrub native to the Mediterranean region that is commonly used as a condiment and as an anti-inflammatory, antioxidant and antimicrobial agent due to its biological activities. Manool is the most abundant micro-metabolite found in Salvia officinalis essential oils and extracts. We therefore decided to evaluate the cytotoxic, genotoxic and antigenotoxic potential of manool in Chinese hamster lung fibroblasts (V79) and human hepatoma cells (HepG2). Cytotoxicity was assessed by the colony-forming assay in V79 cells and toxic effects were observed at concentrations of up to 8.0 μg/mL. The micronucleus test was used to evaluate the genotoxicity and antigenotoxicity of manool in V79 and HepG2 cells at concentrations of 0.5-6.0 μg/mL and 0.5-8.0 μg/mL, respectively. For evaluation of antigenotoxicity, the concentrations of manool were combined with methyl methanesulfonate (MMS, 44 μg/mL). The results showed a significant increase in the frequency of micronuclei in cultures of both cell lines treated with the highest concentration tested, demonstrating a genotoxic effect. On the other hand, manool exhibited a protective effect against chromosome damage induced by MMS in HepG2 cells, but not in V79 cells. These data suggest that some manool metabolite may be responsible for the antigenotoxic effect observed in HepG2 cells.

  20. [3D evaluation model for drug hepatotoxicity testing on HepG2 cells and its application in drug safety evaluation].

    PubMed

    Li, Dan-Dan; Tang, Xiang-Lin; Tan, Hong-Ling; Liang, Qian-de; Wang, Yu-Guang; Ma, Zeng-Chun; Xiao, Cheng-Rong; Gao, Yue

    2016-04-01

    3D in vitro toxicity testing model was developed by magnetic levitation method for culture of the human hepatoma cell line HepG2 and applied to evaluate the drug hepatotoxicity. After formation of stable 3D structure for HepG2 cells, their glycogen storage capacity under 2D and 3D culture conditions were detected by immunohistochemistry technology, and the mRNA expression levels of phase Ⅰ and Ⅱ drug metabolism enzymes, drug transporters, nuclear receptors and liver-specific marker albumin(ALB) were compared between 2D and 3D culture conditions by using RT-PCR method. Immunohistochemistry results showed that HepG2 cells had abundant glycogen storage capacity under 3D culture conditions, which was similar to human liver tissues. The mRNA expression levels of major drug metabolism enzymes, drug transporters, nuclear receptors and ALB in HepG2 cells under 3D culture conditions were up-regulated as compared with 2D culture conditions. For drug hepatotoxicity evaluation, the typical hepatotoxic drug acetaminophen(APAP), and most reported drugs Polygonum multiflorum Thunb.(Chinese name He-shou-wu) and Psoraleae corylifolia L.(Chinese name Bu-gu-zhi) were selected for single dose and repeated dose(7 d) exposure. In the repeated dose exposure test, 3D HepG2 cells showed higher sensitivity. This established 3D HepG2 cells model with magnetic levitation 3D culture techniques was more close to the human liver tissues both in morphology and functions, so it was a better 3D hepatotoxicity evaluation model. Copyright© by the Chinese Pharmaceutical Association.

  1. Free radical generation from an aniline derivative in HepG2 cells: a possible captodative effect.

    PubMed

    Horinouchi, Yuya; Summers, Fiona A; Ehrenshaft, Marilyn; Mason, Ronald P

    2015-01-01

    Xenobiotic metabolism can induce the generation of protein radicals, which are believed to play an important role in the toxicity of chemicals and drugs. It is therefore important to identify chemical structures capable of inducing macromolecular free radical formation in living cells. In this study, we evaluated the ability of four structurally related environmental chemicals, aniline, nitrosobenzene, N,N-dimethylaniline, and N,N-dimethyl-4-nitrosoaniline (DMNA), to induce free radicals and cellular damage in the hepatoma cell line HepG2. Cytotoxicity was assessed using lactate dehydrogenase assays, and morphological changes were observed using phase contrast microscopy. Protein free radicals were detected by immuno-spin trapping using in-cell western experiments and confocal microscopy to determine the subcellular locale of free radical generation. DMNA induced free radical generation, lactate dehydrogenase release, and morphological changes in HepG2 cells, whereas aniline, nitrosobenzene, N,N-dimethylaniline did not. Confocal microscopy showed that DMNA induced free radical generation mainly in the cytosol. Preincubation of HepG2 cells with N-acetylcysteine and 2,2'-dipyridyl significantly prevented free radical generation on subsequent incubation with DMNA, whereas preincubation with apocynin and dimethyl sulfoxide had no effect. These results suggest that DMNA is metabolized to reactive free radicals capable of generating protein radicals which may play a critical role in DMNA toxicity. We propose that the captodative effect, the combined action of the electron-releasing dimethylamine substituent, and the electron-withdrawing nitroso substituent, leads to a thermodynamically stabilized radical, facilitating enhanced protein radical formation by DMNA.

  2. Inhibition of aldose reductase ameliorates ethanol‑induced steatosis in HepG2 cells.

    PubMed

    Qiu, Longxin; Cai, Chengchao; Zhao, Xiangqian; Fang, Yan; Tang, Weibiao; Guo, Chang

    2017-05-01

    Aldose reductase (AR) expression is increased in liver tissue of patients with ethanol‑induced liver disease. However, the exact role of AR in the development of ethanol‑induced liver disease has yet to be elucidated. The present study aimed to determine the effect of an AR inhibitor on ethanol‑induced steatosis in HepG2 cells and to identify possible underlying molecular mechanisms. Steatosis was induced in HepG2 cells by stimulating cells with 100 mM absolute ethanol for 48 h. Oil Red O staining was used to detect the lipid droplet accumulation in cells. Western blot analyses were used to determine protein expression levels and reverse transcription‑quantitative polymerase chain reaction was used to analyze mRNA expression levels. The results showed that AR protein expression was elevated in HepG2 cells stimulated with ethanol. HepG2 cells exhibited marked improvement of ethanol‑induced lipid accumulation following treatment with the AR inhibitor zopolrestat. Phosphorylation levels of 5' adenosine monophosphate‑activated protein kinase (AMPK) were markedly higher, whereas the mRNA expression levels of sterol‑regulatory element‑binding protein (SREBP)‑1c and fatty acid synthase (FAS) were significantly lower in zopolrestat‑treated and ethanol‑stimulated HepG2 cells compared with in untreated ethanol‑stimulated HepG2 cells. In addition, zopolrestat inhibited the ethanol‑induced expression of tumor necrosis factor (TNF)‑α. These results suggested that zopolrestat attenuated ethanol‑induced steatosis by activating AMPK and subsequently inhibiting the expression of SREBP‑1c and FAS, and by suppressing the expression of TNF‑α in HepG2 cells.

  3. A comparison of apoptosis and necrosis induced by hepatotoxins in HepG2 cells.

    PubMed

    O'Brien, T; Babcock, G; Cornelius, J; Dingeldein, M; Talaska, G; Warshawsky, D; Mitchell, K

    2000-05-01

    7H-Dibenzo[c,g]carbazole (DBC), an N-heterocyclic aromatic hydrocarbon, is cytotoxic and carcinogenic in rodent liver. While DBC leads to necrotic lesions in the liver, the induction of apoptosis by DBC has not been investigated. The focus of this study was to determine the degree to which apoptosis and necrosis contributed to DBC cytotoxicity in a human hepatoma cell line (HepG2). To determine if these effects were unique to DBC, the results were compared to another hepatotoxin, aflatoxin B(1) (AFB(1)). DBC produced a distinct biphasic LDH release curve within 24 h of exposure. During the same time period lower concentrations of DBC (<10 microM) induced the formation of DBC-DNA adducts and increased p53 protein levels followed by apoptotic cell death. However, increasing the concentration of DBC to 80 microM led to lower DNA adduct and p53 protein levels. At this concentration, intracellular ATP levels were rapidly depleted followed by cell swelling and loss of membrane integrity consistent with necrotic cell death. In contrast to DBC, a biphasic LDH release curve was not observed for AFB(1). Instead, AFB(1) induced a concentration-dependent increase in apoptosis that reached two- to threefold higher levels than DBC. These results suggest that differences exist in the extent and type of cell death induced by DBC and AFB(1) at equimolar concentrations. Apoptosis and necrosis result from low and high concentrations of DBC, respectively, and may be dependent upon intracellular ATP levels. Copyright 2000 Academic Press.

  4. Altered cellular metabolism of HepG2 cells caused by microcystin-LR.

    PubMed

    Ma, Junguo; Feng, Yiyi; Jiang, Siyu; Li, Xiaoyu

    2017-06-01

    This study aimed to evaluate the possible effects of microcystin-LR (MC-LR) exposure on the metabolism and drug resistance of human hepatocellular carcinoma (HepG2) cells. For this purpose, we first conducted an experiment to make sure that MC-LR could penetrate the HepG2 cell membrane effectively. The transcriptional levels of phase I (such as CYP2E1, CYP3A4, and CYP26B1) and phase II (such as EPHX1, SULTs, and GSTM) enzymes and export pump genes (such as MRP1 and MDR1) were altered by MC-LR-exposure for 24 h, indicating that MC-LR treatment may destabilize the metabolism of HepG2 cells. Further research showed that the CYP inducers omeprazole, ethanol, and rifampicin inhibited cell viability, in particular, ethanol, a CYP2E1 inducer, induced ROS generation, lipid peroxidation, and apoptosis in HepG2 cells treated with MC-LR. The CYP2E1 inhibitor chlormethiazole inhibited ROS generation, mitochondrial membrane potential loss, caspase-3 activity, and cytotoxicity caused by MC-LR. Meanwhile, the results also showed that co-incubation with the ROS scavenger l-ascorbic acid and MC-LR decreased ROS levels and effectively prevented apoptosis. These findings provide an interesting mechanistic explanation of cellular metabolism associated with MC-LR, i.e., MC-LR-exposure exerted toxicity on HepG2 cells and induced apoptosis of HepG2 cells via promoting CYP2E1 expression and inducing excessive ROS in HepG2 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Genistein and daidzein induced apoA-1 transactivation in hepG2 cells expressing oestrogen receptor-alpha.

    PubMed

    Yuen, Yee M; Leung, Lai K

    2008-05-01

    Studies have shown that soya consumption has been associated with low incidence of CVD. Because the chemical structures of soya isoflavones are similar to oestrogen, the beneficial outcome may be attributed to the oestrogenicity of these compounds. In this study, effect of the soya isoflavone genistein on the mRNA expression of apoA-1 in the human hepatoma HepG2 cell was investigated. Without oestrogen receptor (ER) alpha transfection, soya isoflavones in the physiological range had no effect on the apoA-1 transcription. Once ERalpha was ectopically expressed in these cells, soya isoflavone dramatically increased the apoA-1 mRNA abundance quantified by real-time PCR. ApoA-1-reporter assays with plasmid constructed from the 5'-flanking segment upstream to the coding region revealed that the transactivation of the apoA-1 promoter was induced by the soya isoflavone in HepG2 cells expressing ERalpha. This induction was reduced by the anti-oestrogen ICI 182780, but not the inhibitors of protein kinase (PK) C, PKA, or mitogen-activated PK. Based on the previously identified response elements on the promoter, a series of truncated promoter reporter plasmids were then constructed. An induction profile of genistein was built and insulin response core element at -411 to -404 appeared to be a potential site of interaction. This study illustrated that soya isoflavones at physiological concentrations could up regulate apoA-1 mRNA expression in ERalpha-transfected HepG2 cells.

  6. Glucose inhibits the insulin-induced activation of the insulin-degrading enzyme in HepG2 cells.

    PubMed

    Pivovarova, O; Gögebakan, O; Pfeiffer, A F H; Rudovich, N

    2009-08-01

    Hepatic insulin degradation decreases in type 2 diabetes. Insulin-degrading enzyme (IDE) plays a key role in insulin degradation and its gene is located in a diabetes-associated chromosomal region. We hypothesised that IDE may be regulated by insulin and/or glucose in a liver cell model. To validate the observed regulation of IDE in vivo, we analysed biopsies of human adipose tissue during different clamp experiments in men. Human hepatoma HepG2 cells were incubated in normal (1 g/l) or high (4.5 g/l) glucose medium and treated with insulin for 24 h. Catalytic activity, mRNA and protein levels of IDE were assessed. IDE mRNA levels were measured in biopsies of human subcutaneous adipose tissue before and at 240 min of hyperinsulinaemic, euglycaemic and hyperglycaemic clamps. In HepG2 cells, insulin increased IDE activity under normal glucose conditions with no change in IDE mRNA or protein levels. Under conditions of high glucose, insulin increased mRNA levels of IDE without changes in IDE activity. Both in normal and high glucose medium, insulin increased levels of the catalytically more active 15a IDE isoform compared with the 15b isoform. In subcutaneous adipose tissue, IDE mRNA levels were not significantly upregulated after euglycaemic or hyperglycaemic clamps. Insulin increases IDE activity in HepG2 cells in normal but not in high glucose conditions. This disturbance cannot be explained by corresponding alterations in IDE protein levels or IDE splicing. The loss of insulin-induced regulation of IDE activity under hyperglycaemia may contribute to the reduced insulin extraction and peripheral hyperinsulinaemia in type 2 diabetes.

  7. Participation of lipid transport and fatty acid metabolism in valproate sodium-induced hepatotoxicity in HepG2 cells.

    PubMed

    Ji, Qiaoli; Shi, Xiaolian; Lin, Rong; Mao, Youhua; Zhai, Xiaogang; Lin, Qinqin; Zhang, Jiye

    2010-06-01

    The hepatotoxicity induced by valproic acid (VPA) has been described in many clinical studies and the related mechanism has been partly elucidated. The objective of this study is to investigate the hepatotoxicity and its underlying mechanism of valproic acid on human hepatoma carcinoma cell line HepG2. The cell viability was evaluated by 3-(4,5-dimethyltyiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in the medium were detected using spectrophotometry. The gene expressions of cytochrome P450 1 A1 (CYP1A1), ATP-binding cassette transporter G1 (ABCG1) and carnitine palmitoyltransferase 1 (CPT1A), related to lipid transport and fatty acid metabolism, were measured by quantitative real-time reverse transcriptase-PCR. Treatment with valproate sodium obviously decreased the viability of HepG2 cells, accompanied by the increased leakages of ALT, AST and LDH in a dose-dependent manner. Furthermore, the gene expressions of CYP1A1, ABCG1 and CPT1A were almost up-regulated in the treated groups. In conclusion, these data suggest that VPA-induced hepatotoxicity was critically enhanced with the elevation of valproate sodium, which may be correlated with up-regulated gene expressions of CYP1A1, ABCG1 and CPT1A. Copyright 2010 Elsevier Ltd. All rights reserved.

  8. The effect of black seed (Nigella sativa) extract on FOXO3 expression in HepG2 cells.

    PubMed

    Haas, Michael J; Onstead-Haas, Luisa; Naem, Emad; Arnold, Alexis; Rohrbaugh, Nathcelly; Flowers, Megan; Mooradian, Arshag D

    2014-06-01

    Black seed extracts are known to alter cellular metabolism through multiple signaling pathways. Since Forkhead box transcription factor 3 (FOXO3) has a significant role in regulating cellular metabolism, the effect of lipid extracts of black seed (Sativa nigella) on FOXO3 levels and AKT and 5-AMP activated protein kinase α (AMPKα) signaling was measured in HepG2 hepatoma cells. FOXO3 levels, phosphorylation, and nuclear exclusion were measured by Western blot, as were AKT and AMPK expression and activity using phosphorylation-specific antibodies. Apolipoprotein A-I expression, a black seed-responsive gene, was measured by Western blot. Treatment with black seed extract increased FOXO3 phosphorylation and decreased its expression. In contrast to control cells where FOXO3 was located primarily in the nucleus, in black seed-treated HepG2 cells, FOXO3 was localized primarily to the cytoplasm. These changes in FOXO3 phosphorylation, expression, and localization were accompanied by increased AKT activity. Black seed also decreased AMPKα activity but increased AMPKα expression. Lipid extracts from black seeds inhibit FOXO3 activity and thereby modulate the expression of FOXO3-dependent genes. Copyright © 2013 John Wiley & Sons, Ltd.

  9. Induction of apoptosis in HepG2 cells by polysaccharide MEP-II from the fermentation broth of Morchella esculenta.

    PubMed

    Hu, Meili; Chen, Yan; Wang, Cui; Cui, Huali; Duan, Peilu; Zhai, Tianlong; Yang, Yuling; Li, Shaofei

    2013-01-01

    A novel polysaccharide, MEP-II, isolated from the fermentation broth of Morchella esculenta inhibited the proliferation of human hepatoma cell line (HepG2) through an apoptotic pathway. After HepG2 cells were treated with 150-600 μg MEP-II/ml, typical apoptotic characteristics including externalization of phosphatidylserine residues on the cell surface, nuclear fragmentation, chromatin condensation and cytoplasm shrinkage were observed. Furthermore, reactive oxygen species (ROS) burst and the collapse of mitochondrial membrane potential (Δψm) also occurred in HepG2 cells after incubation of 150-600 μg MEP-II/ml. The antioxidant, 1 mM N-acetyl-L-cysteine inhibited MEP-II-induced apoptosis, suggesting that ROS are the key mediators for MEP-II-induced apoptosis. MEP-II is therefore a potential anti-tumor agent that induces apoptosis of HepG2 cells through ROS generation.

  10. Cytotoxic effects of etephon and maleic hydrazide in Vero, Hep2, HepG2 cells.

    PubMed

    Yurdakok, Begum; Baydan, Emine; Okur, Hamza; Gurcan, Ismayil Safa

    2014-10-01

    The toxicity of etephon and maleic hydrazide, used as plant growth regulators in agriculture, were reported as low in mammals in previous studies. However, in vitro cytotoxicity studies in mammalian cells are currently missing to understand their toxicity at molecular level. In the current study, the cytotoxicity of these compounds, were studied in Vero (African green monkey kidney epithelium), HepG2 (human hepatocellular carcinoma), Hep2 (human epidermoid cancer) cells by MTT ((3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromure) and LDH (lactate dehydrogenase) assays. Maleic hydrazide had lower IC50 values for all cell lines compared to ethephon. Least cytotoxic effect treated by ethephon were observed in Vero, followed by HepG2 and Hep2. Similarly maleic hydrazide also showed least cytotoxicity on Vero cells, followed by Hep2 and HepG2 cells (p < 0.05). IC50 values in general were found to be highest in Vero cells, followed by HepG2 and Hep2 cells (p < 0.05). LDH and MTT assays showed correllation and had close relation except HepG2-maleic hydrazide application with the correlation coefficient for all >0.868 (p < 0.05). This study is expected to be a basis to understand the cytotoxic effects of ethephon and maleic hydrazide in mammal cells to be supplemented by further studies.

  11. Pro-apoptotic effects of tectorigenin on human hepatocellular carcinoma HepG2 cells

    PubMed Central

    Jiang, Chun-Ping; Ding, Hui; Shi, Da-Hua; Wang, Yu-Rong; Li, Er-Guang; Wu, Jun-Hua

    2012-01-01

    AIM: To investigate the effects of tectorigenin on human hepatocellular carcinoma (HCC) HepG2 cells. METHODS: Tectorigenin, one of the main components of rhizome of Iris tectorum, was prepared by simple methods, such as extraction, filtration, concentration, precipitation and recrystallization. HepG2 cells were incubated with tectorigenin at different concentrations, and their viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was detected by morphological observation of nuclear change, agarose gel electrophoresis of DNA ladder, and flow cytometry with Hoechst 33342, Annexin V-EGFP and propidium iodide staining. Generation of reactive oxygen species was quantified using DCFH-DA. Intracellular Ca2+ was monitored by Fura 2-AM. Mitochondrial membrane potential was monitored using Rhodamine 123. Release of cytochrome c from mitochondria to cytosol was detected by Western blotting. Activities of caspase-3, -8 and -9 were investigated by Caspase Activity Assay Kit. RESULTS: The viability of HepG2 cells treated by tectorigenin decreased in a concentration- and time-dependent manner. The concentration that reduced the number of viable HepG2 cells by 50% (IC50) after 12, 24 and 48 h of incubation was 35.72 mg/L, 21.19 mg/L and 11.06 mg/L, respectively. However, treatment with tectorigenin at 20 mg/L resulted in a very slight cytotoxicity to L02 cells after incubation for 12, 24 or 48 h. Tectorigenin at a concentration of 20 mg/L greatly inhibited the viability of HepG2 cells and induced the condensation of chromatin and fragmentation of nuclei. Tectorigenin induced apoptosis of HepG2 cells in a time- and dose-dependent manner. Compared with the viability rate, induction of apoptosis was the main mechanism of the anti-proliferation effect of tectorigenin in HepG2 cells. Furthermore, tectorigenin-induced apoptosis of HepG2 cells was associated with the generation of reactive oxygen species, increased intracellular [Ca2+]i

  12. Targeting and molecular imaging of HepG2 cells using surface-functionalized gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Rathinaraj, Pierson; Lee, Kyubae; Choi, Yuri; Park, Soo-Young; Kwon, Oh Hyeong; Kang, Inn-Kyu

    2015-07-01

    Mercaptosuccinic acid (M)-conjugated gold nanoparticles (GM) were prepared and characterized by transmission electron microscope and dynamic light scattering. M was used to improve the monodispersity and non-specific intracellular uptake of nanoparticles. Lactobionic acid (L) was subsequently conjugated to the GM to target preferentially HepG2 cells (liver cancer cells) that express asialoglycoprotein receptors (ASGPR) on their membrane surfaces and facilitate the transit of nanoparticles across the cell membrane. The mean size of lactobionic acid-conjugated gold nanoparticle (GL) was approximately 10 ± 0.2 nm. Finally, the Atto 680 dye (A6) was coupled to the nanoparticles to visualize their internalization into HepG2 cells. The interaction of surface-modified gold nanoparticles with HepG2 cells was studied after culturing cells in media containing the GM or L-conjugated GM (GL).

  13. Selective cytotoxicity of goniothalamin against hepatoblastoma HepG2 cells.

    PubMed

    Al-Qubaisi, Mothanna; Rozita, Rosli; Yeap, Swee-Keong; Omar, Abdul-Rahman; Ali, Abdul-Manaf; Alitheen, Noorjahan B

    2011-04-06

    Liver cancer has become one of the major types of cancer with high mortality and liver cancer is not responsive to the current cytotoxic agents used in chemotherapy. The purpose of this study was to examine the in vitro cytotoxicity of goniothalamin on human hepatoblastoma HepG2 cells and normal liver Chang cells. The cytotoxicity of goniothalamin against HepG2 and liver Chang cell was tested using MTT cell viability assay, LDH leakage assay, cell cycle flow cytometry PI analysis, BrdU proliferation ELISA assay and trypan blue dye exclusion assay. Goniothalamin selectively inhibited HepG2 cells [IC₅₀ = 4.6 (±0.23) µM in the MTT assay; IC₅₀ = 5.20 (±0.01) µM for LDH assay at 72 hours], with less sensitivity in Chang cells [IC₅₀ = 35.0 (±0.09) µM for MTT assay; IC₅₀ = 32.5 (±0.04) µM for LDH assay at 72 hours]. In the trypan blue dye exclusion assay, the Viability Indexes were 52 ± 1.73% for HepG2 cells and 62 ± 4.36% for Chang cells at IC₅₀ after 72 hours. Cytotoxicity of goniothalamin was related to inhibition of DNA synthesis, as revealed by the reduction of BrdU incorporation. At 72 hours, the lowest concentration of goniothalamin (2.3 µL) retained 97.6% of normal liver Chang cells proliferation while it reduced HepG2 cell proliferation to 19.8% as compared to control. Besides, goniothalamin caused accumulation of hypodiploid apoptosis and different degree of G2/M arrested as shown in cell cycle analysis by flow cytometry. Goniothalamin selectively killed liver cancer cell through suppression of proliferation and induction of apoptosis. These results suggest that goniothalamin shows potential cytotoxicity against hepatoblastoma HepG2 cells.

  14. Butyrylcholinesterase expression is regulated by fatty acids in HepG2 cells.

    PubMed

    Gok, Muslum; Zeybek, N Dilara; Bodur, Ebru

    2016-11-25

    Butyrylcholinesterase (BChE) is mostly associated with the detoxification of xenobiotics. In this study to analyze the involvement of BChE in lipid metabolism, linoleic acid (LA) and α-linolenic acid (ALA) were applied to HepG2 cells along with expression of wild type human BChE. After 48 h of these treatments WST-1 cell proliferation assay, FACS analysis, RT-PCR, Oil Red O staining and activity assays were performed. Application of high concentrations of LA to HepG2 cells without BChE transfection lead to detachment of the cells. The IC50 value LA was found as 149.3 μM whereas the IC50 value for ALA could not be calculated. Hence, in order to display minimal effects on cell viability, 5 μM was chosen as appropriate concentration for LA and ALA application to HepG2 cells. Transfection of wild-type BChE plasmid to HepG2 cells yielded increased BChE expression. Application of 5 μM ALA after BChE transfection to HepG2 cells resulted in increased expression of BChE. Although with this low concentration the number of apoptotic cells was decreased with ALA treatments, LA application did not cause a similar result with the same dose. Moreover ghost cell like property was observed in LA-treated cells. Application of ALA, on the other hand, led to an overall increase in cell numbers, BChE expression and activity. Our results indicate that BChE expression might be regulated by ALA in HepG2 cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  15. Liver stage antigen 3 Plasmodium falciparum peptides specifically interacting with HepG2 cells.

    PubMed

    García, Javier E; Curtidor, Hernando; López, Ramses; Rodríguez, Luis; Vera, Ricardo; Valbuena, John; Rosas, Jaiver; Ocampo, Marisol; Puentes, Alvaro; Forero, Martha; Patarroyo, Manuel A; Patarroyo, Manuel Elkin

    2004-09-01

    Binding assays were carried out with 20 amino acid long peptides covering the complete 200-kDa Liver stage antigen (LSA) 3 protein sequence to identify its HepG2 cell binding regions. Seventeen HepG2 cell high-activity binding peptides (HABPs) were identified in the LSA-3 protein. Seven HABPs were found in the nonrepeat (NRA) region A; five of these formed a 100 amino acid long HepG2 cell binding region located between residues 21Ile and 120Thr. Six HABPs were found in the R2 region and another four in the NRB2 region. LSA-3 protein HABPS bound saturably to HepG2 cells having nanomolar affinity constants and bound specifically to 31, 44, and 70 kDa HepG2 cell membrane proteins. Some of them were located in antigenic and immunogenic LSA-3 protein regions. Immunofluorescence and immunoblotting assays using goat sera immunized with LSA-3 protein peptides recognized P. falciparum (FCB-2 strain) erythrocyte stage proteins (58, 68, 72, 81, 86, 160, and 175 kDa). This reactivity was due mainly to the VEESVAEN motif present in some erythrocyte stage proteins. However, our results suggest that antibodies against LSA-3 regions had a crossed reaction with another 86-kDa protein, and that this crossed reaction was due to a motif present in the NRA region.

  16. Curdlan sulphate modulates protein synthesis and enhances NF-κB and C/EBP binding activity in HepG2 cells

    PubMed Central

    Rokita, H.

    1997-01-01

    In human hepatoma HepG2 cell line curdlan sulphate enhances basal and interleukin-6-stimulated fibrinogen and antichymotrypsin (ACT) synthesis, slightly increases basal ceruloplasmin production and exerts only minor effects on alpha-1-proteinase inhibitor and transferrin. Curdlan sulphate may, at least in part, affect protein synthesis at a pretranslational level, as the expression of ACT mRNA was found to be increased, whereas intracellular enzyme, manganese superoxide dismutase mRNA level was decreased in the cell culture treated with curdlan sulphate. Gel mobility shift analysis revealed that curdlan sulphate increases the DNA binding activity of NF-κB and C/EBP, suggesting that these transcription factors may participate in the regulatory effects of curdlan sulphate in HepG2 cells. PMID:18472835

  17. Toxicity evaluation of surface water treated with different disinfectants in HepG2 cells.

    PubMed

    Marabini, Laura; Frigerio, Silvia; Chiesara, Enzo; Radice, Sonia

    2006-01-01

    It is well known that water disinfection through chlorination causes the formation of a mixture of disinfection by-products (DBPs), many of which are genotoxic and carcinogenic. To demonstrate the formation of such compounds, a pilot water plant supplied with water from Lake Trasimeno was set up at the waterworks of Castiglione del Lago (PG, Italy). The disinfectants, continuously added to pre-filtered lake water flowing into three different basins, were sodium hypochlorite, chlorine dioxide and peracetic acid, an alternative disinfectant used until now for disinfecting waste waters, but not yet studied for a possible use in drinking water treatment. The aim of this study was to evaluate the formation during the disinfection processes of some toxic compounds that could explain the genotoxic effects of drinking waters. Differently treated waters were concentrated by solid-phase adsorption on silica C(18) columns and toxicity was assessed in a line of human hepatoma cells (HepG2), a metabolically competent cellular line very useful for human risk evaluation. The seasonal variability of the physical-chemical water characteristics (AOX, UV 254 nm, potential formation of THM, pH and temperature) made indispensable experimentation with water samples taken during the various seasons. Autumn waters cause greater toxicity compared to those of other seasons, in particular dilution of the concentrate at 0.5l equivalent of disinfected waters with chlorine dioxide and peracetic acid causes a 55% reduction in cellular vitality while the cellular vitality is over 80% with the all other water concentrates. Moreover it is very interesting underline that non-cytotoxic quantities of the autumnal water concentrates cause, after 2h treatment, a decrease in GSH and a statistically significant increase in oxygen radicals, while after prolonged treatment (24h) cause a GSH increase, without variations in the oxygen radical content. This phenomenon could be interpreted as the cellular

  18. TGF-β1 promotes human hepatic carcinoma HepG2 cells invasion by upregulating autophagy.

    PubMed

    Ma, C-L; Qiao, S; Li, Y-C; Wang, X-F; Sun, R-J; Zhang, X; Qian, R-K; Song, S-D

    2017-06-01

    To study the role of TGF-β1 in autophagy and invasion ability of human hepatic carcinoma HepG2 cells. Cultured HepG2 cells were treated with different concentrations of TGF-β1 for 24 h. The protein expression levels of autophagy relative marker LC3 and Beclin1 were detected by Western blot. The effect of TGF-β1 on invasion ability of HepG2 cells was detected with transwell method. The results demonstrated that TGF-β1 was able to activate autophagy of HepG2 cells in a dose-dependent manner. Autophagy inhibitor 3-methyladenine (3-MA) could reverse TGF-β1 induced autophagy process. Also, TGF-β1 significantly promotes the invasion ability of HepG2 cells; however, this process could effectively reverse by autophagy inhibitor 3-MA. TGF-β1 enhances HepG2 cells invasion by upregulating autophagy.

  19. The Antiapoptosis Effect of Glycyrrhizate on HepG2 Cells Induced by Hydrogen Peroxide

    PubMed Central

    Su, Miao; Yu, Tengfei; Zhang, Hong; Wu, Yan; Wang, Xiaoqin

    2016-01-01

    This study demonstrated that glycyrrhizate (GAS) could protect HEPG2 cells against damage and apoptosis induced by H2O2 (1600 μM, 4 h). Cell viability assay revealed that GAS was noncytotoxity at concentration 125 µg/mL, and GAS (5 μg/mL, 25 μg/mL, and 125 μg/mL) protected HepG2 cells against H2O2-induced cytotoxicity. H2O2 induced the HepG2 cells apoptosis, obvious morphologic changes were observed after Hochest 33258 staining, and more apoptotic cells were counted in flow cytometry assay compared to that of the natural group. Pretreatment GAS (5 μg/mL, 25 μg/mL, and 125 μg/mL) prior to H2O2 reverses the morphologic changes and reduced the apoptotic cells in HepG2 cells. GAS reduced the release of MDA, increased the activities of superoxide dismutase, and diminished the release of ALT and AST during oxidative stress in HepG2 cells. After Elisa kit detecting, GAS inhibited the caspase activity induced by H2O2, GAS decreased the level of caspase-3 and caspase-9 from mitochondria in dose-dependent manner. Western blot results showed that pretreatment GAS upregulated the expression of Bcl-2 and decreased the expression of Bax. These results reveal that GAS has the cytoprotection in HepG2 cells during ROS exposure by inhibiting the caspase activity in the mitochondria and influencing apoptogenic factors of the expression of Bax and Bcl-2. PMID:27891207

  20. Comparative analysis of 3D culture methods on human HepG2 cells.

    PubMed

    Luckert, Claudia; Schulz, Christina; Lehmann, Nadja; Thomas, Maria; Hofmann, Ute; Hammad, Seddik; Hengstler, Jan G; Braeuning, Albert; Lampen, Alfonso; Hessel, Stefanie

    2017-01-01

    Human primary hepatocytes represent a gold standard in in vitro liver research. Due to their low availability and high costs alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. The human hepatocarcinoma cell line HepG2 is often used as a liver model for toxicity studies. However, under two-dimensional (2D) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers such as albumin is very low. Cultivation for 21 days in a three-dimensional (3D) Matrigel culture system has been reported to strongly increase the metabolic competence of HepG2 cells. In our present study we further compared HepG2 cell cultivation in three different 3D systems: collagen, Matrigel and Alvetex culture. Cell morphology, albumin secretion, cytochrome P450 monooxygenase enzyme activities, as well as gene expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after 3, 7, 14, and 21 days of cultivation. Our results show that the previously reported increase of metabolic competence of HepG2 cells is not primarily the result of 3D culture but a consequence of the duration of cultivation. HepG2 cells grown for 21 days in 2D monolayer exhibit comparable biochemical characteristics, CYP activities and gene expression patterns as all 3D culture systems used in our study. However, CYP activities did not reach the level of HepaRG cells. In conclusion, the increase of metabolic competence of the hepatocarcinoma cell line HepG2 is not due to 3D cultivation but rather a result of prolonged cultivation time.

  1. The Mechanism by Which Amentoflavone Improves Insulin Resistance in HepG2 Cells.

    PubMed

    Zheng, Xiaoke; Ke, Yingying; Feng, Aozi; Yuan, Peipei; Zhou, Jing; Yu, Yang; Wang, Xiaolan; Feng, Weisheng

    2016-05-13

    The aim of this study was to explore the mechanism by which amentoflavone (AME) improves insulin resistance in a human hepatocellular liver carcinoma cell line (HepG2). A model of insulin resistant cells was established in HepG2 by treatment with high glucose and insulin. The glucose oxidase method was used to detect the glucose consumption in each group. To determine the mechanism by which AME improves insulin resistance in HepG2 cells, enzyme-linked immunosorbent assay (ELISA) and western blotting were used to detect the expression of phosphatidyl inositol 3-kinase (PI3K), Akt, and pAkt; the activity of the enzymes involved in glucose metabolism; and the levels of inflammatory cytokines. Insulin resistance was successfully induced in HepG2 cells. After treatment with AME, the glucose consumption increased significantly in HepG2 cells compared with the model group (MG). The expression of PI3K, Akt, and pAkt and the activity of 6-phosphofructokinas (PFK-1), glucokinase (GCK), and pyruvate kinase (PK) increased, while the activity of glycogen synthase kinase-3 (GSK-3), phosphoenolpyruvate carboxylase kinase (PEPCK), and glucose-6-phosphatase (G-6-Pase) as well as the levels of interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and C reactive protein (CRP) decreased. The mechanism by which treatment with AME improves insulin resistance in HepG2 cells may involve the PI3K-Akt signaling pathway, the processes of glucose oxygenolysis, glycogen synthesis, gluconeogenesis and inflammatory cytokine expression.

  2. Demonstration of the presence of the "deleted" MIR122 gene in HepG2 cells.

    PubMed

    Hamad, Ibrahim A Y; Fei, Yue; Kalea, Anastasia Z; Yin, Dan; Smith, Andrew J P; Palmen, Jutta; Humphries, Steve E; Talmud, Philippa J; Walker, Ann P

    2015-01-01

    MicroRNA 122 (miR-122) is highly expressed in the liver where it influences diverse biological processes and pathways, including hepatitis C virus replication and metabolism of iron and cholesterol. It is processed from a long non-coding primary transcript (~7.5 kb) and the gene has two evolutionarily-conserved regions containing the pri-mir-122 promoter and pre-mir-122 hairpin region. Several groups reported that the widely-used hepatocytic cell line HepG2 had deficient expression of miR-122, previously ascribed to deletion of the pre-mir-122 stem-loop region. We aimed to characterise this deletion by direct sequencing of 6078 bp containing the pri-mir-122 promoter and pre-mir-122 stem-loop region in HepG2 and Huh-7, a control hepatocytic cell line reported to express miR-122, supported by sequence analysis of cloned genomic DNA. In contrast to previous findings, the entire sequence was present in both cell lines. Ten SNPs were heterozygous in HepG2 indicating that DNA was present in two copies. Three validation isolates of HepG2 were sequenced, showing identical genotype to the original in two, whereas the third was different. Investigation of promoter chromatin status by FAIRE showed that Huh-7 cells had 6.2 ± 0.19- and 2.7 ± 0.01- fold more accessible chromatin at the proximal (HNF4α-binding) and distal DR1 transcription factor sites, compared to HepG2 cells (p=0.03 and 0.001, respectively). This was substantiated by ENCODE genome annotations, which showed a DNAse I hypersensitive site in the pri-mir-122 promoter in Huh-7 that was absent in HepG2 cells. While the origin of the reported deletion is unclear, cell lines should be obtained from a reputable source and used at low passage number to avoid discrepant results. Deficiency of miR-122 expression in HepG2 cells may be related to a relative deficiency of accessible promoter chromatin in HepG2 versus Huh-7 cells.

  3. Role of salt inducible kinase 1 in high glucose-induced lipid accumulation in HepG2 cells and metformin intervention.

    PubMed

    Zhang, Yue; Takemori, Hiroshi; Wang, Chang; Fu, JiaHui; Xu, MingWang; Xiong, Liang; Li, NingXu; Wen, XiuYing

    2017-03-15

    To investigate the roles of salt inducible kinase (SIK1) in high glucose-induced triglyceride accumulation in human hepatoma HepG2 cells as well as in the molecular mechanism by which metformin, a drug to treat diabetes, suppresses high glucose-induced lipogenesis. A cell model for high glucose-induced hepatic steatosis was prepared by exposing HepG2 cells to high glucose (25mmol) in the absence or presence of metformin (0.5mmol). Intracellular triglycerides were visualized by Oil Red O and measured using a triglyceride assay kit. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. SIK1 overexpression in HepG2 cells was achieved by transient transfection, and the mRNA and protein levels of SIK1 and lipogenic factors were measured using a reverse transcription-polymerase chain reaction and western blotting, respectively. Lipid accumulation in HepG2 cells was obvious after treatment with high glucose for 24h. In response to high glucose, SIK1 expression was negatively correlated with that of lipogenic factors and lipid accumulation in HepG2 cells. We observed that overexpression of SIK1, or treatment with metformin, suppressed lipogenesis, even in high glucose conditions. Furthermore, treatment with metformin upregulated SIK1 mRNA and protein levels, as well as the active form of SIK1. SIK1 plays a vital role in high glucose-induced lipid accumulation, and metformin suppresses lipogenesis via the induction and activation of SIK1. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Nanoceria Attenuated High Glucose-Induced Oxidative Damage in HepG2 Cells

    PubMed Central

    Shokrzadeh, Mohammad; Abdi, Hakimeh; Asadollah-Pour, Azin; Shaki, Fatemeh

    2016-01-01

    Objective Hyperglycemia, a common metabolic disorder in diabetes, can lead to oxidative damage. The use of antioxidants can benefit the control and prevention of diabetes side effects. This study aims to evaluate the effect of nanoceria particles, as an antioxidant, on glucose induced cytotoxicity, reactive oxygen species (ROS), lipid peroxidation (LPO) and glutathione (GSH) content in a human hepatocellular liver carcinoma cell line (HepG2) cell line. Materials and Methods In this experimental study, we divided HepG2 cells into these groups: i. Cells treated with 5 mM D-glucose (control), ii. Cells treated with 45 mM D- mannitol+5 mM D-glucose (osmotic control), iii. Cells treated with 50 mM D-glucose (high glucose), and iv. Cells treated with 50 mM D-glucose+nanoceria. Cell viability, ROS formation, LPO and GSH were measured and analyzed statistically. Results High glucose (50 mM) treatment caused significant cell death and increased oxidative stress markers in HepG2 cells. Interestingly, nanoceria at a concentration of 50 mM significantly decreased the high glucose-induced cytotoxicity, ROS formation and LPO. This concentration of nanoceria increased the GSH content in HepG2 cells (P<0.05). Conclusion The antioxidant feature of nanoceria particles makes it an attractive candidate for attenuation of hyperglycemia oxidative damage in different organs. PMID:27054124

  5. HepG2 cells mount an effective antiviral interferon-lambda based innate immune response to hepatitis C virus infection

    PubMed Central

    Israelow, Benjamin; Narbus, Christopher M.; Sourisseau, Marion; Evans, Matthew J.

    2014-01-01

    Hepatitis C virus (HCV) exposure leads to persistent life-long infections characterized by chronic inflammation often developing into cirrhosis and hepatocellular carcinoma. The mechanism by which HCV remains in the liver while inducing an inflammatory and antiviral response remains unclear. While the innate immune response to HCV in patients seem to be quite active, HCV has been shown in cell culture to employ a diverse array of innate immune antagonists, suggesting that current model systems to study interactions between HCV and the innate immune system are not representative of what is happening in vivo. We recently showed that hepatoma-derived HepG2 cells support the entire HCV life cycle if the liver-specific microRNA miR-122 is expressed along with the entry factor CD81 (HepG2-HFL cells). We found that there was a striking difference in these cells’ ability to sustain HCV infection and spread when compared to Huh-7 and Huh-7.5 cells. Additionally, HepG2-HFL cells produced a more robust antiviral response when challenged with other RNA viruses and viral mimetics than Huh-7 and Huh-7.5 cells. HCV infection elicited a potent IFN-λ (lambda), ISG, and cytokine response in HepG2-HFL cells, but not in Huh-7 cells, suggesting that HepG2-HFL cells more faithfully recapitulate the innate immune response to HCV infection in vivo. Using this new model, we found that blocking the RIG-I like receptor pathway or the IFN-λ signaling pathway, promoted HCV infection and spread in HepG2-HFL cells. Conclusion HepG2-HFL cells represent a promising new system to study the interaction between HCV and the innate immune system, solidifying the importance of IFN-λ in hepatic response to HCV infection and revealing non-redundant roles of RIG-I and MDA5 in HCV recognition and repression of infection. PMID:24833036

  6. Curcumin and (-)-epigallocatechin-3-gallate attenuate acrylamide-induced proliferation in HepG2 cells.

    PubMed

    Shan, Xiaoyun; Li, Yuan; Meng, Xulian; Wang, Pengqi; Jiang, Pan; Feng, Qing

    2014-04-01

    Acrylamide, a proven rodent carcinogen, is present in carbohydrate-rich food heated at high temperatures. It can be metabolized into glycidamide mainly by cytochrome P450 2E1 (CYP2E1). The fact that acrylamide is a potential carcinogen to human-beings draws public attention recently. This study aimed to elucidate the effect of acrylamide at low doses on proliferation of HepG2 cells, and to test whether the two well-studied chemopreventive agents, curcumin and (-)-epigallocatechin-3-gallate (EGCG), would have antagonistic effects against acrylamide. The results showed that lower concentration of acrylamide (⩽100μM) significantly increased the proliferation of HepG2 cells, but not of the other cancer cells (MDA-231, HeLa, A549, and PC-3). Only in HepG2 cells, low concentration of acrylamide was able to induce CYP2E1 expression significantly. Knockdown of CYP2E1 restrained acrylamide to increase viability of HepG2 cells. In addition, acrylamide raised expression of epidermal growth factor receptor (EGFR), cyclin D1 and nuclear factor-κB (NF-κB), which contributed to cell proliferation. Both curcumin and EGCG effectively reduced acrylamide-induced proliferation, as well as protein expression of CYP2E1, EGFR, cyclin D1 and NF-κB. All these results suggest that low concentration of acrylamide may contribute to progression of hepatocellular carcinoma (HCC). Curcumin or EGCG could prevent acrylamide triggering this effect.

  7. Anti-proliferative and cytoskeleton-disruptive effects of icariin on HepG2 cells.

    PubMed

    Wang, Zhi-Min; Song, Nan; Ren, Yan-Ling

    2015-11-01

    Several biological properties of icariin have been identified, including its anticancer effect. However, the potential mechanisms underlying the effect of icariin on HepG2 hepatocellular carcinoma cells remain to be elucidated. The aim of the present study was to examine the effects of icariin on the proliferation and cytoskeleton of HepG2 cells. A 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5 diphenyltetrazolium bromide assay was used to assess the antiproliferative effects of icariin and to determine the optimal concentration and treatment schedule of icariin on the HepG2 cells. Cell cycle analysis was performed using fluorescence activated cell sorting, the protein expression of B‑cell lymphoma (Bcl)‑2 was determined using immunohistochemical and western blot analyses, and F‑actin in the cells was examined using confocal microscopy. The chemotherapeutic drug, oxaliplatin, was used as a positive control. The results demonstrated that the optimal concentration of icarrin to produce an antiproliferative effect on HepG2 cells was 10‑5 mol/l, and the optimal treatment duration was 72 h. The icariin group had a significantly higher proportion of cells in the G0/G1 phase, compared with the control group, treated with high glucose Dulbecco's modified Eagles medium with 10% fetal bovine serum (P<0.05). The proportion of HepG2 cells in the S phase was significantly lower in the oxaliplatin (24.19%; P<0.05) and icariin (21.07%; P<0.01) groups, compared with the control group (28.62%). Icariin markedly decreased the expression of Bcl‑2, compared with the control (P<0.01), and disrupted the polymerization of F‑actin filaments in the HepG2 cells. Therefore, the present study demonstrated that, at an optimum concentration of 10‑5 mol/l, icariin inhibited the proliferation of the HepG2 cells, promoted apoptosis by decreasing the expression of Bcl‑2, and disrupted the actin cytoskeleton.

  8. Silver Nanoparticles Induce HePG-2 Cells Apoptosis Through ROS-Mediated Signaling Pathways

    NASA Astrophysics Data System (ADS)

    Zhu, Bing; Li, Yinghua; Lin, Zhengfang; Zhao, Mingqi; Xu, Tiantian; Wang, Changbing; Deng, Ning

    2016-04-01

    Recently, silver nanoparticles (AgNPs) have been shown to provide a novel approach to overcome tumors, especially those of hepatocarcinoma. However, the anticancer mechanism of silver nanoparticles is unclear. Thus, the purpose of this study was to estimate the effect of AgNPs on proliferation and activation of ROS-mediated signaling pathway on human hepatocellular carcinoma HePG-2 cells. A simple chemical method for preparing AgNPs with superior anticancer activity has been showed in this study. AgNPs were detected by transmission electronic microscopy (TEM) and energy dispersive X-ray (EDX). The size distribution and zeta potential of silver nanoparticles were detected by Zetasizer Nano. The average size of AgNPs (2 nm) observably increased the cellular uptake by endocytosis. AgNPs markedly inhibited the proliferation of HePG-2 cells through induction of apoptosis with caspase-3 activation and PARP cleavage. AgNPs with dose-dependent manner significantly increased the apoptotic cell population (sub-G1). Furthermore, AgNP-induced apoptosis was found dependent on the overproduction of reactive oxygen species (ROS) and affecting of MAPKs and AKT signaling and DNA damage-mediated p53 phosphorylation to advance HePG-2 cells apoptosis. Therefore, our results show that the mechanism of ROS-mediated signaling pathways may provide useful information in AgNP-induced HePG-2 cell apoptosis.

  9. Anti-hepatocarcinoma effects of resveratrol nanoethosomes against human HepG2 cells

    NASA Astrophysics Data System (ADS)

    Meng, Xiang-Ping; Zhang, Zhen; Chen, Tong-sheng; Wang, Yi-fei; Wang, Zhi-ping

    2017-02-01

    Hepatocarcinoma, a malignant cancer, threaten human life badly. It is a current issue to seek the effective natural remedy from plant to treat cancer due to the resistance of the advanced hepatocarcinoma to chemotherapy. Resveratrol (Res) has been widely investigated with its strong anti-tumor activity. However, its low oral bioavailability restricts its wide application. In this study, we prepared resveratrol nanoethosomes (ResN) via ethanol injection method. The in vitro anti-hepatocarcinoma effects of ResN relative to efficacy of bulk Res were evaluated on proliferation and apoptosis of human HepG2 cells. ResN were spherical vesicles and its particle diameter, zeta potential were (115.8 +/- 1.3) nm and (-12.8 +/- 1.9) mV, respectively. ResN exhibited significant inhibitory effects against human HepG2 cells by MTT assay, and the IC50 value was 49.2 μg/ml (105.4 μg/ml of Res bulk solution). By flow cytometry assay, there was an increase in G2/M phase cells treated with ResN. The results demonstrated ResN could effectively block the G2/M phase of HepG2 cells, which can also enhance the inhibitory effect of Res against HepG2 cells.

  10. Cytotoxic and antimigratory effects of Cratoxy formosum extract against HepG2 liver cancer cells.

    PubMed

    Buranrat, Benjaporn; Mairuae, Nootchanat; Kanchanarach, Watchara

    2017-04-01

    The aim of the present study was to investigate the molecular mechanisms underlying Cratoxylum formosum (CF) Dyer-induced cancer cell death and antimigratory effects in HepG2 liver cancer cells. The cytotoxic, antiproliferative and antimigratory effects of CF leaf extract on human liver cancer HepG2 cell lines were evaluated using sulforhodamine B, colony formation, and wound healing assays. In addition, apoptosis induction mechanisms were investigated via reactive oxygen species (ROS) formation, caspase 3 activities, and mitochondrial membrane potential (ΔΨm) disruption. Gene expression and apoptosis-associated protein levels were measured by reverse transcription-quantitative polymerase chain reaction and western blotting. CF induced HepG2 cell death in a time- and dose-dependent manner with half maximal inhibitory concentration values of 219.03±9.96 and 124.90±6.86 µg/ml at 24 and 48 h, respectively. Treatment with CF caused a significant and dose-dependent decrease in colony forming ability and cell migration. Furthermore, the present study demonstrated that CF induced ROS formation, increased caspase 3 activities, decreased the ΔΨm, and caused HepG2 apoptosis. CF marginally decreased the expression level of the cell cycle regulatory protein, ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) and the downstream protein, cyclin dependent kinase 6. Additionally, CF significantly enhanced p21 levels, reduced cyclin D1 protein levels and triggered cancer cell death. CF leaf extracts induced cell death, stimulated apoptosis and inhibited migration in HepG2 cells. Thus, CF may be useful for developing an anticancer drug candidate for the treatment of liver cancer.

  11. Mangiferin: A xanthone attenuates mercury chloride induced cytotoxicity and genotoxicity in HepG2 cells.

    PubMed

    Kaivalya, Mudholkar; Nageshwar Rao, B N; Satish Rao, B S

    2011-01-01

    Mangiferin (MGN), a dietary C-glucosylxanthone present in Mangifera indica, is known to possess a spectrum of beneficial pharmacological properties. This study demonstrates antigenotoxic potential of MGN against mercuric chloride (HgCl2)-induced genotoxicity in HepG2 cell line. Treatment of HepG2 cells with various concentrations of HgCl2 for 3 h caused a dose-dependent increase in micronuclei frequency and elevation in DNA strand breaks (olive tail moment and tail DNA). Pretreatment with MGN significantly (p < 0.01) inhibited HgCl2 -induced (20 µM for 30 h) DNA damage. An optimal antigenotoxic effect of MGN, both in micronuclei and comet assay, was observed at a concentration of 50 µM. Furthermore, HepG2 cells treated with various concentrations of HgCl2 resulted in a dose-dependent increase in the dichlorofluorescein fluorescence, indicating an increase in the generation of reactive oxygen species (ROS). However, MGN by itself failed to generate ROS at a concentration of 50 µM, whereas it could significantly decrease HgCl2 -induced ROS. Our study clearly demonstrates that MGN pretreatment reduced the HgCl2-induced DNA damage in HepG2 cells, thus demonstrating the genoprotective potential of MGN, which is mediated mainly by the inhibition of oxidative stress.

  12. Hyperglycemia and anthocyanin inhibit quercetin metabolism in HepG2 cells

    USDA-ARS?s Scientific Manuscript database

    A high glucose (Glu) milieu promotes generation of reactive oxygen species, which may not only cause cellular damage, but also modulate phase II enzymes that are responsible for the metabolism of flavonoids. Thus, we examined the effect of a high Glu milieu on quercetin (Q) metabolism in HepG2 cells...

  13. 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

  14. Globular adiponectin inhibits ethanol-induced apoptosis in HepG2 cells through heme oxygenase-1 induction.

    PubMed

    Nepal, Saroj; Kim, Mi Jin; Subedi, Amit; Lee, Eung-Seok; Yong, Chul Soon; Kim, Jung-Ae; Kang, WonKu; Kwak, Mi-Kyung; Arya, Dharamvir Singh; Park, Pil-Hoon

    2012-10-01

    Hepatocellular apoptosis is an essential pathological feature of alcoholic liver disease. Adiponectin, an adipokine predominantly secreted from adipose tissue, has been shown to play beneficial roles in alcoholic liver disease against various inflammatory and pro-apoptotic molecules. However, the effects of adiponectin on ethanol-induced apoptosis in liver cells are largely unknown. Herein, we investigated the role of globular adiponectin (gAcrp) in the prevention of ethanol-induced apoptosis and further tried to decipher the potential mechanisms involved. In the present study, we demonstrated that gAcrp significantly inhibits both ethanol-induced increase in Fas ligand expression and activation of caspase-3 in human hepatoma cell lines (HepG2 cells), suggesting that gAcrp plays a protective role against ethanol-induced apoptosis in liver cells. This protective effect of gAcrp was mediated through adiponectin receptor R1 (adipoR1). Further, globular adiponectin treatment caused induction of heme oxygenase-1 (HO-1) through, at least in part, nuclear factor (erythroid-derived 2)-like 2, (Nrf2) signaling. Treatment with SnPP, a pharmacological inhibitor of HO-1, and knockdown of HO-1 with small interfering RNA (siRNA) restored caspase-3 activity suppressed by gAcrp, indicating a critical role of HO-1 in mediating the protective role of gAcrp in ethanol-induced apoptosis in liver cells. In addition, carbon monoxide, a byproduct obtained from the catabolism of free heme was found to contribute to the anti-apoptotic effect of adiponectin. In conclusion, these data demonstrated that globular adiponectin prevents ethanol-induced apoptosis in HepG2 cells via HO-1 induction and revealed a novel biological response of globular adiponectin in the protection of liver injury from alcohol consumption.

  15. Increase of Intracellular Cyclic AMP by PDE4 Inhibitors Affects HepG2 Cell Cycle Progression and Survival.

    PubMed

    Massimi, Mara; Cardarelli, Silvia; Galli, Francesca; Giardi, Maria Federica; Ragusa, Federica; Panera, Nadia; Cinque, Benedetta; Cifone, Maria Grazia; Biagioni, Stefano; Giorgi, Mauro

    2017-06-01

    Type 4 cyclic nucleotide phosphodiesterases (PDE4) are major members of a superfamily of enzymes (PDE) involved in modulation of intracellular signaling mediated by cAMP. Broadly expressed in most human tissues and present in large amounts in the liver, PDEs have in the last decade been key therapeutic targets for several inflammatory diseases. Recently, a significant body of work has underscored their involvement in different kinds of cancer, but with no attention paid to liver cancer. The present study investigated the effects of two PDE4 inhibitors, rolipram and DC-TA-46, on the growth of human hepatoma HepG2 cells. Treatment with these inhibitors caused a marked increase of intracellular cAMP level and a dose- and time-dependent effect on cell growth. The concentrations of inhibitors that halved cell proliferation to about 50% were used for cell cycle experiments. Rolipram (10 μM) and DC-TA-46 (0.5 μM) produced a decrease of cyclin expression, in particular of cyclin A, as well as an increase in p21, p27 and p53, as evaluated by Western blot analysis. Changes in the intracellular localization of cyclin D1 were also observed after treatments. In addition, both inhibitors caused apoptosis, as demonstrated by an Annexin-V cytofluorimetric assay and analysis of caspase-3/7 activity. Results demonstrated that treatment with PDE4 inhibitors affected HepG2 cell cycle and survival, suggesting that they might be useful as potential adjuvant, chemotherapeutic or chemopreventive agents in hepatocellular carcinoma. J. Cell. Biochem. 118: 1401-1411, 2017. © 2016 Wiley Periodicals, Inc.

  16. Inhibition of energy-producing pathways of HepG2 cells by 3-bromopyruvate.

    PubMed

    Pereira da Silva, Ana Paula; El-Bacha, Tatiana; Kyaw, Nattascha; dos Santos, Reinaldo Sousa; da-Silva, Wagner Seixas; Almeida, Fabio C L; Da Poian, Andrea T; Galina, Antonio

    2009-02-01

    3-BrPA (3-bromopyruvate) is an alkylating agent with anti-tumoral activity on hepatocellular carcinoma. This compound inhibits cellular ATP production owing to its action on glycolysis and oxidative phosphorylation; however, the specific metabolic steps and mechanisms of 3-BrPA action in human hepatocellular carcinomas, particularly its effects on mitochondrial energetics, are poorly understood. In the present study it was found that incubation of HepG2 cells with a low concentration of 3-BrPA for a short period (150 microM for 30 min) significantly affected both glycolysis and mitochondrial respiratory functions. The activity of mitochondrial hexokinase was not inhibited by 150 microM 3-BrPA, but this concentration caused more than 70% inhibition of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 3-phosphoglycerate kinase activities. Additionally, 3-BrPA treatment significantly impaired lactate production by HepG2 cells, even when glucose was withdrawn from the incubation medium. Oxygen consumption of HepG2 cells supported by either pyruvate/malate or succinate was inhibited when cells were pre-incubated with 3-BrPA in glucose-free medium. On the other hand, when cells were pre-incubated in glucose-supplemented medium, oxygen consumption was affected only when succinate was used as the oxidizable substrate. An increase in oligomycin-independent respiration was observed in HepG2 cells treated with 3-BrPA only when incubated in glucose-supplemented medium, indicating that 3-BrPA induces mitochondrial proton leakage as well as blocking the electron transport system. The activity of succinate dehydrogenase was inhibited by 70% by 3-BrPA treatment. These results suggest that the combined action of 3-BrPA on succinate dehydrogenase and on glycolysis, inhibiting steps downstream of the phosphorylation of glucose, play an important role in HepG2 cell death.

  17. 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.

  18. Trinitrotoluene Induces Endoplasmic Reticulum Stress and Apoptosis in HePG2 Cells

    PubMed Central

    Song, Li; Wang, Yue; Wang, Jun; Yang, Fan; Li, Xiaojun; Wu, Yonghui

    2015-01-01

    Background This study aims to describe trinitrotoluene (TNT)-induced endoplasmic reticulum stress (ERS) and apoptosis in HePG2 cells. Material/Methods HePG2 cells were cultured in vitro with 0, 6, 12, or 24 μg/ml TNT solution for 12, 24, and 48 h. Western blotting was performed to detect intracellular ERS-related proteins, including glucose-regulated protein (GRP) 78, GRP94, Caspase 4, p-Jun N-terminal kinase (JNK), and C/EBP homologous protein (CHOP). Real-time PCR was used to measure mRNA expression from the respective genes. Results The expressions of ERS-related proteins GRP78 and GRP94 as well as mRNA and protein expression of ERS signaling apoptotic CHOP in the TNT treatment group were significantly increased. In addition, the mRNA and protein expression levels of ERS-induced apoptotic protein Caspase-4 were significantly increased. Flow cytometry revealed that after TNT treatment, the apoptosis rate also significantly increased. Conclusions TNT could increase the expression levels of GRP78, GRP94, Caspase-4, and CHOP in HePG2 cells; this increase in protein expression might be involved in HePG2 apoptosis through the induction of the ERS pathway. PMID:26551326

  19. Esterification of Ginsenoside Rh2 Enhanced Its Cellular Uptake and Antitumor Activity in Human HepG2 Cells.

    PubMed

    Chen, Fang; Deng, Ze-Yuan; Zhang, Bing; Xiong, Zeng-Xing; Zheng, Shi-Lian; Tan, Chao-Li; Hu, Jiang-Ning

    2016-01-13

    Our previous research had indicated that the octyl ester derivative of ginsenoside Rh2 (Rh2-O) might have a higher bioavailability than Rh2 in the Caco-2 cell line. The aim of this study was to investigate the cellular uptake and antitumor effects of Rh2-O in human HepG2 cells as well as its underlying mechanism compared with Rh2. Results showed that Rh2-O exhibited a higher cellular uptake (63.24%) than Rh2 (36.76%) when incubated with HepG2 cells for 24 h. Rh2-O possessed a dose- and time-dependent inhibitory effect against the proliferation of HepG2 cells. The IC50 value of Rh2-O for inhibition of HepG2 cell proliferation was 20.15 μM, which was roughly half the value of Rh2. Rh2-O induced apoptosis of HepG2 cells through a mitochondrial-mediated intrinsic pathway. In addition, the accumulation of ROS was detected in Rh2-O-treated HepG2 cells, which participated in the apoptosis of HepG2 cells. Conclusively, the findings above all suggested that Rh2-O as well as Rh2 inducing HepG2 cells apoptosis might involve similar mechanisms; however, Rh2-O had better antitumor activities than Rh2, probably due to its higher cellular uptake.

  20. HepG2 cells acquire stem cell-like characteristics after immune cell stimulation.

    PubMed

    Wang, Hang; Yang, Miqing; Lin, Ling; Ren, Hongzhen; Lin, Chaotong; Lin, Suling; Shen, Guoying; Ji, Binfeng; Meng, Chun

    2016-02-01

    The presence of cancer stem cells (CSCs) is currently regarded as one of the main culprits of tumor formation and therapy failure. It is known that chronic inflammation is associated with CSCs, but it is not clear yet how inflammation affects the development of CSCs. In the present study we aimed to examine the relationship between cancer cell stimulation mediated by immune cells and the acquisition of a CSC-like phenotype. Cancer cells derived from single hepatocarcinoma HepG2 cells were treated with mouse splenic B cells (MSBCs) and mouse peritoneal macrophage cells (MPMCs), respectively. The stem cell-like characteristics of the resulting HepG2 cells (MSBC-HepG2 and MPMC-HepG2) were evaluated using different assays, including biomarker assays, in vitro tumoroid and colony forming assays, in vivo tumor forming assays and signal transduction pathway activation assays. Various stemness characteristics of HepG2 cells, including self-renewal, proliferation, chemoresistance and tumorigenicity were evaluated. The expression levels of stemness-related genes and its encoded proteins in the MSBC-HepG2 and MPMC-HepG2 cells were assessed using RT-PCR and FACS analyses. We found that MSBC-HepG2 and MPMC-HepG2 cells possess hepatic CSC properties, including persistent self-renewal, extensive proliferation, drug resistance, high tumorigenic capacity and over-expression of CSC-related genes and proteins (i.e., EpCAM, ALDH, CD133 and CD44), compared to the parental cells. We also found that 1x10(3) MSBC-HepG2 and MPMC-HepG2 cells were able to form tumors in NOD/SCID mice and that the Notch and SHH signaling pathways were highly activated in MSBC-HepG2 cells. We conclude that the immune system may have a double-edge effect on cancer development. On one hand, immune cells such as B lymphocytes and macrophages may recognize, attack and eliminate cancer cells, whereas on the other hand, they may promote a subset of cancer cells to acquire stem cell-like characteristics.

  1. Differential genomic effects of six different TiO2 nanomaterials on human liver HepG2 cells

    EPA Science Inventory

    Engineered nanoparticles are reported to cause liver toxicity in vivo. To better assess the mechanism of the in vivo liver toxicity, we used the human hepatocarcinoma cells (HepG2) as a model system. Human HepG2 cells were exposed to 6 TiO2 nanomaterials (with dry primary partic...

  2. Modification of the apolipoprotein B gene in HepG2 cells by gene targeting.

    PubMed Central

    Farese, R V; Flynn, L M; Young, S G

    1992-01-01

    The HepG2 cell line has been used extensively to study the synthesis and secretion of apolipoprotein (apo) B. In this study, we tested whether gene-targeting techniques can be used to inactivate one of the apo B alleles in HepG2 cells by homologous recombination using a transfected gene-targeting vector. Our vector contained exons 1-7 of the apo B gene, in which exon 2 was interrupted by a promoterless neomycin resistance (neo(r)) gene. The recombination of this vector with the cognate gene would inactivate an apo B allele and enable the apo B promoter to activate the transcription of the neo(r) gene. To detect the rare homologous recombinant clone, we developed a novel solid phase RIA that uses the apo B-specific monoclonal antibody MB19 to analyze the apo B secreted by G418-resistant (G418r) clones. Antibody MB19 detects a two-allele genetic polymorphism in apo B by binding to the apo B allotypes MB19(1) and MB19(2) with high and low affinity, respectively. HepG2 cells normally secrete both the apo B MB19 allotypes. Using the MB19 immunoassay, we identified a G418r HepG2 clone that had lost the ability to secrete the MB19(1) allotype. The inactivation of an apo B allele of this clone was confirmed by the polymerase chain reaction amplification of an 865-bp fragment unique to the targeted apo B allele and by Southern blotting of genomic DNA. This study demonstrates that gene-targeting techniques can be used to modify the apo B gene in HepG2 cells and demonstrates the usefulness of a novel solid phase RIA system for detecting apo B gene targeting events in this cell line. Images PMID:1321843

  3. Modification of the apolipoprotein B gene in HepG2 cells by gene targeting.

    PubMed

    Farese, R V; Flynn, L M; Young, S G

    1992-07-01

    The HepG2 cell line has been used extensively to study the synthesis and secretion of apolipoprotein (apo) B. In this study, we tested whether gene-targeting techniques can be used to inactivate one of the apo B alleles in HepG2 cells by homologous recombination using a transfected gene-targeting vector. Our vector contained exons 1-7 of the apo B gene, in which exon 2 was interrupted by a promoterless neomycin resistance (neo(r)) gene. The recombination of this vector with the cognate gene would inactivate an apo B allele and enable the apo B promoter to activate the transcription of the neo(r) gene. To detect the rare homologous recombinant clone, we developed a novel solid phase RIA that uses the apo B-specific monoclonal antibody MB19 to analyze the apo B secreted by G418-resistant (G418r) clones. Antibody MB19 detects a two-allele genetic polymorphism in apo B by binding to the apo B allotypes MB19(1) and MB19(2) with high and low affinity, respectively. HepG2 cells normally secrete both the apo B MB19 allotypes. Using the MB19 immunoassay, we identified a G418r HepG2 clone that had lost the ability to secrete the MB19(1) allotype. The inactivation of an apo B allele of this clone was confirmed by the polymerase chain reaction amplification of an 865-bp fragment unique to the targeted apo B allele and by Southern blotting of genomic DNA. This study demonstrates that gene-targeting techniques can be used to modify the apo B gene in HepG2 cells and demonstrates the usefulness of a novel solid phase RIA system for detecting apo B gene targeting events in this cell line.

  4. Nobiletin, a polymethoxyflavone in citrus fruits, reduces TAFI expression in HepG2 cells through transcriptional inhibition.

    PubMed

    Takada, Kimihiko; Seike, Toru; Sasaki, Tomoyuki; Masuda, Yutaka; Ito, Akira; Ishii, Hidemi

    2013-06-01

    Thrombin-activatable fibrinolysis inhibitor (TAFI, carboxypeptidase B2) is a 58-kDa plasma glycoprotein secreted by hepatocytes as an inactive form. TAFI is activated by the thrombin-thrombomodulin complex, and activated TAFI (TAFIa) plays an important role in regulating the balance between coagulation and fibrinolysis through inhibition of fibrinolysis. It has been suggested that high levels of TAFI in circulating plasma increase the risks of cardiovascular death and acute phase in ischaemic stroke. However, the mechanisms of regulating TAFI expression have been unclear. The present study investigated the effects of nobiletin (a polymethoxy flavonoid contained in the rind of citrus fruits) on TAFI gene (CPB2) and TAFI antigen expression in cultured human hepatoma HepG2 cells. Nobiletin decreased the release of TAFI antigen from HepG2 cells into conditioned medium in parallel with decreased levels of CPB2 mRNA and antigen. The half-life time of CPB2 mRNA in nobiletin-treated cells was unchanged compared to that of untreated control cells. Using nobiletin-treated cells that were transfected with a luciferase CPB2 promoter reporter plasmid, activity decreased to half of that in untreated control cells. A series of luciferase reporter constructs containing 5´-flanking region deletions of the human CPB2 gene showed that the sequences from -150 bp to -50 bp were essential for transcription of CPB2 and contained an AP-1 binding sequence at ~ -119 bp to - 99 bp in the CPB2 promoter. The amount of complexed nuclear protein and sequences from ~ -119 bp to -99 bp was decreased in nobiletin-treated cells. ChIP assays showed that c-Jun bound to the ~ -119 bp to -99 bp region of the CPB2 promoter and that the amount of the immunocomplex decreased after nobiletin treatment. Therefore, nobiletin-induced repression of CPB2 transcription might involve AP-1 inhibition and/or prevention of AP-1 binding in a specific region on the CPB2 gene in HepG2 cells.

  5. Glucosylceramide synthase inhibitors D-PDMP and D-EtDO-P4 decrease the GM3 ganglioside level, differ in their effects on insulin receptor autophosphorylation but increase Akt1 kinase phosphorylation in human hepatoma HepG2 cells.

    PubMed

    Fedoryszak-Kuśka, Natalia; Panasiewicz, Mirosława; Domek, Hanna; Pacuszka, Tadeusz

    2016-01-01

    Gangliosides function as modulators of several cell growth related receptors. It was shown for caveolin-rich adipocytes, that GM3 ganglioside binds to insulin receptor (IR), dissociates its complex with caveolin, and thus lowers IR autophosphorylation following insulin treatment. We extended those studies into human hepatocyte-derived HepG2 cells, characterized by a high level of IR but low of caveolin. To lower the glycosphingolipid content, estimated by GM3 concentration, two glucosylceramide synthase inhibitors d-threo-1-pheny-2-decanoylamino-3-morpholino-1-propanol (d-PDMP) and d-threo-1-(3,4,-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (d-EtDO-P4) were used. d-PDMP at 40 µM or d-EtDO-P4 at 1 µM concentrations in culture medium decreased the GM3 content to 22.3% (17.8-26.1%) and 18.1% (13.7-24.4%), respectively, of the control value. The reduction of GM3 obtained with d-PDMP was accompanied by a 185.1% (153.5-423.8%) significant increase in the level of IR autophosphorylation following cell stimulation with 100 nM insulin. The effect of d-EtDO-P4 on IR autophosphorylation was smaller amounting to an increase by 134.8% (111.3-167.8%) of the control level and statistically non-significant. The effects of d-PDMP and d-EtDO-P4 could also be detected at the level of Akt1 kinase. In cells grown in the presence of d-PDMP the level of phosphorylated Akt1 was 286.0% (151.4%-621.1%) of that in the control. In this case the effect of d-EtDO-P4 was similar: 223.0% (181.4-315.4%) significant increase in phosphorylated Akt1. We assume that glycosphingolipid depletion in HepG2 cells may affect not only IR autophosphorylation but also, independently, the phosphorylation of Akt1, by modifying the membrane microenvironment of this kinase.

  6. Verbesina encelioides: cytotoxicity, cell cycle arrest, and oxidative DNA damage in human liver cancer (HepG2) cell line.

    PubMed

    Al-Oqail, Mai M; Siddiqui, Maqsood A; Al-Sheddi, Ebtesam S; Saquib, Quaiser; Musarrat, Javed; Al-Khedhairy, Abdulaziz A; Farshori, Nida N

    2016-05-10

    Cancer is a major health problem and exploiting natural products have been one of the most successful methods to combat this disease. Verbesina encelioides is a notorious weed with various pharmacological properties. The aim of the present investigation was to screen the anticancer potential of V. encelioides extract against human lung cancer (A-549), breast cancer (MCF-7), and liver cancer (HepG2) cell lines. A-549, MCF-7, and HepG2 cells were exposed to various concentrations of (10-1000 μg/ml) of V. encelioides for 24 h. Further, cytotoxic concentrations (250, 500, and 1000 μg/ml) of V. encelioides induced oxidative stress (GSH and LPO), reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), cell cycle arrest, and DNA damage in HepG2 cells were studied. The exposure of cells to 10-1000 μg/ml of extract for 24 h, revealed the concentrations 250-1000 μg/ml was cytotoxic against MCF-7 and HepG2 cells, but not against A-549 cells. Moreover, the extract showed higher decrease in the cell viability against HepG2 cells than MCF-7 cells. Therefore, HepG2 cells were selected for further studies viz. oxidative stress (GSH and LPO), reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), cell cycle arrest, and DNA damage. The results revealed differential anticancer activity of V. encelioides against A-549, MCF-7 and HepG2 cells. A significant induction of oxidative stress, ROS generation, and MMP levels was observed in HepG2 cells. The cell cycle analysis and comet assay showed that V. encelioides significantly induced G2/M arrests and DNA damage. These results indicate that V. encelioides possess substantial cytotoxic potential and may warrant further investigation to develop potential anticancer agent.

  7. Pentoxifylline induces apoptosis of HepG2 cells by reducing reactive oxygen species production and activating the MAPK signaling.

    PubMed

    Wang, Yan; Dong, Lei; Li, Jing; Luo, Miaosha; Shang, Boxin

    2017-08-15

    Pentoxifylline (PTX) is a methylxanthine derivative and has potent anti-tumor activity. This study aimed at investigating the anti-HCC effects of PTX and associated molecular mechanisms. The effects of varying doses of PTX on viability, cell cycle and apoptosis of HepG2 cells were determined by MTT and flow cytometry, respectively. The effects of PTX on the production of reactive oxygen species (ROS), expression of pro- and anti-apoptotic regulators and activation of the MAPK signaling in HepG2 cells were analyzed by flow cytometry and Western blot assays. The effects of PTX on the growth of implanted HepG2 cells and their apoptosis in mice were examined. Our results indicated that PTX inhibited proliferation of HepG2 cells and induced HepG2 cell cycle arrest at G0/G1 phase and apoptosis in a dose- and time-dependent manner. Treatment with PTX reduced levels of ROS and Bcl-XL expression, but increased caspase 3 and caspase 9 expression and JNK and ERK1/2 phosphorylation in HepG2 cells. Pre-treatment with n-acetyl-l-cysteine (NAC), a ROS scavenger, enhanced PTX-mediated cell cycle arrest, apoptosis and the JNK and ERK MAPK activation, while pre-treatment with SP600125 or PD98509 attenuated PTX-mediated effects in HepG2 cells. Treatment with PTX inhibited the growth of implanted HCC and promoted HCC apoptosis in mice. Our data demonstrate that PTX inhibits proliferation of HepG2 cells and induces HepG2 cell apoptosis by attenuating ROS production and enhancing the MAPK activation in HepG2 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. 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

  9. FOXO1 and LXRα downregulate the apolipoprotein A-I gene expression during hydrogen peroxide-induced oxidative stress in HepG2 cells.

    PubMed

    Shavva, Vladimir S; Bogomolova, Alexandra M; Nikitin, Artemy A; Dizhe, Ella B; Oleinikova, Galina N; Lapikov, Ivan A; Tanyanskiy, Dmitry A; Perevozchikov, Andrej P; Orlov, Sergey V

    2017-01-01

    Reactive oxygen species damage various cell components including DNA, proteins, and lipids, and these impairments could be a reason for severe human diseases including atherosclerosis. Forkhead box O1 (FOXO1), an important metabolic transcription factor, upregulates antioxidant and proapoptotic genes during oxidative stress. Apolipoprotein A-I (ApoA-I) forms high density lipoprotein (HDL) particles that are responsible for cholesterol transfer from peripheral tissues to liver for removal in bile in vertebrates. The main sources for plasma ApoA-I in mammals are liver and jejunum. Hepatic apoA-I transcription depends on a multitude of metabolic transcription factors. We demonstrate that ApoA-I synthesis and secretion are decreased during H2O2-induced oxidative stress in human hepatoma cell line HepG2. Here, we first show that FOXO1 binds to site B of apoA-I hepatic enhancer and downregulates apoA-I gene activity in HepG2 cells. Moreover, FOXO1 and LXRα transcription factors participate in H2O2-triggered downregulation of apoA-I gene together with Src, JNK, p38, and AMPK kinase cascades. Mutations of sites B or C as well as the administration of siRNAs against FOXO1 or LXRα to HepG2 cells abolished the hydrogen peroxide-mediated suppression of apoA-I gene.

  10. Estradiol and Estrogen Receptor Agonists Oppose Oncogenic Actions of Leptin in HepG2 Cells

    PubMed Central

    Shen, Minqian; Shi, Haifei

    2016-01-01

    Obesity is a significant risk factor for certain cancers, including hepatocellular carcinoma (HCC). Leptin, a hormone secreted by white adipose tissue, precipitates HCC development. Epidemiology data show that men have a much higher incidence of HCC than women, suggesting that estrogens and its receptors may inhibit HCC development and progression. Whether estrogens antagonize oncogenic action of leptin is uncertain. To investigate potential inhibitory effects of estrogens on leptin-induced HCC development, HCC cell line HepG2 cells were treated with leptin in combination with 17 β-estradiol (E2), estrogen receptor-α (ER-α) selective agonist PPT, ER-β selective agonist DPN, or G protein-coupled ER (GPER) selective agonist G-1. Cell number, proliferation, and apoptosis were determined, and leptin- and estrogen-related intracellular signaling pathways were analyzed. HepG2 cells expressed a low level of ER-β mRNA, and leptin treatment increased ER-β expression. E2 suppressed leptin-induced HepG2 cell proliferation and promoted cell apoptosis in a dose-dependent manner. Additionally E2 reversed leptin-induced STAT3 and leptin-suppressed SOCS3, which was mainly achieved by activation of ER-β. E2 also enhanced ERK via activating ER-α and GPER and activated p38/MAPK via activating ER-β. To conclude, E2 and its receptors antagonize the oncogenic actions of leptin in HepG2 cells by inhibiting cell proliferation and stimulating cell apoptosis, which was associated with reversing leptin-induced changes in SOCS3/STAT3 and increasing p38/MAPK by activating ER-β, and increasing ERK by activating ER-α and GPER. Identifying roles of different estrogen receptors would provide comprehensive understanding of estrogenic mechanisms in HCC development and shed light on potential treatment for HCC patients. PMID:26982332

  11. Time-course regulation of survival pathways by epicatechin on HepG2 cells.

    PubMed

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

    2009-02-01

    Polyphenols, such as epicatechin, have been reported to exhibit a wide range of biological activities. The objective of the present study was to investigate the time-dependent regulation by epicatechin of survival/proliferation pathways in HepG2 cells. Treatment of HepG2 cells with 10 micromol/L epicatechin did not result in any cell damage up to 18 h, as evaluated by the lactate dehydrogenase assay. Moreover, the enhanced cell death evoked by an oxidative stress induced with tert-butyl hydroperoxide was prevented in the cells pretreated 4 or 18 h with epicatechin. Epicatechin-induced survival was a rapid event that was accompanied by early and sustained activation of major survival signaling proteins, such as AKT/phosphatidylinositol 3-kinase and extracellular-regulated kinase (activated from 5 min to 18 h), as well as protein kinase C (PKC)-alpha (30 min to 18 h), in concert with unaltered c-jun N-amino terminal kinase levels and early inactivation of key death-related signals like PKC-delta (5 min to 18 h). Additionally, reactive oxygen species generation was transiently reduced when cells were treated with 10 micromol/L epicatechin (15-240 min). These data suggest that epicatechin induces cellular survival through a tight regulation of survival/proliferation pathways that requires the integration of different signals and persists over time, the ultimate effect on HepG2 cells being regulated by the balance among these signals.

  12. Protection of human HepG2 cells against oxidative stress by cocoa phenolic extract.

    PubMed

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

    2008-09-10

    Cocoa is a rich source of flavanols and procyanidin oligomers with antioxidative properties, providing protection against oxidation and nitration. The present study investigated the potential protective effect of a polyphenolic extract from cocoa on cell viability and antioxidant defenses of cultured human HepG2 cells submitted to oxidative stress induced by tert-butylhydroperoxide (t-BOOH). Pretreatment of cells with 0.05-50 microg/mL of cocoa polyphenolic extract (CPE) for 2 or 20 h completely prevented cell damage and enhanced activity of antioxidant enzymes induced by a treatment with t-BOOH. Moreover, lower levels of GSH caused by t-BOOH in HepG2 cells were partly recovered by a pretreatment with CPE. Increased reactive oxygen species (ROS) induced by t-BOOH was dose-dependently prevented when cells were pretreated for 2 or 20 h with CPE. These results show that treatment of HepG2 in culture with CPE (within the physiological range of concentrations) confers a significant protection against oxidation to the cells.

  13. Toxicogenetic effects of low concentrations of the pesticides imidacloprid and sulfentrazone individually and in combination in in vitro tests with HepG2 cells and Salmonella typhimurium.

    PubMed

    Bianchi, Jaqueline; Cabral-de-Mello, Diogo Cavalcanti; Marin-Morales, Maria Aparecida

    2015-10-01

    The insecticide imidacloprid and the herbicide sulfentrazone are two different classes of pesticides that are used for pest control in sugarcane agriculture. To evaluate the genotoxic potential of low concentrations of these two pesticides alone and in mixture, the comet assay and the micronucleus (MN) test employing fluorescence in situ hybridization (FISH) with a centromeric probe were applied in human hepatoma cell lines (HepG2), in a 24-h assay. Mutagenicity was assessed by Salmonella/microsome assay with TA98 and TA100 strains in the absence and presence of an exogenous metabolizing system (S9). The results showed significant inductions of MN in HepG2 cells by both pesticides, for all the tested concentrations. As evidenced in the comet assay, only the imidacloprid presented significant responses. When the two pesticides were associated, a significant induction of damage was observed in the HepG2 cells by the comet assay, but not by the MN test. Moreover, the MN induced by the mixtures of the pesticides appeared at lower levels than those induced by sulfentrazone and imidacloprid when tested alone. According to the FISH results, the damage induced by imidacloprid in the HepG2 cells resulted from a clastogenic action of this insecticide (76.6% of the MN did not present a centromeric signal). For the herbicide sulfentrazone and for the mixture of the pesticides, a similar frequency of MN with and without the presence of the centromeric signal (herbicide: 52.45% of the MN without centromeric signal and 47.54% of the MN with centromeric signal; mixture: 48.71% of the MN without centromeric signal and 51.42% of the MN with centromeric signal) was verified. Based on these results, it was concluded that each one of the pesticides evaluated interacts with the DNA of HepG2 cells and causes irreparable alterations in the cells. However, the combination of the pesticides showed an antagonistic effect on the cells and the damage induced was milder and not persistent in

  14. Cytotoxic effects induced by unmodified and organically modified nanoclays in the human hepatic HepG2 cell line.

    PubMed

    Lordan, Sinéad; Kennedy, James E; Higginbotham, Clement L

    2011-01-01

    The term 'nanoclay' generically refers to the natural clay mineral, montmorillonite, with silica and alumina as the dominant constituents. The incorporation of nanoclays into polymeric systems dramatically enhances their barrier properties as well as their thermal and mechanical resistance. Consequently, nanoclays are employed in a wide range of industrial applications with recent studies reporting potential use in the modulation of drug release. With the increase in manufacturing of nanoclay-containing products, information on the toxicological and health effects of nanoclay exposure is warranted. Thus, the objective of the present study was to evaluate the cytotoxicity of two different nanoclays: the unmodified nanoclay, Cloisite Na+ ®, and the organically modified nanoclay, Cloisite 93A®, in human hepatoma HepG2 cells. Following 24 h exposure the nanoclays significantly decreased cell viability. Cloisite Na+ induced intracellular reactive oxygen species (ROS) formation which coincided with increased cell membrane damage, whilst ROS generation did not play a role in Cloisite 93A-induced cell death. Neither of the nanoclays induced caspase-3/7 activation. Moreover, in the cell culture medium the nanoclays aggregated differently and this appeared to have an effect on their mechanisms of toxicity. Taken together, our data demonstrate that nanoclays are highly cytotoxic and as a result pose a possible risk to human health. Copyright © 2010 John Wiley & Sons, Ltd.

  15. Dinitrophenol-induced mitochondrial uncoupling in vivo triggers respiratory adaptation in HepG2 cells.

    PubMed

    Desquiret, Valérie; Loiseau, Dominique; Jacques, Caroline; Douay, Olivier; Malthièry, Yves; Ritz, Patrick; Roussel, Damien

    2006-01-01

    Here, we show that 3 days of mitochondrial uncoupling, induced by low concentrations of dinitrophenol (10 and 50 microM) in cultured human HepG2 cells, triggers cellular metabolic adaptation towards oxidative metabolism. Chronic respiratory uncoupling of HepG2 cells induced an increase in cellular oxygen consumption, oxidative capacity and cytochrome c oxidase activity. This was associated with an upregulation of COXIV and ANT3 gene expression, two nuclear genes that encode mitochondrial proteins involved in oxidative phosphorylation. Glucose consumption, lactate and pyruvate production and growth rate were unaffected, indicating that metabolic adaptation of HepG2 cells undergoing chronic respiratory uncoupling allows continuous and efficient mitochondrial ATP production without the need to increase glycolytic activity. In contrast, 3 days of dinitrophenol treatment did not change the oxidative capacity of human 143B.TK(-) cells, but it increased glucose consumption, lactate and pyruvate production. Despite a large increase in glycolytic metabolism, the growth rate of 143B.TK(-) cells was significantly reduced by dinitrophenol-induced mitochondrial uncoupling. We propose that chronic respiratory uncoupling may constitute an internal bioenergetic signal, which would initiate a coordinated increase in nuclear respiratory gene expression, which ultimately drives mitochondrial metabolic adaptation within cells.

  16. Urotensin II-induced insulin resistance is mediated by NADPH oxidase-derived reactive oxygen species in HepG2 cells

    PubMed Central

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

    2016-01-01

    AIM: To investigated the effects of urotensin II (UII) on hepatic insulin resistance in HepG2 cells and the potential mechanisms involved. METHODS: Human hepatoma HepG2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucose-oxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species (ROS) levels were detected with a multimode reader using a 2′,7′-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase (JNK), insulin signal essential molecules such as insulin receptor substrate -1 (IRS-1), protein kinase B (Akt), glycogen synthase kinase-3β (GSK-3β), and glucose transporter-2 (Glut 2), and NADPH oxidase subunits such as gp91phox, p67phox, p47phox, p40phox, and p22phox were evaluated by Western blot. RESULTS: Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption (P < 0.05) and glycogen content (P < 0.01) in HepG2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression (P < 0.01) and phosphorylation of IRS-1 (P < 0.05), associated with down-regulation of Akt (P < 0.05) and GSK-3β (P < 0.05) phosphorylation levels, and the expression of Glut 2 (P < 0.001), indicating an insulin-resistance state in HepG2 cells. Furthermore, UII enhanced the phosphorylation of JNK (P < 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1 (P < 0.001), phosphorylation of IRS-1 (P < 0.001) and GSK-3β (P < 0.05), and glycogen synthesis (P < 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation (P < 0.05) and NADPH oxidase subunit expression (P < 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production (P < 0.05), JNK phosphorylation (P < 0

  17. Urotensin II-induced insulin resistance is mediated by NADPH oxidase-derived reactive oxygen species in HepG2 cells.

    PubMed

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

    2016-07-07

    To investigated the effects of urotensin II (UII) on hepatic insulin resistance in HepG2 cells and the potential mechanisms involved. Human hepatoma HepG2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucose-oxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species (ROS) levels were detected with a multimode reader using a 2',7'-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase (JNK), insulin signal essential molecules such as insulin receptor substrate -1 (IRS-1), protein kinase B (Akt), glycogen synthase kinase-3β (GSK-3β), and glucose transporter-2 (Glut 2), and NADPH oxidase subunits such as gp91(phox), p67(phox), p47(phox), p40(phox), and p22(phox) were evaluated by Western blot. Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption (P < 0.05) and glycogen content (P < 0.01) in HepG2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression (P < 0.01) and phosphorylation of IRS-1 (P < 0.05), associated with down-regulation of Akt (P < 0.05) and GSK-3β (P < 0.05) phosphorylation levels, and the expression of Glut 2 (P < 0.001), indicating an insulin-resistance state in HepG2 cells. Furthermore, UII enhanced the phosphorylation of JNK (P < 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1 (P < 0.001), phosphorylation of IRS-1 (P < 0.001) and GSK-3β (P < 0.05), and glycogen synthesis (P < 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation (P < 0.05) and NADPH oxidase subunit expression (P < 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production (P < 0.05), JNK phosphorylation (P < 0.05), and insulin

  18. Riboflavin deficiency induces a significant change in proteomic profiles in HepG2 cells

    PubMed Central

    Xin, Zhonghao; Pu, Lingling; Gao, Weina; Wang, Yawen; Wei, Jingyu; Shi, Tala; Yao, Zhanxin; Guo, Changjiang

    2017-01-01

    Riboflavin deficiency is widespread in many regions over the world, especially in underdeveloped countries. In this study, we investigated the effects of riboflavin deficiency on protein expression profiles in HepG2 cells in order to provide molecular information for the abnormalities induced by riboflavin deficiency. HepG2 cells were cultured in media containing different concentrations of riboflavin. Changes of cell viability and apoptosis were assessed. A comparative proteomic analysis was performed using a label-free shotgun method with LC–MS/MS to investigate the global changes of proteomic profiles in response to riboflavin deficiency. Immunoblotting test was used to validate the results of proteomic approach. The cell viability and apoptosis tests showed that riboflavin was vital in maintaining the cytoactivity of HepG2 cells. The label-free proteomic analysis revealed that a total of 37 proteins showing differential expression (±2 fold, p < 0.05) were identified after riboflavin deficiency. Bioinformatics analysis indicated that the riboflavin deficiency caused an up-regulation of Parkinson’s disease pathway, steroid catabolism, endoplasmic reticulum stress and apoptotic process, while the fatty acid metabolism, tricarboxylic citrate cycle, oxidative phosphorylation and iron metabolism were down-regulated. These findings provide a molecular basis for the elucidation of the effects caused by riboflavin deficiency. PMID:28367977

  19. Riboflavin deficiency induces a significant change in proteomic profiles in HepG2 cells.

    PubMed

    Xin, Zhonghao; Pu, Lingling; Gao, Weina; Wang, Yawen; Wei, Jingyu; Shi, Tala; Yao, Zhanxin; Guo, Changjiang

    2017-04-03

    Riboflavin deficiency is widespread in many regions over the world, especially in underdeveloped countries. In this study, we investigated the effects of riboflavin deficiency on protein expression profiles in HepG2 cells in order to provide molecular information for the abnormalities induced by riboflavin deficiency. HepG2 cells were cultured in media containing different concentrations of riboflavin. Changes of cell viability and apoptosis were assessed. A comparative proteomic analysis was performed using a label-free shotgun method with LC-MS/MS to investigate the global changes of proteomic profiles in response to riboflavin deficiency. Immunoblotting test was used to validate the results of proteomic approach. The cell viability and apoptosis tests showed that riboflavin was vital in maintaining the cytoactivity of HepG2 cells. The label-free proteomic analysis revealed that a total of 37 proteins showing differential expression (±2 fold, p < 0.05) were identified after riboflavin deficiency. Bioinformatics analysis indicated that the riboflavin deficiency caused an up-regulation of Parkinson's disease pathway, steroid catabolism, endoplasmic reticulum stress and apoptotic process, while the fatty acid metabolism, tricarboxylic citrate cycle, oxidative phosphorylation and iron metabolism were down-regulated. These findings provide a molecular basis for the elucidation of the effects caused by riboflavin deficiency.

  20. [Establishment of a model for evaluating hypolipidemic effect in HepG2 cells].

    PubMed

    Niu, Yucun; Lü, Na; Li, Ying; Zhao, Dan; Sun, Changhao

    2010-03-01

    To establish a model of evaluating hypolipidemic effect in vitro. Adding cholesterol to the culture medium for HepG2 cells to induce a hypercholesterolemia model. The content of cellular cholesterol and the expression of protein regulating cholesterol metabolism in HepG2 cells were determined. The validation of the model was identified by lovastatin, a widely used cholesterol-lowering drug. Free fatty acid was added to the culture medium for HepG2 cells to induce a hypertriglyceridemia model. The content of cellular triglyceride and the absorption rate of free fatty acid were determined. The validation of the model was identified by fenofibrate, a triglyceride-lowering drug. Cellular cholesterol content was increased and the expression of HMG-CoA redutase, SREBP-2 and LDLR were decreased after adding cholesterol and 25-hydrocholesterol to the culture medium. Cellular cholesterol was decreased and the expression of SREBP-2 and LDLR were up-regulated by Lovastatin. The absorption of oleic acid in cells was up to 40% after adding oleic acid (50 micromol) to the culture medium for 6 h. The absorption of free fatty acid was increased but the content of cellular triglyceride was not increased in cells by Fenofibrate. This model might be an effective method for screening and assessing functional factors for lowing plasma lipids.

  1. 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.

  2. Mechanism of the promotion of steatotic HepG2 cell apoptosis by cholesterol

    PubMed Central

    Zhu, Chunyan; Xie, Ping; Zhao, Fei; Zhang, Lingqiang; An, Wei; Zhan, Yutao

    2014-01-01

    The role of cholesterol in the pathogenesis of non-alcoholic steatohepatitis (NASH) remains unclear. It is known that apoptosis of hepatocytes is an important characteristics of NASH. The objective of this study was to investigate the effects of cholesterol on steatotic HepG2 cell apoptosis and the possible mechanism in vitro. In this study, HepG2 cells were divided into three groups: (1) normal group, (2) steatosis group and (3) cholesterol group. HepG2 cells were treated with oleic acid to establish a steatosis study model. Steatosis was assessed by Oil Red O staining and triglyceride content assay. Cell apoptosis was measured using an apoptosis kit. The expression levels of apoptosis-related proteins (P53, Bcl-2, Bax, caspase-3, cyclin A, cyclin B1 and cyclin E) were determined by western blot analyses. We found that a hepatocyte steatosis model was successfully established by oleic acid (200 μmol/L) induction. The cholesterol (50 mg/L) group had similar amount of lipid droplets and triglyceride content as steatosis group (P > 0.5). However, the apoptosis rate (P < 0.01) of the cholesterol group was significantly higher than that of the normal group or the steatosis group, and the protein expressions of Bax and caspase-3, but not P53, Bcl-2, cyclin A, cyclin B1 and cyclin E, were also increased in the cholesterol group. Those results suggested that cholesterol markedly promoted apoptosis of steatosis HepG2 cells in vitro, likely through the up-regulation of Bax and caspase-3 expression. This study contributes to explain the effect of cholesterol on NASH pathogenesis. PMID:25400762

  3. Biosynthesis of hematite nanoparticles and its cytotoxic effect on HepG2 cancer cells.

    PubMed

    Rajendran, Kumar; Karunagaran, Vithiya; Mahanty, Biswanath; Sen, Shampa

    2015-03-01

    Iron oxide nanoparticles were gaining significant importance in a variety of applications due to its paramagnetic properties and biocompatibility. Various chemical methods were employed for hematite nanoparticle synthesis which require special equipment or a complex production process. In this study, protein capped crystalline hexagonal hematite (α-Fe2O3) nanoparticles were synthesized by green approach using culture supernatant of a newly isolated bacterium, Bacillus cereus SVK1 at ambient conditions. The synthesized nanoparticles were characterized by electron microscopy, X-ray diffraction, UV-visible spectroscopy and Fourier transform infrared spectroscopic analysis. Nanoparticles were evaluated for its possible anticancer activity against HepG2 liver cancer cells by MTT assay. Hematite nanoparticles with an average diameter of 30.2 nm, exhibited a significant cytotoxicity toward HepG2 cells in a concentration-dependent manner (CTC50=704 ng/ml). Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Glycyrrhizin, silymarin, and ursodeoxycholic acid regulate a common hepatoprotective pathway in HepG2 cells.

    PubMed

    Hsiang, Chien-Yun; Lin, Li-Jen; Kao, Shung-Te; Lo, Hsin-Yi; Chou, Shun-Ting; Ho, Tin-Yun

    2015-07-15

    Glycyrrhizin, silymarin, and ursodeoxycholic acid are widely used hepatoprotectants for the treatment of liver disorders, such as hepatitis C virus infection, primary biliary cirrhosis, and hepatocellular carcinoma. The gene expression profiles of HepG2 cells responsive to glycyrrhizin, silymarin, and ursodeoxycholic acid were analyzed in this study. HepG2 cells were treated with 25 µM hepatoprotectants for 24 h. Gene expression profiles of hepatoprotectants-treated cells were analyzed by oligonucleotide microarray in triplicates. Nuclear factor-κB (NF-κB) activities were assessed by luciferase assay. Among a total of 30,968 genes, 252 genes were commonly regulated by glycyrrhizin, silymarin, and ursodeoxycholic acid. These compounds affected the expression of genes relevant various biological pathways, such as neurotransmission, and glucose and lipid metabolism. Genes involved in hepatocarcinogenesis, apoptosis, and anti-oxidative pathways were differentially regulated by all compounds. Moreover, interaction networks showed that NF-κB might play a central role in the regulation of gene expression. Further analysis revealed that these hepatoprotectants inhibited NF-κB activities in a dose-dependent manner. Our data suggested that glycyrrhizin, silymarin, and ursodeoxycholic acid regulated the expression of genes relevant to apoptosis and oxidative stress in HepG2 cells. Moreover, the regulation by these hepatoprotectants might be relevant to the suppression of NF-κB activities. Copyright © 2015 Elsevier GmbH. All rights reserved.

  5. Lovastatin prevents bleomycin-induced DNA damage to HepG2 cells

    PubMed Central

    Nasiri, Marjan; Etebari, Mahmoud; Jafarian-Dehkordi, Abbas; Moradi, Shahla

    2016-01-01

    Lovastatin as a member of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors is used as a lipid-lowering agent. It can also inhibit the formation of hydrogen peroxide and superoxide anion and finally leads to decline in oxidative stress processes. Here, we evaluated whether lovastatin can increase DNA damage resistance of HepG2 cells against genotoxicity of the anticancer drug bleomycin (BLM). HepG2 cells were incubated with different concentrations of lovastatin (0.1, 0.5, 1, 5 µM) before exposure to BLM (0.5 µg/mL for one h). The genotoxic dose of BLM and lovastatin was separately determined and comet assay was used to evaluate the genotoxicity. After trapping cells in agarose coated lames, they were lysed and the electrophoresis was done in alkaline pH, then colored and monitored by florescent microscope. The results of this study indicated that lovastatin in doses lower than 5 µM has genoprotective effect and in doses higher than 50 µM is genotoxic. In conclusion, lovastatin is able to protect genotoxic effects of BLM in HepG2 cells. Further studies are needed to elucidate the mechanism(s) involved in this process. PMID:28003840

  6. Selenocystine against methyl mercury cytotoxicity in HepG2 cells.

    PubMed

    Wang, Han; Chen, Beibei; He, Man; Yu, Xiaoxiao; Hu, Bin

    2017-12-01

    Methyl mercury (MeHg) is a highly toxic substance and the effect of selenium against MeHg toxicity is a hot topic. Until now, no related works have been reported from the view of the point of elemental speciation which is promising to study the mechanism at the molecular level. In this work, to reveal the effect of selenocystine (SeCys2) against MeHg cytotoxicity in HepG2 cells, a comprehensive analytical platform for speciation study of mercury and selenium in MeHg incubated or MeHg and SeCys2 co-incubated HepG2 cells was developed by integrating liquid chromatography (LC) - inductively coupled plasma mass spectrometry (ICP-MS) hyphenated techniques and chip-based pretreatment method. Interesting phenomenon was found that the co-incubation of MeHg with SeCys2 promoted the uptake of MeHg in HepG2 cells, but reduced the cytotoxicity of MeHg. Results obtained by ICP-MS based hyphenated techniques revealed a possible pathway for the incorporation and excretion of mercury species with the coexistence of SeCys2. The formation of MeHg and SeCys2 aggregation promotes the uptake of MeHg; majority of MeHg transforms into small molecular complexes (MeHg-glutathione (GSH) and MeHg-cysteine (Cys)) in HepG2 cells; and MeHg-GSH is the elimination species which results in reducing the cytotoxicity of MeHg.

  7. Molecular mechanisms of methylmercury-induced cell death in human HepG2 cells.

    PubMed

    Cuello, Susana; Goya, Luis; Madrid, Yolanda; Campuzano, Susana; Pedrero, Maria; Bravo, Laura; Cámara, Carmen; Ramos, Sonia

    2010-05-01

    Methylmercury (MeHg) has been suggested to exert cytotoxicity through multiple mechanisms, but the precise biochemical machinery has not been fully defined. This study was aimed at investigating the time-course (0-24h) effect of 2mg/L MeHg on cell death in human HepG2 cells. MeHg decreased cell viability in a time-dependent manner, which was concomitant with increased LDH leakage, reduced GSH levels, CAT activity and altered activity of the antioxidant enzymes GPx and GR at the longest times of incubation (16 and 24h). Activity of the detoxifying enzyme GST was also early enhanced (2h). Caspase-3 activity reached a maximum value at 8h and continued increased up to 24h. This feature was preceded by an enhancement in the caspase-9 activity (2h), whereas caspase-8 activity remained unchanged. MeHg early diminished Bcl-x(L)/Bcl-x(S) ratio and increased levels of the pro-apoptotic Bax and Bad. Moreover, MeHg-induced cytotoxicity was completely inhibited by the antioxidants (GSH and NAC) and notably by the mitochondrial complex I inhibitor rotenone, but not by the NADH oxidase inhibitor DPI. In summary, MeHg induced an oxidative stress responsible for apoptosis in HepG2 cells through direct activation of the caspase cascade and altered the cellular antioxidant and detoxificant enzymatic system to later provoke necrosis at later stages. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  8. Protective effects of quercetin on nicotine induced oxidative stress in 'HepG2 cells'.

    PubMed

    Yarahmadi, Amir; Zal, Fatemeh; Bolouki, Ayeh

    2017-10-01

    Nicotine is a natural component of tobacco plants and is responsible for the addictive properties of tobacco. Nicotine has been recognized to result in oxidative stress by inducing the generation of reactive oxygen species (ROS). The purpose of this work was to estimate the hepatotoxicity effect of nicotine on viability and on antioxidant defense system in cultures of HepG2 cell line and the other hand, ameliorative effect of quercetin (Q) as an antioxidant was analyzed. Nicotine induced concentration dependent loss in HepG2 cell line viability. The results indicated that nicotine decreased activity of superoxide dismutase (SOD) and glutathione reductase (GR) and increased activities of catalase (CAT) and glutathione peroxidase (GPx) and glutathione (GSH) content in the HepG2 cells. Q significantly increased activity of SOD, GR and GSH content and decreased activity of GPX in nicotine + Q groups. Our data demonstrate that Q plays a protective role against the imbalance elicited by nicotine between the production of free radicals and antioxidant defense systems, and suggest that administration of this antioxidant may find clinical application where cellular damage is a consequence of ROS.

  9. Downregulation of human paraoxonase 1 (PON1) by organophosphate pesticides in HepG2 cells.

    PubMed

    Medina-Díaz, Irma Martha; Ponce-Ruiz, Néstor; Ramírez-Chávez, Bryana; Rojas-García, Aurora Elizabeth; Barrón-Vivanco, Briscia S; Elizondo, Guillermo; Bernal-Hernández, Yael Y

    2017-02-01

    Paraoxonase 1 (PON1) is a calcium-dependent esterase synthesized primarily in the liver and secreted into the plasma where it is associated with high-density lipoproteins (HDL). PON1 hydrolyzes and detoxifies some toxic metabolites of organophosphorus compounds (OPs) such as methyl parathion and chlorpyrifos. Thus, PON1 activity and expression levels are important for determining susceptibility against OPs poisoning. Some studies have demonstrated that OPs can modulate gene expression through interactions with nuclear receptors. In this study, we evaluated the effects of methyl parathion and chlorpyrifos on the modulation of PON1 in Human Hepatocellular Carcinoma (HepG2) cells by real-time PCR, PON1 activity assay, and western blot. The results showed that the treatments with methyl parathion and chlorpyrifos decreased PON1 mRNA and immunoreactive protein and increased inflammatory cytokines in HepG2 cells. The effects of methyl parathion and chlorpyrifos on the downregulation of PON1 gene expression in HepG2 cells may provide evidence of OPs cytotoxicity related to oxidative stress and an inflammatory response. A decrease in the expression of the PON1 gene may increase the susceptibility to OPs intoxication and the risk of diseases related to inflammation and oxidative stress. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 490-500, 2017.

  10. [Experimental study on the immune response of fusion tumor vaccine of HepG2 and dendritic cells in vitro].

    PubMed

    Pang, Y B; Cui, B Y; He, J; Huang, X P; Liang, W; Li, L Q; Luo, X L

    2017-02-21

    Objective: To estimate the immune response of HepG2/dendritic cell (DC) fusion cells vaccines against HepG2 cells in vitro. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Hypaque density-gradient centrifugation.Then DC were obtain from PBMCs by culturing in medium containing granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 5 days.DC and HepG2 fusion cells were induced by polythyleneglycol (PEG). The fusion cells were examined under fluorescence microscope by labeling DCs and HepG2 with green and red fluorescein, respectively, and then the fusion rates were analyzed by flow cytometry.The capacity of fusion cells to secrete interleukin (IL)-12 and stimulate the proliferation of T lymphocyte was assessed by ELISA and Flow cytometry, respectively.ELISPOT was used to assess the interferon gamma (IFN-γ) produced by cytotoxicity T lymphocyte (CTL), and the specific killing ability of fusion cells induce-CTL targeting HepG2 was estimated. Results: The fusion rate of HepG2/DC was 54.5%, and the fusion cells expressed a higher levels of DC mature marker CD80 and costimulatory molecules CD83, CD86 and MHC-Ⅰ, MHC-Ⅱ molecules HLA-ABC and HLA-DR than those in immature DCs (P<0.01). HepG2/DC showed a greater capacity to secrete high level of IL-12 (P<0.05) and activate proliferation of lymphocytes in vitro, as compared with DCs alone and DCs mix HepG2 (P<0.01). The HepG2/DC -activated CTL generated higher IFN-γ level and had a specific killing ability against HepG2 cells at the effecter/target ratio 30∶1 (31.4%±2.4%) and 100∶1 (57.6%±7.3%) (P<0.01). Conclusions: HepG2/DC fusion cells could efficiently stimulate T lymphocytes to generate specific CTL targeting HepG2 cells.It might be a promising strategy of immunotherapy for HCC.

  11. 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.

  12. Induction of Apoptosis by Berberine in Hepatocellular Carcinoma HepG2 Cells via Downregulation of NF-κB.

    PubMed

    Li, Min; Zhang, Mao; Zhang, Zhi-Lang; Liu, Ning; Han, Xiao-Yu; Liu, Qin-Cheng; Deng, Wei-Jun; Liao, Cai-Xian

    2017-01-26

    Hepatocellular carcinoma (HCC) is highly resistant to traditional chemotherapeutic approaches, which causes difficulty in the development of effective drugs for the treatment of HCC. Berberine, a major ingredient of Rhizoma coptidis, is a natural alkaloid used in traditional Chinese medicine. Berberine exhibits potent antitumor activity against HCC due to its high efficiency and low toxicity. In the present study, we found that berberine sensitized HepG cells to NF-κB-mediated apoptosis. Berberine exhibited a significant antiproliferation effect on the HepG2 cells and promoted apoptosis. Both qRT-PCR and immunofluorescence staining revealed that berberine reduced the NF-κB p65 levels in HepG2 cells. Moreover, p65 overexpression rescued berberine-induced cell proliferation and prevented HepG2 cells from undergoing apoptosis. These results suggest that berberine inhibits the growth of HepG2 cells by promoting apoptosis through the NF-κB p65 pathway.

  13. Quercetin reduces cyclin D1 activity and induces G1 phase arrest in HepG2 cells.

    PubMed

    Zhou, Jin; Li, L U; Fang, L I; Xie, Hua; Yao, Wenxiu; Zhou, Xiang; Xiong, Zhujuan; Wang, L I; Li, Zhixi; Luo, Feng

    2016-07-01

    Quercetin is able to inhibit proliferation of malignant tumor cells; however, the exact mechanism involved in this biological process remains unclear. The current study utilized a quantitative proteomic analysis to explore the antitumor mechanisms of quercetin. The leucine of HepG2 cells treated with quercetin was labeled as d3 by stable isotope labeling by amino acids in cell culture (SILAC). The isotope peaks of control HepG2 cells were compared with the d3-labeled HepG2 cells by mass spectrometry (MS) to identify significantly altered proteins. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses were subsequently employed to verify the results of the MS analysis. A flow cytometry assay was designed to observe the influence of various quercetin treatment concentrations on the cell cycle distribution of HepG2 cells. The results indicated that quercetin is able to substantially inhibit proliferation of HepG2 cells and induce an obvious morphological alteration of cells. According to the MS results, the 70 credibly-changed proteins that were identified may play important roles in multiple cellular processes, including protein synthesis, signaling, cytoskeletal processes and metabolism. Among these functional proteins, the expression of cyclin D1 (CCND1) was found to be significantly decreased. RT-PCR and western blot analyses verified the SILAC-MS results of decreased CCND1 expression. In summary, flow cytometry revealed that quercetin is able to induce G1 phase arrest in HepG2 cells. Based on the aforementioned observations, it is suggested that quercetin exerts antitumor activity in HepG2 cells through multiple pathways, including interfering with CCND1 gene expression to disrupt the cell cycle and proliferation of HepG2 cells. In the future, we aim to explore this effect in vivo.

  14. [Biological function and molecular mechanism of URI in HepG2 cells].

    PubMed

    Zhou, Wei; Zhong, Yanyu; Wang, Hongmin; Yang, Sijun; Wei, Wenxiang

    2014-11-01

    To explore the effect and molecular mechanism of the unconventional prefoldin RPB5 interactor (URI) in hepatocellular carcinoma HepG2 cells. The cDNA sequence and shRNA of URI were obtained and sub-cloned into eukaryotic expression vectors. Then those vectors were transfected into HepG2 cells to obtain stable transfection cell line. The cell proliferation and anchor-independent growth in URI-overexpressing and knockdown HepG2 cells were determined by CCK-8 and soft agar colony assay. Flow cytometry was applied to detect the cell cycle and apoptosis of γ-ray irradiated cells. Apoptosis related genes were detected by Western blot. The pCDNA3.1-URI and pGPU6-URIi eukaryotic expression vectors were constructed successfully and corresponding stable transfection cell lines were obtained. Cell proliferation rates of the HepG2, pCDNA3.1-URI-HepG2 and pGPU6-URIi-HepG2 cells were (588.78 ± 32.12)%, (959.33 ± 58.8)% and (393.93 ± 39.7)%, respectively (P < 0.05). The number of cell clones of HepG2, pCDNA3.1-URI-HepG2 and pGPU6-URIi-HepG2 cells were 43 ± 7, 85 ± 5 and 20 ± 4 (P < 0.05), respectively. After γ-ray irradiation, the URI-overexpressing cell line showed a significantly lower apoptosis rate and G(2)/M phase arrest than those in the URI-depleted cell line (P < 0.05). In the HepG2 cells, the relative protein expression levels of URI, Bax and Bcl-2 were 0.92 ± 0.03, 1.11 ± 0.13 and 0.82 ± 0.01 (P < 0.05). In the pCDNA3.1-URI-HepG2 cells, the relative protein expression levels of URI, Bax and Bcl-2 were 1.79 ± 0.12, 0.48 ± 0.01 and 2.20 ± 0.30 (P < 0.05), respectively. In the pGPU6-URIi-HepG2 cells, the relative protein expression levels of URI, Bax and Bcl-2 were 0.50 ± 0.04, 1.52 ± 0.20 and 0.38 ± 0.01 (P < 0.05), respectively. The expression of Bax was down-regulated and Bcl-2 was up-regulated in the URI-overexpressing cell line. However, on the contrary, expression of Bax was up-regulated and Bcl-2 was down-regulated in the URI-depleted cell line. URI

  15. Investigation of quercetin-induced HepG2 cell apoptosis-associated cellular biophysical alterations by atomic force microscopy.

    PubMed

    Pi, Jiang; Li, Baole; Tu, Lvying; Zhu, Haiyan; Jin, Hua; Yang, Fen; Bai, Haihua; Cai, Huaihong; Cai, Jiye

    2016-01-01

    Quercetin, a wildly distributed bioflavonoid, has been proved to possess excellent antitumor activity on hepatocellular carcinoma (HCC). In the present study, the biophysical properties of HepG2 cells were qualitatively and quantitatively determined using high resolution atomic force microscopy (AFM) to understand the anticancer effects of quercetin on HCC cells at nanoscale. The results showed that quercetin could induce severe apoptosis in HepG2 cells through arrest of cell cycle and disruption of mitochondria membrane potential. Additionally, the nuclei and F-actin structures of HepG2 cells were destroyed by quercetin treatment as well. AFM morphological data showed some typical apoptotic characterization of HepG2 cells with increased particle size and roughness in the ultrastructure of cell surface upon quercetin treatment. As an important biophysical property of cells, the membrane stiffness of HepG2 cells was further quantified by AFM force measurements, which indicated that HepG2 cells became much stiffer after quercetin treatment. These results collectively suggest that quercetin can be served as a potential therapeutic agent for HCC, which not only extends our understanding of the anticancer effects of quercetin against HCC cells into nanoscale, but also highlights the applications of AFM for the investigation of anticancer drugs.

  16. Cytotoxic effect of oxaloacetate on HepG2-human hepatic carcinoma cells via apoptosis and ROS accumulation.

    PubMed

    Jiao, Y; Ji, L; Kuang, Y; Yang, Q

    2017-01-01

    Oxaloacetate (OA) is one of the intermediates of the Krebs cycle. In addition to its role in energy production, OA may have other effects on the cell. We report here that OA could have a cell type dependent cytotoxic effect on the human hepatic carcinoma cell line HepG2 through induction of apoptosis and reactive oxygen species (ROS) accumulation. In our study, OA decreased the viability and colony formation of HepG2 cells and induced cell death. Caspase-3 activity was increased, the pro-apoptotic protein Bax was up-regulated, and the anti-apoptotic protein Bcl-2 was down-regulated in OA-treated HepG2 cells indicating that apoptosis through the intrinsic pathway was involved in the cell death. The ROS level in OA-treated HepG2 cells was increased. The anti-oxidant N-acetylcysteine (NAC) and glutathione (GSH) prevented the OA-induced decrease in cell but did not alter the enhanced apoptotic Bax/Bcl-2 mRNA ratio. These results suggest that the OA-induced apoptosis of HepG2 cell is not driven by oxidative damage and at least two distinct mechanisms, one mediated by ROS and one involving apoptosis, result in the cytotoxic effects of OA on HepG2 cells. These studies expand the biological functional repertoire of OA and provide a mechanism by which hepatocellular carcinoma may be targeted by OA.

  17. Selective killing of hepatocellular carcinoma HepG2 cells by three-dimensional nanographene nanoparticles based on triptycene.

    PubMed

    Xiong, Xiaoqin; Gan, Lu; Liu, Ying; Zhang, Chun; Yong, Tuying; Wang, Ziyi; Xu, Huibi; Yang, Xiangliang

    2015-03-12

    Carbon-based materials have been widely used in the biomedical fields including drug delivery and cancer therapies. In this paper, a recently synthesized three-dimensional nanographene (NG) based on triptycene self-assembles into nanoparticles which selectively kill human hepatocellular carcinoma HepG2 cells as compared to human normal liver HL7702 cells. Obvious differences in cellular accumulation, the endocytic pathway and intracellular trafficking of NG nanoparticles are observed in HepG2 cells and HL7702 cells. Further studies reveal that NG nanoparticles significantly increase the levels of reactive oxygen species (ROS) in HepG2 cells, but not in HL7702 cells. NG nanoparticle-induced ROS result in apoptosis induction and the decrease in mitochondrial membrane potential in HepG2 cells. Moreover, IKK/nuclear factor-κB (NF-κB) signaling is found to be activated by NG nanoparticle-induced ROS and serves to antagonize NG nanoparticle-induced apoptosis in HepG2 cells. Our studies show that the distinct behaviors of cellular uptake and ROS-mediated cytotoxicity are responsible for the selective killing of HepG2 cells. This study provides a foundation for understanding the mechanism of selective induction of apoptosis in cancer cells by NG nanoparticles and designing more effective chemotherapeutical agents.

  18. Autophagy in anti-apoptotic effect of augmenter of liver regeneration in HepG2 cells

    PubMed Central

    Shi, Hong-Bo; Sun, Hai-Qing; Shi, Hong-Lin; Ren, Feng; Chen, Yu; Chen, De-Xi; Lou, Jin-Li; Duan, Zhong-Ping

    2015-01-01

    AIM: To investigate the role of autophagy in the anti-apoptotic effect of augmenter of liver regeneration (ALR). METHODS: Autophagy was induced through serum deprivation. An ALR-expressing plasmid was transfected into HepG2 cells, and autophagic flux was determined using fluorescence microscopy, electron microscopy, Western blot and quantitative polymerase chain reaction (qPCR) assays. After ALR-expressing plasmid transfection, an autophagy inhibitor [3-methyladenine (3-MA)] was added to HepG2 cells, and apoptosis was observed using fluorescence microscopy and flow cytometry. RESULTS: Autophagy was activated in HepG2 cells, peaking at 24 h after serum deprivation. Microtubule-associated protein light chain three-II levels were higher in HepG2 cells treated with ALR than in control cells, fluorescence microscopy, electron microscopy and qPCR studies showed the similar trend, and p62 levels showed the opposite trend, which indicated that ALR may play an important role in increasing autophagy flux. The numbers of apoptotic cells were substantially higher in HepG2 cells treated with both ALR and 3-MA than in cells treated with ALR alone. Therefore, the protective effect of ALR was significantly attenuated or abolished when autophagy was inhibited, indicating that the anti-apoptotic effect of ALR may be related to autophagy. CONCLUSION: ALR protects cells from apoptosis partly through increased autophagy in HepG2 cells and may be valuable as a new therapeutic treatment for liver disease. PMID:25954098

  19. Autophagy in anti-apoptotic effect of augmenter of liver regeneration in HepG2 cells.

    PubMed

    Shi, Hong-Bo; Sun, Hai-Qing; Shi, Hong-Lin; Ren, Feng; Chen, Yu; Chen, De-Xi; Lou, Jin-Li; Duan, Zhong-Ping

    2015-05-07

    To investigate the role of autophagy in the anti-apoptotic effect of augmenter of liver regeneration (ALR). Autophagy was induced through serum deprivation. An ALR-expressing plasmid was transfected into HepG2 cells, and autophagic flux was determined using fluorescence microscopy, electron microscopy, Western blot and quantitative polymerase chain reaction (qPCR) assays. After ALR-expressing plasmid transfection, an autophagy inhibitor [3-methyladenine (3-MA)] was added to HepG2 cells, and apoptosis was observed using fluorescence microscopy and flow cytometry. Autophagy was activated in HepG2 cells, peaking at 24 h after serum deprivation. Microtubule-associated protein light chain three-II levels were higher in HepG2 cells treated with ALR than in control cells, fluorescence microscopy, electron microscopy and qPCR studies showed the similar trend, and p62 levels showed the opposite trend, which indicated that ALR may play an important role in increasing autophagy flux. The numbers of apoptotic cells were substantially higher in HepG2 cells treated with both ALR and 3-MA than in cells treated with ALR alone. Therefore, the protective effect of ALR was significantly attenuated or abolished when autophagy was inhibited, indicating that the anti-apoptotic effect of ALR may be related to autophagy. ALR protects cells from apoptosis partly through increased autophagy in HepG2 cells and may be valuable as a new therapeutic treatment for liver disease.

  20. Whole genome analysis and microRNAs regulation in HepG2 cells exposed to cadmium.

    PubMed

    Fabbri, Marco; Urani, Chiara; Sacco, Maria Grazia; Procaccianti, Claudio; Gribaldo, Laura

    2012-01-01

    Cadmium (Cd) is a metal known to be toxic and carcinogenic, but its mechanism of action remains to be fully elucidated. We investigated the gene expression modulation in the human hepatoma cell line HepG2 after exposure to 2 μM and 10 μM Cd using an Agilent microarray. Furthermore, we evaluated the microRNA modulation after exposure to 10 μM Cd with a Low Density Array. At the low concentration only eleven genes belonging to the metallothionein familiy were regulated. At the higher concentration the pathway enrichment analysis for the 536 up-regulated genes showed a large number of pathways related to cancer, whereas the 424 down-regulated genes were enriched on pathways correlated to liver function. A large percentage of modified microRNAs belonged to the let-7 family, which is considered to have oncosuppressor functions. Several pathways connected to cancer were regulated at the transcription level, and miRNAs had a potential impact on the modulation of this regulation.

  1. Induction of micronuclei and alteration of gene expression by an organomodified clay in HepG2 cells.

    PubMed

    Maisanaba, Sara; Hercog, Klara; Ortuño, Natalia; Jos, Ángeles; Žegura, Bojana

    2016-07-01

    Clay2 is an organomodified montmorillonite developed by the Technological Institute of Packaging, Transport and Logistic (ITENE) in order to improve polymeric materials used in food packaging. There is not much known on Clay2 toxic potential, particularly at DNA level, therefore it is mandatory to assess its toxicity prior to its commercialization. In the present study the human hepatoma cell line (HepG2) was exposed to non-cytotoxic concentrations of Clay2 and the genomic stability was studied with the Cytokinesis block micronucleus cytome assay, by determining the formation of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs). Moreover, the expression of various genes involved in the mechanisms of its action using the real-time quantitative PCR was studied. The results obtained provide the evidence that Clay2 is potentially genotoxic as it increased the frequency of micronuclei. In addition it deregulated genes involved in the metabolism, immediate-early response/signaling, DNA damage and oxidative stress showing new valuable information on the cellular response to Clay2. Nonetheless, further studies are highly needed to elucidate the molecular mechanisms of clays toxicity.

  2. Use of high concentrations of dimethyl sulfoxide for cryopreservation of HepG2 cells adhered to glass and polydimethylsiloxane matrices.

    PubMed

    Nagahara, Yukitoshi; Sekine, Hiroaki; Otaki, Mari; Hayashi, Masakazu; Murase, Norio

    2016-02-01

    Animal cells are generally cryopreserved in cryovials in a cell suspension state containing 5%-10% v/v dimethyl sulfoxide (DMSO) used as a cryoprotective agent. However, cryopreservation of cells in an attached state has not been intensively studied, and the effective freezing solution remains unknown. Here we determined the suitable DMSO concentration for the cryopreservation of human hepatoma HepG2 cells attached to glass and polydimethylsiloxane (PDMS) matrices coated with poly-l-lysine. With the use of the glass matrix, the rate of cell adhesion increased with the DMSO concentration up to 30% v/v in the freezing solution. In contrast, the cell-adhesion rate remained constant in the case of the PDMS matrix irrespective of the DMSO concentration between 10% v/v and 30% v/v. The viability of post-thawed cells attached to glass or PDMS matrix was also investigated. The viability was highest at the DMSO concentration of 20% v/v in the freezing solution. The DMSO concentration of 30% v/v, however, had a cytotoxic effect on the cell viability. Thus, the 20% v/v DMSO concentration was found to be most suitable for the cryopreservation of HepG2 cells in the attached state. This dose is high compared to the DMSO concentration used for the cryopreservation of cells in the suspended state.

  3. Growth inhibition effect of HMME-mediated PDT on hepatocellular carcinoma HepG2 cells.

    PubMed

    Liu, Lifeng; Song, Yuanjian; Ma, Limin; Zang, Lixin; Tao, Lili; Zhang, Zhiguo; Han, Jiwu

    2014-09-01

    Photodynamic therapy (PDT) is considered a promising new strategy for liver cancer treatment. Three elements of PDT--optical output power, irradiation time, and photosensitizer concentration--play important roles in promoting cell death. This research aimed to characterize the effects of hematoporphyrin monomethyl ether (HMME)-based PDT on hepatocellular carcinoma cells HepG2 and thus elucidate the relationship between cell death and the three elements mentioned earlier. Furthermore, in this study, we present a parameter that represents the cumulative effects of these elements. The accumulation of HMME in HepG2 cells was observed by fluorescence microscopy. The absorption spectrum of HMME was detected using fluorescence spectral analysis. The viability of the treated cells was determined using the MTT assay, and cell apoptosis was evaluated using flow cytometry. We found that the fluorescence intensity was positively correlated with the incubation time for up to 2 h. The cell growth inhibition rate was significantly high and gradually increased with increasing concentrations of HMME or increasing light intensity, which was calculated as optical output power × irradiation time. Further analysis revealed an e-exponential decay of the cell survival rate to the product of the HMME concentration and the light intensity. We defined the product as parameter B (B = optical output power × irradiation time × HMME concentration). Similarly, the rate of cell apoptosis showed roughly e-exponential growth to parameter B. In conclusion, HMME-mediated PDT can significantly kill HepG2 cells, and the killing effect was related to the cumulative effects of the optical output power, the irradiation time, and the HMME concentration. Therefore, the newly defined parameter B, as a comprehensive physical quantity, may be of great significance for the regulation of light and photosensitizer according to patient-specific conditions in clinical practice.

  4. Glutathione and thioredoxin type 1 cooperatively denitrosate HepG2 cells-derived cytosolic S-nitrosoproteins

    PubMed Central

    Stoyanovsky, Detcho A.; Scott, Melanie J.; Billiar, Timothy R.

    2013-01-01

    In this study, we present experimental evidence that glutathione acts in concert with human thioredoxin type 1 in the denitrosation of cytosolic S-nitrosoproteins (PSNOs) from HepG2 cells. PMID:23743503

  5. Cytotoxicity, oxidative stress, and apoptosis in HepG2 cells induced by ionic liquid 1-methyl-3-octylimidazolium bromide.

    PubMed

    Li, Xiaoyu; Ma, Junguo; Wang, Jianji

    2015-10-01

    The present study aimed to determine the cytotoxicity of 1-methyl-3-octylimidazolium bromide ([C8mim]Br) on the human hepatocellular carcinoma (HepG2) cells in order to elucidate the biochemical and molecular mechanism of [C8mim]Br-cytotoxicity. For this purpose, cell viability, oxidative stress, apoptosis, caspase activity, and apoptosis-related gene expression in HepG2 cells following [C8mim]Br-exposure were evaluated. The results showed that viability of HepG2 cells was decreased by [C8mim]Br-exposure in a concentration-dependent pattern. Moreover, biochemical assays reveal that [C8mim]Br-exposure can induce apoptosis, cause overproduction of reactive oxygen species (ROS), inhibit superoxide dismutase and catalase, reduce glutathione content, and increase the cellular malondialdehyde level of HepG2 cells. The transcriptions of p53 and bax were markedly up-regulated while bcl-2 was significantly down-regulated in HepG2 cells after [C8mim]Br-exposure, suggesting that p53 and bcl-2 family may be involved in the cytotoxicity and apoptosis of HepG2 cells caused by [C8mim]Br. In addition, we also found that caspase-3, caspase-8, and caspase-9 were significantly activated in HepG2 cells following [C8mim]Br-exposure. Our results suggest that ROS may be a key early signal of [C8mim]Br-induced apoptosis and caspases play a key role in the initiation and execution of apoptosis of HepG2 cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Induction of apoptosis in HepG2 cells by solanine and Bcl-2 protein.

    PubMed

    Ji, Y B; Gao, S Y; Ji, C F; Zou, X

    2008-01-17

    The nightshade (Solanum nigrum Linn.) has been widely used in Chinese traditional medicine as a remedy for the treatment of digestive system cancer. The anti-tumor activity of solanine, a steroid alkaloid isolated from the nightshade has been demonstrated. To observe the effect of anti-tumor and mechanism of solanine. The MTT assay was used to evaluate the IC(50) on the three digestive system tumor cell lines. The effect on the morphology was observed with a laser confocal microscopy; the rate of apoptosis and the cell cycle were measured using flow cytometry (FCM); the expression of Bcl-2 protein was measured by Western blot. The results show that the IC(50) for HepG(2), SGC-7901, and LS-174 were 14.47, >50, and >50 microg/ml, respectively; the morphology of cells in the negative control was normal; for the treated groups, typical signs for apoptosis were found. The rate of apoptosis in HepG(2) cells induced by solanine was found to be 6.0, 14.4, 17.3, 18.9, and 32.2%, respectively. Observation of the cell cycle showed that cells in the G(2)/M phases disappeared while the number of cells in the S phase increased significantly for treated groups. Western blot showed that solanine decreased the expression of Bcl-2 protein. Therefore, the target of solanine in inducing apoptosis in HepG(2) cells seems to be mediated by the inhibition in the expression of Bcl-2 protein.

  7. Eurycomanone induce apoptosis in HepG2 cells via up-regulation of p53

    PubMed Central

    Zakaria, Yusmazura; Rahmat, Asmah; Pihie, Azimahtol Hawariah Lope; Abdullah, Noor Rain; Houghton, Peter J

    2009-01-01

    Background Eurycomanone is a cytotoxic compound found in Eurycoma longifolia Jack. Previous studies had noted the cytotoxic effect against various cancer cell lines. The aim of this study is to investigate the cytotoxicity against human hepato carcinoma cell in vitro and the mode of action. The cytotoxicity of eurycomanone was evaluated using MTT assay and the mode of cell death was detected by Hoechst 33258 nuclear staining and flow cytometry with Annexin-V/propidium iodide double staining. The protein expression Bax, Bcl-2, p53 and cytochrome C were studied by flow cytometry using a spesific antibody conjugated fluorescent dye to confirm the up-regulation of p53 and Bax in cancer cells. Results The findings suggested that eurycomanone was cytotoxic on cancerous liver cell, HepG2 and less toxic on normal cells Chang's liver and WLR-68. Furthermore, various methods proved that apoptosis was the mode of death in eurycomanone-treated HepG2 cells. The characteristics of apoptosis including chromatin condensation, DNA fragmentation and apoptotic bodies were found following eurycomanone treatment. This study also found that apoptotic process triggered by eurycomanone involved the up-regulation of p53 tumor suppressor protein. The up-regulation of p53 was followed by the increasing of pro-apoptotic Bax and decreasing of anti-apoptotic Bcl-2. The increased of cytochrome C levels in cytosol also results in induction of apoptosis. Conclusion The data suggest that eurycomanone was cytotoxic on HepG2 cells by inducing apoptosis through the up-regulation of p53 and Bax, and down-regulation of Bcl-2. PMID:19508737

  8. Effects of usnic acid exposure on human hepatoblastoma HepG2 cells in culture.

    PubMed

    Sahu, Saura C; Amankwa-Sakyi, Margaret; O'Donnell, Michael W; Sprando, Robert L

    2012-09-01

    Usnic acid, a natural botanical product, is a constituent of some dietary supplements used for weight loss. It has been associated with clinical hepatotoxicity leading to liver failure in humans. The present study was undertaken for metabolism and toxicity evaluations of usnic acid in human hepatoblastoma HepG2 cells in culture. The cells were treated with the vehicle control and usnic acid at concentrations of 0-100 µm for 24 h at 37 °C in 5% CO2 . Following the treatment period, the cells were evaluated by biochemical and toxicogenomic endpoints of toxicity that included cytochrome P450 activity, cytotoxicity, oxidative stress, mitochondrial dysfunction and changes in pathway focused gene expression profiles. Usnic acid exposure resulted in increased P450 activity, cytotoxicity, oxidative stress and mitochondrial dysfunction in HepG2 cells. The pathway-focused gene expression analysis resulted in significantly altered expression of six genes out of a total of 84 genes examined. Of the six altered genes, three genes were up-regulated and three genes down-regulated. A marked up-regulation of one gene CCL21 associated with inflammation, one gene CCNC associated with proliferation and carcinogenesis and one gene UGT1A4 associated with metabolism as well as DNA damage and repair were observed in the usnic acid-treated cells compared with the vehicle control. Also a marked down-regulation of one gene CSF2 associated with inflammation and two genes (CYP7A1 and CYP2E1) associated with oxidative metabolic stress were observed in the usnic acid-treated cells compared with the control. The biomarkers used in this study demonstrate the toxicity of usnic acid in human hepatoblastoma HepG2 cells, suggesting an oxidative mechanism of action. Published 2011. This article is a US Government work and is in the public domain in the USA.

  9. DNA fragmentation is not associated with apoptosis in zerumbone-induced HepG2 cells.

    PubMed

    Kamalidehghan, Behnam; Ahmadipour, Fatemeh; Noordin, Mohamed Ibrahim

    2012-01-01

    Zerumbone is a cytotoxic compound isolated from the herbal plant, Zingiber zerumbet Smith, which exhibits antitumor activity [1-2], anti-inflammatory effects and possesses anti-proliferative potentials in a variety of cell lines [3-4]. DNA fragmentation indicates an early event of apoptosis leading to cell death due to the absence of new cellular proteins synthesizing for cell survival. Previous studies indicated that the cleavage of double-stranded DNA in apoptotic DNA degradation occurs via the activation of endogenous Ca2+/Mg2+-dependent endonuclease that specifically cleaves between nucleosomes to produce DNA fragments that are multiples of ~180 base pairs [5]. In order to investigate DNA fragmentation, we treated HepG2 cells with zerumbone (IC50: 3.45 ± 0.026 µg/mL) in both dose-dependent (2, 4, 6 and 8 µg/mL) and time-dependent manner (4, 8, 12, 16, 24, 48 and 72 h). The assay was performed using the Suicide Track™ DNA Ladder Isolation Kit (Calbio-chem, CA, USA), according to the manufacturer's instructions. DNA was analyzed using 1.5% agarose gel electrophoresis, observed under UV illumination and visualized using a gel documentation system (UVP Biospectrum HR410, USA). To furthur confirm the induction of apoptosis, the protein of zerumbone-induced HepG2 cells using Western-blotting indicated a low and high expression of Bcl2 and Bax proteins, respectively. In conclusion, these results indicate that no DNA fragmentation in the human hepatocellular liver carcinoma (HepG2) cells was observed even in the presence of caspase-3 during apoptosis. Therefore, we hypothesize that not all compounds necessairly indicate fragmentation of condensed chromatin into several discrete mass in cell lines as in vitro condition.

  10. Induction of apoptosis by FFJ-5, a novel naphthoquinone compound, occurs via downregulation of PKM2 in A549 and HepG2 cells

    PubMed Central

    Wei, Xiaoli; Li, Ming; Ma, Mingming; Jia, Huina; Zhang, Yu; Kang, Wenyi; Wang, Tianxiao; Shi, Xiaoyan

    2017-01-01

    Pyruvate kinase isoenzyme M2 (PKM2) has previously been identified as a tumor biomarker and as a potential target for cancer therapy. In this study, F§FJ-5, a characterized naphthoquinone modifier of mollugin, was synthesized in order to investigate its anticancer activity and the potential mechanisms. It was observed that FFJ-5 inhibited the cell growth of human lung adenocarcinoma cells A549 and human hepatoma cells HepG2 by MTT assays. FFJ-5 arrested cell cycle at the G2/M phase. Further analyses demonstrated that FFJ-5 attenuated the expression of PKM2 and reduced the production of adenosine triphosphate (ATP). Reduced expression and activity of epidermal growth factor receptor (EGFR) and Akt were observed in A549 and HepG2 cells exposed to FFJ-5. FFJ-5 exposure also resulted in cell apoptosis, in association with decreased intracellular pH level and mitochondrial membrane potential. In addition, FFJ-5 activated the caspase-3 cascade. In conclusion, FFJ-5 inhibited cancer cell growth via the blocking the EGFR-Akt-PKM2 pathway or through the synergistic action of EGFR, Akt and PKM2 proteins, alongside a decrease in ATP production. In addition, FFJ-5 induced cancer cell apoptosis by decreasing the intracellular pH level and the mitochondrial apoptosis pathway. The present results suggest a potential role of FFJ-5 on the therapy of human cancer. PMID:28356960

  11. Galactomannan from Schizolobium amazonicum seed and its sulfated derivatives impair metabolism in HepG2 cells.

    PubMed

    Cunha de Padua, Monique Meyenberg; Suter Correia Cadena, Silvia Maria; de Oliveira Petkowicz, Carmen Lucia; Martinez, Glaucia Regina; Rodrigues Noleto, Guilhermina

    2017-08-01

    This study evaluated the effects of native galactomannan from Schizolobium amazonicum seeds and its sulfated forms on certain metabolic parameters of HepG2 cells. Aqueous extraction from S. amazonicum seeds furnished galactomannan with 3.2:1 Man:Gal ratio (SAGM) and molar mass of 4.34×10(5)g/mol. The SAGM fraction was subjected to sulfation using chlorosulfonic acid to obtain SAGMS1 and SAGMS2 with DS of 0.4 and 0.6, respectively. Cytotoxicity of SAGM, SAGMS1, and SAGMS2 was evaluated in human hepatocellular carcinoma cells (HepG2). After 72h, SAGM decreased the viability of HepG2 cells by 50% at 250μg/mL, while SAGMS1 reduced it by 30% at the same concentration. SAGM, SAGMS1, and SAGMS2 promoted a reduction in oxygen consumption and an increase in lactate production in non-permeabilized HepG2 cells after 72h of treatment. These results suggest that SAGM, SAGMS1, and SAGMS2 could be recognized by HepG2 cells and might trigger alterations that impair its survival. These effects could be implicated in the modification of the oxidative phosphorylation process in HepG2 cells and activation of the glycolytic pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Silencing of Wnt10B reduces viability of heptocellular carcinoma HepG2 cells

    PubMed Central

    Wu, Guohui; Fan, Xiaoli; Sun, Li

    2015-01-01

    Dysregulation of Wnt-mediated β-catenin signaling is associated with carcinogenesis and progression of hepatocellular carcinoma (HCC). Our previous studies showed that the Wnt10B gene, a member of Wnt gene family, over-activated in HCC tissues and cells. Here we demonstrate that stable silencing of Wnt10B reduces the viability of HCC cells in culture. HepG2, a human HCC cell line, was cultured in vitro and Wnt10B gene in the cells stably silenced, as showed in Western blotting analysis, by the shRNA interference with lentivirus plasmid transfection. Compared to the control (HepG2 cells without Wnt10B silencing), the Wnt10B-silencing cells showed significant reductions in proliferation, colony formation, migration and invasion. Furthermore, serum deprivation-induced apoptotic death, assessed by Hoechst 33342 staining and fluorescent microscopy, increased significantly in the Wnt10B-silencing cells. FACScan analysis indicated an arrest of the cell cycle in the Wnt10B-silencing HCC cells, with significant increases in the number of cells in G0-G1 and S phases. Thus, we hypothesize that Wnt10B plays an oncogenic role in HCC and is a potential therapeutic target. PMID:26269753

  13. Nobiletin attenuates metastasis via both ERK and PI3K/Akt pathways in HGF-treated liver cancer HepG2 cells.

    PubMed

    Shi, Ming-Der; Liao, Yi-Chen; Shih, Yuan-Wei; Tsai, Li-Yu

    2013-06-15

    Hepatocyte growth factor (HGF), and its receptor, c-Met activation has recently been shown to play important roles in cancer invasion and metastasis in a wide variety of tumor cells. We use HGF as an invasive inducer of human HepG2 cells to investigate the effect of four flavones including apigenin, tricetin, tangeretin, and nobiletin on HGF/c-Met-mediated tumor invasion and metastasis. Among them, nobiletin markedly inhibited HGF-induced the abilities of the adhesion, invasion, and migration by cell-matrix adhesion assay and transwell-chamber invasion/migration assay under non-cytotoxic concentrations. Data also showed nobiletin inhibited HGF-induced cell scattering and cytoskeleton changed such as filopodia and lamellipodia. Furthermore, nobiletin could inhibit HGF-induced the membrane localization of phosphorylated c-Met, ERK2, and Akt, but not phosphorylated JNK1/2 and p38. Next, nobiletin significantly decreased the levels of phospho-ERK2 and phospho-Akt in ERK2 or Akt siRNA-transfected cells concomitantly with a marked reduction on cell invasion and migration. In conclusion, nobiletin attenuates HGF-induced HepG2 cells metastasis involving both ERK and PI3K/Akt pathways and are potentially useful as anti-metastatic agents for the treatment of hepatoma.

  14. Condition medium of HepG-2 cells induces the transdifferentiation of human umbilical cord mesenchymal stem cells into cancerous mesenchymal stem cells

    PubMed Central

    Yang, Juan; Miao, Yinglei; Chang, Yefei; Zhang, Fan; Wang, Yubo; Zheng, Sheng

    2016-01-01

    This study aimed to investigate the transdifferentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) into cancer-associated mesenchymal stem cells (CA-MSCs) after incubation with condition medium (CM) from liver cancer HepG-2 cells, and the biobehaviors (proliferation and migration) of these CA-MSCs were further evaluated. The supernatant of HepG-2 cells was collected and mixed with equal volume of low glucose DMEM. The resultant medium was used to treat hUCMSCs for 48 h. The expression of CA-MSCs related proteins and miR-221 was detected in cells. The supernatant of induced hUCMSCs was mixed with equal volume of high glucose DMEM, and the resultant medium was used treat HepG-2 cells for 48 h and the proliferation and migration of HepG-2 cells were evaluated. Moreover, HepG-2 cells were co-cultured with hUCMSCs and then the proliferation and migration of HepG-2 cells were assessed. After incubation with the supernatant from HepG-2 cells, hUCMSCs showed significantly elevated expression of vimentin, fibroblast activation protein (FAP) and miR-221. The supernatant of induced hUCMSCs was able to significantly increase the proliferation and migration of HepG-2 cells. Following co-culture, the proliferation and migration of HepG-2 cells increased dramatically. These findings suggest that the supernatant of HepG-2 cells is able to induce the phenotype of CA-MSCs and the supernatant of CA-MSCs may promote the proliferation and migration of HepG-2 cells. These findings provide experimental evidence for the cellular remodeling in tumor microenvironment and the safety of clinical use of hUCMSCs. PMID:27648133

  15. Campomanesia adamantium (Myrtaceae) fruits protect HEPG2 cells against carbon tetrachloride-induced toxicity.

    PubMed

    de Oliveira Fernandes, Thaís; de Ávila, Renato Ivan; de Moura, Soraia Santana; de Almeida Ribeiro, Gerlon; Naves, Maria Margareth Veloso; Valadares, Marize Campos

    2015-01-01

    Campomanesia adamantium (Myrtaceae) is an antioxidant compounds-rich Brazilian fruit popularly known as gabiroba. In view of this, it was evaluated the hepatoprotective effects of pulp (GPE) or peel/seed (GPSE) hydroalcoholic extracts of gabiroba on injured liver-derived HepG2 cells by CCl4 (4 mM). The results showed the presence of total phenolic in GPSE was (60%) higher when compared to GPE, associated with interesting antioxidant activity using DPPH• assay. Additionally, HPLC chromatograms and thin layer chromatography of GPE and GPSE showed the presence of flavonoids. Pretreatment of HepG2 cells with GPE or GPSE (both at 800-1000 μg/mL) significantly (p < 0.0001) protected against cytotoxicity induced by CCl4. Additionally, the cells treated with both extracts (both at 1000 μg/mL) showed normal morphology (general and nuclear) contrasting with apoptotic characteristics in the cells only exposed to CCl4. In these experiments, GPSE also was more effective than GPE. In addition, CCl4 induced a marked increase in AST (p < 0.05) and ALT (p < 0.0001) levels, while GPE or GPSE significantly (p < 0.0001) reduced these levels, reaching values found in the control group. In conclusion, the results suggest that gabiroba fruits exert hepatoprotective effects on HepG2 cells against the CCl4-induced toxicity, probably, at least in part, associated with the presence of antioxidant compounds, especially flavonoids.

  16. Metallomics Study of CdSe/ZnS Quantum Dots in HepG2 Cells.

    PubMed

    Peng, Lu; He, Man; Chen, Beibei; Qiao, Yu; Hu, Bin

    2015-10-27

    Toxicity of quantum dots (QDs) has been a hot research concern in the past decade, and there is a lot of challenge in this field. The physicochemical characteristics of QDs can affect their toxicity, while little is known about the specific chemical form of QDs in living cells after incubation so far. In this work, speciation of four CdSe/ZnS QDs in HepG2 cells was carried out from the metallomics' point of view for the first time by using size exclusion chromatography (SEC) coupled with inductively coupled plasma-mass spectrometry (ICP-MS). On the basis of the signal of Cd, two kinds of chemical forms, named as QD-1 and QD-2, were observed in HepG2 cells incubated with CdSe/ZnS QDs. QD-1 was demonstrated to be a kind of QD-like nanoparticles, confirmed by chromatographic retention time, transmission electron microscopy (TEM) characterization, and fluorescence detection. QD-2 was demonstrated to be cadmium-metallothioneins complex (Cd-MTs) by reversed phase liquid chromatography (RPLC) synchronously coupled with ICP-MS and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) analysis. Meanwhile, speciation of QDs in HepG2 cells incubated with different conditions was analyzed. With the variation of QDs incubation concentration/time, and elimination time, the species of QD-1 and QD-2 were also observed without other obvious species, and both the amount of QD-1 and QD-2 increased with incubation concentration and time. The obtained results provide valuable information and a strategy for the study of existing chemical form of QDs, greatly benefiting the understanding of QDs toxicity in living cells.

  17. Lipid rafts are essential for peroxisome biogenesis in HepG2 cells.

    PubMed

    Woudenberg, Jannes; Rembacz, Krzysztof P; Hoekstra, Mark; Pellicoro, Antonella; van den Heuvel, Fiona A J; Heegsma, Janette; van Ijzendoorn, Sven C D; Holzinger, Andreas; Imanaka, Tsuneo; Moshage, Han; Faber, Klaas Nico

    2010-08-01

    Peroxisomes are particularly abundant in the liver and are involved in bile salt synthesis and fatty acid metabolism. Peroxisomal membrane proteins (PMPs) are required for peroxisome biogenesis [e.g., the interacting peroxisomal biogenesis factors Pex13p and Pex14p] and its metabolic function [e.g., the adenosine triphosphate-binding cassette transporters adrenoleukodystrophy protein (ALDP) and PMP70]. Impaired function of PMPs is the underlying cause of Zellweger syndrome and X-linked adrenoleukodystrophy. Here we studied for the first time the putative association of PMPs with cholesterol-enriched lipid rafts and their function in peroxisome biogenesis. Lipid rafts were isolated from Triton X-100-lysed or Lubrol WX-lysed HepG2 cells and analyzed for the presence of various PMPs by western blotting. Lovastatin and methyl-beta-cyclodextrin were used to deplete cholesterol and disrupt lipid rafts in HepG2 cells, and this was followed by immunofluorescence microscopy to determine the subcellular location of catalase and PMPs. Cycloheximide was used to inhibit protein synthesis. Green fluorescent protein-tagged fragments of PMP70 and ALDP were analyzed for their lipid raft association. PMP70 and Pex14p were associated with Triton X-100-resistant rafts, ALDP was associated with Lubrol WX-resistant rafts, and Pex13p was not lipid raft-associated in HepG2 cells. The minimal peroxisomal targeting signals in ALDP and PMP70 were not sufficient for lipid raft association. Cholesterol depletion led to dissociation of PMPs from lipid rafts and impaired sorting of newly synthesized catalase and ALDP but not Pex14p and PMP70. Repletion of cholesterol to these cells efficiently reestablished the peroxisomal sorting of catalase but not ALDP. Human PMPs are differentially associated with lipid rafts independently of the protein homology and/or their functional interaction. Cholesterol is required for peroxisomal lipid raft assembly and peroxisome biogenesis.

  18. Lead enhances fluoride influence on apoptotic processes in the HepG2 liver cell line.

    PubMed

    Gutowska, Izabela; Baranowska-Bosiacka, Irena; Siwiec, Ewa; Szczuko, Małgorzata; Kolasa, Agnieszka; Kondarewicz, Anna; Rybicka, Marta; Dunaj-Stańczyk, Małgorzata; Wiernicki, Ireneusz; Chlubek, Dariusz; Stachowska, Ewa

    2016-03-01

    Chronic long-term exposure to high levels of fluoride leads to fluorosis, manifested by skeletal fluorosis and damage to internal organs, including kidneys, liver, parathyroid glands, and brain. Excess fluoride can also cause DNA damage, trigger apoptosis, and change cell cycle. The effect of fluoride may be exacerbated by lead (Pb), a potent inhibitor of many enzymes and a factor causing apoptosis, still present in the environment in excessive amounts. Therefore, in this study, we investigated the effects of sodium fluoride (NaF) and/or lead acetate (PbAc) on development of apoptosis, cell vitality, and proliferation in the liver cell line HepG2. We examined hepatocytes from the liver cell line HepG2, incubated for 48 h with NaF, PbAc, and their mixture (NaF + PbAc), and used for measuring apoptosis, index of proliferation, and vitality of cells. Incubation of the hepatocytes with NaF or PbAc increased apoptosis, more when fluoride and Pb were used simultaneously. Vitality of the cells depended on the compound used and its concentration. Proliferation slightly increased and then decreased in a high fluoride environment; it decreased significantly after addition of Pb in a dose-dependent manner. When used together, fluoride inhibited the decreasing effect of Pb on cell proliferation. © The Author(s) 2013.

  19. Protection of human HepG2 cells against oxidative stress by the flavonoid epicatechin.

    PubMed

    Martín, María Angeles; Ramos, Sonia; Mateos, Raquel; Izquierdo-Pulido, María; Bravo, Laura; Goya, Luis

    2010-04-01

    Flavanols, such as epicatechin (EC), constitute an important part of the human diet; they can be found in green tea, grapes and cocoa and possess different biological activities such as antioxidant, anti-inflammatory and anticarcinogenic. This study investigated the potential chemo-protective effect of EC against oxidative stress induced by tert-butylhydroperoxide (t-BOOH) on human HepG2 cells. Cell viability by lactate dehydrogenase assay and markers of oxidative status: reduced glutathione (GSH), malondialdehyde (MDA), reactive oxygen species (ROS), glutathione peroxidase (GPx) and glutathione reductase (GR) were evaluated. Pretreatment of cells with EC for 20 h prevented the enhanced cell damage and GPx and GR activities as well as the decrease in GSH induced by t-BOOH. The increased ROS generation induced by t-BOOH was also partly prevented by a pretreatment for 20 h with EC. In addition, pretreatment of cells with EC for 20 h recovered the t-BOOH-induced MDA concentration to control values. A pretreatment for 2 h with EC did not reduce cell damage but partly recovered GSH, reduced ROS levels and muffled the increase of GPx and GR after exposure to t-BOOH. Treatment of HepG2 cells with concentrations of EC in the micromolar range confers a significant protection against oxidative stress.

  20. Pinolenic Acid Downregulates Lipid Anabolic Pathway in HepG2 Cells.

    PubMed

    Lee, Ah Ron; Han, Sung Nim

    2016-07-01

    Pine nut oil (PNO) was reported to reduce lipid accumulation in the liver. However, the specific effect of pinolenic acid (18:3, all-cis-Δ5,9,12), a unique component of PNO, on lipid metabolism has not been studied. We hypothesized that pinolenic acid downregulates the lipid anabolic pathway in HepG2 cells. HepG2 cells were incubated in serum-free medium supplemented with 50 μM bovine serum albumin (BSA), palmitic acid, oleic acid, γ-linolenic acid, pinolenic acid, eicosapentaenoic acid (EPA), or α-linolenic acid for 24 h. Lipid accumulation was determined by Oil Red O (ORO) staining. The mRNA levels of genes related to fatty acid biosynthesis (SREBP1c, FAS, SCD1, and ACC1), fatty acid oxidation (ACC2, PPARα, CPT1A, and ACADL), cholesterol synthesis (SREBP2 and HMGCR), and lipoprotein uptake (LDLr) and of genes that may be involved in the downregulation of the lipogenic pathway (ACSL3, ACSL4, and ACSL5) were determined by qPCR. LDLR protein levels were measured by Western blot analysis. The mRNA levels of SREBP1c, FAS, and SCD1 were significantly downregulated by pinolenic acid treatment compared to BSA control (53, 54, and 38 % lower, respectively). In addition, the mRNA levels of HMGCR, ACSL3, and LDLr were significantly lower (30, 30, and 43 % lower, respectively), and ACSL4 tended to be lower in the pinolenic acid group (20 % lower, P = 0.082) relative to the control group. In conclusion, pinolenic acid downregulated the lipid anabolic pathway in HepG2 cells by reducing expression of genes related to lipid synthesis, lipoprotein uptake, and the regulation of the lipogenic pathway.

  1. A Novel Anti-Hepatitis C Virus and Antiproliferative Agent Alters Metabolic Networks in HepG2 and Hep3B Cells

    PubMed Central

    Keogh, Adrian; Şenkardeş, Sevil; Idle, Jeffrey R.; Küçükgüzel, Ş. Güniz; Beyoğlu, Diren

    2017-01-01

    A series of novel diflunisal hydrazide-hydrazones has been reported together with their anti-hepatitis C virus and antiproliferative activities in a number of human hepatoma cell lines. However, the mechanisms underlying the efficacy of these agents remain unclear. It was chosen to investigate the lead diflunisal hydrazide-hydrazone, 2′,4′-difluoro-4-hydroxy-N′- [(pyridin-2-yl)methylidene]biphenyl-3-carbohydrazide (compound 3b), in two cultured human hepatoma cell lines—HepG2 and Hep3B—using a metabolomic protocol aimed at uncovering any effects of this agent on cellular metabolism. One sub-therapeutic concentration (2.5 μM) and one close to the IC50 for antimitotic effect (10 μM), after 72 h in cell culture, were chosen for both compound 3b and its inactive parent compound diflusinal as a control. A GCMS-based metabolomic investigation was performed on cell lysates after culture for 24 h. The intracellular levels of a total of 42 metabolites were found to be statistically significantly altered in either HepG2 or Hep3B cells, only eight of which were affected in both cell lines. It was concluded that compound 3b affected the following pathways—purine and pyrimidine catabolism, the glutathione cycle, and energy metabolism through glycolysis and the pentose phosphate pathway. Although the metabolomic findings occurred after 24 h in culture, significant cytotoxicity of compound 3b to both HepG2 and Hep3B cells at 10 μM were reported not to occur until 72 h in culture. These observations show that metabolomics can provide mechanistic insights into the efficacy of novel drug candidates prior to the appearance of their pharmacological effect. PMID:28574427

  2. Protective effects of garlic sulfur compounds against DNA damage induced by direct- and indirect-acting genotoxic agents in HepG2 cells.

    PubMed

    Belloir, C; Singh, V; Daurat, C; Siess, M H; Le Bon, A M

    2006-06-01

    The aim of this study was to assess the antigenotoxic activity of several garlic organosulfur compounds (OSC) in the human hepatoma cell line HepG2, using comet assay. The OSC selected were allicin (DADSO), diallyl sulfide (DAS), diallyl disulfide (DADS), S-allyl cysteine (SAC) and allyl mercaptan (AM). To explore their potential mechanisms of action, two approaches were performed: (i) a pre-treatment protocol which allowed study of the possible modulation of drug metabolism enzymes by OSC before treatment of the cells with the genotoxic agent; (ii) a co-treatment protocol by which the ability of OSC to scavenge direct-acting compounds was assessed. Preliminary studies showed that, over the concentration range tested (5-100 microM), the studied OSC neither affected cell viability nor induced DNA damage by themselves. In the pre-treatment protocol, aflatoxin B1 genotoxicity was significantly reduced by all the OSC tested except AM. DADS was the most efficient OSC in reducing benzo(a)pyrene genotoxicity. SAC and AM significantly decreased DNA breaks in HepG2 cells treated with dimethylnitrosamine. Additionally, all the OSC studied were shown to decrease the genotoxicity of the direct-acting compounds, hydrogen peroxide and methyl methanesulfonate. This study demonstrated that garlic OSC displayed antigenotoxic activity in human metabolically competent cells.

  3. Distinct Nongenomic Signal Transduction Pathways Controlled by 17β-Estradiol Regulate DNA Synthesis and Cyclin D1 Gene Transcription in HepG2 Cells

    PubMed Central

    Marino, Maria; Acconcia, Filippo; Bresciani, Francesco; Weisz, Alessandro; Trentalance, Anna

    2002-01-01

    Estrogens induce cell proliferation in target tissues by stimulating progression through the G1 phase of the cell cycle. Activation of cyclin D1 gene expression is a critical feature of this hormonal action. The existence of rapid/nongenomic estradiol-regulated protein kinase C (PKC-α) and extracellular signal-regulated kinase (ERK) signal transduction pathways, their cross talk, and role played in DNA synthesis and cyclin D1 gene transcription have been studied herein in human hepatoma HepG2 cells. 17β-Estradiol was found to rapidly activate PKC-α translocation and ERK-2/mitogen-activated protein kinase phosphorylation in this cell line. These actions were independent of each other, preceding the increase of thymidine incorporation into DNA and cyclin D1 expression, and did not involve DNA binding by estrogen receptor. The results obtained with specific inhibitors indicated that PKC-α pathway is necessary to mediate the estradiol-induced G1-S progression of HepG2 cells, but it does not exert any effect(s) on cyclin D1 gene expression. On the contrary, ERK-2 cascade was strongly involved in both G1-S progression and cyclin D1 gene transcription. Deletion of its activating protein-1 responsive element motif resulted in attenuation of cyclin D1 promoter responsiveness to estrogen. These results indicate that estrogen-induced cyclin D1 transcription can occur in HepG2 cells independently of the transcriptional activity of estrogen receptor, sustaining the pivotal role played by nongenomic pathways of estrogen action in hormone-induced proliferation. PMID:12388769

  4. Crambescin C1 Exerts a Cytoprotective Effect on HepG2 Cells through Metallothionein Induction

    PubMed Central

    Roel, María; Rubiolo, Juan A.; Ternon, Eva; Thomas, Olivier P.; Vieytes, Mercedes R.; Botana, Luis M.

    2015-01-01

    The Mediterranean marine sponge Crambe crambe is the source of two families of guanidine alkaloids known as crambescins and crambescidins. Some of the biological effects of crambescidins have been previously reported while crambescins have undergone little study. Taking this into account, we performed comparative transcriptome analysis to examine the effect of crambescin-C1 (CC1) on human tumor hepatocarcinoma cells HepG2 followed by validation experiments to confirm its predicted biological activities. We report herein that, while crambescin-A1 has a minor effect on these cells, CC1 protects them against oxidative injury by means of metallothionein induction even at low concentrations. Additionally, at high doses, CC1 arrests the HepG2 cell cycle in G0/G1 and thus inhibits tumor cell proliferation. The findings presented here provide the first detailed approach regarding the different effects of crambescins on tumor cells and provide a basis for future studies on other possible cellular mechanisms related to these bioactivities. PMID:26225985

  5. Quercetin induces HepG2 cell apoptosis by inhibiting fatty acid biosynthesis

    PubMed Central

    ZHAO, PENG; MAO, JUN-MIN; ZHANG, SHU-YUN; ZHOU, ZE-QUAN; TAN, YANG; ZHANG, YU

    2014-01-01

    Quercetin can inhibit the growth of cancer cells with the ability to act as a ‘chemopreventer’. Its cancer-preventive effect has been attributed to various mechanisms, including the induction of cell-cycle arrest and/or apoptosis, as well as its antioxidant functions. Quercetin can also reduce adipogenesis. Previous studies have shown that quercetin has potent inhibitory effects on animal fatty acid synthase (FASN). In the present study, activity of quercetin was evaluated in human liver cancer HepG2 cells. Intracellular FASN activity was calculated by measuring the absorption of NADPH via a spectrophotometer. MTT assay was used to test the cell viability, immunoblot analysis was performed to detect FASN expression levels and the apoptotic effect was detected by Hoechst 33258 staining. In the present study, it was found that quercetin could induce apoptosis in human liver cancer HepG2 cells with overexpression of FASN. This apoptosis was accompanied by the reduction of intracellular FASN activity and could be rescued by 25 or 50 μM exogenous palmitic acids, the final product of FASN-catalyzed synthesis. These results suggested that the apoptosis induced by quercetin was via the inhibition of FASN. These findings suggested that quercetin may be useful for preventing human liver cancer. PMID:25009654

  6. Crambescin C1 Exerts a Cytoprotective Effect on HepG2 Cells through Metallothionein Induction.

    PubMed

    Roel, María; Rubiolo, Juan A; Ternon, Eva; Thomas, Olivier P; Vieytes, Mercedes R; Botana, Luis M

    2015-07-27

    The Mediterranean marine sponge Crambe crambe is the source of two families of guanidine alkaloids known as crambescins and crambescidins. Some of the biological effects of crambescidins have been previously reported while crambescins have undergone little study. Taking this into account, we performed comparative transcriptome analysis to examine the effect of crambescin-C1 (CC1) on human tumor hepatocarcinoma cells HepG2 followed by validation experiments to confirm its predicted biological activities. We report herein that, while crambescin-A1 has a minor effect on these cells, CC1 protects them against oxidative injury by means of metallothionein induction even at low concentrations. Additionally, at high doses, CC1 arrests the HepG2 cell cycle in G0/G1 and thus inhibits tumor cell proliferation. The findings presented here provide the first detailed approach regarding the different effects of crambescins on tumor cells and provide a basis for future studies on other possible cellular mechanisms related to these bioactivities.

  7. Biscuit melanoidins of different molecular masses protect human HepG2 cells against oxidative stress.

    PubMed

    Martín, María Angeles; Ramos, Sonia; Mateos, Raquel; Rufián-Henares, José Angel; Morales, Francisco José; Bravo, Laura; Goya, Luis

    2009-08-26

    Soluble melanoidins from biscuits were enzymatically solubilized and isolated by sequential ultrafiltration and separated by molecular mass in three different fractions, below 3 kDa, between 3 and 10 kDa, and over 10 kDa; the latter was subsequently digested by simulating gastric plus pancreatic digestive conditions. The four fractions were investigated for their protective effect against an oxidative challenge in HepG2 cells. Pretreatment of cells for 20 h with 0.5-10 microg/mL of any of the four fractions prevented the increased cell damage evoked by the challenge but, except for the intermediate size fraction, did not suppress the increased reactive oxygen species. Antioxidant defenses were rapidly restored after the challenge, and the increase of the oxidative stress biomarker malondialdehyde was prevented by the pretreatment with all but the undigested high molecular mass fraction. The results show that treatment of HepG2 cells with concentrations of biscuit melanoidins within the expected physiological range confers on the cells a significant protection against an oxidative challenge.

  8. Quercetin induces HepG2 cell apoptosis by inhibiting fatty acid biosynthesis.

    PubMed

    Zhao, Peng; Mao, Jun-Min; Zhang, Shu-Yun; Zhou, Ze-Quan; Tan, Yang; Zhang, Yu

    2014-08-01

    Quercetin can inhibit the growth of cancer cells with the ability to act as a 'chemopreventer'. Its cancer-preventive effect has been attributed to various mechanisms, including the induction of cell-cycle arrest and/or apoptosis, as well as its antioxidant functions. Quercetin can also reduce adipogenesis. Previous studies have shown that quercetin has potent inhibitory effects on animal fatty acid synthase (FASN). In the present study, activity of quercetin was evaluated in human liver cancer HepG2 cells. Intracellular FASN activity was calculated by measuring the absorption of NADPH via a spectrophotometer. MTT assay was used to test the cell viability, immunoblot analysis was performed to detect FASN expression levels and the apoptotic effect was detected by Hoechst 33258 staining. In the present study, it was found that quercetin could induce apoptosis in human liver cancer HepG2 cells with overexpression of FASN. This apoptosis was accompanied by the reduction of intracellular FASN activity and could be rescued by 25 or 50 μM exogenous palmitic acids, the final product of FASN-catalyzed synthesis. These results suggested that the apoptosis induced by quercetin was via the inhibition of FASN. These findings suggested that quercetin may be useful for preventing human liver cancer.

  9. Immobilization of native type I collagen on polypropylene fabrics as a substrate for HepG2 cell culture.

    PubMed

    Peng, Gongze; Li, Saina; Peng, Qing; Li, Yang; Weng, Jun; Jia, Zhidong; Kang, Jiyao; Lei, Xiongxin; Zhang, Guifeng; Gao, Yi

    2017-07-01

    Background/aims The critical part of a bio-artificial liver device is establishment of a bioreactor filled with liver cells. However, it is still unclear how to maintain benign cell function while achieving the sufficient cell quantity. In the current study, we aim to establish a novel carrier for the culture of HepG2 cells, a liver cell line, by modifying polypropylene nonwoven fabrics with native type I collagen. Methods "Piranha" solution, KH-550 and glutaraldehyde subsequently were used to bridge native type I collagen and polypropylene nonwoven fabrics. The type I collagen-coupled polypropylene nonwoven fabric was characterized by XPS, SEM, ATR-FTIR and water contact angle measurement. Furthermore, the biocompatibility between HepG2 cells and fiber film is evaluated by the ability of cell proliferation, albumin secretion, as well as urea synthesis. Results The coating of collagen onto polypropylene fabrics was more efficient using the chemical covalent binding method than direct immersion, which was validated by the presence of collagen-related elements and chemical bond. The adding of collagen in polypropylene fabrics promoted hydrophilicity and HepG2 cell adherence. Additionally, enhanced cell proliferation, increased albumin secretion and urea synthesis were observed in HepG2 cells growing on collagen-coated polypropylene fabrics. Conclusions The collagen coated polypropylene nonwoven fabrics, acting as a feasible substrate for HepG2 cell culture, may be used as a promising liver cell carrier for artificial liver reactor.

  10. Chromate Reductase YieF from Escherichia coli Enhances Hexavalent Chromium Resistance of Human HepG2 Cells

    PubMed Central

    Liu, Xuan; Wu, Gaofeng; Zhang, Yanli; Wu, Dan; Li, Xiangkai; Liu, Pu

    2015-01-01

    Hexavalent chromium (Cr(VI)) is a serious environmental pollutant and human toxicant. Mammalian cells are very sensitive to chromate as they lack efficient chromate detoxifying strategy, e.g., chromate-reducing genes that are widely present in prokaryotes. To test whether introduction of prokaryotic chromate-reducing gene into mammalian cells could render higher chromate resistance, an Escherichia coli chromate-reducing gene yieF was transfected into human HepG2 cells. The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI) induction. In chromate-reducing ability test, HepG2-YieF cells that harbored the reductase showed significantly higher reducing ability of Cr(VI) than HepG2 control cells. This result was further supported by the evidence of increased Cr(VI)-removing ability of crude cell extract of HepG2-YieF. Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI) stress. Subcellular localization of YieF was determined by tracing GFP-YieF fusion protein that was detected in both nucleus and cytoplasm by laser confocal microscopy. Altogether, this study successfully demonstrated that the expression of a prokaryotic Cr(VI)-reducing gene yieF endowed mammalian cell HepG2 with enhanced chromate resistance, which brought new insight of Cr(VI) detoxification in mammalian cells. PMID:26016500

  11. Chromate Reductase YieF from Escherichia coli Enhances Hexavalent Chromium Resistance of Human HepG2 Cells.

    PubMed

    Liu, Xuan; Wu, Gaofeng; Zhang, Yanli; Wu, Dan; Li, Xiangkai; Liu, Pu

    2015-05-26

    Hexavalent chromium (Cr(VI)) is a serious environmental pollutant and human toxicant. Mammalian cells are very sensitive to chromate as they lack efficient chromate detoxifying strategy, e.g., chromate-reducing genes that are widely present in prokaryotes. To test whether introduction of prokaryotic chromate-reducing gene into mammalian cells could render higher chromate resistance, an Escherichia coli chromate-reducing gene yieF was transfected into human HepG2 cells. The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI) induction. In chromate-reducing ability test, HepG2-YieF cells that harbored the reductase showed significantly higher reducing ability of Cr(VI) than HepG2 control cells. This result was further supported by the evidence of increased Cr(VI)-removing ability of crude cell extract of HepG2-YieF. Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI) stress. Subcellular localization of YieF was determined by tracing GFP-YieF fusion protein that was detected in both nucleus and cytoplasm by laser confocal microscopy. Altogether, this study successfully demonstrated that the expression of a prokaryotic Cr(VI)-reducing gene yieF endowed mammalian cell HepG2 with enhanced chromate resistance, which brought new insight of Cr(VI) detoxification in mammalian cells.

  12. Protective Effects of Sweet Orange, Unshiu Mikan, and Mini Tomato Juice Powders on t-BHP-Induced Oxidative Stress in HepG2 Cells.

    PubMed

    Jannat, Susoma; Ali, Md Yousof; Kim, Hyeung-Rak; Jung, Hyun Ah; Choi, Jae Sue

    2016-09-01

    The aim of this study was to investigate the protective effects of juice powders from sweet orange [Citrus sinensis (L.) Osbeck], unshiu mikan (Citrus unshiu Marcow), and mini tomato (Solanum lycopersicum L.), and their major flavonoids, hesperidin, narirutin, and rutin in tert-butyl hydroperoxide (t-BHP)-induced oxidative stress in HepG2 cells. The increased reactive oxygen species and decreased glutathione levels observed in t-BHP-treated HepG2 cells were ameliorated by pretreatment with juice powders, indicating that the hepatoprotective effects of juice powders and their major flavonoids are mediated by induction of cellular defense against oxidative stress. Moreover, pretreatment with juice powders up-regulated phase-II genes such as heme oxygenase-1 (HO-1), thereby preventing cellular damage and the resultant increase in HO-1 expression. The high-performance liquid chromatography profiles of the juice powders confirmed that hesperidin, narirutin, and rutin were the key flavonoids present. Our results suggest that these fruit juice powders and their major flavonoids provide a significant cytoprotective effect against oxidative stress, which is most likely due to the flavonoid-related bioactive compounds present, leading to the normal redox status of cells. Therefore, these fruit juice powders could be advantageous as bioactive sources for the prevention of oxidative injury in hepatoma cells.

  13. Protective Effects of Sweet Orange, Unshiu Mikan, and Mini Tomato Juice Powders on t-BHP-Induced Oxidative Stress in HepG2 Cells

    PubMed Central

    Jannat, Susoma; Ali, Md Yousof; Kim, Hyeung-Rak; Jung, Hyun Ah; Choi, Jae Sue

    2016-01-01

    The aim of this study was to investigate the protective effects of juice powders from sweet orange [Citrus sinensis (L.) Osbeck], unshiu mikan (Citrus unshiu Marcow), and mini tomato (Solanum lycopersicum L.), and their major flavonoids, hesperidin, narirutin, and rutin in tert-butyl hydroperoxide (t-BHP)-induced oxidative stress in HepG2 cells. The increased reactive oxygen species and decreased glutathione levels observed in t-BHP-treated HepG2 cells were ameliorated by pretreatment with juice powders, indicating that the hepatoprotective effects of juice powders and their major flavonoids are mediated by induction of cellular defense against oxidative stress. Moreover, pretreatment with juice powders up-regulated phase-II genes such as heme oxygenase-1 (HO-1), thereby preventing cellular damage and the resultant increase in HO-1 expression. The high-performance liquid chromatography profiles of the juice powders confirmed that hesperidin, narirutin, and rutin were the key flavonoids present. Our results suggest that these fruit juice powders and their major flavonoids provide a significant cytoprotective effect against oxidative stress, which is most likely due to the flavonoid-related bioactive compounds present, leading to the normal redox status of cells. Therefore, these fruit juice powders could be advantageous as bioactive sources for the prevention of oxidative injury in hepatoma cells. PMID:27752497

  14. A proteomic analysis of mushroom polysaccharide-treated HepG2 cells

    PubMed Central

    Chai, Yangyang; Wang, Guibin; Fan, Lili; Zhao, Min

    2016-01-01

    The anti-tumor properties of fungal polysaccharides have gained significant recognition in Asia and tropical America. In this study, the differential expression of proteins in normal HepG2 cells and those treated with polysaccharides that had been isolated from Phellinus linteus (PL), Ganoderma lucidum (GL) and Auricularia auricula (AA) was investigated. Using two-dimensional electrophoresis (2DE), a total of 104 protein spots were determined to be overexpressed in these cells compared with noncancerous regions. A total of 59 differentially expressed proteins were identified through MALDI-TOF-MS. In addition, 400 biological processes (BP), 133 cell components (CC) and 146 molecular functions (MF) were enriched by Gene Ontology (GO) analysis, and 78 KEGG pathways were enriched by pathway enrichment. Protein-Protein Interaction (PPI) analysis demonstrated the interaction networks affected by polysaccharides in HepG2 cells. Then, DJ-1 and 14-3-3 were identified as the key proteins in the networks, and the expression of the mRNA and proteins were evaluated using Real-time quantitative PCR (qRT-PCR) and Western blotting (WB), respectively. The results were in agreement with the 2DE. These results provided information on significant proteins of hepatocellular carcinoma (HCC) and form an important basis for the future development of valuable medicinal mushroom resources. PMID:27020667

  15. A proteomic analysis of mushroom polysaccharide-treated HepG2 cells.

    PubMed

    Chai, Yangyang; Wang, Guibin; Fan, Lili; Zhao, Min

    2016-03-29

    The anti-tumor properties of fungal polysaccharides have gained significant recognition in Asia and tropical America. In this study, the differential expression of proteins in normal HepG2 cells and those treated with polysaccharides that had been isolated from Phellinus linteus (PL), Ganoderma lucidum (GL) and Auricularia auricula (AA) was investigated. Using two-dimensional electrophoresis (2DE), a total of 104 protein spots were determined to be overexpressed in these cells compared with noncancerous regions. A total of 59 differentially expressed proteins were identified through MALDI-TOF-MS. In addition, 400 biological processes (BP), 133 cell components (CC) and 146 molecular functions (MF) were enriched by Gene Ontology (GO) analysis, and 78 KEGG pathways were enriched by pathway enrichment. Protein-Protein Interaction (PPI) analysis demonstrated the interaction networks affected by polysaccharides in HepG2 cells. Then, DJ-1 and 14-3-3 were identified as the key proteins in the networks, and the expression of the mRNA and proteins were evaluated using Real-time quantitative PCR (qRT-PCR) and Western blotting (WB), respectively. The results were in agreement with the 2DE. These results provided information on significant proteins of hepatocellular carcinoma (HCC) and form an important basis for the future development of valuable medicinal mushroom resources.

  16. Insulin resistance reduces sensitivity to Cis-platinum and promotes adhesion, migration and invasion in HepG2 cells.

    PubMed

    Li, Lin-Jing; Li, Guang-Di; Wei, Hu-Lai; Chen, Jing; Liu, Yu-Mei; Li, Fei; Xie, Bei; Wang, Bei; Li, Cai-Li

    2014-01-01

    The liver is normally the major site of glucose metabolism in intact organisms and the most important target organ for the action of insulin. It has been widely accepted that insulin resistance (IR) is closely associated with postoperative recurrence of hepatocellular carcinoma (HCC). However, the relationship between IR and drug resistance in liver cancer cells is unclear. In the present study, IR was induced in HepG2 cells via incubation with a high concentration of insulin. Once the insulin-resistant cell line was established, the instability of HepG2/ IR cells was further tested via incubation in insulin-free medium for another 72h. Afterwards, the biological effects of insulin resistance on adhesion, migration, invasion and sensitivity to cis-platinum (DDP) of cells were determined. The results indicated that glucose consumption was reduced in insulin-resistant cells. In addition, the expression of the insulin receptor and glucose transportor-2 was downregulated. Furthermore, HepG2/IR cells displayed markedly enhanced adhesion, migration, and invasion. Most importantly, these cells exhibited a lower sensitivity to DDP. By contrast, HepG2/IR cells exhibited decreased adhesion and invasion after treatment with the insulin sensitizer pioglitazone hydrochloride. The results suggest that IR is closely related to drug resistance as well as adhesion, migration, and invasion in HepG2 cells. These findings may help explain the clinical observation of limited efficacy for chemotherapy on a background of IR, which promotes the invasion and migration of cancer cells.

  17. Regulation of the Lactobacillus Strains on HMGCoA Reductase Gene Transcription in Human HepG2 Cells via Nuclear Factor-κB.

    PubMed

    Chen, Kun; Li, Shaocong; Chen, Fang; Li, Jun; Luo, Xuegang

    2016-02-01

    Lactic acid bacteria have been identified to be effective in reducing cholesterol levels. Most of the mechanistic studies were focused on the bile salt deconjugation ability of bile salt hydrolase in lactic acid bacteria. However, the mechanism by which Lactobacillus decreases cholesterol levels has not been thoroughly studied in intact primate cells. 3-Hydroxy-3- methyl-glutaryl-coenzyme A reductase (HMGCR) is the vital enzyme in cholesterol synthesis. To confirm the effect of probiotic Lactobacillus strains on HMGCR level, in the present study, human hepatoma HepG2 cells were treated with Lactobacillus strains, and then the HMGCR level was illustrated by luciferase reporter assay and RT-PCR. The results showed that the level of HMGCR was suppressed after being treated with the live Lactobacillus strains. These works might set a foundation for the following study of the antihyperlipidemic effects of L. acidophilus, and contribute to the development of functional foods or drugs that benefit patients suffering from hyperlipidemia diseases.

  18. Glibenclamide induces apoptosis through inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels and intracellular Ca(2+) release in HepG2 human hepatoblastoma cells.

    PubMed

    Kim, J A; Kang, Y S; Lee, S H; Lee, E H; Yoo, B H; Lee, Y S

    1999-08-11

    Glibenclamide, an inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels, induced apoptosis in a dose- and time-dependent manner in HepG2 human hepatoblastoma cells. Glibenclamide increased intracellular Ca(2+) concentration, which was significantly inhibited by Ca(2+) release blockers dantrolene and TMB-8. BAPTA/AM, an intracellular Ca(2+) chelator, and the Ca(2+) release blockers significantly inhibited glibenclamide-induced apoptosis. Glibanclamide also increased intracellular Cl(-) concentration, which was significantly blocked by CFTR Cl(-) channel activators levamisole and bromotetramisole. These activators also significantly inhibited both intracellular Ca(2+) release and apoptosis induced by glibenclamide. The expression of CFTR protein in the cells was confirmed by Western blot analysis. These results suggest that glibenclamide induced apoptosis through inhibition of CFTR Cl(-) channels and intracellular Ca(2+) release and that this protein may be a good target for treatment of human hepatomas.

  19. Toxicity Effect of Silver Nanoparticles on Mice Liver Primary Cell Culture and HepG2 Cell Line.

    PubMed

    Faedmaleki, Firouz; H Shirazi, Farshad; Salarian, Amir-Ahmad; Ahmadi Ashtiani, Hamidreza; Rastegar, Hossein

    2014-01-01

    Nano-silver (AgNP) has biological properties which are significant for consumer products, food technology, textiles and medical applications (e.g. wound care products, implantable medical devices, in diagnosis, drug delivery, and imaging). For their antibacterial activity, silver nanoparticles are largely used in various commercially available products. Thus, the use of nano-silver is becoming more and more widespread in medicine. In this study we investigated the cytotoxic effects of AgNPs on liver primary cells of mice, as well as the human liver HepG2 cell. Cell viability was examined with MTT assay after HepG2 cells exposure to AgNPs at 1, 2, 3, 4, 5, 7.5, 10 ppm compared to mice primary liver cells at 1, 10, 50, 100, 150, 200, 400 ppm for 24h. AgNPs caused a concentration-dependent decrease of cell viability in both cells. IC50 value of 2.764 ppm (µg/mL) was calculated in HepG2 cell line and IC50 value of 121.7 ppm (µg/mL) was calculated in primary liver cells of mice. The results of this experiment indicated that silver nanoparticles had cytotoxic effects on HepG2 cell line and primary liver cells of mice. The results illustrated that nano-silver had 44 times stronger inhibitory effect on the growth of cancerous cells (HepG2 cell line) compared to the normal cells (primary liver cells of mice). which might further justify AgNPs as a cytotoxic agents and a potential anticancer candidate which needs further studies in this regard.

  20. HCMV Activates the IL-6-JAK-STAT3 Axis in HepG2 Cells and Primary Human Hepatocytes

    PubMed Central

    Kumar, Amit; Tripathy, Manoj K.; Herbein, Georges

    2013-01-01

    Objectives There has been increased interest in the possible role of human cytomegalovirus (HCMV) in carcinogenesis during the last decade. HCMV seroprevalence was enhanced in patients with hepatocellular carcinoma (HCC) but a possible relationship between HCC and HCMV infection remained to be assessed. The aim of this work was to investigate the pro-tumor influence of HCMV on primary human hepatocytes (PHH) and HepG2 cells. Methods Following infection of PHH and HepG2 cells by two different strains of HCMV, we measured the production of IL-6 in culture supernatants by ELISA and the protein levels of STAT3, pSTAT3, JAK, cyclin D1, survivin, p53, p21, and Mdm2 by western Blotting in infected and uninfected cells. Cell proliferation and transformation were investigated using Ki67Ag expression measurement and soft-agar colony formation assay respectively. Results Infection of HepG2 cells and PHH by HCMV resulted in the production of IL-6 and the subsequent activation of the IL-6R-JAK-STAT3 pathway. HCMV increased the expression of cyclin D1 and survivin. Cell proliferation was enhanced in HepG2 and PHH infected with HCMV, despite a paradoxical overexpression of p53 and p21. More importantly, we observed the formation of colonies in soft agar seeded with PHH infected with HCMV and when we challenged the HepG2 cultures to form tumorspheres, we found that the HCMV-infected cultures formed 2.5-fold more tumorspheres than uninfected cultures. Conclusion HCMV activated the IL-6-JAK-STAT3 pathway in PHH and HepG2 cells, favored cellular proliferation, induced PHH transformation and enhanced HepG2 tumorsphere formation. Our observations raise the possibility that HCMV infection might be involved in the genesis of hepatocellular carcinoma. PMID:23555719

  1. Metabolic basis of ethanol-induced cytotoxicity in recombinant HepG2 cells: Role of nonoxidative metabolism

    SciTech Connect

    Wu Hai; Cai Ping; Clemens, Dahn L.; Jerrells, Thomas R.; Ansari, G.A. Shakeel; Kaphalia, Bhupendra S. . E-mail: bkaphali@utmb.edu

    2006-10-15

    Chronic alcohol abuse, a major health problem, causes liver and pancreatic diseases and is known to impair hepatic alcohol dehydrogenase (ADH). Hepatic ADH-catalyzed oxidation of ethanol is a major pathway for the ethanol disposition in the body. Hepatic microsomal cytochrome P450 (CYP2E1), induced in chronic alcohol abuse, is also reported to oxidize ethanol. However, impaired hepatic ADH activity in a rat model is known to facilitate a nonoxidative metabolism resulting in formation of nonoxidative metabolites of ethanol such as fatty acid ethyl esters (FAEEs) via a nonoxidative pathway catalyzed by FAEE synthase. Therefore, the metabolic basis of ethanol-induced cytotoxicity was determined in HepG2 cells and recombinant HepG2 cells transfected with ADH (VA-13), CYP2E1 (E47) or ADH + CYP2E1 (VL-17A). Western blot analysis shows ADH deficiency in HepG2 and E47 cells, compared to ADH-overexpressed VA-13 and VL-17A cells. Attached HepG2 cells and the recombinant cells were incubated with ethanol, and nonoxidative metabolism of ethanol was determined by measuring the formation of FAEEs. Significantly higher levels of FAEEs were synthesized in HepG2 and E47 cells than in VA-13 and VL-17A cells at all concentrations of ethanol (100-800 mg%) incubated for 6 h (optimal time for the synthesis of FAEEs) in cell culture. These results suggest that ADH-catalyzed oxidative metabolism of ethanol is the major mechanism of its disposition, regardless of CYP2E1 overexpression. On the other hand, diminished ADH activity facilitates nonoxidative metabolism of ethanol to FAEEs as found in E47 cells, regardless of CYP2E1 overexpression. Therefore, CYP2E1-mediated oxidation of ethanol could be a minor mechanism of ethanol disposition. Further studies conducted only in HepG2 and VA-13 cells showed lower ethanol disposition and ATP concentration and higher accumulation of neutral lipids and cytotoxicity (apoptosis) in HepG2 cells than in VA-13 cells. The apoptosis observed in HepG2 vs

  2. Study of the efficacy of photofrin®-Mediated PDT on human hepatocellular carcinoma (HepG2) cell line

    NASA Astrophysics Data System (ADS)

    Atif, M.; Fakhar-e-Alam, M.; Zaidi, S. S. Z.; Suleman, R.

    2011-06-01

    The present study evaluates the effects of photodynamic therapy (PDT) with Photofrin® using human liver cancer cells (HepG2) as an experimental model. We optimized the different PDT parameters, e.g. (time of incubation, optimal dose of light and drug concentration), cytotoxicity, phototoxicity, and cellular viability of the HepG2 cells has also been investigated in this experimental work. The effect of light on the viability of cells without the photosensitizer was examined firstly, HepG2 cell line was irradiated with red light (a diode laser, λ = 635 nm). The toxicity of the photosensitizer in the absence of light in current cell line was investigated secondly, Photofrin® has been used as photosensitizing agent. Optimal dose of light along with suitable concentration of Photofrin® were traced into HepG2 cell line, by means of spectrophotometric measurement. Cells viability was determined by means of neutral red assay (NRA). Finally, it was observed that no toxic effects with the absence of light, and no significant photodamage effect on the cells without the presence of photosensitizer were found, when studied independently. Our results showed that light doses of 100 J/cm2 gives effective PDT outcome for HepG2 cell line at photosensitizer concentration of 100 μg/ml.

  3. Dehydroepiandrosterone-induces miR-21 transcription in HepG2 cells through estrogen receptor β and androgen receptor

    PubMed Central

    Teng, Yun; Litchfield, Lacey M.; Ivanova, Margarita M.; Prough, Russell A.; Clark, Barbara J.; Klinge, Carolyn M.

    2014-01-01

    Although oncomiR miR-21 is highly expressed in liver and overexpressed in hepatocellular carcinoma (HCC), its regulation is uncharacterized. We examined the effect of physiologically relevant nanomolar concentrations of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEA-S) on miR-21 expression in HepG2 human hepatoma cells. 10 nM DHEA and DHEA-S increase pri-miR-21 transcription in HepG2 cells. Dietary DHEA increased miR-21 in vivo in mouse liver. siRNA and inhibitor studies suggest that DHEA-S requires desulfation for activity and that DHEA-induced pri-miR-21 transcription involves metabolism to androgen and estrogen receptor (AR and ER) ligands. Activation of ERβ and AR by DHEA metabolites androst-5-ene-3,17-dione (ADIONE), androst-5-ene-3β,17β-diol (ADIOL), dihydrotestosterone (DHT), and 5α-androstane-3β,17β-diol (3β-Adiol) increased miR-21 transcription. DHEA-induced miR-21 increased cell proliferation and decreased Pdcd4 protein, a bona fide miR-21. Estradiol (E2) inhibited miR-21 expression via ERα. DHEA increased ERβ and AR recruitment to the miR-21 promoter within the VMP1/TMEM49 gene, with possible significance in hepatocellular carcinoma. PMID:24845419

  4. Effects of nitric oxide on the biological behavior of HepG2 human hepatocellular carcinoma cells.

    PubMed

    Zhou, Lei; Zhang, Heng; Wu, Jie

    2016-05-01

    Many studies have found the function of nitric oxide (NO) in cancer as a pro-neoplastic vs. an anti-neoplastic effector, but the role of NO in hepatocellular carcinoma (HCC) remains unclear. The present study aimed to investigate the effects of nitric oxide (NO) on the biological behavior of the human hepatocellular carcinoma cell line HepG2. HepG2 cell was cultured in vitro and treated with or without sodium nitroprusside (SNP), a NO donor. Subsequently, we evaluated the effects of NO in cell proliferation, cell cycle, apoptosis, migration and invasion by MTT assay, flow cytometry, wound healing assay and Matrigel invasion assay. We demonstrate that NO significantly inhibited HepG2 cell proliferation by inducing G0/G1 phase arrest in a dose-dependent manner. In addition, compared to the control group, cells treated with SNP showed obviously higher apoptosis ratios in a dose-dependent manner. Furthermore, we revealed that NO effectively inhibited the ability of migration and invasion of HepG2 cells. Taken together, our results suggested that NO has an important role in the regulation of biological behavior in HepG2 cells and the potential for use in the prevention and treatment of HCC.

  5. Cytotoxicity of mequindox and its metabolites in HepG2 cells in vitro and murine hepatocytes in vivo.

    PubMed

    Liu, Yingchun; Jiang, Wei; Chen, Yongjun; Liu, Yanyan; Zeng, Peng; Xue, Feiqun; Wang, Quan

    2016-02-01

    Mequindox, a quinoxaline 1,4-dioxide, is widely used as a feed additive in the Chinese livestock industry because of its effective antibacterial properties. Many recent studies have found that mequindox is rapidly metabolized to numerous metabolites following administration to animals. There have, however, been few reports describing the cytotoxicity of mequindox metabolites. In this study, HepG2 cells were treated with mequindox (0, 2, 10, 50 or 100 μg/ml) or its major metabolites (0, 40, 100, 250 or 500 μg/ml) for 24h. Mice were administrated with mequindox (0, 50, 200 or 500 mg/kg.bw) for five days. DNA damage in the HepG2 cells and mouse hepatocytes was then assessed using an SCGE assay. The cell cycle of the HepG2 cells was also determined by flow cytometry. Mequindox was found to induce cell cycle arrest to the G2/M phase and cause dose-dependent DNA damage in HepG2 cells in vitro and in murine hepatocytes in vivo. Compared with mequindox, the major metabolites had much smaller effects on the cell cycle and caused much less DNA damage in HepG2 cells. And the results indicated that the process of metabolites formed by reduction of the MEQ acetyl group or reduction of the N → O groups could contribute to DNA damage in murine hepatocytes in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. An 'activatable' aptamer-based fluorescence probe for the detection of HepG2 cells.

    PubMed

    Lai, Zongqiang; Tan, Juntao; Wan, Ruirong; Tan, Jie; Zhang, Zhenghua; Hu, Zixi; Li, Jieping; Yang, Wei; Wang, Yiwei; Jiang, Yafeng; He, Jian; Yang, Nuo; Lu, Xiaoling; Zhao, Yongxiang

    2017-05-01

    It is significant to develop a probe with sensitivity and specificity for the detection of cancer cells. The present study aimed to develop an 'activatable' aptamer-based fluorescence probe (AAFP) to detect cancer cells and frozen cancer tissue. This AAFP consisted of two fragments: aptamer TLS11a that targets HepG2 cells, and two short extending complementary DNA sequences with a 5'- and 3'-terminus that make the aptamer in hairpin structure a capable quencher to fluorophore. The ability of the AAFP to bind specifically to cancer cells was assessed using flow cytometry, fluorescence spectroscopy and fluorescence microscopy. Its ability to bind to frozen cancer tissue was assessed using fluorescence microscopy. As a result, in the absence of cancer cells, AAFP showed minimal fluorescence, reflecting auto-quenching. In the presence of cancer cells, however, AAFP showed a strong fluorescent signal. Therefore, this AAFP may be a promising tool for sensitive and specific detection of cancer.

  7. Constitutive Effects of Lead on Aryl Hydrocarbon Receptor Gene Battery and Protection by β-carotene and Ascorbic Acid in Human HepG2 Cells.

    PubMed

    Darwish, Wageh S; Ikenaka, Yoshinori; Nakayama, Shouta M M; Mizukawa, Hazuki; Ishizuka, Mayumi

    2016-01-01

    Lead (Pb) is an environmental pollutant that can get entry into human body through contaminated foods, drinks, and inhaled air leading to severe biological consequences, and has been responsible for many deaths worldwide. The objectives of this study were 1st to investigate the modulatory effects of environmentally relevant concentrations of Pb on AhR gene battery, which is controlling xenobiotics metabolism. 2nd, trials to reduce Pb-induced adverse effects were done using some phytochemicals like β-carotene or ascorbic acid. Human hepatoma (HepG2) cell lines were exposed to a wide range of Pb concentrations varying from physiological to toxic levels (0 to 10 mg/L) for 24 h. High Pb concentrations (1 to 10 mg/L) significantly reduced phase I (CYP1A1 and 1A2) and phase II (UGT1A6 and NQO1) xenobiotic metabolizing enzyme mRNA expression in a mechanistic manner through the AhR regulation pathway. Additionally, these Pb concentrations induced oxidative stress in HepG2 cells in terms of production of reactive oxygen species (ROS) and induced heme oxygenase-1 mRNA expression in a concentration-dependent phenomenon. Coexposure of HepG2 cells to physiological concentrations of some micronutrients, like β-carotene (10 μM) or ascorbic acid (0.1 mM), along with Pb (1 mg/L) for 24 h significantly reduced the levels of ROS production and recovered AhR mRNA expression into the normal levels. Thus, consumption of foods rich in these micronutrients may help to reduce the adverse effects of lead in areas with high levels of pollution.

  8. Dichlorodiphenyldichloroethylene exposure reduces r-GCS via suppressed Nrf2 in HepG2 cells.

    PubMed

    Jin, Xiaoting; Song, Li; Li, Zhuoyu; Newton, Ian P; Zhao, Meirong; Liu, Weiping

    2016-03-01

    p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE), the major isomer of persistent 1,1-Bis(4-chlorophenyl)-2,2,2-trichloroethane metabolite, is highly associated with the risk of liver cancer. γ-glutamyl-cysteine synthetase (γ-GCS), which is the rate-limiting enzyme of glutathione (GSH) biosynthesis and an important scavenger of reactive oxygen species (ROS), is considered as a potential therapeutic target for many cancers. However, the association between the body burden of p,p'-DDE and γ-GCS has not been fully established. Here, we indicated that low doses of p,p'-DDE exposure promoted the proliferation and decreased γ-GCS activity of HepG2 cells in a dose- and time-dependent manner. In addition, p,p'-DDE elevated ROS content and attenuated glutathione peroxidase activity. This was accompanied with inhibitions of NF-E2-related factor 2 (Nrf2) at the mRNA and protein levels. ROS inhibitor supplement could significantly reverse these effects. Moreover, the addition of the proteasome inhibitor, MG132, strongly reversed the p,p'-DDE-reduced Nrf2 expression and γ-GCS activity. Consistently, GSH content was in line with the alteration of γ-GCS. Collectively, the results indicate that p,p'-DDE treatment downregulates γ-GCS activity in HepG2 cells by inducing ROS-mediated Nrf2 loss. © 2014 Wiley Periodicals, Inc.

  9. Basic apoptotic and necrotic cell death in human liver carcinoma (HepG2 ) cells induced by synthetic azamacrocycle.

    PubMed

    Yedjou, Clement G; Saeed, Musabbir A; Hossain, Md Alamgir; Dorsey, Waneene; Yu, Hongtao; Tchounwou, Paul B

    2014-06-01

    Treatment of diseases with synthetic materials has been an aspiration of mankind since the dawn of human development. In this research, three complex compounds of azamacrocycle (TD1, TD2, and TD3) were synthesized, and experiments were conducted to determine whether their toxicity to human liver carcinoma (HepG2 ) cells is associated with apoptotic and/or necrotic cell death. Cell survival was determined by MTT assay. Apoptosis and necrosis were measured by annexin V FITC/PI assay using the flow cytometry and by propidium iodide (PI) assay using the cellometer vision. HepG2 cells were treated with different concentrations of azamacrocycles for 48 h. Results from MTT assay indicated that all the three azamacrocycles significantly (p < 0.05) reduce cell viability in a dose-dependent manner, showing 48 h-LD50 values of about 37.97, 33.60, and 19.29 μM, for TD3, TD1 and TD2, respectively. Among the three compounds tested, TD2 showed the most pronounced cytotoxic activity against HepG2 cells, being about twofold more potent than TD3. The order of toxicity was TD2 > TD1 > TD3. Because TD2 exerted the most cytotoxic activity against HepG2 cells, it was used in the subsequent apoptosis and necrosis-related experiments. The flow cytometry assessment showed a strong dose-response relationship with regard to TD2 exposure and annexin V/PI positive cells. PI assay data indicated that TD2 exposure increased the proportion of fluorescence positive cells. Overall, our results indicate that azamacrocycle toxicity to HepG2 cells is associated with apoptotic and necrotic cell death resulting from phosphatidylserine externalization and loss of membrane integrity. Copyright © 2012 Wiley Periodicals, Inc.

  10. Endoplasmic reticulum stress mediates sulforaphane-induced apoptosis of HepG2 human hepatocellular carcinoma cells.

    PubMed

    Zou, Xiang; Qu, Zhongyuan; Fang, Yueni; Shi, Xin; Ji, Yubin

    2017-01-01

    Sulforaphane (SFN) is a naturally occurring chemopreventive agent, which effectively inhibits proliferation of HepG2 human hepatocellular carcinoma cells via mitochondria‑mediated apoptosis. Endoplasmic reticulum stress is considered the most important cause of cell apoptosis; therefore, the present study aimed to determine whether the endoplasmic reticulum pathway was involved in SFN-induced apoptosis of HepG2 cells. An MTT assay was used to detect the inhibitory effects of SFN on HepG2 cells. Fluorescence microscopy was used to observe the morphological changes in apoptotic cells, and western blot analysis was conducted to detect the expression of binding immunoglobulin protein (Bip)/glucose-regulated protein 78 (GRP78), X‑box binding protein‑1 (XBP‑1) and BH3 interacting domain death agonist (Bid). Furthermore, flow cytometry was used to determine the apoptotic rate of HepG2 cells, and the protein expression of C/EBP homologous protein (CHOP)/growth arrest‑ and DNA damage‑inducible gene 153 (GADD153) and caspase-12 in HepG2 cells. The results indicated that SFN significantly inhibited the proliferation of HepG2 cells; the half maximal inhibitory concentration values were 32.03±0.96, 20.90±1.96 and 13.87±0.44 µmol/l, following treatment with SFN for 24, 48 and 72 h, respectively. Following 48 h of SFN treatment (10, 20 and 40 µmol/l), the apoptotic rates of HepG2 cells were 31.8, 61.3 and 77.1%, respectively. Furthermore, after 48 h of exposure to SFN, the cells presented typical morphological alterations of apoptosis, as detected under fluorescence microscopy. Treatment with SFN for 48 h also significantly upregulated the protein expression levels of Bip/GRP78, XBP‑1, caspase‑12, CHOP/GADD153 and Bid in HepG2 cells. In conclusion, endoplasmic reticulum stress may be considered the most important mechanism underlying SFN-induced apoptosis in HepG2 cells.

  11. MSC(TRAIL)-mediated HepG2 cell death in direct and indirect co-cultures.

    PubMed

    Sun, Xu-Yong; Nong, Jiang; Qin, Ke; Lu, Hong; Moniri, Mani R; Dai, Long-Jun; Warnock, Garth L

    2011-11-01

    Mesenchymal stem cells (MSCs) have attracted great interest in cancer therapy since the discovery of their tumor tropism. This study was performed to investigate the effects of TNF-related apoptosis-inducing ligand (TRAIL)-engineered MSCs on hepatocellular carcinoma (HCC) cells (HepG2) under different culture conditions. MSCs engineered with non-secreting TRAIL (MSC(TRAIL-GFP)) (GFP, green fluorescence protein) and secreting TRAIL (MSC(stTRAIL)) were used for the direct co-cultures, and conditioned media (CM) from corresponding cultures were applied to HepG2 as indirect co-cultures. Immunoblotting, ELISA and FACS analysis were used to detect the expression of TRAIL and TRAIL receptors. Cell death was assessed using live/dead assay. Death receptor (DR) 5 was identified on the HepG2 cells. The expression of TRAIL was confirmed in the cell lysates (MSC(TRAIL-GFP) >MSC(stTRAIL)) and the conditioned media (MSC(stTRAIL) >MSC(TRAIL-GFP)). Higher cell death was observed in high MSC/HepG2 ratio co-cultures. HepG2 cell death was proportionally related to CM from MSC(TRAIL-GFP) and MSC(stTRAIL). MSCs exhibit intrinsic inhibition of HepG2 which is potentiated by TRAIL-transfection.

  12. A combination of transcriptomics and metabolomics uncovers enhanced bile acid biosynthesis in HepG2 cells expressing CCAAT/enhancer-binding protein β (C/EBPβ), hepatocyte nuclear factor 4α (HNF4α), and constitutive androstane receptor (CAR).

    PubMed

    Blazquez, Marina; Carretero, Aitor; Ellis, James K; Athersuch, Toby J; Cavill, Rachel; Ebbels, Timothy M D; Keun, Hector C; Castell, José V; Lahoz, Agustín; Bort, Roque

    2013-06-07

    The development of hepatoma-based in vitro models to study hepatocyte physiology is an invaluable tool for both industry and academia. Here, we develop an in vitro model based on the HepG2 cell line that produces chenodeoxycholic acid, the main bile acid in humans, in amounts comparable to human hepatocytes. A combination of adenoviral transfections for CCAAT/enhancer-binding protein β (C/EBPβ), hepatocyte nuclear factor 4α (HNF4α), and constitutive androstane receptor (CAR) decreased intracellular glutamate, succinate, leucine, and valine levels in HepG2 cells, suggestive of a switch to catabolism to increase lipogenic acetyl CoA and increased anaplerosis to replenish the tricarboxylic acid cycle. Transcripts of key genes involved in bile acid synthesis were significantly induced by approximately 160-fold. Consistently, chenodeoxycholic acid production rate was increased by more than 20-fold. Comparison between mRNA and bile acid levels suggest that 12-alpha hydroxylation of 7-alpha-hydroxy-4-cholesten-3-one is the limiting step in cholic acid synthesis in HepG2 cells. These data reveal that introduction of three hepatocyte-related transcription factors enhance anabolic reactions in HepG2 cells and provide a suitable model to study bile acid biosynthesis under pathophysiological conditions.

  13. Fucoidan from Fucus vesiculosus protects against alcohol-induced liver damage by modulating inflammatory mediators in mice and HepG2 cells.

    PubMed

    Lim, Jung Dae; Lee, Sung Ryul; Kim, Taeseong; Jang, Seon-A; Kang, Se Chan; Koo, Hyun Jung; Sohn, Eunsoo; Bak, Jong Phil; Namkoong, Seung; Kim, Hyoung Kyu; Song, In Sung; Kim, Nari; Sohn, Eun-Hwa; Han, Jin

    2015-02-16

    Fucoidan is an l-fucose-enriched sulfated polysaccharide isolated from brown algae and marine invertebrates. In this study, we investigated the protective effect of fucoidan from Fucus vesiculosus on alcohol-induced murine liver damage. Liver injury was induced by oral administration of 25% alcohol with or without fucoidan (30 mg/kg or 60 mg/kg) for seven days. Alcohol administration increased serum aspartate aminotransferase and alanine aminotransferase levels, but these increases were suppressed by the treatment of fucoidan. Transforming growth factor beta 1 (TGF-β1), a liver fibrosis-inducing factor, was highly expressed in the alcohol-fed group and human hepatoma HepG2 cell; however, the increase in TGF-β1 expression was reduced following fucoidan administration. Treatment with fucoidan was also found to significantly reduce the production of inflammation-promoting cyclooygenase-2 and nitric oxide, while markedly increasing the expression of the hepatoprotective enzyme, hemeoxygenase-1, on murine liver and HepG2 cells. Taken together, the antifibrotic and anti-inflammatory effects of fucoidan on alcohol-induced liver damage may provide valuable insights into developing new therapeutics or interventions.

  14. Fucoidan from Fucus vesiculosus Protects against Alcohol-Induced Liver Damage by Modulating Inflammatory Mediators in Mice and HepG2 Cells

    PubMed Central

    Lim, Jung Dae; Lee, Sung Ryul; Kim, Taeseong; Jang, Seon-A; Kang, Se Chan; Koo, Hyun Jung; Sohn, Eunsoo; Bak, Jong Phil; Namkoong, Seung; Kim, Hyoung Kyu; Song, In Sung; Kim, Nari; Sohn, Eun-Hwa; Han, Jin

    2015-01-01

    Fucoidan is an l-fucose-enriched sulfated polysaccharide isolated from brown algae and marine invertebrates. In this study, we investigated the protective effect of fucoidan from Fucus vesiculosus on alcohol-induced murine liver damage. Liver injury was induced by oral administration of 25% alcohol with or without fucoidan (30 mg/kg or 60 mg/kg) for seven days. Alcohol administration increased serum aspartate aminotransferase and alanine aminotransferase levels, but these increases were suppressed by the treatment of fucoidan. Transforming growth factor beta 1 (TGF-β1), a liver fibrosis-inducing factor, was highly expressed in the alcohol-fed group and human hepatoma HepG2 cell; however, the increase in TGF-β1 expression was reduced following fucoidan administration. Treatment with fucoidan was also found to significantly reduce the production of inflammation-promoting cyclooygenase-2 and nitric oxide, while markedly increasing the expression of the hepatoprotective enzyme, hemeoxygenase-1, on murine liver and HepG2 cells. Taken together, the antifibrotic and anti-inflammatory effects of fucoidan on alcohol-induced liver damage may provide valuable insights into developing new therapeutics or interventions. PMID:25690093

  15. Borax-induced apoptosis in HepG2 cells involves p53, Bcl-2, and Bax.

    PubMed

    Wei, Y; Yuan, F J; Zhou, W B; Wu, L; Chen, L; Wang, J J; Zhang, Y S

    2016-06-21

    Borax, a boron compound and a salt of boric acid, is known to inhibit the growth of tumor cells. HepG2 cells have been shown to be clearly susceptible to the anti-proliferative effects of borax. However, the specific mechanisms regulating this effect are poorly understood. This study aimed to investigate the pathways underlying the growth inhibition induced by borax in HepG2 cells. The effects of borax on HepG2 cell viability were characterized using MTT. Apoptosis was also verified by annexin V/propidium iodide staining. JC-1 dye and western blotting techniques were used to measure mitochondrial membrane potential and p53, Bax, and Bcl-2 protein expression, respectively. Relevant mRNA levels were measured by qRT-PCR. Borax inhibited the proliferation of HepG2 cells in a time- and dose-dependent manner in vitro. The apoptotic process triggered by borax involved the upregulation of p53 and Bax and the downregulation of Bcl-2, which was confirmed by a change in the mitochondrial membrane potential. These results elucidate a borax-induced apoptotic pathway in HepG2 cells that involves the upregulation of p53 and Bax and the downregulation of Bcl-2.

  16. The role of alkaline phosphatase in intracellular lipid accumulation in the human hepatocarcinoma cell line, HepG2.

    PubMed

    Chirambo, George M; van Niekerk, Chantal; Crowther, Nigel J

    2017-04-01

    Inhibition of tissue non-specific alkaline phosphatase (TNALP) decreases intracellular lipid accumulation in human preadipocytes and the murine preadipocyte cell line, 3T3-L1. Therefore, the current study was performed to determine if TNALP is required for intracellular lipid deposition in the human hepatocyte cell line, HepG2. Intracellular lipid accumulation, TNALP activity and peroxisome proliferator activated receptor (PPAR) γ gene expression were measured in HepG2 and 3T3-L1 cells in the presence and absence of the TNALP inhibitors levamisole and histidine. Sub-cellular TNALP activity was localized using cytochemical analysis. Both PPARγ gene expression and TNALP activity increased during intracellular lipid accumulation in HepG2 and 3T3-L1 cells. Inhibition of TNALP blocked intracellular lipid accumulation but did not alter expression of the PPARγ gene. In HepG2 cells, TNALP co-localized with adipophilin on the lipid droplet membrane. These data suggest a role for TNALP in lipid droplet formation, possibly downstream from PPARγ, within HepG2 and 3T3-L1 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Effect of taurine on the proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells.

    PubMed

    Tu, Shuo; Zhang, Xiali; Luo, Daya; Liu, Zhuoqi; Yang, Xiaohong; Wan, Huifang; Yu, Lehan; Li, Hua; Wan, Fusheng

    2015-07-01

    The aim of the present study was to observe the effect and molecular mechanism of taurine (Tau) on the cell proliferation and apoptosis of human hepatocellular carcinoma (HHCC) HepG2 cells. HHCC HepG2 cells were used as target cells, and the cell survival rate was assessed using a multi-time-step method. The p53 upregulated modulator of apoptosis (PUMA) gene was transiently transfected by lipofection and subsequently silenced with specific small interfering (si)RNA. The cell apoptosis rate was detected by flow cytometry, and protein expression levels were analyzed with western blotting. Addition of 20-160 mM Tau was shown to have a significant inhibitory effect on cell proliferation, while promoting the induction of HHCC HepG2 cell apoptosis (P<0.05). Transfection of the PUMA gene significantly enhanced the ability of Tau to inhibit proliferation and induce apoptosis of HepG2 cells. In addition, transfection of the PUMA gene increased the protein expression of B-cell lymphoma-2-associated X and reduced the expression of B-cell lymphoma-2 (P<0.05). Silencing the PUMA gene with specific siRNA was demonstrated to significantly reduce the ability of Tau to inhibit proliferation and induce the apoptosis of HHCC HepG2 cells (P<0.01). Therefore, the PUMA gene was shown to have an important role in mechanism underlying the effect that Tau exerts on cell proliferation and apoptosis in HHCC HepG2 cells.

  18. [Anti-proliferation Effect of Taraxacum mongolicum Extract in HepG2 Cells and Its Mechanism].

    PubMed

    Guo, Jun-bin; Ye, Hai-hong; Chen, Jian-feng

    2015-10-01

    To study the anti-proliferation effect of Taraxacum mongolicum extract in HepG2 cells and its mechanism. The total proteins of HepG2 cells treated with Taraxacum mongolicum extract were. extracted and mitochondria-mediated apoptosis-related proteins (Survivin, Mcl-1, BCL-xL, BCL-2, Smac, BAX, Bad, Cytochrome c and Caspase-3/7/9) were detected by Western blot. Taraxacum mongolicum extract obviously inhibited the proliferation of HepG2 cells and the expression of anti-apoptotic proteins (Survivin, BCL-xL and BCL-2), increased the expression of pro-apoptotic proteins (Smac and Caspase-3/7/9), and promoted the release of Cytochrome c from mitochondria to cytoplasm in HepG2 cells. The effects were in a dose-independent mode. Taraxacum mongolicum extract can inhibit the proliferation of HepG2 cells and the anti-proliferation mechanism is related to mitochondria-mediated apoptosis.

  19. Selective killing of hepatocellular carcinoma HepG2 cells by three-dimensional nanographene nanoparticles based on triptycene

    NASA Astrophysics Data System (ADS)

    Xiong, Xiaoqin; Gan, Lu; Liu, Ying; Zhang, Chun; Yong, Tuying; Wang, Ziyi; Xu, Huibi; Yang, Xiangliang

    2015-03-01

    Carbon-based materials have been widely used in the biomedical fields including drug delivery and cancer therapies. In this paper, a recently synthesized three-dimensional nanographene (NG) based on triptycene self-assembles into nanoparticles which selectively kill human hepatocellular carcinoma HepG2 cells as compared to human normal liver HL7702 cells. Obvious differences in cellular accumulation, the endocytic pathway and intracellular trafficking of NG nanoparticles are observed in HepG2 cells and HL7702 cells. Further studies reveal that NG nanoparticles significantly increase the levels of reactive oxygen species (ROS) in HepG2 cells, but not in HL7702 cells. NG nanoparticle-induced ROS result in apoptosis induction and the decrease in mitochondrial membrane potential in HepG2 cells. Moreover, IKK/nuclear factor-κB (NF-κB) signaling is found to be activated by NG nanoparticle-induced ROS and serves to antagonize NG nanoparticle-induced apoptosis in HepG2 cells. Our studies show that the distinct behaviors of cellular uptake and ROS-mediated cytotoxicity are responsible for the selective killing of HepG2 cells. This study provides a foundation for understanding the mechanism of selective induction of apoptosis in cancer cells by NG nanoparticles and designing more effective chemotherapeutical agents.Carbon-based materials have been widely used in the biomedical fields including drug delivery and cancer therapies. In this paper, a recently synthesized three-dimensional nanographene (NG) based on triptycene self-assembles into nanoparticles which selectively kill human hepatocellular carcinoma HepG2 cells as compared to human normal liver HL7702 cells. Obvious differences in cellular accumulation, the endocytic pathway and intracellular trafficking of NG nanoparticles are observed in HepG2 cells and HL7702 cells. Further studies reveal that NG nanoparticles significantly increase the levels of reactive oxygen species (ROS) in HepG2 cells, but not in HL7702

  20. Anticancer effects of imperatorin isolated from Angelica dahurica: induction of apoptosis in HepG2 cells through both death-receptor- and mitochondria-mediated pathways.

    PubMed

    Luo, Ke-wang; Sun, Jian-guo; Chan, Judy Yuet-Wa; Yang, Lu; Wu, Shi-hua; Fung, Kwok-Pui; Liu, Fei-yan

    2011-01-01

    Imperatorin (IM) is a furanocoumarin isolated from the root of Angelica dahurica, which is reported to have anticonvulsant and anticancer effects. In this study, the antiproliferative effect of IM on 9 human cancer cell lines was examined, and human hepatoma HepG2 cells were chosen as the target for preferential killing by IM. Particularly, the mechanism of IM-induced apoptosis and in vivo animal effects were also studied. Cell viability was measured using MTT assay, and apoptosis was detected by Hoechst staining, annexin V-PI staining, and DNA laddering assay. Mitochondrial membrane potential was detected by JC-1 staining. Western blot analysis was employed to detect the expression of apoptosis-related proteins. In addition, the in vivo anticancer effect of IM was examined in nude mice bearing HepG2 cells. IM inhibited the proliferation of HepG2 cells through apoptosis induction in a time- and dose-dependent manner by observation of the nuclear morphology, DNA fragmentation, phosphatidylserine externalization, loss of mitochondrial membrane potential, release of cytochrome c into cytosol, and activation of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase cleavage. As cell death could partly be prevented by the caspase-8 or caspase-9 inhibitor and was evidenced by the results of Western blot analysis, our results also suggest that IM-induced apoptosis is mediated through both death receptor and mitochondrial pathways. In the animal model, IM was found to effectively suppress tumor growth by 31.93 and 63.18% at dosages of 50 and 100 mg/kg, respectively, after treatment for 14 days. No significant weight loss or toxicity to the hosts was found. IM can function as a cancer suppressor by inducing apoptosis in HepG2 cells through both death-receptor- and mitochondria-mediated pathways. Furthermore, the in vivo antitumor activities of IM are significant with negligible weight loss and damage to the host. Copyright © 2012 S. Karger AG, Basel.

  1. [Arginase inhibitor nor-NOHA induces apoptosis and inhibits invasion and migration of HepG2 cells].

    PubMed

    Li, Xiangnan; Zhu, Fangyu; He, Yongsong; Luo, Fang

    2017-04-01

    Objective To investigate the cell inhibitory effect of arginase inhibitor nor-NOHA on HepG2 hepatocellular carcinoma cells and related mechanism. Methods CCK-8 assay was used to detect the cell proliferation and flow cytometry to detect the apoptosis of HepG2 cells treated with (0, 0.5, 1.0, 2.0, 3.0) ng/μL nor-NOHA. The protein levels of arginase 1 (Arg1), P53, matrix metalloproteinase-2 (MMP-2), E-cadherin (ECD) were determined by Western blotting. Real time quantitative PCR was employed to examine the changes in the mRNA level of inducible nitric oxide synthase (iNOS). Griess assay was used to measure the concentration of nitric oxide (NO) in HepG2 cells. Transwell(TM) assay and wound-healing assay were performed to evaluate the changes of the cell invasion and migration ability, respectively. Results nor-NOHA inhibited the proliferation and induced the apoptosis of HepG2 cells. It also decreased the expression levels of Arg1 and MMP-2, increased the expression levels of P53 and ECD as well as the production of NO; in addition, nor-NOHA inhibited the invasion and migration of HepG2 cells. Conclusion Nor-NOHA can induce cell apoptosis and inhibit the ability of invasion and migration of HepG2 cells by inhibiting Arg1, which is related with the increase of iNOS expression and the high concentration of NO.

  2. Indoxyl sulfate suppresses hepatic fetuin-A expression via the aryl hydrocarbon receptor in HepG2 cells.

    PubMed

    Ochi, Akinobu; Mori, Katsuhito; Nakatani, Shinya; Emoto, Masanori; Morioka, Tomoaki; Motoyama, Koka; Fukumoto, Shinya; Imanishi, Yasuo; Shoji, Tetsuo; Ishimura, Eiji; Inaba, Masaaki

    2015-10-01

    Fetuin-A is a liver-derived circulating protein that has potent calcification-inhibitory activity. Uraemic patients exhibit decreased serum fetuin-A levels, increased vascular calcification and elevated cardiovascular mortality. Because the mechanisms for fetuin-A deficiency are unknown, we hypothesized that some uraemic toxins suppressed hepatic fetuin-A production, which resulted in accelerated vascular calcification and poor outcome. Among these potential candidates, indoxyl sulfate (IS) has highly toxic properties. We examined the direct effects of IS on hepatic fetuin-A expression using the human hepatoma HepG2 cell line. IS, but not p-cresyl sulfate, suppressed the mRNA and protein expression of fetuin-A in a dose- and time-dependent manner. As reported previously, IS stimulated p38 MAPK phosphorylation and reactive oxygen species (ROS) production, although the knockdown of p38 and inhibition of ROS generation had no effect on IS-induced fetuin-A suppression. Then, because IS is a potent endogenous ligand of the aryl hydrocarbon receptor (AhR), we assessed whether IS suppresses fetuin-A production via AhR. The knockdown of AhR prevented IS-induced fetuin-A suppression. However, some attention should be paid to no effect of IS on fetuin-A expression in mouse and human primary cultured hepatocytes. These findings suggest that IS could suppress hepatic fetuin-A expression by activating AhR, suggesting a relationship between uraemia and fetuin-A deficiency. © The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

  3. Sodium valproate induces mitochondrial respiration dysfunction in HepG2 in vitro cell model.

    PubMed

    Komulainen, Tuomas; Lodge, Tiffany; Hinttala, Reetta; Bolszak, Maija; Pietilä, Mika; Koivunen, Peppi; Hakkola, Jukka; Poulton, Joanna; Morten, Karl J; Uusimaa, Johanna

    2015-05-04

    Sodium valproate (VPA) is a potentially hepatotoxic antiepileptic drug. Risk of VPA-induced hepatotoxicity is increased in patients with mitochondrial diseases and especially in patients with POLG1 gene mutations. We used a HepG2 cell in vitro model to investigate the effect of VPA on mitochondrial activity. Cells were incubated in glucose medium and mitochondrial respiration-inducing medium supplemented with galactose and pyruvate. VPA treatments were carried out at concentrations of 0-2.0mM for 24-72 h. In both media, VPA caused decrease in oxygen consumption rates and mitochondrial membrane potential. VPA exposure led to depleted ATP levels in HepG2 cells incubated in galactose medium suggesting dysfunction in mitochondrial ATP production. In addition, VPA exposure for 72 h increased levels of mitochondrial reactive oxygen species (ROS), but adversely decreased protein levels of mitochondrial superoxide dismutase SOD2, suggesting oxidative stress caused by impaired elimination of mitochondrial ROS and a novel pathomechanism related to VPA toxicity. Increased cell death and decrease in cell number was detected under both metabolic conditions. However, immunoblotting did not show any changes in the protein levels of the catalytic subunit A of mitochondrial DNA polymerase γ, the mitochondrial respiratory chain complexes I, II and IV, ATP synthase, E3 subunit dihydrolipoyl dehydrogenase of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glutathione peroxidase. Our results show that VPA inhibits mitochondrial respiration and leads to mitochondrial dysfunction, oxidative stress and increased cell death, thus suggesting an essential role of mitochondria in VPA-induced hepatotoxicity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  4. Liv.52 protects HepG2 cells from oxidative damage induced by tert-butyl hydroperoxide.

    PubMed

    Vidyashankar, S; K Mitra, S; Nandakumar, Krishna S

    2010-01-01

    Oxidative stress induced by toxicants is known to cause various complications in the liver. Herbal drug such as Liv.52 is found to have hepatoprotective effect. However, the biochemical mechanism involved in the Liv.52 mediated protection against toxicity is not well elucidated using suitable in vitro models. Hence, in the present study, the hepatoprotective effect of Liv.52 against oxidative damage induced by tert-butyl hydroperoxide (t-BHP) in HepG2 cells was evaluated in order to relate in vitro antioxidant activity with cytoprotective effects. Cytotoxicity was measured by MTT assay. Antioxidant effect of Liv.52 was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, ferric-reducing antioxidant power (FRAP) assay, and lipid peroxidation and measurement of non-enzymic and antioxidant enzymes in HepG2 cells exposed to t-BHP over a period of 24 h. The results obtained indicate that t-BHP induced cell damage in HepG2 cells as shown by significant increase in lipid peroxidation as well as decreased levels of reduced glutathione (GSH). Liv.52 significantly decreased toxicity induced by t-BHP in HepG2 cells. Liv.52 was also significantly decreased lipid peroxidation and prevented GSH depletion in HepG2 cells induced by t-BHP. Therefore, Liv.52 appeared to be important for cell survival when exposed to t-BHP. The protective effect of Liv.52 against cell death evoked by t-BHP was probably achieved by preventing intracellular GSH depletion and lipid peroxidation. The results showed protective effect of Liv.52 against oxidative damage induced in HepG2 cells. Hence, taken together, these findings derived from the present study suggest the beneficial effect of Liv.52 in regulating oxidative stress induced in liver by toxicants.

  5. 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

  6. Transferrin-cisplatin specifically deliver cisplatin to HepG2 cells in vitro and enhance cisplatin cytotoxicity.

    PubMed

    Luo, Lian-Zhong; Jin, Hong-Wei; Huang, He-Qing

    2012-12-21

    Cisplatin is a major broad-spectrum chemotherapeutic agent, however, its dose-dependent side effects limit the administration of large doses. Presently, developing a drug targeted delivery system is suggested as one of the most promising approaches to minimize the side effects of cisplatin. Here, we found that each human serum transferrin (HTf) has the potential to bind with over 22 cisplatins, and the complex of apo-HTf-cisplatin can specifically deliver cisplatin to HepG2 cells (human hepatocellular liver carcinoma cell line) in vitro, and facilitate HepG2 cells to apoptosis. Moreover, proteomics methods revealed that the abundances of 23 proteins in HepG2 cells were remarkably altered in response to cisplatin/apo-HTf-cisplatin exposure, and Realtime-PCR revealed that a number of important genes related to chemotherapeutic cytotoxicity and chemotherapeutic resistance are differentially transcribed between the HepG2 cells of cisplatin exposed and HTf-cisplatin exposed. The pathway analysis of the differentially expressed proteins and gene transcriptions indicated that those regulated proteins and gene transcriptions are involved in apoptosis regulation, transcription, cell cycle control, protein biosynthesis, energy metabolism, signal transduction, protein binding and other functions. It indicated that the cisplatin toxicity in HepG2 cell is diverse, the transport process has an effect on the cisplatin cytotoxicity, and the mechanism of the apoptosis of HepG2 cells induced by apo-HTf-cisplatin is different from that of cisplatin. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Inhibitory effect of FSLLRY-NH2 on inflammatory responses induced by hydrogen peroxide in HepG2 cells.

    PubMed

    Lee, Yeon Joo; Kim, Su Jin; Kwon, Kyoung Wan; Lee, Won Mo; Im, Wi Joon; Sohn, Uy Dong

    2017-07-01

    Proteinase activated receptor 2 (PAR2), which is localized in the GI tract, the respiratory system, and the kidney tubules is a G protein-coupled receptor associated with inflammation, metabolism, and disease. The aim of this study was to explore the role of PAR2 in hydrogen peroxide (H2O2)-induced HepG2 cells by using FSLLRY-NH2 a PAR2 antagonist. H2O2 treatment resulted in induction of PAR2 in esophageal, gastric, and liver cells, with the most robust response being in HepG2 cells. Furthermore, this effect was dose-dependent in HepG2 cells. Treatment with H2O2 at concentrations above 400 μM for 24 h also reduced HepG2 cell viability. H2O2 treatment increased both the protein and mRNA levels of IL-1β, IL-8, and TNF-α, as well as those of SAPK/JNK. The increased levels of these pro-inflammatory genes and SAPK/JNK induced by H2O2 were attenuated in a dose-dependent manner when cells were co-treated with H2O2 and FSLLRY-NH2. In summary, the PAR2 antagonist peptide, FSLLRY-NH2, reduces the level of the pro-inflammatory genes IL-8, IL-1β, and TNF-α induced by H2O2, through the SAPK/JNK pathways in HepG2 cells. These data suggest that a PAR2 antagonist could be an anti-inflammatory agent in HepG2 cells.

  8. Serum metabolites of proanthocyanidin-administered rats decrease lipid synthesis in HepG2 cells.

    PubMed

    Guerrero, Ligia; Margalef, Maria; Pons, Zara; Quiñones, Mar; Arola, Lluis; Arola-Arnal, Anna; Muguerza, Begoña

    2013-12-01

    The regular consumption of flavonoids has been associated with reduced mortality and a decreased risk of cardiovascular diseases. The proanthocyanidins found in plasma are very different from the original flavonoids in food sources. The use of physiologically appropriate conjugates of proanthocyanidins is essential for the in vitro analysis of flavonoid bioactivity. In this study, the effect of different proanthocyanidin-rich extracts, which were obtained from cocoa (CCX), French maritime pine bark (Pycnogenol extract, PYC) and grape seed (GSPE), on lipid homeostasis was evaluated. Hepatic human cells (HepG2 cells) were treated with 25 mg/L of CCX, PYC or GSPE. We also performed in vitro experiments to assess the effect on lipid synthesis that is induced by the bioactive GSPE proanthocyanidins using the physiological metabolites that are present in the serum of GSPE-administered rats. For this, Wistar rats were administered 1 g/kg of GSPE, and serum was collected after 2 h. The semipurified serum of GSPE-administered rats was fully characterized by liquid chromatography tandem triple quadrupole mass spectrometry (LC-QqQ/MS(2)). The lipids studied in the analyses were free cholesterol (FC), cholesterol ester (CE) and triglycerides (TG). All three proanthocyanidin-rich extracts induced a remarkable decrease in the de novo lipid synthesis in HepG2 cells. Moreover, GSPE rat serum metabolites reduced the total percentage of CE, FC and particularly TG; this reduction was significantly higher than that observed in the cells directly treated with GSPE. In conclusion, the bioactivity of the physiological metabolites that are present in the serum of rats after their ingestion of a proanthocyanidin-rich extract was demonstrated in Hep G2 cells. © 2013.

  9. 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.

  10. Inflammation response at the transcriptional level of HepG2 cells induced by multi-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Piret, Jean-Pascal; Vankoningsloo, Sébastien; Noël, Florence; Mejia Mendoza, Jorge; Lucas, Stéphane; Saout, Christelle; Toussaint, Olivier

    2011-07-01

    Poor information are currently available about the biological effects of multi-walled carbon nanotubes (MWCNT) on the liver. In this study, we evaluated the effects of MWCNT at the transcriptional level on the classical in vitro model of HepG2 hepatocarcinoma cells. The expression levels of 96 transcript species implicated in the inflammatory and immune responses was studied after a 24h incubation of HepG2 cells in presence of raw MWCNT dispersed in water by stirring. Among the 46 transcript species detected, only a few transcripts including mRNA coding for interleukine-7, chemokines receptor of the C-C families CCR7, as well as Endothelin-1, were statistically more abundant after treatment with MWCNT. Altogether, these data indicate that MWCNT can only induce a weak inflammatory response in HepG2 cells.

  11. Sasa quelpaertensis and p-coumaric acid attenuate oleic acid-induced lipid accumulation in HepG2 cells.

    PubMed

    Kim, Jeong-Hwan; Kang, Seong-Il; Shin, Hye-Sun; Yoon, Seon-A; Kang, Seung-Woo; Ko, Hee-Chul; Kim, Se-Jae

    2013-01-01

    In this study, we examined the effects of Jeju dwarf bamboo (Sasa quelpaertensis Nakai) extract (JBE) and p-coumaric acid (CA) on oleic acid (OA)-induced lipid accumulation in HepG2 cells. JBE and CA increased the phosphorylation of AMP-activated protein kinase (AMPK), and acetyl-CoA carboxylase (ACC) and the expression of carnitine palmitoyl transferase 1a (CPT1a) in OA-treated HepG2 cells. Additionally, these compounds decreased sterol regulatory element-binding protein-1c (SREBP-1c), fatty acid synthase (FAS), and OA-induced lipid accumulation, suggesting that JBE and CA modulate lipid metabolism in HepG2 cells via the AMPK activation pathway.

  12. Comparison of HepG2 feeder cells generated by exposure to gamma-rays, X-rays, UV-C light or mitomycin C for ability to activate 7,12-dimethylbenz[a]anthracene in a cell-mediated Chinese hamster V79/HGPRT mutation assay.

    PubMed

    Schrader, T J

    1999-01-25

    The cell-mediated Chinese hamster V79/HGPRT mutagenicity assay is an established in vitro testing method. Although gamma-irradiated human HepG2 hepatoma cells have been used recently for chemical activation, an alternative is now needed due to scheduled retirement of the available gamma-source. X-irradiation, 254 nm UV-C light and mitomycin C were examined as possible HepG2 mitotic inhibitors, and treated cells compared for activation of 7, 12-dimethylbenz[a]anthracene (DMBA). In colony-forming assays, V79 and HepG2 cells differed in sensitivity to DMBA, with V79 survival declining sharply between 1-2.5 microM (LD50=1.75 microM) while HepG2 survival decreased gradually, beginning at 0.01 microM DMBA (LD50=0.045 microM). When HepG2 feeder cells generated by each method were included in V79/HGPRT mutation assays, activation of 1 microM DMBA was found to vary according to the mitotic inhibitor used, with mutation frequencies decreasing in the order 4000 rads gamma-rays>25 microg/ml mitomycin C>4000 rads X-rays>25 J/m2 UV-C light. Only assays containing gamma-irradiated HepG2 cells generated an increase (2-3-fold) in mutation frequency when DMBA exposure was extended from 24 to 48 h. The effect of HepG2 preincubation with either Aroclor 1254 or DMBA on feeder cell activation of DMBA was also assessed using concentrations of Aroclor 1254 (10 microg/ml) or DMBA (1.0 microM) which were found to produce optimum induction of ethoxyresorufin-O-deethylase (EROD) activity (3.1-fold and 2-fold increases, respectively). Compared to results obtained with uninduced HepG2 cells, assays incorporating HepG2 cells activated by either Aroclor 1254 or DMBA produced slightly increased V79/HGPRT mutation frequencies after 24 h of exposure to mutagen; however, a 48 h incubation with mutagen in the presence of HepG2 preincubated with either Aroclor 1254 or DMBA resulted in higher mutation frequencies regardless of the mitotic inhibitor treatment. EROD activity was also induced 1.4-fold

  13. Inhibition of net HepG2 cell apolipoprotein B secretion by the citrus flavonoid naringenin involves activation of phosphatidylinositol 3-kinase, independent of insulin receptor substrate-1 phosphorylation.

    PubMed

    Borradaile, Nica M; de Dreu, Linda E; Huff, Murray W

    2003-10-01

    The flavonoid naringenin improves hyperlipidemia and hyperglycemia in streptozotocin-treated rats. In HepG2 human hepatoma cells, naringenin inhibits apolipoprotein B (apoB) secretion primarily by inhibiting microsomal triglyceride transfer protein and enhances LDL receptor (LDLr)-mediated apoB-containing lipoprotein uptake. Phosphatidylinositol 3-kinase (PI3K) activation by insulin increases sterol regulatory element-binding protein (SREBP)-1 and LDLr expression and inhibits apoB secretion in hepatocytes. Thus, we determined whether naringenin activates this pathway. Insulin and naringenin induced PI3K-dependent increases in cytosolic and nuclear SREBP-1 and LDLr expression. Similar PI3K-mediated increases in SREBP-1 were observed in McA-RH7777 rat hepatoma cells, which express predominantly SREBP-1c. Reductions in HepG2 cell media apoB with naringenin were partially attenuated by wortmannin, whereas the effect of insulin was completely blocked. Both treatments reduced apoB100 secretion in wild-type and LDLr(-/-) mouse hepatocytes to the same extent. Insulin and naringenin increased HepG2 cell PI3K activity and decreased insulin receptor substrate (IRS)-2 levels. In sharp contrast to insulin, naringenin did not induce tyrosine phosphorylation of IRS-1. We conclude that naringenin increases LDLr expression in HepG2 cells via PI3K-mediated upregulation of SREBP-1, independent of IRS-1 phosphorylation. Although this pathway may not regulate apoB secretion in primary hepatocytes, PI3K activation by this novel mechanism may explain the insulin-like effects of naringenin in vivo.

  14. Dietary catechins and procyanidins modulate zinc homeostasis in human HepG2 cells.

    PubMed

    Quesada, Isabel M; Bustos, Mario; Blay, Mayte; Pujadas, Gerard; Ardèvol, Anna; Salvadó, M Josepa; Bladé, Cinta; Arola, Lluís; Fernández-Larrea, Juan

    2011-02-01

    Catechins and their polymers procyanidins are health-promoting flavonoids found in edible vegetables and fruits. They act as antioxidants by scavenging reactive oxygen species and by chelating the redox-active metals iron and copper. They also behave as signaling molecules, modulating multiple cell signalling pathways and gene expression, including that of antioxidant enzymes. This study aimed at determining whether catechins and procyanidins interact with the redox-inactive metal zinc and at assessing their effect on cellular zinc homeostasis. We found that a grape-seed procyanidin extract (GSPE) and the green tea flavonoid (-)-epigallocatechin-3-gallate (EGCG) bind zinc cations in solution with higher affinity than the zinc-specific chelator Zinquin, and dose-dependently prevent zinc-induced toxicity in the human hepatocarcinoma cell line HepG2, evaluated by the lactate dehydrogenase test. GSPE and EGCG hinder intracellular accumulation of total zinc, measured by atomic flame absorption spectrometry, concomitantly increasing the level of cytoplasmic labile zinc detectable by Zinquin fluorescence. Concurrently, GSPE and EGCG inhibit the expression, evaluated at the mRNA level by quantitative reverse transcriptase-polymerase chain reaction, of zinc-binding metallothioneins and of plasma membrane zinc exporter ZnT1 (SLC30A1), while enhancing the expression of cellular zinc importers ZIP1 (SLC39A1) and ZIP4 (SLC39A4). GSPE and EGCG also produce all these effects when HepG2 cells are stimulated to import zinc by treatment with supplemental zinc or the proinflammatory cytokine interleukin-6. We suggest that extracellular complexation of zinc cations and the elevation of cytoplasmic labile zinc may be relevant mechanisms underlying the modulation of diverse cell signaling and metabolic pathways by catechins and procyanidins.

  15. Altered gene expression in HepG2 cells exposed to a methanolic coal dust extract.

    PubMed

    Guerrero-Castilla, Angelica; Olivero-Verbel, Jesus

    2014-11-01

    Exposure to coal dust has been associated with different chronic diseases and mortality risk. This airborne pollutant is produced during coal mining and transport activities, generating environmental and human toxicity. The aim of this study was to determine the effects of a coal dust methanolic extract on HepG2, a human liver hepatocellular carcinoma cell line. Cells were exposed to 5-100ppm methanolic coal extract for 12h, using DMSO as control. MTT and comet assays were used for the evaluation of cytotoxicity and genotoxicity, respectively. Real time PCR was utilized to quantify relative expression of genes related to oxidative stress, xenobiotic metabolism and DNA damage. Coal extract concentrations did not induce significant changes in HepG2 cell viability after 12h exposure; however, 50 and 100ppm of the coal extract produced a significant increase in genetic damage index with respect to negative control. Compared to vehicle control, mRNA CYP1A1 (up to 163-fold), NQO1 (up to 4.7-fold), and GADD45B (up to 4.7-fold) were up regulated, whereas PRDX1, SOD, CAT, GPX1, XPA, ERCC1 and APEX1 remained unaltered. This expression profile suggests that cells exposed to coal dust extract shows aryl hydrocarbon receptor-mediated alterations, changes in cellular oxidative status, and genotoxic effects. These findings share some similarities with those observed in liver of mice captured near coal mining areas, and add evidence that living around these industrial operations may be negatively impacting the biota and human health.

  16. IRE1α links Nck1 deficiency to attenuated PTP1B expression in HepG2 cells.

    PubMed

    Li, Hui; Li, Bing; Larose, Louise

    2017-08-01

    PTP1B, a prototype of the non-receptor subfamily of the protein tyrosine phosphatase superfamily, plays a key role in regulating intracellular signaling from various receptor and non-receptor protein tyrosine kinases. Previously, we reported that silencing Nck1 in human hepatocellular carcinoma HepG2 cells enhances basal and growth factor-induced activation of the PI3K-Akt pathway through attenuating PTP1B expression. However, the underlying mechanism by which Nck1 depletion represses PTP1B expression remains unclear. In this study, we found that silencing Nck1 attenuates PTP1B expression in HepG2 cells through down-regulation of IRE1α. Indeed, we show that silencing Nck1 in HepG2 cells leads to decreased IRE1α expression and signaling. Accordingly, IRE1α depletion using siRNA in HepG2 cells enhances PI3K-dependent basal and growth factor-induced Akt activation, reproducing the effects of silencing Nck1 on activation of this pathway. In addition, depletion of IRE1α also leads to reduced PTP1B expression, which was rescued by ectopic expression of IRE1α in Nck1-depleted cells. Mechanistically, we found that silencing either Nck1 or IRE1α in HepG2 cells decreases PTP1B mRNA levels and stability. However, despite miR-122 levels, a miRNA targeting PTP1B 3' UTR and inducing PTP1B mRNA degradation in HepG2 cells, are increased in both Nck1- and IRE1α-depleted HepG2 cells, a miR-122 antagomir did not rescue PTP1B expression in these cells. Overall, this study highlights an important role for Nck1 in fine-tuning IRE1α expression and signaling that regulate PTP1B expression and subsequent activation of the PI3K-Akt pathway in HepG2 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Synergistic complex from plants Solanaceae exhibits cytotoxicity for the human hepatocellular carcinoma cell line HepG2.

    PubMed

    Schwarzlin, Romina; Pušenjak, Nika; Makuc, Damjan; Križman, Mitja; Vovk, Irena; Plavec, Janez; Švajger, Urban

    2016-10-18

    It had been demonstrated that sugars from various plants can act as potent agents, which induce apoptosis of cancer cells. Using HPLC, we fractionated a mixture of two plant extracts from the plant family Solanaceae, namely Capsicum chinense and the plant family Amaryllidaceae namely Allium sativum. We evaluated the effect of different fractions on apoptosis of HepG2 cell line. The most effective fraction was further studied to determine its molecular composition using mass spectrometry (MS) and NMR. We further evaluated the effect of determined molecular composition found in the selected fraction by using a mixture of commercially available substances, which were found in the fraction and tested its pro-apoptotic effect on HepG2 cells. To get some insight into potential apoptotic mechanisms we studied caspase-3 activity and mitochondrial integrity in treated cells. Out of 93 fractions obtained by HPLC from the plant extract we found HPLC fraction 10 (10 min elution) was the most effective. MS and NMR studies revealed high presence of cellobiose together with vitamin C, sulphur (S) and trace amounts of selenium (Se). HPLC fraction 10 triggered apoptosis of HepG2 within 3 h in the 0.01-1.0 mg/mL concentration range. Furthermore, a mixture of pure cellobiose, vitamin C, S and Se (complex cellobiose/C/S/Se) had a very similar capacity in inducing apoptosis of HepG2 cells compared to HPLC fraction 10. Complex cellobiose/C/S/Se was capable of inducing caspase-3 activity and led to loss of mitochondrial integrity. The capacity of cellobiose alone to induce apoptosis of HepG2 was approximately 1000-fold lower compared to complex cellobiose/C/S/Se. In this study we present the highly synergistic effect of a unique complex consisting of cellobiose, vitamin C, sulphur and selenium on triggering the apoptosis of human hepatocellular carcinoma (HepG2) cell line.

  18. Cisplatin combined with hyperthermia kills HepG2 cells in intraoperative blood salvage but preserves the function of erythrocytes.

    PubMed

    Yang, Jin-ting; Tang, Li-hui; Liu, Yun-qing; Wang, Yin; Wang, Lie-ju; Zhang, Feng-jiang; Yan, Min

    2015-05-01

    The safe use of intraoperative blood salvage (IBS) in cancer surgery remains controversial. Here, we investigated the killing effect of cisplatin combined with hyperthermia on human hepatocarcinoma (HepG2) cells and erythrocytes from IBS in vitro. HepG2 cells were mixed with concentrated erythrocytes and pretreated with cisplatin (50, 100, and 200 μg/ml) alone at 37 °C for 60 min and cisplatin (25, 50, 100, and 200 μg/ml) combined with hyperthermia at 42 °C for 60 min. After pretreatment, the cell viability, colony formation and DNA metabolism in HepG2 and the Na(+)-K(+)-ATPase activity, 2,3-diphosphoglycerate (2,3-DPG) concentration, free hemoglobin (Hb) level, osmotic fragility, membrane phosphatidylserine externalization, and blood gas variables in erythrocytes were determined. Pretreatment with cisplatin (50, 100, and 200 μg/ml) combined with hyperthermia (42 °C) for 60 min significantly decreased HepG2 cell viability, and completely inhibited colony formation and DNA metabolism when the HepG2 cell concentration was 5×10(4) ml(-1) in the erythrocyte (P<0.01). Erythrocytic Na(+)-K(+)-ATPase activity, 2,3-DPG level, phosphatidylserine externalization, and extra-erythrocytic free Hb were significantly altered by hyperthermia plus high concentrations of cisplatin (100 and 200 μg/ml) (P<0.05), but not by hyperthermia plus 50 μg/ml cisplatin (P>0.05). In conclusion, pretreatment with cisplatin (50 μg/ml) combined with hyperthermia (42 °C) for 60 min effectively eliminated HepG2 cells from IBS but did not significantly affect erythrocytes in vitro.

  19. Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells

    PubMed Central

    Hamad, Ibrahim A. Y.; Fei, Yue; Kalea, Anastasia Z.; Yin, Dan; Smith, Andrew J. P.; Palmen, Jutta; Humphries, Steve E.; Talmud, Philippa J.; Walker, Ann P.

    2015-01-01

    MicroRNA 122 (miR-122) is highly expressed in the liver where it influences diverse biological processes and pathways, including hepatitis C virus replication and metabolism of iron and cholesterol. It is processed from a long non-coding primary transcript (~7.5 kb) and the gene has two evolutionarily-conserved regions containing the pri-mir-122 promoter and pre-mir-122 hairpin region. Several groups reported that the widely-used hepatocytic cell line HepG2 had deficient expression of miR-122, previously ascribed to deletion of the pre-mir-122 stem-loop region. We aimed to characterise this deletion by direct sequencing of 6078 bp containing the pri-mir-122 promoter and pre-mir-122 stem-loop region in HepG2 and Huh-7, a control hepatocytic cell line reported to express miR-122, supported by sequence analysis of cloned genomic DNA. In contrast to previous findings, the entire sequence was present in both cell lines. Ten SNPs were heterozygous in HepG2 indicating that DNA was present in two copies. Three validation isolates of HepG2 were sequenced, showing identical genotype to the original in two, whereas the third was different. Investigation of promoter chromatin status by FAIRE showed that Huh-7 cells had 6.2 ± 0.19- and 2.7 ± 0.01- fold more accessible chromatin at the proximal (HNF4α-binding) and distal DR1 transcription factor sites, compared to HepG2 cells (p=0.03 and 0.001, respectively). This was substantiated by ENCODE genome annotations, which showed a DNAse I hypersensitive site in the pri-mir-122 promoter in Huh-7 that was absent in HepG2 cells. While the origin of the reported deletion is unclear, cell lines should be obtained from a reputable source and used at low passage number to avoid discrepant results. Deficiency of miR-122 expression in HepG2 cells may be related to a relative deficiency of accessible promoter chromatin in HepG2 versus Huh-7 cells. PMID:25811611

  20. 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

  1. SiC nanoparticles cyto- and genotoxicity to Hep-G2 cells

    NASA Astrophysics Data System (ADS)

    Barillet, Sabrina; Jugan, Mary-Line; Simon-Deckers, Angélique; Leconte, Yann; Herlin-Boime, Nathalie; Mayne-l'Hermite, Martine; Reynaud, Cécile; Carrière, Marie

    2009-05-01

    While emerging nanotechnologies have seen significant development in recent years, knowledge on exposure levels as well as data on toxicity of nanoparticles are still quite limited. Indeed, there is a general agreement that development of nanotechnologies may lead to considerable dissemination of nanoparticles in the environment. Nevertheless, questions relative to toxicity versus innocuousness of such materials still remain. Our present study has thus been carried out with the purpose of assessing some aspects of toxicological capacities of three kinds of nano-sized particles: TiO2 and SiC nanoparticles, as well as multi-walled carbon nanotubes (CNT). In order to address the question of their potential toxicity toward living cells, we chose several cellular models. Assuming inhalation as the most probable exposure scenario, we used A549 alveolar epithelial cells as a model for mammalian primary target organ (lung). Furthermore, we considered that nanoparticles that would deposit into the pulmonary system may be translocated to the circulatory system. Thus, we decided to study the effect of nanoparticles on potentially secondary target organs: liver (WIF-B9, Can-10, HepG2) and kidneys (NRK-52E, LLC-PK1). Herein, we will focus our attention on results obtained on the HepG2 cell line exposed to SiC nanoparticles. Scarce literature exists on SiC nanotoxicology. According to the authors that have already carried out studies on this particular nanoparticle, it would seem that SiC nanoparticles do not induce cytotoxicity. That is one of the reasons of the potential use of these nanoparticles as biological labels [1]. We thus were interested in acquiring more data on biological effects induced by SiC nanoparticles. Furthermore, one of the particular aspects of the present study lies in the fact that we tried to specify the influence of physico-chemical characteristics of nanoparticles on toxicological endpoints (cytotoxicity and genotoxicity).

  2. Prooxidant and antioxidant properties of salicylaldehyde isonicotinoyl hydrazone iron chelators in HepG2 cells

    PubMed Central

    Caro, Andres A.; Commissariat, Ava; Dunn, Caroline; Kim, Hyunjoo; García, Salvador Lorente; Smith, Allen; Strang, Harrison; Stuppy, Jake; Desrochers, Linda P.; Goodwin, Thomas E.

    2015-01-01

    Background Salicylaldehyde isonicotinoyl hydrazone (SIH) is an iron chelator of the aroylhydrazone class that displays antioxidant or prooxidant effects in different mammalian cell lines. Because the liver is the major site of iron storage, elucidating the effect of SIH on hepatic oxidative metabolism is critical for designing effective hepatic antioxidant therapies. Methods Hepatocyte-like HepG2 cells were exposed to SIH or to analogs showing greater stability, such as N′-[1-(2-Hydroxyphenyl)ethyliden]isonicotinoyl hydrazide (HAPI), or devoid of iron chelating properties, such as benzaldehyde isonicotinoyl hydrazone (BIH), and toxicity, oxidative stress and antioxidant (glutathione) metabolism were evaluated. Results Autoxidation of Fe2+ in vitro increased in the presence of SIH or HAPI (but not BIH), an effect partially blocked by Fe2+ chelation. Incubation of HepG2 cells with SIH or HAPI (but not BIH) was non-toxic and increased reactive oxygen species (ROS) levels, activated the transcription factor Nrf2, induced the catalytic subunit of γ-glutamate cysteine ligase (Gclc), and increased glutathione concentration. Fe2+ chelation decreased ROS and inhibited Nrf2 activation, and Nrf2 knock-down inhibited the induction of Gclc in the presence of HAPI. Inhibition of γ-glutamate cysteine ligase enzymatic activity inhibited the increase in glutathione caused by HAPI, and increased oxidative stress. Conclusions SIH iron chelators display both prooxidant (increasing the autoxidation rate of Fe2+) and antioxidant (activating Nrf2 signaling) effects. General significance Activation by SIH iron chelators of a hormetic antioxidant response contributes to its antioxidant properties and modulates the anti- and pro-oxidant balance. PMID:26275495

  3. Cyclosporine A and palmitic acid treatment synergistically induce cytotoxicity in HepG2 cells.

    PubMed

    Luo, Yi; Rana, Payal; Will, Yvonne

    2012-06-01

    Immunosuppressant cyclosporine A (CsA) treatment can cause severe side effects. Patients taking immunosuppressant after organ transplantation often display hyperlipidemia and obesity. Elevated levels of free fatty acids have been linked to the etiology of metabolic syndromes, nonalcoholic fatty liver and steatohepatitis. The contribution of free fatty acids to CsA-induced toxicity is not known. In this study we explored the effect of palmitic acid on CsA-induced toxicity in HepG2 cells. CsA by itself at therapeutic exposure levels did not induce detectible cytotoxicity in HepG2 cells. Co-treatment of palmitic acid and CsA resulted in a dose dependent increase in cytotoxicity, suggesting that fatty acid could sensitize cells to CsA-induced cytotoxicity at the therapeutic doses of CsA. A synergized induction of caspase-3/7 activity was also observed, indicating that apoptosis may contribute to the cytotoxicity. We demonstrated that CsA reduced cellular oxygen consumption which was further exacerbated by palmitic acid, implicating that impaired mitochondrial respiration might be an underlying mechanism for the enhanced toxicity. Inhibition of c-Jun N-terminal kinase (JNK) attenuated palmitic acid and CsA induced toxicity, suggesting that JNK activation plays an important role in mediating the enhanced palmitic acid/CsA-induced toxicity. Our data suggest that elevated FFA levels, especially saturated FFA such as palmitic acid, may be predisposing factors for CsA toxicity, and patients with underlying diseases that would elevate free fatty acids may be susceptible to CsA-induced toxicity. Furthermore, hyperlipidemia/obesity resulting from immunosuppressive therapy may aggravate CsA-induced toxicity and worsen the outcome in transplant patients.

  4. Cytotoxicity of thiazolidinedione-, oxazolidinedione- and pyrrolidinedione-ring containing compounds in HepG2 cells.

    PubMed

    Keil, Alyssa M; Frederick, Douglas M; Jacinto, Erina Y; Kennedy, Erica L; Zauhar, Randy J; West, Nathan M; Tchao, Ruy; Harvison, Peter J

    2015-10-01

    Liver damage occurred in some patients who took troglitazone (TGZ) for type II diabetes. The 2,4-thiazolidinedione (TZD) ring in TGZ's structure has been implicated in its hepatotoxicity. To further examine the potential role of a TZD ring in toxicity we used HepG2 cells to evaluate two series of compounds containing different cyclic imides. N-phenyl analogues comprised 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT); 3-(3,5-dichlorophenyl)-2,4-oxazolidinedione (DCPO) and N-(3,5-dichlorophenyl)succinimide (NDPS). Benzylic compounds, which closely resemble TGZ, included 5-(3,5-dichlorophenylmethyl)-2,4-thiazolidinedione (DCPMT); 5-(4-methoxyphenylmethyl)-2,4-thiazolidinedione (MPMT); 5-(4-methoxyphenylmethylene)-2,4-thiazolidinedione (MPMT-I); 5-(4-methoxyphenylmethyl)-2,4-oxazolidinedione (MPMO); 3-(4-methoxyphenylmethyl)succinimide (MPMS) and 3-(4-methoxyphenylmethylene)succinimide (MPMS-I). Cytotoxicity was assessed using the MTS assay after incubating the compounds (0-250μM) with HepG2 cells for 24h. Only certain TZD derivatives (TGZ, DCPT, DCPMT and MPMT-I) markedly decreased cell viability, whereas MPMT had low toxicity. In contrast, analogues without a TZD ring (DCPO, NDPS, MPMO, MPMS and MPMS-I) were not cytotoxic. These findings suggest that a TZD ring may be an important determinant of toxicity, although different structural features, chemical stability, cellular uptake or metabolism, etc., may also be involved. A simple clustering approach, using chemical fingerprints, assigned each compound to one of three classes (each containing one active compound and close homologues), and provided a framework for rationalizing the activity in terms of structure.

  5. Chemically induced hepatotoxicity in human stem cell-induced hepatocytes compared with primary hepatocytes and HepG2.

    PubMed

    Kang, Seok-Jin; Lee, Hyuk-Mi; Park, Young-Il; Yi, Hee; Lee, Hunjoo; So, ByungJae; Song, Jae-Young; Kang, Hwan-Goo

    2016-10-01

    Stem cell-induced hepatocytes (SC-iHeps) have been suggested as a valuable model for evaluating drug toxicology. Here, human-induced pluripotent stem cells (QIA7) and embryonic stem cells (WA01) were differentiated into hepatocytes, and the hepatotoxic effects of acetaminophen (AAP) and aflatoxin B1 (AFB1) were compared with primary hepatocytes (p-Heps) and HepG2. In a cytotoxicity assay, the IC50 of SC-iHeps was similar to that in p-Heps and HepG2 in the AAP groups but different from that in p-Heps of the AFB1 groups. In a multi-parameter assay, phenotypic changes in mitochondrial membrane potential, calcium influx and oxidative stress were similar between QIA7-iHeps and p-Heps following AAP and AFB1 treatment but relatively low in WA01-iHeps and HepG2. Most hepatic functional markers (hepatocyte-specific genes, albumin/urea secretion, and the CYP450 enzyme activity) were decreased in a dose-dependent manner following AAP and AFB1 treatment in SC-iHeps and p-Heps but not in HepG2. Regarding CYP450 inhibition, the cell viability of SC-iHeps and p-Heps was increased by ketoconazole, a CYP3A4 inhibitor. Collectively, SC-iHeps and p-Heps showed similar cytotoxicity and hepatocyte functional effects for AAP and AFB1 compared with HepG2. Therefore, SC-iHeps have phenotypic characteristics and sensitivity to cytotoxic chemicals that are more similar to p-Heps than to HepG2 cells.

  6. Impairment of oxidative phosphorylation increases the toxicity of SYD-1 on hepatocarcinoma cells (HepG2).

    PubMed

    Brandt, Anna Paula; Gozzi, Gustavo Jabor; Pires, Amanda do Rocio Andrade; Martinez, Glaucia Regina; Dos Santos Canuto, André Vinícius; Echevarria, Aurea; Di Pietro, Attilio; Cadena, Sílvia Maria Suter Correia

    2016-08-25

    Toxicity of the SYD-1 mesoionic compound (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) was evaluated on human liver cancer cells (HepG2) grown in either high glucose (HG) or galactose (GAL) medium, and also on suspended cells kept in HG medium. SYD-1 was able to decrease the viability of cultured HepG2 cells in a dose-dependent manner, as assessed by MTT, LDH release and dye with crystal violet assays, but no effect was observed on suspended cells after 1-40 min of treatment. Respiration analysis was performed after 2 min (suspended cells) or 24 h (cultured cells) of treatment: no change was observed in suspended cells, whereas SYD-1 inhibited as well basal, leak and uncoupled states of the respiration in cultured cells with HG medium. These inhibitions were consistent with the decrease in pyruvate level and increase in lactate level. Even more extended results were obtained with HepG2 cells grown in GAL medium where, additionally, the ATP amount was reduced. Furthermore, SYD-1 appears not to be transported by the main ABC multidrug transporters. These results show that SYD-1 is able to change the metabolism of HepG2 cells, and suggest that its cytotoxicity is related to impairment of mitochondrial metabolism. Therefore, we may propose that SYD-1 is a potential candidate for hepatocarcinoma treatment. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. Classification of hepatotoxicants using HepG2 cells: A proof of principle study.

    PubMed

    Van den Hof, Wim F P M; Coonen, Maarten L J; van Herwijnen, Marcel; Brauers, Karen; Wodzig, Will K W H; van Delft, Joost H M; Kleinjans, Jos C S

    2014-03-17

    With the number of new drug candidates increasing every year, there is a need for high-throughput human toxicity screenings. As the liver is the most important organ in drug metabolism and thus capable of generating relatively high levels of toxic metabolites, it is important to find a reliable strategy to screen for drug-induced hepatotoxicity. Microarray-based transcriptomics is a well-established technique in toxicogenomics research and is an ideal approach to screen for drug-induced injury at an early stage. The aim of this study was to prove the principle of classifying known hepatotoxicants and nonhepatotoxicants using their distinctive gene expression profiles in vitro in HepG2 cells. Furthermore, we undertook to subclassify the hepatotoxic compounds by investigating the subclass of cholestatic compounds. Prediction analysis for microarrays was used for classification of hepatotoxicants and nonhepatotoxicants, which resulted in an accuracy of 92% on the training set and 91% on the validation set, using 36 genes. A second model was set up with the goal of finding classifiers for cholestasis, resulting in 12 genes that appeared capable of correctly classifying 8 of the 9 cholestatic compounds, resulting in an accuracy of 93%. We were able to prove the principle that transcriptomic analyses of HepG2 cells can indeed be used to classify chemical entities for hepatotoxicity. Genes selected for classification of hepatotoxicity and cholestasis indicate that endoplasmic reticulum stress and the unfolded protein response may be important cellular effects of drug-induced liver injury. However, the number of compounds in both the training set and the validation set should be increased to improve the reliability of the prediction.

  8. [Apoptosis and activity changes of telomerase induced by essential oil from pine needles in HepG2 cell line].

    PubMed

    Wei, Feng-xiang; Li, Mei-yu; Song, Yu-hong; Li, Hong-zhi

    2008-08-01

    To study the effects of essential oil extracted from pine needles on HepG2 cell line. HepG2 cells were treated with essential oil extracted from pine needles. Cell growth rate was determined with MTF assay, cell morphologic changes were examined under transmission electromicroscope and HE straining. Flow cytometry was used to exmine apoptotic cells. Bcl-2 gene expression was determined by flow cytometry and telomerase activity by TRAP assay. Essential oils from pine needles could not only repress the growth of HepG2 cells significantly, but also induce apoptosis to them. Both dose-effect and time-effect relationship could be confirmed. Typical morphology changes of apoptosis such as nuclear enrichment and karyorrhexis were observed through transmission electromicroscope and HE straining. Telomerase activity was down regulated in the essential oil extracted from pine needles induced apoptotic cells. The expression of bcl-2 gene was suppressed after the essential oil from pine needles treatement. The essential oil extracted from pine needles can inhibit cell growth of HepG2 cell line and induce apoptosis, which may associate with inhibition of telomerase activity and bcl-2 may be involved in the regulation of telomerase activity.

  9. Effect of copper overload on the survival of HepG2 and A-549 human-derived cells.

    PubMed

    Arnal, N; de Alaniz, M J T; Marra, C A

    2013-03-01

    We investigated the effect of copper (Cu) overload (20-160 µM/24 h) in two cell lines of human hepatic (HepG2) and pulmonary (A-549) origin by determining lipid and protein damage and the response of the antioxidant defence system. A-549 cells were more sensitive to Cu overload than HepG2 cells. A marked increase was observed in both the cell lines in the nitrate plus nitrite concentration, protein carbonyls and thiobarbituric acid reactive substances (TBARS). The TBARS increase was consistent with an increment in saturated fatty acids at the expense of polyunsaturated acids in a Cu concentration-dependent fashion. Antioxidant enzymes were stimulated by Cu overload. Superoxide dismutase activity increased significantly in both the cell lines, with greater increases in HepG2 than in A-549 cells. A marked increase in ceruloplasmin and metallothionein content in both the cell types was also observed. Dose-dependent decreases in α-tocopherol and ferric reducing ability were observed. Total glutathione content was lower in A-549 cells and higher in HepG2. Calpain and caspase-3 were differentially activated in a dose-dependent manner under copper-induced reactive oxygen species production. We conclude that Cu exposure of human lung- and liver-derived cells should be considered a reliable experimental system for detailed study of mechanism/mechanisms by which Cu overload exerts its deleterious effects.

  10. 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

  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. Flavonoids of Korean Citrus aurantium L. Induce Apoptosis via Intrinsic Pathway in Human Hepatoblastoma HepG2 Cells.

    PubMed

    Lee, Seung Hwan; Yumnam, Silvia; Hong, Gyeong Eun; Raha, Suchismita; Saralamma, Venu Venkatarame Gowda; Lee, Ho Jeong; Heo, Jeong Doo; Lee, Sang Joon; Lee, Won-Sup; Kim, Eun-Hee; Park, Hyeon Soo; Kim, Gon Sup

    2015-12-01

    Korean Citrus aurantium L. has long been used as a medicinal herb for its anti-inflammatory, antioxidant, and anticancer properties. The present study investigates the anticancer role of flavonoids extracted from C. aurantium on human hepatoblastoma cell, HepG2. The Citrus flavonoids inhibit the proliferation of HepG2 cells in a dose-dependent manner. This result was consistent with the in vivo xenograft results. Apoptosis was detected by cell morphology, cell cycle analysis, and immunoblot. Flavonoids decreased the level of pAkt and other downstream targets of phosphoinositide-3-kinase/Akt pathway - P-4EBP1 and P-p70S6K. The expressions of cleaved caspase 3, Bax, and Bak were increased, while those of Bcl-2 and Bcl-xL were decreased with an increase in the expression of Bax/Bcl-xL ratio in treated cells. Loss of mitochondrial membrane potential was also observed in flavonoid-treated HepG2 cells. It was also observed that the P-p38 protein level was increased both dose and time dependently in flavonoid-treated cells. Collectively, these results suggest that flavonoid extracted from Citrus inhibits HepG2 cell proliferation by inducing apoptosis via an intrinsic pathway. These findings suggest that flavonoids extracted from C. aurantium L. are potential chemotherapeutic agents against liver cancer.

  14. Inferring Toxicological Responses of HepG2 Cells from ToxCast High Content Imaging Data (SOT)

    EPA Science Inventory

    Understanding the dynamic perturbation of cell states by chemicals can aid in for predicting their adverse effects. High-content imaging (HCI) was used to measure the state of HepG2 cells over three time points (1, 24, and 72 h) in response to 976 ToxCast chemicals for 10 differe...

  15. Data on HepG2 cells changes following exposure to cadmium sulphide quantum dots (CdS QDs).

    PubMed

    Paesano, Laura; Perotti, Alessio; Buschini, Annamaria; Carubbi, Cecilia; Marmiroli, Marta; Maestri, Elena; Iannotta, Salvatore; Marmiroli, Nelson

    2017-04-01

    The data included in this paper are associated with the research article entitled "Markers for toxicity to HepG2 exposed to cadmium sulphide quantum dots; damage to mitochondria" (Paesano et al.) [1]. The article concerns the cytotoxic and genotoxic effects of CdS QDs in HepG2 cells and the mechanisms involved. In this dataset, changes in expression levels of candidate genes are reported, together with details concerning synthesis and properties of CdS QDs, additional information obtained through literature survey, measures of the mitochondrial membrane potential and the glutathione redox state.

  16. [The expression of AQP9 in HepG2 cells affects cell biological behaviors and sensitivity to As2O3].

    PubMed

    Tang, Jie; Wang, Chuan; Jiang, Zheng

    2015-06-01

    To investigate the effect of aquaporin 9 (AQP9) expression level on the apoptosis and biological behaviors of HepG2 cells induced by arsenic trioxide (As2O3). The effects of different concentration As2O3 on the cell proliferation were measured by MTT assay, and IC50 was calculated. The recombinant plasmids pEGFP-N1-AQP9 and pshRNA-AQP9 were transfected into HepG2 cells. The expression of AQP9 mRNA and protein were detected by reverse transcription PCR and Western blotting, respectively. Then As2O3 was added to plasmid-transfected cells and untreated HepG2 cells. Cell proliferation was detected by MTT assay, cell cycle and apoptosis rate were determined by flow cytometry, and cell invasion and migration ability were examined by Transwell(TM) (Boyden Chamber) assay. The activity of caspase-3 was detected by microplate reader. The recombinant plasmids remarkably influenced the expression of AQP9. Compared with the group treated with As2O3 alone (control group), the cell proliferation, invasion and migration of HepG2 cells transfected with pEGFP-N1-AQP9 were attenuated significantly, while the cells transfected with pshRNA-AQP9 increased. The increasing population of apoptotic cells, augmented caspase 3 activity and highest percentage of cells stagnating at G0/G1 phase were observed in HepG2 cells transfected with pEGFP-N1-AQP9 as compared with the control HepG2 cells; in contrast, the HepG2 cells transfected with pshRNA-AQP9 presented with lower caspase 3 activity and stronger invasion and migration ability than the control HepG2 cells did. The alterations in the expression of AQP9 could affect the biological behaviors of HepG2 cells, but also their sensitivity to As2O3.

  17. Solanine-induced reactive oxygen species inhibit the growth of human hepatocellular carcinoma HepG2 cells

    PubMed Central

    MENG, XUE-QIN; ZHANG, WEI; ZHANG, FENG; YIN, SHENG-YONG; XIE, HAI-YANG; ZHOU, LIN; ZHENG, SHU-SEN

    2016-01-01

    The aim of the present study was to investigate the effect of solanine on promoting human hepatocellular carcinoma HepG2 cells to produce reactive oxygen species (ROS), and the molecular mechanisms leading to tumor cell apoptosis. Solanine was administered to HepG2 cells in vitro. A selection of probes targeting various cellular localizations of ROS were used to detect ROS expression using flow cytometry. The expression levels of apoptosis-associated proteins, including apoptosis signal-regulating kinase 1 (ASK1) and thioredoxin binding protein 2 (TBP-2), and proliferation-associated proteins, including histone deacetylase 1 (HDAC1), were detected using western blotting. The percentage of cells undergoing apoptosis was measured using an Annexin V-fluorescein isothiocyanate/propidium iodide assay, and cell morphology was examined using Wright's stain followed by inverted microscopy analysis. ROS detection probes 2′,7′-dichlorofluorescin diacetate and dihydrorhodamine 123 identified that abundant ROS, including hydroxyl radical (OH−) and hydrogen peroxide (H2O2), were produced in the cytoplasm and mitochondria of the solanine-treated HepG2 cells compared with the control cells (P<0.05). Superoxide anion specific probes dihydroethidium and MitoSOX™ demonstrated that there were no significant alterations in the HepG2 cells following solanine treatment compared with the control cells (P>0.05). Western blotting results revealed that solanine upregulated the expression levels of ASK1 and TBP-2 and enhanced their kinase activities, whereas solanine decreased the expression level of the proliferation-associated protein, HDAC1. The cell apoptotic rate was significantly increased (P<0.0001) in the solanine-treated HepG2 cells compared with the control cells. (P<0.05). Overall, the study indicated that solanine induces HepG2 cells to produce ROS, mainly OH− and H2O2, in a mitochondria-dependent and -independent manner. In addition, solanine stimulates the expression

  18. Solanine-induced reactive oxygen species inhibit the growth of human hepatocellular carcinoma HepG2 cells.

    PubMed

    Meng, Xue-Qin; Zhang, Wei; Zhang, Feng; Yin, Sheng-Yong; Xie, Hai-Yang; Zhou, Lin; Zheng, Shu-Sen

    2016-03-01

    The aim of the present study was to investigate the effect of solanine on promoting human hepatocellular carcinoma HepG2 cells to produce reactive oxygen species (ROS), and the molecular mechanisms leading to tumor cell apoptosis. Solanine was administered to HepG2 cells in vitro. A selection of probes targeting various cellular localizations of ROS were used to detect ROS expression using flow cytometry. The expression levels of apoptosis-associated proteins, including apoptosis signal-regulating kinase 1 (ASK1) and thioredoxin binding protein 2 (TBP-2), and proliferation-associated proteins, including histone deacetylase 1 (HDAC1), were detected using western blotting. The percentage of cells undergoing apoptosis was measured using an Annexin V-fluorescein isothiocyanate/propidium iodide assay, and cell morphology was examined using Wright's stain followed by inverted microscopy analysis. ROS detection probes 2',7'-dichlorofluorescin diacetate and dihydrorhodamine 123 identified that abundant ROS, including hydroxyl radical (OH(-)) and hydrogen peroxide (H2O2), were produced in the cytoplasm and mitochondria of the solanine-treated HepG2 cells compared with the control cells (P<0.05). Superoxide anion specific probes dihydroethidium and MitoSOX™ demonstrated that there were no significant alterations in the HepG2 cells following solanine treatment compared with the control cells (P>0.05). Western blotting results revealed that solanine upregulated the expression levels of ASK1 and TBP-2 and enhanced their kinase activities, whereas solanine decreased the expression level of the proliferation-associated protein, HDAC1. The cell apoptotic rate was significantly increased (P<0.0001) in the solanine-treated HepG2 cells compared with the control cells. (P<0.05). Overall, the study indicated that solanine induces HepG2 cells to produce ROS, mainly OH(-) and H2O2, in a mitochondria-dependent and -independent manner. In addition, solanine stimulates the expression of

  19. Garcinia dulcis Fruit Extract Induced Cytotoxicity and Apoptosis in HepG2 Liver Cancer Cell Line

    PubMed Central

    Abu Bakar, Mohd Fadzelly; Ahmad, Nor Ezani; Suleiman, Monica; Rahmat, Asmah; Isha, Azizul

    2015-01-01

    Garcinia dulcis or locally known in Malaysia as “mundu” belongs to the family of Clusiaceae. The study was conducted to investigate the anticancer potential of different parts of G. dulcis fruit extracts and their possible mechanism of action in HepG2 liver cancer cell line. MTT assay showed that the peel, flesh, and seed extracts of G. dulcis induced cytotoxicity in HepG2 cell line with IC50 values of 46.33 ± 4.51, 38.33 ± 3.51, and 7.5 ± 2.52 µg/mL, respectively. The flesh extract of G. dulcis induced cell cycle arrest at sub-G1 (apoptosis) phase in a time-dependent manner. Staining with Annexin V-FITC and propidium iodide showed that 41.2% of the cell population underwent apoptosis after 72 hours of exposure of the HepG2 cell line to G. dulcis flesh extract. Caspase-3 has been shown to be activated which finally leads to the death of HepG2 cell (apoptosis). GC-MS analysis showed that the highest percentage of compound identified in the extract of G. dulcis flesh was hydroxymethylfurfural and 3-methyl-2,5-furandione, together with xanthones and flavonoids (based on literature), could synergistically contribute to the observed effects. This finding suggested that the flesh extract of G. dulcis has its own potential as cancer chemotherapeutic agent against liver cancer cell. PMID:26557713

  20. Polyethylenimine-functionalized silver nanoparticle-based co-delivery of paclitaxel to induce HepG2 cell apoptosis.

    PubMed

    Li, Yinghua; Guo, Min; Lin, Zhengfang; Zhao, Mingqi; Xiao, Misi; Wang, Changbing; Xu, Tiantian; Chen, Tianfeng; Zhu, Bing

    Hepatocarcinoma is the third leading cause of cancer-related deaths around the world. Recently, a novel emerging nanosystem as anticancer therapeutic agents with intrinsic therapeutic properties has been widely used in various medical applications. In this study, surface decoration of functionalized silver nanoparticles (AgNPs) by polyethylenimine (PEI) and paclitaxel (PTX) was synthesized. The purpose of this study was to evaluate the effect of Ag@ PEI@PTX on cytotoxic and anticancer mechanism on HepG2 cells. The transmission electron microscope image and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that Ag@PEI@PTX had satisfactory size distribution and high stability and selectivity between cancer and normal cells. Ag@PEI@PTX-induced HepG2 cell apoptosis was confirmed by accumulation of the sub-G1 cells population, translocation of phosphatidylserine, depletion of mitochondrial membrane potential, DNA fragmentation, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. Furthermore, Ag@PEI@PTX enhanced cytotoxic effects on HepG2 cells and triggered intracellular reactive oxygen species; the signaling pathways of AKT, p53, and MAPK were activated to advance cell apoptosis. In conclusion, the results reveal that Ag@ PEI@PTX may provide useful information on Ag@PEI@PTX-induced HepG2 cell apoptosis and as appropriate candidate for chemotherapy of cancer.

  1. Garcinia dulcis Fruit Extract Induced Cytotoxicity and Apoptosis in HepG2 Liver Cancer Cell Line.

    PubMed

    Abu Bakar, Mohd Fadzelly; Ahmad, Nor Ezani; Suleiman, Monica; Rahmat, Asmah; Isha, Azizul

    2015-01-01

    Garcinia dulcis or locally known in Malaysia as "mundu" belongs to the family of Clusiaceae. The study was conducted to investigate the anticancer potential of different parts of G. dulcis fruit extracts and their possible mechanism of action in HepG2 liver cancer cell line. MTT assay showed that the peel, flesh, and seed extracts of G. dulcis induced cytotoxicity in HepG2 cell line with IC50 values of 46.33 ± 4.51, 38.33 ± 3.51, and 7.5 ± 2.52 µg/mL, respectively. The flesh extract of G. dulcis induced cell cycle arrest at sub-G1 (apoptosis) phase in a time-dependent manner. Staining with Annexin V-FITC and propidium iodide showed that 41.2% of the cell population underwent apoptosis after 72 hours of exposure of the HepG2 cell line to G. dulcis flesh extract. Caspase-3 has been shown to be activated which finally leads to the death of HepG2 cell (apoptosis). GC-MS analysis showed that the highest percentage of compound identified in the extract of G. dulcis flesh was hydroxymethylfurfural and 3-methyl-2,5-furandione, together with xanthones and flavonoids (based on literature), could synergistically contribute to the observed effects. This finding suggested that the flesh extract of G. dulcis has its own potential as cancer chemotherapeutic agent against liver cancer cell.

  2. Effects of matrine on HepG2 cell proliferation and expression of tumor relevant proteins in vitro.

    PubMed

    Qin, Xue-Gong; Hua, Zhang; Shuang, Wang; Wang, Yan-Hong; Cui, Yu-Dong

    2010-03-01

    Matrine, one of the main active components extracted from dry roots of Sophora flavescens Ait (Leguminosae), has been reported to have anticancer effects on a number of cancer cell lines, but the anticancer mechanism of matrine remains elusive. This study shows that matrine also displays anticancer activity on human hepatocellular carcinoma (HepG2) cells. In this work, the optimal cultivation condition for HepG2 cells was determined using the combinatorial orthogonal test design [L18 (21 x 37)]. Exposure of HepG2 cells to matrine resulted in inhibition of proliferation in both a time- and dose-dependent manner, as measured by morphology observation, hematoxylin and eosin (H&E) staining, and MTT assay (p<0.05). Further immunohistochemical analyses revealed that the expression of alpha fetal protein (AFP), proliferating cell nuclear antigen (PCNA), C-myc and Bcl-2 was down-regulated significantly, but the expression of Bax was up-regulated higher than untreated cells. The results demonstrated that matrine inhibited HepG2 cells proliferation primarily via up-regulating or down-regulating expression of the tumor relevant proteins.

  3. Polyethylenimine-functionalized silver nanoparticle-based co-delivery of paclitaxel to induce HepG2 cell apoptosis

    PubMed Central

    Li, Yinghua; Guo, Min; Lin, Zhengfang; Zhao, Mingqi; Xiao, Misi; Wang, Changbing; Xu, Tiantian; Chen, Tianfeng; Zhu, Bing

    2016-01-01

    Hepatocarcinoma is the third leading cause of cancer-related deaths around the world. Recently, a novel emerging nanosystem as anticancer therapeutic agents with intrinsic therapeutic properties has been widely used in various medical applications. In this study, surface decoration of functionalized silver nanoparticles (AgNPs) by polyethylenimine (PEI) and paclitaxel (PTX) was synthesized. The purpose of this study was to evaluate the effect of Ag@ PEI@PTX on cytotoxic and anticancer mechanism on HepG2 cells. The transmission electron microscope image and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that Ag@PEI@PTX had satisfactory size distribution and high stability and selectivity between cancer and normal cells. Ag@PEI@PTX-induced HepG2 cell apoptosis was confirmed by accumulation of the sub-G1 cells population, translocation of phosphatidylserine, depletion of mitochondrial membrane potential, DNA fragmentation, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. Furthermore, Ag@PEI@PTX enhanced cytotoxic effects on HepG2 cells and triggered intracellular reactive oxygen species; the signaling pathways of AKT, p53, and MAPK were activated to advance cell apoptosis. In conclusion, the results reveal that Ag@ PEI@PTX may provide useful information on Ag@PEI@PTX-induced HepG2 cell apoptosis and as appropriate candidate for chemotherapy of cancer. PMID:27994465

  4. Expression of CAR in SW480 and HepG2 cells during G1 is associated with cell proliferation

    SciTech Connect

    Osabe, Makoto; Sugatani, Junko Takemura, Akiko; Yamazaki, Yasuhiro; Ikari, Akira; Kitamura, Naomi; Negishi, Masahiko; Miwa, Masao

    2008-05-16

    Constitutive androstane receptor (CAR) is a transcription factor to regulate the expression of several genes related to drug-metabolism. Here, we demonstrate that CAR protein accumulates during G1 in human SW480 and HepG2 cells. After the G1/S phase transition, CAR protein levels decreased, and CAR was hardly detected in cells by the late M phase. CAR expression in both cell lines was suppressed by RNA interference-mediated suppression of CDK4. Depletion of CAR by RNA interference in both cells and by hepatocyte growth factor treatment in HepG2 cells resulted in decreased MDM2 expression that led to p21 upregulation and repression of HepG2 cell growth. Thus, our results demonstrate that CAR expression is an early G1 event regulated by CDK4 that contributes to MDM2 expression; these findings suggest that CAR may influence the expression of genes involved in not only the metabolism of endogenous and exogenous substances but also in the cell proliferation.

  5. Expression of CAR in SW480 and HepG2 cells during G1 is associated with cell proliferation.

    PubMed

    Osabe, Makoto; Sugatani, Junko; Takemura, Akiko; Yamazaki, Yasuhiro; Ikari, Akira; Kitamura, Naomi; Negishi, Masahiko; Miwa, Masao

    2008-05-16

    Constitutive androstane receptor (CAR) is a transcription factor to regulate the expression of several genes related to drug-metabolism. Here, we demonstrate that CAR protein accumulates during G1 in human SW480 and HepG2 cells. After the G1/S phase transition, CAR protein levels decreased, and CAR was hardly detected in cells by the late M phase. CAR expression in both cell lines was suppressed by RNA interference-mediated suppression of CDK4. Depletion of CAR by RNA interference in both cells and by hepatocyte growth factor treatment in HepG2 cells resulted in decreased MDM2 expression that led to p21 upregulation and repression of HepG2 cell growth. Thus, our results demonstrate that CAR expression is an early G1 event regulated by CDK4 that contributes to MDM2 expression; these findings suggest that CAR may influence the expression of genes involved in not only the metabolism of endogenous and exogenous substances but also in the cell proliferation.

  6. 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.

  7. Quantification of the uptake of silver nanoparticles and ions to HepG2 cells.

    PubMed

    Yu, Su-juan; Chao, Jing-bo; Sun, Jia; Yin, Yong-guang; Liu, Jing-fu; Jiang, Gui-bin

    2013-04-02

    The toxic mechanism of silver nanoparticles (AgNPs) is still debating, partially because of the common co-occurrence and the lack of methods for separation of AgNPs and Ag(+) in biological matrices. For the first time, Triton-X 114-based cloud point extraction (CPE) was proposed to separate AgNPs and Ag(+) in the cell lysates of exposed HepG2 cells. Cell lysates were subjected to CPE after adding Na2S2O3, which facilitated the transfer of AgNPs into the nether Triton X-114-rich phase by salt effect and the preserve of Ag(+) in the upper aqueous phase through the formation of hydrophilic complex. Then the AgNP and Ag(+) contents in the exposed cells were determined by ICP-MS after microwave digestion of the two phases, respectively. Under the optimized conditions, over 67% of AgNPs in cell lysates were extracted into the Triton X-114-rich phase while 94% of Ag(+) remained in the aqueous phase, and the limits of detection for AgNPs and Ag(+) were 2.94 μg/L and 2.40 μg/L, respectively. This developed analytical method was applied to quantify the uptake of AgNPs to the HepG2 cells. After exposure to 10 mg/L AgNPs for 24 h, about 67.8 ng Ag were assimilated per 10(4) cells, in which about 10.3% silver existed as Ag(+). Compared to the pristine AgNPs (with 5.2% Ag(+)) for exposure, the higher ratio of Ag(+) to AgNPs in the exposed cells (10.3% Ag(+)) suggests the transformation of AgNPs into Ag(+) in the cells and/or the higher uptake rate of Ag(+) than that of AgNPs. Given that the toxicity of Ag(+) is much higher than that of AgNPs, the substantial content of Ag(+) in the exposed cells suggests that the contribution of Ag(+) should be taken into account in evaluating the toxicity of AgNPs to organisms, and previous results obtained by regarding the total Ag content in organisms as AgNPs should be reconsidered.

  8. Effects of TLR4 gene silencing on the proliferation and apotosis of hepatocarcinoma HEPG2 cells.

    PubMed

    Liu, Yating; Li, Tao; Xu, Yuanhong; Xu, Enjun; Zhou, Min; Wang, Baolong; Shen, Jilong

    2016-05-01

    Toll-like receptors (TLRs) are key factors in the innate immune system and initiate an inflammatory response to foreign pathogens, such as bacteria, fungi and viruses. TLR4-mediated signaling has been implicated in tumor cell proliferation and apoptosis in numerous cancers. The present study aimed to investigate the biological effect of TLR4 on the proliferation and apoptosis of human liver cancer cells and the mechanisms responsible for the regulation of cellular responses following TLR4 gene knockdown. Three TLR4 small interfering (si)RNA constructs, consisting of TLR4-siRNA-1, TLR4-siRNA-2 and TLR4-siRNA-3, were transiently transfected into HepG2 cells using Lipofectamine 2000. TLR4 knockdown was confirmed using reverse transcription-polymerase chain reaction and western blotting. The effect of the TLR4 siRNA on tumor cell proliferation was monitored by methyl thiazolyl tetrazolium assay and cell apoptosis was observed by flow cytometry. The expression of TLR4-associated proteins, consisting of myeloid differentiation primary response 88 (MyD88), Toll-interleukin-1R-domain-containing adapter-inducing interferon-β (TRIF), interferon regulatory factor-3 (IRF3), nuclear factor (NF)-κB, NF-κB inhibitor α (IκBα), phosphorylated IκBα (p-IκBα), extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), was detected by western blot analysis. TLR4-siRNA-1 had the strongest knockdown effect and inhibited TLR4 messenger RNA and protein expression. TLR4 knockdown with TLR4-siRNA-1 reduced cell proliferation and promoted cell apoptosis. MyD88, TRIF, IRF3, IκBα, JNK and ERK were markedly suppressed in the cells transfected with TLR4 siRNA. However, nuclear expression of NF-κB and p-IκBα increased in HepG2 cells with TLR4 gene knockdown. The present study revealed that TLR4-mediated signaling plays a key role in the proliferation and apoptosis of cultured hepatocarcinoma cells. Therefore, RNA interference-directed targeting of TLR4 may raise

  9. [Role of reactive oxygen species in sodium selenite induced DNA damage in HepG2 cells].

    PubMed

    Zou, Yun-Feng; Niu, Pi-Ye; Gong, Zhi-Yong; Yuan, Jing

    2006-05-01

    To investigate the mechanisms of sodium selenite induced DNA damage in HepG2 cells. HepG2 cells were treated with the designed concentrations of sodium selenite and the selenite (10 micromol/L) added simultaneously with GSH (10 mmol) and NAC (5 mmol). Then the cell viability was detected by MTT, and the flurescent intensity of reactive oxygen species (ROS) was determined by flow cytometry, and DNA damage was detected by commet assay. The level of ROS was increased after HepG2 was treated with 5, 10, 20 micromol/L sodium selenite for one hour, and the cell viability was decreased after 12 hours, and the DNA damage was enhanced. Compared with the control group, the difference was statistically significant (P < 0.05) . GSH and NAC effectively inhibited the ROS increased and cell viability decreased and DNA damage weakened. ROS may be the important reason that sodium selenite induced HepG2 cells DNA damage.

  10. [Evaluation of the infectivity of dengue 1 strains in the HepG2 and Vero cell lines].

    PubMed

    Aguilar Barroso, Alicia; Amin Blanco, Nevis; Morier Díaz, Luis; Pérez Hernández, Ela María

    2005-01-01

    Viral infectivity of Hawaii, 3 Peri and Riberao Pretto dengue 1 strains was evaluated in Vero and HepG2 cell lines by indirect immunofluorescence techniques. Dengue virus cellular tropism in vitro is diverse. They may replicate themselves in a great variety of cellular cultures, whose sensibility to viral infection is variable. The greatest percentage of infected cells in the HepG2 cell line was obtained with the highest multiplicities of infection (0.04 for Hawaii and Riberao Pretto strains and 0.01 for 3 Perú). The highest percentage of infected HepG2 and Vero cells for the studied strains and the greatest titer in the viral overnadant was obtained on the 5th day. Vero cell line was more sensitive to viral infection, since for the same multiplicity values it was detected a higher number of fluorescent cells and a better viral titre in the line of this overnadant than in the HepG2. The best result was obtained with the Hawaii strain that allowed to confirm faster the infection of the studied cellular lines.

  11. Zebularine upregulates expression of CYP genes through inhibition of DNMT1 and PKR in HepG2 cells

    PubMed Central

    Nakamura, Kazuaki; Aizawa, Kazuko; Aung, Kyaw Htet; Yamauchi, Junji; Tanoue, Akito

    2017-01-01

    Drug-induced hepatotoxicity is one of the major reasons cited for drug withdrawal. Therefore, it is of extreme importance to detect human hepatotoxic candidates as early as possible during the drug development process. In this study, we aimed to enhance hepatocyte functions such as CYP gene expression in HepG2 cells, one of the most extensively used cell lines in evaluating hepatotoxicity of chemicals and drugs. We found that zebularine, a potent inhibitor of DNA methylation, remarkably upregulates the expression of CYP genes in HepG2 cells. In addition, we revealed that the upregulation of CYP gene expression by zebularine was mediated through the inhibition of both DNA methyltransferase 1 (DNMT1) and double-stranded RNA-dependent protein kinase (PKR). Furthermore, HepG2 cells treated with zebularine were more sensitive than control cells to drug toxicity. Taken together, our results show that zebularine may make HepG2 cells high-functioning and thus could be useful for evaluating the hepatotoxicity of chemicals and drugs speedily and accurately in in-vitro systems. The finding that zebularine upregulates CYP gene expression through DNMT1 and PKR modulation sheds light on the mechanisms controlling hepatocyte function and thus may aid in the development of new in-vitro systems using high-functioning hepatocytes. PMID:28112215

  12. Cytotoxicity assessments of Portulaca oleracea and Petroselinum sativum seed extracts on human hepatocellular carcinoma cells (HepG2).

    PubMed

    Farshori, Nida Nayyar; Al-Sheddi, Ebtesam Saad; Al-Oqail, Mai Mohammad; Musarrat, Javed; Al-Khedhairy, Abdulaziz Ali; Siddiqui, Maqsood Ahmed

    2014-01-01

    The Pharmacological potential, such as antioxidant, anti-inflammatory, and antibacterial activities of Portulaca oleracea (PO) and Petroselinum sativum (PS) extracts are well known. However, the preventive properties against hepatocellular carcinoma cells have not been explored so far. Therefore, the present investigation was designed to study the anticancer activity of seed extracts of PO and PS on the human hepatocellular carcinoma cells (HepG2). The HepG2 cells were exposed with 5-500 μg/ml of PO and PS for 24 h. After the exposure, cell viability by 3-(4,5-dimethylthiazol-2yl)-2,5-biphenyl tetrazolium bromide (MTT) assay, neutral red uptake (NRU) assay, and cellular morphology by phase contrast inverted microscope were studied. The results showed that PO and PS extracts significantly reduced the cell viability of HepG2 in a concentration dependent manner. The cell viability was recorded to be 67%, 31%, 21%, and 17% at 50, 100, 250, and 500 μg/ml of PO, respectively by MTT assay and 91%, 62%, 27%, and 18% at 50, 100, 250, and 500 μg/ml of PO, respectively by NRU assay. PS exposed HepG2 cells with 100 μg/ml and higher concentrations were also found to be cytotoxic. The decrease in the cell viability at 100, 250, and 500 μg/ml of PS was recorded as 70%, 33%, and 15% by MTT assay and 63%, 29%, and 17%, respectively by NRU assay. Results also showed that PO and PS exposed cells reduced the normal morphology and adhesion capacity of HepG2 cells. HepG2 cells exposed with 50 μg/ml and higher concentrations of PO and PS lost their typical morphology, become smaller in size, and appeared in rounded bodies. Our results demonstrated preliminary screening of anticancer activity of Portulaca oleracea and Petroselinum sativum extracts against HepG2 cells, which can be further used for the development of a potential therapeutic anticancer agent.

  13. GRK2 negatively regulates IGF-1R signaling pathway and cyclins' expression in HepG2 cells.

    PubMed

    Wei, Zhengyu; Hurtt, Reginald; Gu, Tina; Bodzin, Adam S; Koch, Walter J; Doria, Cataldo

    2013-09-01

    G protein coupled receptor kinase 2 (GRK2) plays a central role in the regulation of a variety of important signaling pathways. Alternation of GRK2 protein level and activity casts profound effects on cell physiological functions and causes diseases such as heart failure, rheumatoid arthritis, and obesity. We have previously reported that overexpression of GRK2 has an inhibitory role in cancer cell growth. To further examine the role of GRK2 in cancer, in this study, we investigated the effects of reduced protein level of GRK2 on insulin-like growth factor 1 receptor (IGF-1R) signaling pathway in human hepatocellular carcinoma (HCC) HepG2 cells. We created a GRK2 knockdown cell line using a lentiviral vector mediated expression of GRK2 specific short hairpin RNA (shRNA). Under IGF-1 stimulation, HepG2 cells with reduced level of GRK2 showed elevated total IGF-1R protein expression as well as tyrosine phosphorylation of receptor. In addition, HepG2 cells with reduced level of GRK2 also demonstrated increased tyrosine phosphorylation of IRS1 at the residue 612 and increased phosphorylation of Akt, indicating a stronger activation of IGF-1R signaling pathway. However, HepG2 cells with reduced level of GRK2 did not display any growth advantage in culture as compared with the scramble control cells. We further detected that reduced level of GRK2 induced a small cell cycle arrest at G2/M phase by enhancing the expression of cyclin A, B1, and E. Our results indicate that GRK2 has contrasting roles on HepG2 cell growth by negatively regulating the IGF-1R signaling pathway and cyclins' expression.

  14. NOX1 supports the metabolic remodeling of HepG2 cells.

    PubMed

    Bertram, Katharina; Valcu, Cristina-Maria; Weitnauer, Michael; Linne, Uwe; Görlach, Agnes

    2015-01-01

    NADPH oxidases are important sources of reactive oxygen species (ROS) which act as signaling molecules in the regulation of protein expression, cell proliferation, differentiation, migration and cell death. The NOX1 subunit is over-expressed in several cancers and NOX1 derived ROS have been repeatedly linked with tumorigenesis and tumor progression although underlying pathways are ill defined. We engineered NOX1-depleted HepG2 hepatoblastoma cells and employed differential display 2DE experiments in order to investigate changes in NOX1-dependent protein expression profiles. A total of 17 protein functions were identified to be dysregulated in NOX1-depleted cells. The proteomic results support a connection between NOX1 and the Warburg effect and a role for NOX in the regulation of glucose and glutamine metabolism as well as of lipid, protein and nucleotide synthesis in hepatic tumor cells. Metabolic remodeling is a common feature of tumor cells and understanding the underlying mechanisms is essential for the development of new cancer treatments. Our results reveal a manifold involvement of NOX1 in the metabolic remodeling of hepatoblastoma cells towards a sustained production of building blocks required to maintain a high proliferative rate, thus rendering NOX1 a potential target for cancer therapy.

  15. NOX1 Supports the Metabolic Remodeling of HepG2 Cells

    PubMed Central

    Weitnauer, Michael; Linne, Uwe; Görlach, Agnes

    2015-01-01

    NADPH oxidases are important sources of reactive oxygen species (ROS) which act as signaling molecules in the regulation of protein expression, cell proliferation, differentiation, migration and cell death. The NOX1 subunit is over-expressed in several cancers and NOX1 derived ROS have been repeatedly linked with tumorigenesis and tumor progression although underlying pathways are ill defined. We engineered NOX1-depleted HepG2 hepatoblastoma cells and employed differential display 2DE experiments in order to investigate changes in NOX1-dependent protein expression profiles. A total of 17 protein functions were identified to be dysregulated in NOX1-depleted cells. The proteomic results support a connection between NOX1 and the Warburg effect and a role for NOX in the regulation of glucose and glutamine metabolism as well as of lipid, protein and nucleotide synthesis in hepatic tumor cells. Metabolic remodeling is a common feature of tumor cells and understanding the underlying mechanisms is essential for the development of new cancer treatments. Our results reveal a manifold involvement of NOX1 in the metabolic remodeling of hepatoblastoma cells towards a sustained production of building blocks required to maintain a high proliferative rate, thus rendering NOX1 a potential target for cancer therapy. PMID:25806803

  16. Citreoviridin Induces Autophagy-Dependent Apoptosis through Lysosomal-Mitochondrial Axis in Human Liver HepG2 Cells.

    PubMed

    Wang, Yuexia; Liu, Yanan; Liu, Xiaofang; Jiang, Liping; Yang, Guang; Sun, Xiance; Geng, Chengyan; Li, Qiujuan; Yao, Xiaofeng; Chen, Min

    2015-08-06

    Citreoviridin (CIT) is a mycotoxin derived from fungal species in moldy cereals. In our previous study, we reported that CIT stimulated autophagosome formation in human liver HepG2 cells. Here, we aimed to explore the relationship of autophagy with lysosomal membrane permeabilization and apoptosis in CIT-treated cells. Our data showed that CIT increased the expression of LC3-II, an autophagosome biomarker, from the early stage of treatment (6 h). After treatment with CIT for 12 h, lysosomal membrane permeabilization occurred, followed by the release of cathepsin D in HepG2 cells. Inhibition of autophagosome formation with siRNA against Atg5 attenuated CIT-induced lysosomal membrane permeabilization. In addition, CIT induced collapse of mitochondrial transmembrane potential as assessed by JC-1 staining. Furthermore, caspase-3 activity assay showed that CIT induced apoptosis in HepG2 cells. Inhibition of autophagosome formation attenuated CIT-induced apoptosis, indicating that CIT-induced apoptosis was autophagy-dependent. Cathepsin D inhibitor, pepstatin A, relieved CIT-induced apoptosis as well, suggesting the involvement of the lysosomal-mitochondrial axis in CIT-induced apoptosis. Taken together, our data demonstrated that CIT induced autophagy-dependent apoptosis through the lysosomal-mitochondrial axis in HepG2 cells. The study thus provides essential mechanistic insight, and suggests clues for the effective management and treatment of CIT-related diseases.

  17. Citreoviridin Induces Autophagy-Dependent Apoptosis through Lysosomal-Mitochondrial Axis in Human Liver HepG2 Cells

    PubMed Central

    Wang, Yuexia; Liu, Yanan; Liu, Xiaofang; Jiang, Liping; Yang, Guang; Sun, Xiance; Geng, Chengyan; Li, Qiujuan; Yao, Xiaofeng; Chen, Min

    2015-01-01

    Citreoviridin (CIT) is a mycotoxin derived from fungal species in moldy cereals. In our previous study, we reported that CIT stimulated autophagosome formation in human liver HepG2 cells. Here, we aimed to explore the relationship of autophagy with lysosomal membrane permeabilization and apoptosis in CIT-treated cells. Our data showed that CIT increased the expression of LC3-II, an autophagosome biomarker, from the early stage of treatment (6 h). After treatment with CIT for 12 h, lysosomal membrane permeabilization occurred, followed by the release of cathepsin D in HepG2 cells. Inhibition of autophagosome formation with siRNA against Atg5 attenuated CIT-induced lysosomal membrane permeabilization. In addition, CIT induced collapse of mitochondrial transmembrane potential as assessed by JC-1 staining. Furthermore, caspase-3 activity assay showed that CIT induced apoptosis in HepG2 cells. Inhibition of autophagosome formation attenuated CIT-induced apoptosis, indicating that CIT-induced apoptosis was autophagy-dependent. Cathepsin D inhibitor, pepstatin A, relieved CIT-induced apoptosis as well, suggesting the involvement of the lysosomal-mitochondrial axis in CIT-induced apoptosis. Taken together, our data demonstrated that CIT induced autophagy-dependent apoptosis through the lysosomal-mitochondrial axis in HepG2 cells. The study thus provides essential mechanistic insight, and suggests clues for the effective management and treatment of CIT-related diseases. PMID:26258792

  18. ssDNA Aptamer Specifically Targets and Selectively Delivers Cytotoxic Drug Doxorubicin to HepG2 Cells.

    PubMed

    Yu, Ge; Li, Huan; Yang, Shuanghui; Wen, Jianguo; Niu, Junqi; Zu, Youli

    2016-01-01

    Hepatocellular carcinoma (HCC) is the third leading cause of death due to cancer worldwide with over 500,000 people affected annually. Although chemotherapy has been widely used to treat patients with HCC, alternate modalities to specifically deliver therapeutic cargos to cancer cells have been sought in recent years due to the severe side effects of chemotherapy. In this respect, aptamer-based tumor targeted drug delivery has emerged as a promising approach to increase the efficacy of chemotherapy and reduce or eliminate drug toxicity. In this study, we developed a new HepG2-specific aptamer (HCA#3) by a procedure known as systematic evolution of ligands by exponential enrichment (SELEX) and exploited its role as a targeting ligand to deliver doxorubicin (Dox) to HepG2 cells in vitro. The selected 76-base nucleotide aptamer preferentially bound to HepG2 hepatocellular carcinoma cells but not to control cells. The aptamer HCA#3 was modified with paired CG repeats at the 5'-end to carry and deliver a high payload of intercalated Dox molecules at the CG sites. Four Dox molecules (mol/mol) were fully intercalated in each conjugate aptamer-Dox (ApDC) molecule. Biostability analysis showed that the ApDC molecules are stable in serum. Functional analysis showed that ApDC specifically targeted and released Dox within HepG2 cells but not in control cells, and treatment with HCA#3 ApDC induced HepG2 cell apoptosis but had minimal effect on control cells. Our study demonstrated that HCA#3 ApDC is a promising aptamer-targeted therapeutic that can specifically deliver and release a high doxorubicin payload in HCC cells.

  19. Cytotoxic effect of Agaricus bisporus and Lactarius rufus β-D-glucans on HepG2 cells.

    PubMed

    Pires, Amanda do Rocio Andrade; Ruthes, Andrea Caroline; Cadena, Silvia Maria Suter Correia; Acco, Alexandra; Gorin, Philip Albert James; Iacomini, Marcello

    2013-07-01

    The cytotoxic activity of β-D-glucans isolated from Agaricus bisporus and Lactarius rufus fruiting bodies was evaluated on human hepatocellular carcinoma cells (HepG2). NMR and methylation analysis suggest that these β-d-glucans were composed of a linear (1→6)-linked and a branched (1→3), (1→6)-linked backbone, respectively. They both decreased cell viability at concentrations of up to 100 μg mL(-1), as shown by MTT assay. The amount of LDH released and the analysis of cell morphology corroborated these values and also showed that the β-D-glucan of L. rufus was more cytotoxic to HepG2 cells than that of A. bisporus. The treatment of HepG2 cells with L. rufus and A. bisporus β-D-glucans at a dose of 200 μg mL(-1) for 24h promoted an increase of cytochrome c release and a decrease of ATP content, suggesting that these polysaccharides could promote cell death by apoptosis. Both β-D-glucans were tested against murine primary hepatocytes at a dose of 200 μg mL(-1). The results suggest that the L. rufus β-d-glucan was as cytotoxic for hepatocytes as for HepG2 cells, whereas the A. bisporus β-D-glucan, under the same conditions, was cytotoxic only for HepG2 cells, suggesting cell selectivity. These results open new possibilities for use of mushroom β-D-glucans in cancer therapy.

  20. ssDNA Aptamer Specifically Targets and Selectively Delivers Cytotoxic Drug Doxorubicin to HepG2 Cells

    PubMed Central

    Yu, Ge; Li, Huan; Yang, Shuanghui; Wen, Jianguo; Niu, Junqi; Zu, Youli

    2016-01-01

    Hepatocellular carcinoma (HCC) is the third leading cause of death due to cancer worldwide with over 500,000 people affected annually. Although chemotherapy has been widely used to treat patients with HCC, alternate modalities to specifically deliver therapeutic cargos to cancer cells have been sought in recent years due to the severe side effects of chemotherapy. In this respect, aptamer-based tumor targeted drug delivery has emerged as a promising approach to increase the efficacy of chemotherapy and reduce or eliminate drug toxicity. In this study, we developed a new HepG2-specific aptamer (HCA#3) by a procedure known as systematic evolution of ligands by exponential enrichment (SELEX) and exploited its role as a targeting ligand to deliver doxorubicin (Dox) to HepG2 cells in vitro. The selected 76-base nucleotide aptamer preferentially bound to HepG2 hepatocellular carcinoma cells but not to control cells. The aptamer HCA#3 was modified with paired CG repeats at the 5′-end to carry and deliver a high payload of intercalated Dox molecules at the CG sites. Four Dox molecules (mol/mol) were fully intercalated in each conjugate aptamer-Dox (ApDC) molecule. Biostability analysis showed that the ApDC molecules are stable in serum. Functional analysis showed that ApDC specifically targeted and released Dox within HepG2 cells but not in control cells, and treatment with HCA#3 ApDC induced HepG2 cell apoptosis but had minimal effect on control cells. Our study demonstrated that HCA#3 ApDC is a promising aptamer-targeted therapeutic that can specifically deliver and release a high doxorubicin payload in HCC cells. PMID:26808385

  1. 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

  2. [CXCR3 monoclonal antibody inhibits the proliferation and migration of MCF-7 cells and HepG2 cells in vitro].

    PubMed

    Zhu, Yuqiang; Wang, Zhiyao; Zhu, Ying; Wang, Jing; Cai, Lei; Shen, Hui; Kong, Yong; Qiu, Yuhua

    2015-11-01

    To study the effects of chemokine (C-X-C motif) receptor 3 (CXCR3) monoclonal antibody (mAb) on the proliferation and migration of MCF-7 and HepG2 cells in vitro. Ascites of CXCR3 mAb was prepared at first. MCF-7 and HepG2 cells with high expressions of CXCR3 were screened by flow cytometry. MTT assay was used to detect the effects of CXCR3 mAb on the proliferation of MCF-7 and HepG2 cells in vitro in the absence/presence of interferon-inducible T-cell alpha chemoattractant (I-TAC). Transwell™ assay was performed to investigate the effects of CXCR3 mAb on the migration of MCF-7 and HepG2 cells in vitro in the absence/presence of I-TAC. The expression rate of CXCR3 on MCF-7 and HepG2 cells were 83.5% and 96.2%, respectively. 50 mg/mL CXCR3 mAb significantly inhibited the proliferation and migration of MCF-7 and HepG2 cells, and also inhibited the promoting effect of I-TAC on the proliferation and migration of MCF-7 and HepG2 cells in vitro. CXCR3 mAb can significantly inhibit the proliferation and migration of the tumor cells highly expressing CXCR3 in vitro.

  3. An autophagic process is activated in HepG2 cells to mediate BDE-100-induced toxicity.

    PubMed

    Pereira, Lilian Cristina; Duarte, Filipe Valente; Varela, Ana Teresa Inácio Ferreira; Rolo, Anabela Pinto; Palmeira, Carlos Manuel Marques; Dorta, Daniel Junqueira

    2017-02-01

    To reduce flammability and meet regulatory requirements, Brominated Flame Retardants (BFRs) are added to a wide variety of consumer products including furniture, textiles, electronics, and construction materials. Exposure to polybrominated phenyl ethers (PBDEs) adversely affects the human health. Bearing in mind that (i) PBDEs are potentially toxic, (ii) the mechanism of PBDE toxicity is unclear, and (iii) the importance of the autophagy to the field of toxicology is overlooked, this study investigates whether an autophagic process is activated in HepG2 cells (human hepatoblastoma cell line) to mediate BDE-100-induced toxicity. HepG2 cells were exposed with BDE-100 at three concentrations (0.1, 5, and 25μM), selected from preliminary toxicity tests, for 24 and 48h. To assess autophagy, immunocytochemistry was performed after exposure of HepG2 cells to BDE-100. Labeling of HepG2 cells with 100nM LysoTracker Red DND-99 aided examination of lysosome distribution. Proteins that are key to the autophagic process (p62 and LC3) were evaluated by western blotting. DNA was isolated and quantified to assess mitochondrial DNA copy number by qPCR on the basis of the number of DNA copies of a mitochondrial encoded gene normalized against a nuclear encoded gene. Conversion of LC3-I to LC3-II increased in HepG2 cells. Pre-addition of 100nM wortmannin decreased the amount of LC3 in the punctuate form and increased nuclear fragmentation (apoptotic feature). HepG2 cells exposed to BDE-100 presented increased staining with the lysosomal dye and had larger LC3 and p62 content after pre-treatment with ammonium chloride. The mitochondrial DNA copy number decreased, which probably constituted an attempt of the cell to manage mitochondrial damage by selective mitochondrial degradation (mitophagy). In conclusion, an autophagic process is activated in HepG2 cells to mediate BDE-100-induced toxicity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. Ovothiol Isolated from Sea Urchin Oocytes Induces Autophagy in the Hep-G2 Cell Line

    PubMed Central

    Russo, Gian Luigi; Russo, Maria; Castellano, Immacolata; Napolitano, Alessandra; Palumbo, Anna

    2014-01-01

    Ovothiols are histidine-derived thiols isolated from sea urchin eggs, where they play a key role in the protection of cells toward the oxidative burst associated with fertilization by controlling the cellular redox balance and recycling oxidized glutathione. In this study, we show that treatment of a human liver carcinoma cell line, Hep-G2, with ovothiol A, isolated from Paracentrotus lividus oocytes, results in a decrease of cell proliferation in a dose-dependent manner. The activation of an autophagic process is revealed by phase contrast and fluorescence microscopy, together with the expression of the specific autophagic molecular markers, LC3 II and Beclin-1. The effect of ovothiol is not due to its antioxidant capacity or to hydrogen peroxide generation. The concentration of ovothiol A in the culture media, as monitored by HPLC analysis, decreased by about 24% within 30 min from treatment. The proliferation of normal human embryonic lung cells is not affected by ovothiol A. These results hint at ovothiol as a promising bioactive molecule from marine organisms able to inhibit cell proliferation in cancer cells. PMID:25003791

  5. Dihydrotestosterone regulating apolipoprotein M expression mediates via protein kinase C in HepG2 cells

    PubMed Central

    2012-01-01

    Background Administration of androgens decreases plasma concentrations of high-density lipid cholesterol (HDL-C). However, the mechanisms by which androgens mediate lipid metabolism remain unknown. This present study used HepG2 cell cultures and ovariectomized C57BL/6 J mice to determine whether apolipoprotein M (ApoM), a constituent of HDL, was affected by dihydrotestosterone (DHT). Methods HepG2 cells were cultured in the presence of either DHT, agonist of protein kinase C (PKC), phorbol-12-myristate-13-acetate (PMA), blocker of androgen receptor flutamide together with different concentrations of DHT, or DHT together with staurosporine at different concentrations for 24 hrs. Ovariectomized C57BL/6 J mice were treated with DHT or vehicle for 7d or 14d and the levels of plasma ApoM and livers ApoM mRNA were measured. The mRNA levels of ApoM, ApoAI were determined by real-time RT-PCR. ApoM and ApoAI were determined by western blotting analysis. Results Addition of DHT to cell culture medium selectively down-regulated ApoM mRNA expression and ApoM secretion in a dose-dependent manner. At 10 nM DHT, the ApoM mRNA levels were about 20% lower than in untreated cells and about 40% lower at 1000 nM DHT than in the control cells. The secretion of ApoM into the medium was reduced to a similar extent. The inhibitory effect of DHT on ApoM secretion was not blocked by the classical androgen receptor blocker flutamide but by an antagonist of PKC, Staurosporine. Agonist of PKC, PMA, also reduced ApoM. At 0.5 μM PMA, the ApoM mRNA levels and the secretion of ApoM into the medium were about 30% lower than in the control cells. The mRNA expression levels and secretion of another HDL-associated apolipoprotein AI (ApoAI) were not affected by DHT. The levels of plasma ApoM and liver ApoM mRNA of DHT-treated C57BL/6 J mice were lower than those of vehicle-treated mice. Conclusions DHT directly and selectively down-regulated the level of ApoM mRNA and the secretion of ApoM by protein

  6. Dihydrotestosterone regulating apolipoprotein M expression mediates via protein kinase C in HepG2 cells.

    PubMed

    Yi-zhou, Ye; Bing, Cao; Ming-qiu, Li; Wei, Wang; Ru-xing, Wang; Jun, Rui; Liu-yan, Wei; Zhao-hui, Jing; Yong, Ji; Guo qing, Jiao; Jian, Zou

    2012-12-05

    Administration of androgens decreases plasma concentrations of high-density lipid cholesterol (HDL-C). However, the mechanisms by which androgens mediate lipid metabolism remain unknown. This present study used HepG2 cell cultures and ovariectomized C57BL/6 J mice to determine whether apolipoprotein M (ApoM), a constituent of HDL, was affected by dihydrotestosterone (DHT). HepG2 cells were cultured in the presence of either DHT, agonist of protein kinase C (PKC), phorbol-12-myristate-13-acetate (PMA), blocker of androgen receptor flutamide together with different concentrations of DHT, or DHT together with staurosporine at different concentrations for 24 hrs. Ovariectomized C57BL/6 J mice were treated with DHT or vehicle for 7d or 14d and the levels of plasma ApoM and livers ApoM mRNA were measured. The mRNA levels of ApoM, ApoAI were determined by real-time RT-PCR. ApoM and ApoAI were determined by western blotting analysis. Addition of DHT to cell culture medium selectively down-regulated ApoM mRNA expression and ApoM secretion in a dose-dependent manner. At 10 nM DHT, the ApoM mRNA levels were about 20% lower than in untreated cells and about 40% lower at 1000 nM DHT than in the control cells. The secretion of ApoM into the medium was reduced to a similar extent. The inhibitory effect of DHT on ApoM secretion was not blocked by the classical androgen receptor blocker flutamide but by an antagonist of PKC, Staurosporine. Agonist of PKC, PMA, also reduced ApoM. At 0.5 μM PMA, the ApoM mRNA levels and the secretion of ApoM into the medium were about 30% lower than in the control cells. The mRNA expression levels and secretion of another HDL-associated apolipoprotein AI (ApoAI) were not affected by DHT. The levels of plasma ApoM and liver ApoM mRNA of DHT-treated C57BL/6 J mice were lower than those of vehicle-treated mice. DHT directly and selectively down-regulated the level of ApoM mRNA and the secretion of ApoM by protein kinase C but independently of the

  7. The toxicity of extracts of plant parts of Moringa stenopetala in HEPG2 cells in vitro.

    PubMed

    Mekonnen, Negussu; Houghton, Peter; Timbrell, John

    2005-10-01

    The cytotoxicity of extracts from a widely used species of plant, Moringa stenopetala, was assessed in HEPG2 cells, by measuring the leakage of lactate dehydrogenase (LDH) and cell viability. The functional integrity of extract-exposed cells was determined by measuring intracellular levels of ATP and glutathione (GSH). The ethanol extracts of leaves and seeds increased significantly (p < 0.01) LDH leakage in a dose- and time-dependent manner. The water extract of leaves and the ethanol extract of the root did not increase LDH leakage. A highly significant (p < 0.001) decrease in HEPG2 viability was found after incubating the cells with the highest concentration (500 microg/mL) of the ethanol leaf and seed extracts. At a concentration of 500 microg/mL, the water extract of leaves increased (p < 0.01), while the ethanol extract of the same plant part decreased (p < 0.01), ATP levels. The root and seed extracts had no significant effect on ATP levels. The ethanol leaf extract decreased GSH levels at a concentration of 500 microg/mL (p < 0.01), as did the ethanol extract of the seeds at 250 microg/mL and 500 microg/mL (p < 0.05). The water extract of the leaves did not alter GSH or LDH levels or affect cell viability, suggesting that it may be non-toxic, and is consistent with its use as a vegetable. The data obtained from the studies with the ethanol extract of the leaves and seeds from Moringa stenopetala show that they contain toxic substances that are extractable with organic solvents or are formed during the process of extraction with these solvents. The significant depletion of ATP and GSH only occurred at concentrations of extract that caused leakage of LDH. Further investigation with this plant in order to identify the constituents extracted and their individual toxic effects both in vivo and in vitro is warranted. This study also illustrates the utility of cell culture for screening plant extracts for potential toxicity.

  8. Biochemical Effects of six Ti02 and four Ce02 Nanomaterials in HepG2 cells

    EPA Science Inventory

    Abstract The potential mammalian hepatotoxicity of nanomaterials were explored in dose-response and structure-activity studies with human hepatic HepG2 cells exposed to between 10 and 1000 ug/ml of six different TiO2 and four CeO2 nanomaterials for 3 days. Var...

  9. Differential genomic effects on signaling pathways by two different CeO2 nanoparticles in HepG2 cells

    EPA Science Inventory

    To investigate genomic effects, human liver hepatocellular carcinoma (HepG2) cells were exposed for three days to two different forms of nanoparticles both composed of Ce02 (0.3, 3 and 30 µg/mL). The two Ce02 nanopartices had dry primary particle sizes of 8 nanometers {(M) ...

  10. Biochemical Effects of six Ti02 and four Ce02 Nanomaterials in HepG2 cells

    EPA Science Inventory

    Abstract The potential mammalian hepatotoxicity of nanomaterials were explored in dose-response and structure-activity studies with human hepatic HepG2 cells exposed to between 10 and 1000 ug/ml of six different TiO2 and four CeO2 nanomaterials for 3 days. Var...

  11. Differential genomic effects on signaling pathways by two different CeO2 nanoparticles in HepG2 cells

    EPA Science Inventory

    To investigate genomic effects, human liver hepatocellular carcinoma (HepG2) cells were exposed for three days to two different forms of nanoparticles both composed of Ce02 (0.3, 3 and 30 µg/mL). The two Ce02 nanopartices had dry primary particle sizes of 8 nanometers {(M) ...

  12. N-Acetyl-Serotonin Protects HepG2 Cells from Oxidative Stress Injury Induced by Hydrogen Peroxide

    PubMed Central

    Jiang, Jiying; Yu, Shuna; Jiang, Zhengchen; Liang, Cuihong; Yu, Wenbo; Li, Jin; Du, Xiaodong; Wang, Hailiang; Gao, Xianghong; Wang, Xin

    2014-01-01

    Oxidative stress plays an important role in the pathogenesis of liver diseases. N-Acetyl-serotonin (NAS) has been reported to protect against oxidative damage, though the mechanisms by which NAS protects hepatocytes from oxidative stress remain unknown. To determine whether pretreatment with NAS could reduce hydrogen peroxide- (H2O2-) induced oxidative stress in HepG2 cells by inhibiting the mitochondrial apoptosis pathway, we investigated the H2O2-induced oxidative damage to HepG2 cells with or without NAS using MTT, Hoechst 33342, rhodamine 123, Terminal dUTP Nick End Labeling Assay (TUNEL), dihydrodichlorofluorescein (H2DCF), Annexin V and propidium iodide (PI) double staining, immunocytochemistry, and western blot. H2O2 produced dramatic injuries in HepG2 cells, represented by classical morphological changes of apoptosis, increased levels of malondialdehyde (MDA) and intracellular reactive oxygen species (ROS), decreased activity of superoxide dismutase (SOD), and increased activities of caspase-9 and caspase-3, release of cytochrome c (Cyt-C) and apoptosis-inducing factor (AIF) from mitochondria, and loss of membrane potential (ΔΨm). NAS significantly inhibited H2O2-induced changes, indicating that it protected against H2O2-induced oxidative damage by reducing MDA levels and increasing SOD activity and that it protected the HepG2 cells from apoptosis through regulating the mitochondrial apoptosis pathway, involving inhibition of mitochondrial hyperpolarization, release of mitochondrial apoptogenic factors, and caspase activity. PMID:25013541

  13. Cytotoxicity of various chemicals and mycotoxins in fresh primary duck embryonic fibroblasts: a comparison to HepG2 cells.

    PubMed

    Chen, Xi; Murdoch, Rhonda; Shafer, Daniel J; Ajuwon, Kolapo M; Applegate, Todd J

    2016-11-01

    To screen cost-effectively the overall toxicity of a sample, particularly in the case of food and feed ingredient quality control, a sensitive bioassay is necessary. With the wide variety of cytotoxicity assays, performance comparison between assays using different cells has become of interest. Fresh primary duck embryonic fibroblasts (DEF) were hypothesized to be a sensitive tool for in vitro cytotoxicity screening; cell viability of DEF in response to various cytotoxins was determined and compared with response of HepG2 cells. The IC50 values by the alamar blue assay in the DEF cells had a high correlation (R(2)  = 0.96) with those obtained in HepG2 cells. Within the same toxin, primary DEF yielded significantly lower IC50 values than that obtained from HepG2 cells using the MTT and alamar blue assay. Additionally, primary DEF responded to all mycotoxins tested using the alamar blue assay, while HepG2 was less sensitive, particularly at short exposure times. The estimated IC50 for aflatoxin B1 , fumonisins B1 and deoxynivalenol in DEF after 72 h incubation were 3.69, 4.19 and 1.26 μg ml(-1) , respectively. Results from the current study suggest that primary DEF are more sensitive to cytotoxins and mycotoxins compared to HepG2, and thus may have great potential as an effective tool for cytotoxicity assessment. The question remains whether in vitro IC50 values can accurately predict in vivo toxicity; however, the current study accentuates the need for further attention to identify sensitive cell models for in vitro cytotoxicity screening and subsequent exploration of species-specific prediction models for in vivo toxicity. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  14. Recombinant production of native human α-1-antitrypsin protein in the liver HepG2 cells.

    PubMed

    Jaberie, Hajar; Naghibalhossaini, Fakhraddin

    2016-10-01

    Alpha-1 antitrypsin (A1AT) deficiency is associated with emphysema and liver disease. Only plasma-derived A1AT protein is available for augmentation therapy. Recombinant A1AT (recA1AT) protein expressed in various types of available hosts are either non-glycosylated or aberrantly glycosylated resulting into reduced stability and biological activity. To overcome these limitations, we have used the human liver HepG2 cell line to produce recA1AT protein. HepG2 cells were transfected by A1AT cDNA and cell populations were generated that stably overexpressed A1AT protein. Real-time RT-PCR and rocket immunoelectrophoresis of cell culture supernatants indicated that the transfection resulted more than two-fold increase in A1AT production compared to that of control parental cells. Immunoblot analysis showed that both plasma and HepG2-produced A1AT proteins have identical molecular weight in either glycosylated or deglycosylated form. Partial digestion with PNGase F indicated that the three N-glycosylation sites of recA1AT, like the native A1AT protein in plasma, are occupied. Recombinant A1AT also like the native A1AT was thermostable and could efficiently inhibit trypsin proteolytic activity against BSA and BAPNA chromogenic substrate. The recombinant HepG2 cells cultured in media containing B27 serum free supplement released recA1AT at the same level as in the serum containing media. RecA1AT production in HepG2 cells grown under serum free condition at a large scale could provide a reliable source of the native protein suitable for therapeutic use in human.

  15. The color and size of chili peppers (Capsicum annuum) influence Hep-G2 cell growth.

    PubMed

    Popovich, David G; Sia, Sharon Y; Zhang, Wei; Lim, Mon L

    2014-11-01

    Four types of chili (Capsicum annuum) extracts, categorized according to color; green and red, and size; small and large were studied in Hep-G2 cells. Red small (RS) chili had an LC50 value of 0.378 ± 0.029 compared to green big (GB) 1.034 ± 0.061 and green small (GS) 1.070 ± 0.21 mg/mL. Red big (RB) was not cytotoxic. Capsaicin content was highest in RS and produced a greater percentage sub-G1 cells (6.47 ± 1.8%) after 24 h compared to GS (2.96 ± 1.3%) and control (1.29 ± 0.8%) cells. G2/M phase was reduced by GS compared to RS and control cells. RS at the LC50 concentration contained 1.6 times the amount of pure capsaicin LC50 to achieve the same effect of capsaicin alone. GS and GB capsaicin content at the LC50 value was lower (0.2 and 0.66, respectively) compared to the amount of capsaicin to achieve a similar reduction in cell growth.

  16. Bile acids reduce endocytosis of high-density lipoprotein (HDL) in HepG2 cells.

    PubMed

    Röhrl, Clemens; Eigner, Karin; Fruhwürth, Stefanie; Stangl, Herbert

    2014-01-01

    High-density lipoprotein (HDL) transports lipids to hepatic cells and the majority of HDL-associated cholesterol is destined for biliary excretion. Cholesterol is excreted into the bile directly or after conversion to bile acids, which are also present in the plasma as they are effectively reabsorbed through the enterohepatic cycle. Here, we provide evidence that bile acids affect HDL endocytosis. Using fluorescent and radiolabeled HDL, we show that HDL endocytosis was reduced in the presence of high concentrations of taurocholate, a natural non-cell-permeable bile acid, in human hepatic HepG2 and HuH7 cells. In contrast, selective cholesteryl-ester (CE) uptake was increased. Taurocholate exerted these effects extracellularly and independently of HDL modification, cell membrane perturbation or blocking of endocytic trafficking. Instead, this reduction of endocytosis and increase in selective uptake was dependent on SR-BI. In addition, cell-permeable bile acids reduced HDL endocytosis by farnesoid X receptor (FXR) activation: chenodeoxycholate and the non-steroidal FXR agonist GW4064 reduced HDL endocytosis, whereas selective CE uptake was unaltered. Reduced HDL endocytosis by FXR activation was independent of SR-BI and was likely mediated by impaired expression of the scavenger receptor cluster of differentiation 36 (CD36). Taken together we have shown that bile acids reduce HDL endocytosis by transcriptional and non-transcriptional mechanisms. Further, we suggest that HDL endocytosis and selective lipid uptake are not necessarily tightly linked to each other.

  17. Kaempferol induces apoptosis in HepG2 cells via activation of the endoplasmic reticulum stress pathway.

    PubMed

    Guo, Haiqing; Ren, Feng; Zhang, Li; Zhang, Xiangying; Yang, Rongrong; Xie, Bangxiang; Li, Zhuo; Hu, Zhongjie; Duan, Zhongping; Zhang, Jing

    2016-03-01

    Kaempferol is a flavonoid compound that has gained importance due to its antitumor properties; however, the underlying mechanisms remain to be fully understood. The present study aimed to investigate the molecular mechanisms of the antitumor function of kaempferol in HepG2 hepatocellular carcinoma cells. Kaempferol was determined to reduce cell viability, increase lactate dehydrogenase activity and induce apoptosis in a concentration‑ and time‑dependent manner in HepG2 cells. Additionally, kaempferol‑induced apoptosis possibly acts via the endoplasmic reticulum (ER) stress pathway, due to the significant increase in the protein expression levels of glucose‑regulated protein 78, glucose‑regulated protein 94, protein kinase R‑like ER kinase, inositol‑requiring enzyme 1α, partial activating transcription factor 6 cleavage, caspase‑4, C/EBP homologous protein (CHOP) and cleaved caspase‑3. The pro‑apoptotic activity of kaempferol was determined to be due to induction of the ER stress‑CHOP pathway, as: i) ER stress was blocked by 4‑phenyl butyric acid (4‑PBA) pretreatment and knockdown of CHOP with small interfering RNA, which resulted in alleviation of kaempferol‑induced HepG2 cell apoptosis; and ii) transfection with plasmid overexpressing CHOP reversed the protective effect of 4‑PBA in kaempferol‑induced HepG2 cells and increased the apoptotic rate. Thus, kaempferol promoted HepG2 cell apoptosis via induction of the ER stress‑CHOP signaling pathway. These observations indicate that kaempferol may be used as a potential chemopreventive treatment strategy for patients with hepatocellular carcinoma.

  18. Cyclosporine A and palmitic acid treatment synergistically induce cytotoxicity in HepG2 cells

    SciTech Connect

    Luo, Yi Rana, Payal; Will, Yvonne

    2012-06-01

    Immunosuppressant cyclosporine A (CsA) treatment can cause severe side effects. Patients taking immunosuppressant after organ transplantation often display hyperlipidemia and obesity. Elevated levels of free fatty acids have been linked to the etiology of metabolic syndromes, nonalcoholic fatty liver and steatohepatitis. The contribution of free fatty acids to CsA-induced toxicity is not known. In this study we explored the effect of palmitic acid on CsA-induced toxicity in HepG2 cells. CsA by itself at therapeutic exposure levels did not induce detectible cytotoxicity in HepG2 cells. Co-treatment of palmitic acid and CsA resulted in a dose dependent increase in cytotoxicity, suggesting that fatty acid could sensitize cells to CsA-induced cytotoxicity at the therapeutic doses of CsA. A synergized induction of caspase-3/7 activity was also observed, indicating that apoptosis may contribute to the cytotoxicity. We demonstrated that CsA reduced cellular oxygen consumption which was further exacerbated by palmitic acid, implicating that impaired mitochondrial respiration might be an underlying mechanism for the enhanced toxicity. Inhibition of c-Jun N-terminal kinase (JNK) attenuated palmitic acid and CsA induced toxicity, suggesting that JNK activation plays an important role in mediating the enhanced palmitic acid/CsA-induced toxicity. Our data suggest that elevated FFA levels, especially saturated FFA such as palmitic acid, may be predisposing factors for CsA toxicity, and patients with underlying diseases that would elevate free fatty acids may be susceptible to CsA-induced toxicity. Furthermore, hyperlipidemia/obesity resulting from immunosuppressive therapy may aggravate CsA-induced toxicity and worsen the outcome in transplant patients. -- Highlights: ► Palmitic acid and cyclosporine (CsA) synergistically increased cytotoxicity. ► The impairment of mitochondrial functions may contribute to the enhanced toxicity. ► Inhibition of JNK activity attenuated

  19. Antioxidant Activity of Oat Proteins Derived Peptides in Stressed Hepatic HepG2 Cells

    PubMed Central

    Du, Yichen; Esfandi, Ramak; Willmore, William G.; Tsopmo, Apollinaire

    2016-01-01

    The purpose of this study was to determine, for the first time, antioxidant activities of seven peptides (P1–P7) derived from hydrolysis of oat proteins in a cellular model. In the oxygen radical absorbance capacity (ORAC) assay, it was found that P2 had the highest radical scavenging activity (0.67 ± 0.02 µM Trolox equivalent (TE)/µM peptide) followed by P5, P3, P6, P4, P1, and P7 whose activities were between 0.14–0.61 µM TE/µM). In the hepatic HepG2 cells, none of the peptides was cytotoxic at 20–300 µM. In addition to having the highest ORAC value, P2 was also the most protective (29% increase in cell viability) against 2,2′-azobis(2-methylpropionamidine) dihydrochloride -induced oxidative stress. P1, P6, and P7 protected at a lesser extent, with an 8%–21% increase viability of cells. The protection of cells was attributed to several factors including reduced production of intracellular reactive oxygen species, increased cellular glutathione, and increased activities of three main endogenous antioxidant enzymes. PMID:27775607

  20. Inhibition of MEK/ERK activation attenuates autophagy and potentiates pemetrexed-induced activity against HepG2 hepatocellular carcinoma cells.

    PubMed

    Tong, Yongxi; Huang, Haijun; Pan, Hongying

    2015-01-02

    Identification of efficient chemo-therapeutic/chemo-preventive agents for treatment of hepatocellular carcinoma (HCC) is important. In this study, we examined the activity of pemetrexed, an anti-folate chemotherapy drug, against HepG2 human HCC cells. Pemetrexed treatment in vitro exerted weak but significant cytotoxic activity against HepG2 cells. When analyzing the possible pemetrexed-resistance factors, we indentified that pemetrexed treatment in HepG2 cells induced cyto-protective autophagy activation, evidenced by GFP-light chain 3B (LC3B) puncta formation, p62 downregulation and Beclin-1/LC3B-II upregulation. Correspondingly, autophagy inhibitors, including bafliomycin A1, 3-methyladenine and chloroquine, enhanced pemetrexed-induced cytotoxicity against HepG2 cells. Further, RNAi-mediated knockdown of Beclin-1 in HepG2 cells also increased pemetrexed sensitivity. Pemetrexed activated MEK (mitogen-activated protein kinase/ERK kinase)/ERK (extracellular-signal-regulated kinase) signaling in HepG2 cells, which was required for autophagy induction. Pharmacological inhibition of MEK/ERK activation attenuated pemetrexed-induced autophagy, enhanced HepG2 cell death and apoptosis. In summary, pemetrexed activates MEK/ERK-dependent cyto-protective autophagy, and inhibition of this pathway potentiates pemetrexed's activity in HepG2 cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Investigation of the anti-cancer effect of quercetin on HepG2 cells in vivo.

    PubMed

    Zhou, Jin; Fang, Li; Liao, Jiaxu; Li, Lin; Yao, Wenxiu; Xiong, Zhujuan; Zhou, Xiang

    2017-01-01

    Quercetin, a natural polyphenolic flavonoid compound, can inhibit the growth of several malignant cancers. However, the mechanism still remains unclear. Our previous findings have suggested that quercetin can significantly inhibit HepG2 cell proliferation and induce cell apoptosis in vitro. It can also affect cell cycle distribution and significantly decrease cyclin D1 expression. In this study, we investigated the anti-cancer effect of quercetin on HepG2 tumor-bearing nude mice and its effect on cyclin D1 expression in the tumor tissue. First, the nude murine tumor model was established by subcutaneous inoculation of HepG2 cells, then quercetin was administered intraperitoneally, and the mice injected with saline solution were used as controls. The daily behavior of the tumor-bearing mice was observed and differences in tumor growth and survival rate were monitored. The expression of cyclin D1 in isolated tumor sections was evaluated by immunohistochemistry. We found that HepG2 tumor became palpable in the mice one-week post-inoculation. Tumors in the control group grew rapidly and the daily behavior of the mice changed significantly, including listlessness, poor feeding and ataxia. The mice in quercetin-treated group showed delayed tumor growth, no significant changes in daily behavior, and the survival rate was significantly improved. Finally, we observed increased tumor necrosis and a lighter cyclin D1 staining with reduced staining areas. Our findings thus suggest that quercetin can significantly inhibit HepG2 cell proliferation, and this effect may be achieved through the regulation of cyclin D1 expression.

  2. Investigation of the anti-cancer effect of quercetin on HepG2 cells in vivo

    PubMed Central

    Li, Lin; Yao, Wenxiu; Xiong, Zhujuan; Zhou, Xiang

    2017-01-01

    Quercetin, a natural polyphenolic flavonoid compound, can inhibit the growth of several malignant cancers. However, the mechanism still remains unclear. Our previous findings have suggested that quercetin can significantly inhibit HepG2 cell proliferation and induce cell apoptosis in vitro. It can also affect cell cycle distribution and significantly decrease cyclin D1 expression. In this study, we investigated the anti-cancer effect of quercetin on HepG2 tumor-bearing nude mice and its effect on cyclin D1 expression in the tumor tissue. First, the nude murine tumor model was established by subcutaneous inoculation of HepG2 cells, then quercetin was administered intraperitoneally, and the mice injected with saline solution were used as controls. The daily behavior of the tumor-bearing mice was observed and differences in tumor growth and survival rate were monitored. The expression of cyclin D1 in isolated tumor sections was evaluated by immunohistochemistry. We found that HepG2 tumor became palpable in the mice one-week post-inoculation. Tumors in the control group grew rapidly and the daily behavior of the mice changed significantly, including listlessness, poor feeding and ataxia. The mice in quercetin-treated group showed delayed tumor growth, no significant changes in daily behavior, and the survival rate was significantly improved. Finally, we observed increased tumor necrosis and a lighter cyclin D1 staining with reduced staining areas. Our findings thus suggest that quercetin can significantly inhibit HepG2 cell proliferation, and this effect may be achieved through the regulation of cyclin D1 expression. PMID:28264020

  3. HMGB1 release by human liver L02 and HepG2 cells induced by lipopolysaccharide.

    PubMed

    Huang, Ze-Bing; Dai, Xia-Hong; Xiao, Mei-Fang; Zhou, Rong-Rong; Zhao, Shu-Shan; Zhang, Bao-Xin; Yi, Pan-Pan; Chen, Ruo-Chan; Li, Wen-Ting; Yaser, Ai-Madhagi; Huang, Yan; Fan, Xue-Gong

    2013-07-01

    Liver cells release the high mobility group box-1 (HMGB1) protein when exposed to lipopolysaccharides (LPSs). However, the timing and levels of protein released remain unclear. The present study aimed to characterize the secretion of the late pro-inflammatory cytokine HMGB1 by liver L02 and HepG2 cells. The human mononuclear macrophage cell line U937 was used as a control. Various concentrations of LPS were added to human U937, L02 and HepG2 cells for different durations, and the cells were analyzed at different time-points following this addition. Reverse transcription polymerase chain reaction (RT-PCR) was used to measure cellular HMGB1 mRNA levels, western blotting was performed to detect HMGB1 in cellular supernatants and the translocation of HMGB1 from the nucleus to the cytosol was examined using immunofluorescence staining. L02 and HepG2 cells exhibited higher HMGB1 mRNA levels compared with the control U937 cells 20 and 24 h following continuous exposure to LPS. U937 cells exhibited higher HMGB1 mRNA levels compared with the corresponding L02 and HepG2 cells 16 h following LPS exposure. The phase of HMGB1 protein detected in the cellular supernatants of L02 and HepG2 cells (16 h) was later than that of U937 cells (8 h). For the three cell lines, HMGB1 levels demonstrated a time dependency; however, the protein level was the highest in U937 cells. In the three cell lines, translocation of HMGB1 from the nucleus to the cytosol occurred; however, the phases of HMGB1 translocation in L02 and HepG2 cells occurred later than in U937 cells. LPS-induced secretion of the late pro‑inflammatory cytokine HMGB1 by liver cells is characterized by a late phase of release and smaller quantity, and the process of HMGB1 secretion appears to be associated with HMGB1 translocation.

  4. Soya phytoestrogens, genistein and daidzein, decrease apolipoprotein B secretion from HepG2 cells through multiple mechanisms.

    PubMed

    Borradaile, Nica M; de Dreu, Linda E; Wilcox, Lisa J; Edwards, Jane Y; Huff, Murray W

    2002-09-01

    Diets containing the soya-derived phytoestrogens, genistein and daidzein, decrease plasma cholesterol in humans and experimental animals. The mechanisms responsible for the hypocholesterolaemic effects of these isoflavones are unknown. The present study was conducted to determine if genistein and daidzein regulate hepatocyte cholesterol metabolism and apolipoprotein (apo) B secretion in cultured human hepatoma (HepG2) cells. ApoB secretion was decreased dose-dependently by up to 63% and 71% by genistein and daidzein (100 microM; P<0.0001) respectively. In contrast, no effect on apoAI secretion was observed. Cellular cholesterol synthesis was inhibited 41% by genistein (100 microM; P<0.005) and 18% by daidzein (100 microM; P<0.05), which was associated with significant increases in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA. Cellular cholesterol esterification was decreased 56% by genistein (100 microM; P<0.04) and 29% by daidzein (100 microM; P<0.04); however, mRNA levels for acyl-CoA:cholesterol acyltransferase (ACAT) 1 and ACAT2 were unaffected. At 100 microM, both isoflavones equally inhibited the activities of both forms of ACAT in cells transfected with either ACAT1 or ACAT2. Genistein (100 microM) and daidzein (100 microM) significantly decreased the activity of microsomal triacylglycerol transfer protein (MTP) by 30% and 24% respectively, and significantly decreased MTP mRNA levels by 35% and 55%. Both isoflavones increased low-density lipoprotein (LDL)-receptor mRNA levels by 3- to 6-fold (100 microM; P<0.03) and significantly increased the binding, uptake and degradation of (125)I-labelled LDL, suggesting that enhanced reuptake of newly secreted apoB-containing lipoproteins contributed to the net decrease in apoB secretion. These results indicate that genistein and daidzein inhibit hepatocyte apoB secretion through several mechanisms, including inhibition of cholesterol synthesis and esterification, inhibition of MTP activity and expression and

  5. Soya phytoestrogens, genistein and daidzein, decrease apolipoprotein B secretion from HepG2 cells through multiple mechanisms.

    PubMed Central

    Borradaile, Nica M; de Dreu, Linda E; Wilcox, Lisa J; Edwards, Jane Y; Huff, Murray W

    2002-01-01

    Diets containing the soya-derived phytoestrogens, genistein and daidzein, decrease plasma cholesterol in humans and experimental animals. The mechanisms responsible for the hypocholesterolaemic effects of these isoflavones are unknown. The present study was conducted to determine if genistein and daidzein regulate hepatocyte cholesterol metabolism and apolipoprotein (apo) B secretion in cultured human hepatoma (HepG2) cells. ApoB secretion was decreased dose-dependently by up to 63% and 71% by genistein and daidzein (100 microM; P<0.0001) respectively. In contrast, no effect on apoAI secretion was observed. Cellular cholesterol synthesis was inhibited 41% by genistein (100 microM; P<0.005) and 18% by daidzein (100 microM; P<0.05), which was associated with significant increases in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA. Cellular cholesterol esterification was decreased 56% by genistein (100 microM; P<0.04) and 29% by daidzein (100 microM; P<0.04); however, mRNA levels for acyl-CoA:cholesterol acyltransferase (ACAT) 1 and ACAT2 were unaffected. At 100 microM, both isoflavones equally inhibited the activities of both forms of ACAT in cells transfected with either ACAT1 or ACAT2. Genistein (100 microM) and daidzein (100 microM) significantly decreased the activity of microsomal triacylglycerol transfer protein (MTP) by 30% and 24% respectively, and significantly decreased MTP mRNA levels by 35% and 55%. Both isoflavones increased low-density lipoprotein (LDL)-receptor mRNA levels by 3- to 6-fold (100 microM; P<0.03) and significantly increased the binding, uptake and degradation of (125)I-labelled LDL, suggesting that enhanced reuptake of newly secreted apoB-containing lipoproteins contributed to the net decrease in apoB secretion. These results indicate that genistein and daidzein inhibit hepatocyte apoB secretion through several mechanisms, including inhibition of cholesterol synthesis and esterification, inhibition of MTP activity and expression and

  6. 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.

  7. 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.

  8. Cytotoxicity evaluation of symmetrically branched glycerol trimer in human hepatocellular carcinoma HepG2 cells.

    PubMed

    Miyamoto, Licht; Watanabe, Masashi; Kono, Mai; Matsushita, Tsuyoshi; Hattori, Hatsuhiko; Ishizawa, Keisuke; Nemoto, Hisao; Tsuchiya, Koichiro

    2012-01-01

    An appropriate balance between lipophilicity and hydrophilicity is necessary for pharmaceuticals to achieve fine Absorption, Distribution, Metabolism and Excretion (ADME) properties including absorption and distribution, in particular. We have designed and proposed symmetrically branched oligoglycerols (BGL) as an alternative approach to improve the lipophilic-hydrophilic balance. We have previously shown that stability in circulation and water-solubility of such molecules as proteins, liposomes and hydrophobic compounds are much improved by conjugation to BGL. Albeit these successful applications of BGL, little was known whether BGL could be used in safety. Thus we conducted evaluation of the cytotoxicity of a representative BGL, symmetrically branched glycerol trimer (BGL003) in the cultured cells to clarify its biological safeness. Here we demonstrate that water-solubility of an extremely hydrophobic agent, fenofibrate was more than 2,000-fold improved just by conjugated with BGL003. BGL003 did not exhibit any significant cytotoxicity in human hepatocarcinoma HepG2 cells. Thus BGL003 should be safe and suitable strategy to endow hydrophobic molecules with much hydrophilicity.

  9. Enhanced cytotoxicity of pentachlorophenol by perfluorooctane sulfonate or perfluorooctanoic acid in HepG2 cells.

    PubMed

    Shan, Guoqiang; Ye, Minqiang; Zhu, Benzhan; Zhu, Lingyan

    2013-11-01

    Chlorinated phenols and perfluoroalkyl acids (PFAAs) are two kinds of pollutants which are widely present in the environment. Considering liver is the primary toxic target organ for these two groups of chemicals, it is interesting to evaluate the possible joint effects of them on liver. In this work, the combined toxicity of pentachlorophenol (PCP) and perfluorooctane sulfonate (PFOS) or perfluorooctanoic acid (PFOA) were investigated using HepG2 cells. The results indicated that PFOS and PFOA could strengthen PCP's hepatotoxicity. Further studies showed that rather than intensify the oxidative stress or promote the biotransformation of PCP, PFOS (or PFOA) might lead to strengthening of the oxidative phosphorylation uncoupling of PCP. By measuring the intracellular PCP concentration and the cell membrane properties, it was suggested that PFOS and PFOA could disrupt the plasma membrane and increase the membrane permeability. Thus, more cellular accessibility of PCP was induced when they were co-exposed to PCP and PFOS (or PFOA), leading to increased cytotoxicity. Further research is warranted to better understand the combined toxicity of PFAAs and other environmental pollutants. Copyright © 2013. Published by Elsevier Ltd.

  10. β3-Adrenoceptor activation upregulates apolipoprotein A-I expression in HepG2 cells, which might further promote cholesterol efflux from macrophage foam cells

    PubMed Central

    Gao, Xia-qing; Li, Yan-fang; Jiang, Zhi-li

    2017-01-01

    Objective The aim of this study was to explore the effects of β3-adrenoceptor (β3-AR) activation on HepG2 cells and its influence on cholesterol efflux from macrophage foam cells. Materials and methods HepG2 cells were cultured and treated with the β3-AR agonist, BRL37344, and antagonist, SR52390A, and the expression of apolipoprotein (Apo) A-I, ApoA-II, ApoB, and β3-AR in the supernatants and cells was determined. The expression of peroxisome proliferator-activated receptor (PPAR) γ and PPARα in the HepG2 cells was also assessed. Next, using the RAW264.7 macrophage foam cell model, we also assessed the influence of the HepG2 cell supernatants on lipid efflux. The cholesterol content of the foam cells was also measured, and the cholesterol efflux from the macrophages was examined by determining 3H-labeled cholesterol levels. Expression of ATP-binding cassette transporter (ABC) A1 and ABCG1 of the macrophage foam cells was also assessed. Results β3-AR activation increased ApoA-I expression in both the HepG2 cells and the supernatants; PPARγ expression was upregulated, but PPARα expression was not. Treatment with GW9662 abolished the increased expression of ApoA-I induced by the β3-AR agonist. The HepG2 cell supernatants decreased the lipid accumulation and increased the cholesterol efflux from the macrophage foam cells. ABCA1 expression, but not ABCG1 expression, increased in the macrophage foam cells treated with BRL37344-treated HepG2 cell supernatants. Conclusion Activation of β3-AR in HepG2 cells upregulates ApoA-I expression, which might further promote cholesterol efflux from macrophage foam cells. PPARγ might be required for the induction of ApoA-I expression. PMID:28424539

  11. β3-Adrenoceptor activation upregulates apolipoprotein A-I expression in HepG2 cells, which might further promote cholesterol efflux from macrophage foam cells.

    PubMed

    Gao, Xia-Qing; Li, Yan-Fang; Jiang, Zhi-Li

    2017-01-01

    The aim of this study was to explore the effects of β3-adrenoceptor (β3-AR) activation on HepG2 cells and its influence on cholesterol efflux from macrophage foam cells. HepG2 cells were cultured and treated with the β3-AR agonist, BRL37344, and antagonist, SR52390A, and the expression of apolipoprotein (Apo) A-I, ApoA-II, ApoB, and β3-AR in the supernatants and cells was determined. The expression of peroxisome proliferator-activated receptor (PPAR) γ and PPARα in the HepG2 cells was also assessed. Next, using the RAW264.7 macrophage foam cell model, we also assessed the influence of the HepG2 cell supernatants on lipid efflux. The cholesterol content of the foam cells was also measured, and the cholesterol efflux from the macrophages was examined by determining (3)H-labeled cholesterol levels. Expression of ATP-binding cassette transporter (ABC) A1 and ABCG1 of the macrophage foam cells was also assessed. β3-AR activation increased ApoA-I expression in both the HepG2 cells and the supernatants; PPARγ expression was upregulated, but PPARα expression was not. Treatment with GW9662 abolished the increased expression of ApoA-I induced by the β3-AR agonist. The HepG2 cell supernatants decreased the lipid accumulation and increased the cholesterol efflux from the macrophage foam cells. ABCA1 expression, but not ABCG1 expression, increased in the macrophage foam cells treated with BRL37344-treated HepG2 cell supernatants. Activation of β3-AR in HepG2 cells upregulates ApoA-I expression, which might further promote cholesterol efflux from macrophage foam cells. PPARγ might be required for the induction of ApoA-I expression.

  12. Effect of acetone extract from stem bark of Acacia species (A. dealbata, A. ferruginea and A. leucophloea) on antioxidant enzymes status in hydrogen peroxide-induced HepG2 cells.

    PubMed

    Sowndhararajan, Kandhasamy; Hong, Sunghyun; Jhoo, Jin-Woo; Kim, Songmun; Chin, Nyuk Ling

    2015-11-01

    Acacia species are multipurpose trees, widely used in the traditional systems of medicine to treat various ailments. The major objective of the present study was to determine the gene expression of enzymatic antioxidants by acetone extract from the stem bark of three Acacia species (Acacia dealbata, Acacia ferruginea and Acacia leucophloea) in hydrogen peroxide (H2O2)-induced human hepatoma (HepG2) cells. The expression of antioxidant enzymes such as superoxide dismutase containing copper-zinc (CuZnSOD)/manganese (MnSOD), catalase (CAT) and glutathione peroxidase (GPx) in HepG2 cells was evaluated by real-time PCR. The results of antioxidant enzyme expression in real-time PCR study revealed that the H2O2 (200 μM) challenged HepG2 cells reduced the expression of enzymes such as SOD, GPx and CAT. However, the cells pre-treated with acetone extracts of all the three Acacia species significantly (P > 0.05) up-regulated the expression of antioxidant enzymes in a concentration dependent manner (25, 50 and 75 μg/mL). In conclusion, the findings of our study demonstrated that the acetone extract of Acacia species effectively inhibited H2O2 mediated oxidative stress and may be useful as a therapeutic agent in preventing oxidative stress mediated diseases.

  13. Effect of acetone extract from stem bark of Acacia species (A. dealbata, A. ferruginea and A. leucophloea) on antioxidant enzymes status in hydrogen peroxide-induced HepG2 cells

    PubMed Central

    Sowndhararajan, Kandhasamy; Hong, Sunghyun; Jhoo, Jin-Woo; Kim, Songmun; Chin, Nyuk Ling

    2015-01-01

    Acacia species are multipurpose trees, widely used in the traditional systems of medicine to treat various ailments. The major objective of the present study was to determine the gene expression of enzymatic antioxidants by acetone extract from the stem bark of three Acacia species (Acacia dealbata, Acacia ferruginea and Acacia leucophloea) in hydrogen peroxide (H2O2)-induced human hepatoma (HepG2) cells. The expression of antioxidant enzymes such as superoxide dismutase containing copper–zinc (CuZnSOD)/manganese (MnSOD), catalase (CAT) and glutathione peroxidase (GPx) in HepG2 cells was evaluated by real-time PCR. The results of antioxidant enzyme expression in real-time PCR study revealed that the H2O2 (200 μM) challenged HepG2 cells reduced the expression of enzymes such as SOD, GPx and CAT. However, the cells pre-treated with acetone extracts of all the three Acacia species significantly (P > 0.05) up-regulated the expression of antioxidant enzymes in a concentration dependent manner (25, 50 and 75 μg/mL). In conclusion, the findings of our study demonstrated that the acetone extract of Acacia species effectively inhibited H2O2 mediated oxidative stress and may be useful as a therapeutic agent in preventing oxidative stress mediated diseases. PMID:26586994

  14. Insulin resistance contributes to multidrug resistance in HepG2 cells via activation of the PERK signaling pathway and upregulation of Bcl-2 and P-gp.

    PubMed

    Liu, Xinyue; Li, Linjing; Li, Jing; Cheng, Yan; Chen, Jing; Shen, Minghui; Zhang, Shangdi; Wei, Hulai

    2016-05-01

    Liver tumorigenesis frequently causes insulin resistance which may be used as an independent risk factor for evaluation of survival and post-surgery relapse of liver cancer patients. In the present study, HepG2/IR, an insulin resistant HepG2 cell line, was established by exposing HepG2 cells to 0.5 µmol/l of insulin for 72 h, and comparison of HepG2/IR with the parental HepG2 cells indicated that the HepG2/IR cells showed significantly enhanced resistance to the most frequently used chemotherapeutics for solid tumors, such as cisplatin, 5-fluorouracil, vincristine and mitomycin. Flow cytometric analysis of cisplatin-treated HepG2/IR cells showed a significantly decreased hypodiploid peak and a significantly downregulated expression level of pro-apoptotic protein caspase-3 compared with the parental HepG2 cells. Our data further showed swollen endoplasmic reticulum (ER) in the cisplatin-treated HepG2/IR cells with significantly increased levels of glucose-regulated protein 78 (GRP78), phosphorylated protein kinase R-like ER kinase (p-PERK) and P-glycoprotein (P-gp). There was also an upregulated expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) whereas no significant change was observed for CCAAT-enhancer-binding protein homologous protein (CHOP), which is known to be induced by ER stress and to mediate apoptosis. Our results demonstrated that insulin resistance in HepG2 cells promoted a protective unfolded protein response and upregulated the expression of ER chaperone protein GRP78, which resulted in the phosphorylation of PERK kinase to activate the PERK-mediated ER stress signal transduction pathway and the upregulation of Bcl-2 and P-gp, leading to the inhibition of the caspase-3-dependent apoptosis pathway and to the survival of liver tumor cells.

  15. A Homogeneous Polysaccharide from Fructus Schisandra chinensis (Turz.) Baill Induces Mitochondrial Apoptosis through the Hsp90/AKT Signalling Pathway in HepG2 Cells.

    PubMed

    Chen, Yonglin; Shi, Songshan; Wang, Huijun; Li, Ning; Su, Juan; Chou, Guixin; Wang, Shunchun

    2016-06-28

    According to the potential anti-hepatoma therapeutic effect of Schisandra chinensis polysaccharides presented in previous studies, a bioactive constituent, homogeneous Schisandra chinensis polysaccharide-0-1 (SCP-0-1), molecular weight (MW) circa 69.980 kDa, was isolated and purified. We assessed the efficacy of SCP-0-1 against human hepatocellular liver carcinoma (HepG2) cells to investigate the effects of its antitumour activity and molecular mechanisms. Anticancer activity was evaluated using microscopy, 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT) assay, Hoechst 33258 staining, acridine orange (AO) staining, flow cytometry (FCM), and cell-cycle analysis. SCP-0-1 inhibited the HepG2 cells' growth via inducing apoptosis and second gap/mitosis (G2/M) arrest dose-dependently, with a half maximal inhibitory concentration (IC50) value of 479.63 µg/mL. Western blotting of key proteins revealed the apoptotic and autophagic potential of SCP-0-1. Besides, SCP-0-1 upregulated Bcl-2 Associated X Protein (Bax) and downregulated B-cell leukemia/lymphoma 2 (Bcl-2) in the HepG2 cells. The expression of caspase-3, -8, and -9; poly (ADP-ribose) polymerase (PARP); cytochrome c (Cyt C); tumor protein 53 (p53); survivin; sequestosome 1 (p62); microtubule-associated protein 1 light chain-3B (LC3B); mitogen-activated protein kinase p38 (p38); extracellular regulated protein kinases (ERK); c-Jun N-terminal kinase (JNK); protein kinase B (AKT); and heat shock protein 90 (Hsp90) were evaluated using Western blotting. Our findings demonstrate a novel mechanism through which SCP-0-1 exerts its antiproliferative activity and induces mitochondrial apoptosis rather than autophagy. The induction of mitochondrial apoptosis was attributed to the inhibition of the Hsp90/AKT signalling pathway in an extracellular signal-regulated kinase-independent manner. The results also provide initial evidence on a molecular basis that SCP-0-1 can be used as an anti

  16. Ursolic acid sensitizes cisplatin-resistant HepG2/DDP cells to cisplatin via inhibiting Nrf2/ARE pathway

    PubMed Central

    Wu, Shouhai; Zhang, Tianpeng; Du, Jingsheng

    2016-01-01

    Background Combinations of adjuvant sensitizers with anticancer drugs is a promising new strategy to reverse chemoresistance. Ursolic acid (UA) is one of the natural pentacyclic triterpene compounds known to have many pharmacological characteristics such as anti-inflammatory and anticancer properties. This study investigates whether UA can sensitize hepatocellular carcinoma cells to cisplatin. Materials and methods Cells were transfected with nuclear factor erythroid-2-related factor 2 (Nrf2) small interfering RNA and Nrf2 complementary DNA by using Lipofectin 2000. The cytotoxicity of cells was investigated by Cell Counting Kit 8 assay. Cell apoptosis, cell cycle, reactive oxygen species, and mitochondrial membrane potential were detected by flow cytometry fluorescence-activated cell sorting. The protein level of Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), and heme oxygenase-1 (HO-1) was detected by Western blot analysis. Results The results showed that the reverse index was 2.9- and 9.69-fold by UA of 1.125 μg/mL and 2.25 μg/mL, respectively, for cisplatin to HepG2/DDP cells. UA–cisplatin combination induced cell apoptosis and reactive oxygen species, blocked the cell cycle in G0/G1 phase, and reduced the mitochondrial membrane potential. Mechanistically, UA–cisplatin dramatically decreased the expression of Nrf2 and its downstream genes. The sensibilization of UA–cisplatin combination was diminished in Nrf2 small interfering RNA-transfected HepG2/DDP cells, as well as in Nrf2 complementary DNA-transfected HepG2/DDP cells. Conclusion The results confirmed the sensibilization of UA on HepG2/DDP cells to cisplatin, which was possibly mediated via the Nrf2/antioxidant response element pathway. PMID:27822011

  17. 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.

  18. Cholesterol-lowing effect of taurine in HepG2 cell.

    PubMed

    Guo, Junxia; Gao, Ya; Cao, Xuelian; Zhang, Jing; Chen, Wen

    2017-03-16

    A number of studies indicate that taurine promotes cholesterol conversion to bile acids by upregulating CYP7A1 gene expression. Few in vitro studies are concerned the concentration change of cholesterol and its product of bile acids, and the molecular mechanism of CYP7A1 induction by taurine. The levels of intracellular total cholesterol (TC), free cholesterol (FC), cholesterol ester (EC), total bile acids (TBA) and medium TBA were determined after HepG2 cells were cultured for 24/48 h in DMEM supplemented with taurine at the final concentrations of 1/10/20 mM respectively. The protein expressions of CYP7A1, MEK1/2, c-Jun, p-c-Jun and HNF-4α were detected. Taurine significantly reduced cellular TC and FC in dose -and time-dependent ways, and obviously increased intracellular/medium TBA and CYP7A1 expressions. There was no change in c-Jun expression, but the protein expressions of MEK1/2 and p-c-Jun were increased at 24 h and inhibited at 48 h by 20 mM taurine while HNF4α was induced after both of the 24 h and 48 h treatment. Taurine could enhance CYP7A1 expression by inducing HNF4α and inhibiting MEK1/2 and p-c-Jun expressions to promote intracellular cholesterol metabolism.

  19. Evaluation of anti-hepatocarcinoma capacity of puerarin nanosuspensions against human HepG2 cells

    NASA Astrophysics Data System (ADS)

    Meng, Xiang-Ping; Zhang, Zhen; Wang, Yi-Fei; Wang, Zhi-ping; Chen, Tong-sheng

    2017-02-01

    Hepatocarcinoma, a malignant cancer, threaten human life badly. It is a current issue to seek the effective natural remedy from plant to treat cancer due to the resistance of the advanced hepatocarcinoma to chemotherapy. Puerarin (Pue), a major active ingredient in the traditional Chinese medicine Gegen, has a wide range of pharmacological properties and is considered to have anti-hepatocarcinoma effects. However its low oral bioavailability restricts its wide application. In this report, Pue nanosuspension (Pue-NS) composed of Pue and poloxamer 188 was prepared by high pressure homogenization technique. The in vitro anti-hepatocarcinoma effects of Pue-NS relative to efficacy of bulk Pue were evaluated. The particle size and zeta potential of Pue-NS were 218.5 nm and -18.8 mV, respectively. MTT assay showed that Pue-NS effectively inhibited the proliferation of HepG2 cells, and the corresponding IC50 values of Pue-NS and bulk Pue were 3.39 and 5.73 μg/ml. These results suggest that the delivery of Pue-NS is a promising approach for treating tumors.

  20. Does Resveratrol Improve Insulin Signalling in HepG2 Cells?

    PubMed

    Norouzzadeh, Marjan; Amiri, Fatemehsadat; Saboor-Yaraghi, Ali Akbar; Shemirani, Farnoosh; Kalikias, Yas; Sharifi, Loghman; Seyyedsalehi, Monireh Sadat; Mahmoudi, Maryam

    2017-04-01

    Diabetes mellitus is a common metabolic disorder with high global prevalence. It is characterized by a decrease in insulin secretion or a decrease in insulin sensitivity or both. The aim of the present study was to investigate the effects of resveratrol treatment on the expression of the genes involved in insulin signalling cascade, such as Forkhead box protein O1 (FoxO1), 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and mammalian target of rapamycin (mTOR). HepG2 cells were cultured in serum-free medium with high concentrations of glucose and insulin and then were treated with resveratrol (5, 10 and 20 µM) for 24 and 48 hours. Complementary deoxyribonucleic acids (cDNAs) were synthesized followed by RNA extraction. Real-time quantitative reverse transcription polymerase chain reaction was used to analyze the expression of FoxO1, PDPK1 and mTOR. Resveratrol increased the expression of PDPK1, mTOR and FoxO1. No significant difference was seen among differing dosages of resveratrol, but treatments for 48 hours exerted the greatest effectiveness. Our results were consistent with other studies showing the beneficial effects of resveratrol on diabetes. However, considering the effects of resveratrol in increasing FoxO1 and gluconeogenic gene expression, long-term usage of resveratrol should be investigated in greater depth in future studies. Copyright © 2016 Canadian Diabetes Association. Published by Elsevier Inc. All rights reserved.

  1. Crosstalk between microRNA-122 and FOX family genes in HepG2 cells.

    PubMed

    Kumar, Subodh; Batra, Ankita; Kanthaje, Shruthi; Ghosh, Sujata; Chakraborti, Anuradha

    2017-02-01

    MicroRNA-122 (miR-122) is liver specific and plays an important role in physiology as well as diseases including hepatocellular carcinoma (HCC). Downregulation of miR-122 in HCC modulates apoptosis. Similarly, the putative targets of miR-122, the forkhead box (FOX) family genes also play an important role in the regulation of apoptosis. Hence, an interplay between miR-122 and FOX family genes has been explored in this study. Initially, an augmentation of apoptosis was noticed in HepG2 cells after transfection with miR-122. Further, the predicted miR-122 targets, the FOX family genes ( FOXM1b, FOXP1, and FOXO4) were selected via in silico analysis based on their role in apoptosis. We checked the expression of all these genes at transcript level after the transfection of miR-122 and found that the relative expression of FOXP1 and FOXM1b was significantly downregulated (p < 0.005) and that of FOXO4 was upregulated (p < 0.005). Thus, the finding indicates deregulation of these FOX genes as a result of miR-122 augmentation might be involved in the modulation of apoptosis.

  2. Black soybean seed coat polyphenols prevent B(a)P-induced DNA damage through modulating drug-metabolizing enzymes in HepG2 cells and ICR mice.

    PubMed

    Zhang, Tianshun; Jiang, Songyan; He, Chao; Kimura, Yuki; Yamashita, Yoko; Ashida, Hitoshi

    2013-04-15

    Black soybean seed coat is a rich source of polyphenols that have been reported to have various physiological functions. The present study investigated the potential protective effects of polyphenolic extracts from black soybean seed coat on DNA damage in human hepatoma HepG2 cells and ICR mice. The results from micronucleus (MN) assay revealed that black soybean seed coat extract (BE) at concentrations up to 25μg/mL was non-genotoxic. It is noteworthy that BE (at 4.85μg/mL) and its main components, procyanidins (PCs) and cyanidin 3-glucoside (C3G), at 10μM significantly reduced the genotoxic effect induced by benzo[a]pyrene [B(a)P]. To obtain insights into the underlying mechanism, we investigated BE and its main components on drug-metabolizing enzyme expression. The results of this study demonstrate that BE and its main components, PCs and C3G, down-regulated B(a)P-induced cytochrome P4501A1 (CYP1A1) expression by inhibiting the transformation of aryl hydrocarbon receptor. Moreover, they increased expression of detoxifying defense enzymes, glutathione S-transferases (GSTs) via increasing the binding of nuclear factor-erythroid-2-related factor 2 to antioxidant response elements. Collectively, we found that PCs and C3G, which are the main active compounds of BE, down-regulated CYP1A1 and up-regulated GST expression to protect B(a)P-induced DNA damage in HepG2 cells and ICR mice effectively. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. ROS generation and JNK activation contribute to 4-methoxy-TEMPO-induced cytotoxicity, autophagy, and DNA damage in HepG2 cells.

    PubMed

    Zhang, Zhuhong; Ren, Zhen; Chen, Si; Guo, Xiaoqing; Liu, Fang; Guo, Lei; Mei, Nan

    2017-10-09

    4-Methoxy-TEMPO, a derivative of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), is a stable nitroxide radical and is generally used in organic and pharmaceutical syntheses for the oxidation of alcohols. Previously, we reported the involvement of reactive oxygen species (ROS) and c-Jun N-terminal kinases (JNK) in TEMPO-induced apoptosis in mouse L5178Y cells. In this study, we investigated 4-methoxy-TEMPO induced toxicity in human HepG2 hepatoma cells and its underlying mechanisms. Treatments with 4-methoxy-TEMPO (0.5-5 mM for 2-6 h) caused oxidative stress as demonstrated by increased intensity of the ROS indicator H2DCF-DA, decreased levels of glutathione. 4-Methoxy-TEMPO treatment also induced DNA damage as characterized by increased levels of DNA tail intensity in the Comet assay, increased phosphorylation of related proteins including γ-H2A.X, p-Chk1, and p-Chk2, and activation of MAPK signaling pathways. In addition, 4-methoxy-TEMPO also induced autophagy as demonstrated by the conversion of LC3B-I to II, decreased level of p62, and the appearance of GFP-LC3B punctae. To investigate the crosstalk between different signaling pathways, pretreatment of HepG2 with N-acetylcysteine, an ROS scavenger, attenuated 4-methoxy-TEMPO-induced DNA damage, suppressed JNK activation, and diminished autophagy induction. Furthermore, inhibiting JNK activation by a JNK-specific inhibitor, SP600125, decreased DNA damage levels induced by 4-methoxy-TEMPO. These results suggest that multiple mechanisms including ROS generation, DNA damage, and MAPK activation contribute to 4-methoxy-TEMPO-induced toxicity.

  4. [PCR analysis of the absolute number of copies of human chromosome 18 transcripts in liver and HepG2 cells].

    PubMed

    Kiseleva, Y Y; Ptitsyn, K G; Tikhonova, O V; Radko, S P; Kurbatov, L K; Vakhrushev, I V; Zgoda, V G; Ponomarenko, E A; Lisitsa, A V; Archakov, A I

    2017-03-01

    Using reverse transcription in conjunction with the quantitative real-time PCR or digital droplet PCR, the transcriptome profiling of human chromosome 18 has been carried out in liver hepatocytes and hepatoblastoma cells (HepG2 cell line) in terms of the absolute number of each transcript per cell. The transcript abundance varies within the range of 0.006 to 9635 and 0.011 to 4819 copies per cell for HepG2 cell line and hepatocytes, respectively. The expression profiles for genes of chromosome 18 in hepatocytes and HepG2 cells were found to significantly correlate: the Spearman's correlation coefficient was equal to 0.81. The distribution of frequency of transcripts over their abundance was bimodal for HepG2 cells and unimodal for liver hepatocytes. Bioinformatic analysis of the differential gene expression has revealed that genes of chromosome 18, overexpressed in HepG2 cells compared to hepatocytes, are associated with cell division and cell adhesion processes. It is assumed that the enhanced expression of those genes in HepG2 cells is related to the proliferation activity of cultured cells. The differences in transcriptome profiles have to be taken into account when modelling liver hepatocytes with cultured HepG2 cells.

  5. Alcohol Dehydrogenase 5 Is a Source of Formate for De Novo Purine Biosynthesis in HepG2 Cells.

    PubMed

    Bae, Sajin; Chon, James; Field, Martha S; Stover, Patrick J

    2017-04-01

    Background: Formate provides one-carbon units for de novo purine and thymidylate (dTMP) synthesis and is produced via both folate-dependent and folate-independent pathways. Folate-independent pathways are mediated by cytosolic alcohol dehydrogenase 5 (ADH5) and mitochondrial aldehyde dehydrogenase 2 (ALDH2), which generate formate by oxidizing formaldehyde. Formate is a potential biomarker of B-vitamin-dependent one-carbon metabolism.Objective: This study investigated the contributions of ADH5 and ALDH2 to formate production and folate-dependent de novo purine and dTMP synthesis in HepG2 cells.Methods:ADH5 knockout and ALDH2 knockdown HepG2 cells were cultured in folate-deficient [0 nM (6S) 5-formyltetrahydrofolate] or folate-sufficient [25 nM (6S) 5-formyltetrahydrofolate] medium. Purine biosynthesis was quantified as the ratio of [(14)C]-formate to [(3)H]-hypoxanthine incorporated into genomic DNA, which indicates the contribution of the de novo purine synthesis pathway relative to salvage synthesis. dTMP synthesis was quantified as the ratio of [(14)C]-deoxyuridine to [(3)H]-thymidine incorporation into genomic DNA, which indicates the capacity of de novo dTMP synthesis relative to salvage synthesis.Results: The [(14)C]-formate-to-[(3)H]-hypoxanthine ratio was greater in ADH5 knockout than in wild-type HepG2 cells, under conditions of both folate deficiency (+30%; P < 0.001) and folate sufficiency (+22%; P = 0.02). These data indicate that ADH5 deficiency increases the use of exogenous formate for de novo purine biosynthesis. The [(14)C]-deoxyuridine-to-[(3)H]-thymidine ratio did not differ between ADH5 knockout and wild-type cells, indicating that ADH5 deficiency does not affect de novo dTMP synthesis capacity relative to salvage synthesis. Under folate deficiency, ALDH2 knockdown cells exhibited a 37% lower ratio of [(14)C]-formate to [(3)H]-hypoxanthine (P < 0.001) compared with wild-type HepG2 cells, indicating decreased use of exogenous formate, or

  6. Human hepatitis B virus X protein induces apoptosis in HepG2 cells: Role of BH3 domain

    SciTech Connect

    Lu, Y.W.; Chen, W.N. . E-mail: WNChen@ntu.edu.sg

    2005-12-23

    The smallest protein of hepatitis B virus, HBX, has been implicated in the development of liver diseases by interfering with normal cellular processes. Its role in cell proliferation has been unclear as both pro-apoptotic and anti-apoptotic activities have been reported. We showed molecular evidence that HBX induced apoptosis in HepG2 cells. A Bcl-2 Homology Domain 3 was identified in HBX, which interacted with anti-apoptotic but not pro-apoptotic members of the Bcl-2 family of proteins. HBX induced apoptosis when transfected into HepG2 cells, as demonstrated by both flow cytometry and caspase-3 activity. However, HBX protein may not be stable in apoptotic cells triggered by its own expression as only its mRNA or the fusion protein with the glutathione-S-transferase was detected in transfected cells. Our results suggested that HBX behaved as a pro-apoptotic protein and was able to induce apoptosis.

  7. A Homogeneous Polysaccharide from Fructus Schisandra chinensis (Turz.) Baill Induces Mitochondrial Apoptosis through the Hsp90/AKT Signalling Pathway in HepG2 Cells

    PubMed Central

    Chen, Yonglin; Shi, Songshan; Wang, Huijun; Li, Ning; Su, Juan; Chou, Guixin; Wang, Shunchun

    2016-01-01

    According to the potential anti-hepatoma therapeutic effect of Schisandra chinensis polysaccharides presented in previous studies, a bioactive constituent, homogeneous Schisandra chinensis polysaccharide-0-1 (SCP-0-1), molecular weight (MW) circa 69.980 kDa, was isolated and purified. We assessed the efficacy of SCP-0-1 against human hepatocellular liver carcinoma (HepG2) cells to investigate the effects of its antitumour activity and molecular mechanisms. Anticancer activity was evaluated using microscopy, 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT) assay, Hoechst 33258 staining, acridine orange (AO) staining, flow cytometry (FCM), and cell-cycle analysis. SCP-0-1 inhibited the HepG2 cells’ growth via inducing apoptosis and second gap/mitosis (G2/M) arrest dose-dependently, with a half maximal inhibitory concentration (IC50) value of 479.63 µg/mL. Western blotting of key proteins revealed the apoptotic and autophagic potential of SCP-0-1. Besides, SCP-0-1 upregulated Bcl-2 Associated X Protein (Bax) and downregulated B-cell leukemia/lymphoma 2 (Bcl-2) in the HepG2 cells. The expression of caspase-3, -8, and -9; poly (ADP-ribose) polymerase (PARP); cytochrome c (Cyt C); tumor protein 53 (p53); survivin; sequestosome 1 (p62); microtubule-associated protein 1 light chain-3B (LC3B); mitogen-activated protein kinase p38 (p38); extracellular regulated protein kinases (ERK); c-Jun N-terminal kinase (JNK); protein kinase B (AKT); and heat shock protein 90 (Hsp90) were evaluated using Western blotting. Our findings demonstrate a novel mechanism through which SCP-0-1 exerts its antiproliferative activity and induces mitochondrial apoptosis rather than autophagy. The induction of mitochondrial apoptosis was attributed to the inhibition of the Hsp90/AKT signalling pathway in an extracellular signal-regulated kinase-independent manner. The results also provide initial evidence on a molecular basis that SCP-0-1 can be used as an anti

  8. Tat-DJ-1 enhances cell survival by inhibition of oxidative stress, NF-κB and MAPK activation in HepG2 cells.

    PubMed

    Jo, Hyo Sang; Yeo, Eun Ji; Shin, Min Jea; Choi, Yeon Joo; Yeo, Hyeon Ji; Cho, Su Bin; Park, Jung Hwan; Lee, Chi Hern; Eum, Won Sik; Choi, Soo Young

    2017-04-01

    To identify the protective effect of DJ-1 protein against oxidative stress-induced HepG2 cell death, we used cell-permeable wild type (WT) and a mutant (C106A Tat-DJ-1) protein. By using western blotting and fluorescence microscopy, we observed WT and C106A Tat-DJ-1 proteins were efficiently transduced into HepG2 cells. Transduced WT Tat-DJ-1 proteins increased cell survival and protected against DNA fragmentation and intracellular ROS generation levels in H2O2-exposed HepG2 cells. At the same time, transduced WT Tat-DJ-1 protein significantly inhibited NF-κB and MAPK (JNK and p38) activation as well as regulated the Bcl-2 and Bax expression levels. However, C106A Tat-DJ-1 protein did not show any protective effect against cell death responses in H2O2-exposed HepG2 cells. Oxidative stress-induced HepG2 cell death was significantly reduced by transduced WT Tat-DJ-1 protein, not by C106A Tat-DJ-1 protein. Thus, transduction of WT Tat-DJ-1 protein could be a novel strategy for promoting cell survival in situations of oxidative stress-induced HepG2 cell death.

  9. Effect of sevoflurane on human hepatocellular carcinoma HepG2 cells under conditions of high glucose and insulin.

    PubMed

    Nishiwada, Tadashi; Kawaraguchi, Yoshitaka; Uemura, Keiko; Sugimoto, Hiroshi; Kawaguchi, Masahiko

    2015-10-01

    Diabetes mellitus is associated with morbidity and progression of some cancers, such as hepatocellular carcinoma. It has been reported that sevoflurane, a volatile anesthetic agent commonly used in cancer surgery, can lead to lower overall survival rates than those observed when propofol is used to treat cancer patients, and sevoflurane increases cancer cell proliferation in in vitro studies. It has been also reported that glucose levels in rats anesthetized with sevoflurane were higher than those in rats anesthetized with propofol. We investigated the effect of sevoflurane, under conditions of high glucose and insulin, on cell proliferation in the human hepatocellular carcinoma cell line, HepG2. First, we exposed HepG2 cells to sevoflurane at 1 or 2 % concentration for 6 h in various glucose concentrations and then evaluated cell proliferation using the MTT assay. Subsequently, to mimic diabetic conditions observed during surgery, HepG2 cells were exposed to sevoflurane at 1 or 2 % concentration in high glucose concentrations at various concentrations of insulin for 6 h. One-percent sevoflurane exposure enhanced cell proliferation under conditions of high glucose, treated with 0.05 mg/l insulin. Our study implies that sevoflurane may affect cell proliferation in human hepatocellular carcinoma cells in a physiological situation mimicking that of diabetes.

  10. Restoration of miR-20a expression suppresses cell proliferation, migration, and invasion in HepG2 cells

    PubMed Central

    Chen, Guang Shun; Zhou, Ning; Li, Jie-Qun; Li, Ting; Zhang, Zhong-Qiang; Si, Zhong-Zhou

    2016-01-01

    Objective To study microRNA (miR)-20a expression in hepatocellular carcinoma (HCC) and its effects on the proliferation, migration, and invasion of HepG2. Methods The real-time polymerase chain reaction was used to detect the expression of miR-20a in HCC tissue and normal tissue, as well as in HCC cell lines and normal liver cells. miR-20a mimic and miR negative control (NC) were transfected into HepG2 cells. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay was used to detect cell proliferation. Annexin fluorescein isothiocyanate/propidium iodide assay was run to examine the early apoptosis of cells. Transwell chamber assay was carried out to investigate the cell invasion and migration abilities. Results miR-20a was lowly expressed both in HCC tissues and HCC cell lines. After transfection of exogenous miR-20 mimics, miR-20a expression in HepG2 cells was significantly increased by 61.29% compared to the blank group (P<0.01). MTT assay showed that the growth of HepG2 cells in the miR-20a mimics group was significantly inhibited, and optical density values during the 36–96 hour time period were dramatically decreased compared to the blank group (P<0.01). Apoptosis rates of the miR-20a mimics group were higher than those of the blank and NC groups (both P<0.01). The number of HCC cells after transfection by miR-20a mimics in the G1 and S phases were 15.88% and 7.89%, respectively, which were lower than in the blank and NC groups (both P<0.05). Transwell assay showed that in the miR-20a mimics group the number of cell migration and invasion were 0.459 and 0.501 times that of the blank group (both P<0.01), and the migration and inhibition rates were 54.1% and 51.4%, respectively. After closing target gene CCND1 in HepG2 cells, the number of cell migration and invasion in the small interfering (si)-CCND1 group were 0.444 and 0.435 times that of the si-NC group (P<0.05); and compared to the si-NC group, the migration and inhibition rates

  11. Restoration of miR-20a expression suppresses cell proliferation, migration, and invasion in HepG2 cells.

    PubMed

    Chen, Guang Shun; Zhou, Ning; Li, Jie-Qun; Li, Ting; Zhang, Zhong-Qiang; Si, Zhong-Zhou

    2016-01-01

    To study microRNA (miR)-20a expression in hepatocellular carcinoma (HCC) and its effects on the proliferation, migration, and invasion of HepG2. The real-time polymerase chain reaction was used to detect the expression of miR-20a in HCC tissue and normal tissue, as well as in HCC cell lines and normal liver cells. miR-20a mimic and miR negative control (NC) were transfected into HepG2 cells. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay was used to detect cell proliferation. Annexin fluorescein isothiocyanate/propidium iodide assay was run to examine the early apoptosis of cells. Transwell chamber assay was carried out to investigate the cell invasion and migration abilities. miR-20a was lowly expressed both in HCC tissues and HCC cell lines. After transfection of exogenous miR-20 mimics, miR-20a expression in HepG2 cells was significantly increased by 61.29% compared to the blank group (P<0.01). MTT assay showed that the growth of HepG2 cells in the miR-20a mimics group was significantly inhibited, and optical density values during the 36-96 hour time period were dramatically decreased compared to the blank group (P<0.01). Apoptosis rates of the miR-20a mimics group were higher than those of the blank and NC groups (both P<0.01). The number of HCC cells after transfection by miR-20a mimics in the G1 and S phases were 15.88% and 7.89%, respectively, which were lower than in the blank and NC groups (both P<0.05). Transwell assay showed that in the miR-20a mimics group the number of cell migration and invasion were 0.459 and 0.501 times that of the blank group (both P<0.01), and the migration and inhibition rates were 54.1% and 51.4%, respectively. After closing target gene CCND1 in HepG2 cells, the number of cell migration and invasion in the small interfering (si)-CCND1 group were 0.444 and 0.435 times that of the si-NC group (P<0.05); and compared to the si-NC group, the migration and inhibition rates were 55.6% and 56

  12. Prevention of phosphine-induced cytotoxicity by nutrients in HepG2 cells

    PubMed Central

    Rashedinia, Marzieh; Jamshidzadeh, Akram; Mehrabadi, Abbas Rezaiean; Niknahad, Hossein

    2016-01-01

    Background & objectives: Phosphides used as an insecticide and rodenticide, produce phosphine (PH3) which causes accidental and intentional poisoning cases and deaths. There is no specific treatment or antidote available for PH3 poisoning. It is suggested that PH3-induced toxicity is associated with adenosine triphosphate (ATP) depletion; therefore, in this study the effect of some nutrients was evaluated on PH3 cytotoxicity in a cell culture model. Methods: PH3 was generated from reaction of zinc phosphide (10 mM) with water in the closed culture medium of HepG2 cells, and cytotoxicity was measured after one and three hours of incubation. ATP, glutathione (GSH) and lipid peroxidation were also assessed at one or three hours post-incubation. ATP suppliers including dihydroxyacetone, glyceraldehyde and fructose were added to the culture medium 10 min before PH3 generation to prevent or reduce phosphine-induced cytotoxicity. Results: Phosphine caused about 30 and 66 per cent cell death at one and three hours of incubation, respectively. ATP content of the cells was depleted to 14.7 per cent of control at one hour of incubation. ATP suppliers were able to prevent cytotoxicity and ATP depletion induced by PH3. Dihydroxyacetone, α-ketoglutarate, fructose and mannitol restored the ATP content of the cells from 14.7 per cent to about 40, 34, 32 and 30 per cent, respectively. Lipid peroxidation and GSH depletion were not significantly induced by zinc phosphide in this study. Interpretation & conclusions: The results supported the hypothesis that phosphine-induced cytotoxicity was due to decrease of ATP levels. ATP suppliers could prevent its toxicity by generating ATP through glycolysis. α-keto compounds such as dihydroxyacetone and α-ketoglutarate may bind to phosphine and restore mitochondrial respiration. PMID:28256464

  13. Prevention of phosphine-induced cytotoxicity by nutrients in HepG2 cells.

    PubMed

    Rashedinia, Marzieh; Jamshidzadeh, Akram; Mehrabadi, Abbas Rezaiean; Niknahad, Hossein

    2016-10-01

    Phosphides used as an insecticide and rodenticide, produce phosphine (PH3) which causes accidental and intentional poisoning cases and deaths. There is no specific treatment or antidote available for PH3poisoning. It is suggested that PH3-induced toxicity is associated with adenosine triphosphate (ATP) depletion; therefore, in this study the effect of some nutrients was evaluated on PH3cytotoxicity in a cell culture model. PH3was generated from reaction of zinc phosphide (10 mM) with water in the closed culture medium of HepG2 cells, and cytotoxicity was measured after one and three hours of incubation. ATP, glutathione (GSH) and lipid peroxidation were also assessed at one or three hours post-incubation. ATP suppliers including dihydroxyacetone, glyceraldehyde and fructose were added to the culture medium 10 min before PH3generation to prevent or reduce phosphine-induced cytotoxicity. Phosphine caused about 30 and 66 per cent cell death at one and three hours of incubation, respectively. ATP content of the cells was depleted to 14.7 per cent of control at one hour of incubation. ATP suppliers were able to prevent cytotoxicity and ATP depletion induced by PH3. Dihydroxyacetone, α-ketoglutarate, fructose and mannitol restored the ATP content of the cells from 14.7 per cent to about 40 , 34 , 32 and 30 per cent, respectively. Lipid peroxidation and GSH depletion were not significantly induced by zinc phosphide in this study. The results supported the hypothesis that phosphine-induced cytotoxicity was due to decrease of ATP levels. ATP suppliers could prevent its toxicity by generating ATP through glycolysis. α-keto compounds such as dihydroxyacetone and α-ketoglutarate may bind to phosphine and restore mitochondrial respiration.

  14. Influence of diquat on growth and death of HepG2 cells using quartz crystal and micro CCD camera.

    PubMed

    Kang, Hyen-Wook; Lee, Dong-Yun; Muramatsu, Hiroshi; Lee, Burm-Jong; Kwon, Young-Soo

    2011-05-01

    Diquat is widely used agent which produces toxicity in human and implicated as an environmental toxicity. HepG2 cell was cultured onto an indium tin oxide (ITO) surface of quartz crystal modified a collagen film. In this paper, we investigated the physical properties and the morphological change of the HepG2 cells cultured onto the ITO electrode of the quartz crystal sensor with micro CCD camera. The resonance responses of the quartz crystal and the morphological change were directly monitored. After seeding the cells and diquat injection into the chamber, the resonance frequency and the resonance resistance were obtained with real time morphologies. From the resonance characteristics and the series of morphologies, we could know the diquat to be death and weakening of the cells.

  15. Quercetin Induces Antiproliferative Activity Against Human Hepatocellular Carcinoma (HepG2) Cells by Suppressing Specificity Protein 1 (Sp1).

    PubMed

    Lee, Ra Ham; Cho, Jin Hyoung; Jeon, Young-Joo; Bang, Woong; Cho, Jung-Jae; Choi, Nag-Jin; Seo, Kang Seok; Shim, Jung-Hyun; Chae, Jung-Il

    2015-02-01

    Preclinical Research Quercetin, found in red onions and red apple skin can induce apoptosis insome malignant cells. However, the apoptotic effect of quercetin in hepatocellular carcinoma HepG2 cells via regulation of specificity protein 1 (Sp1) has not been studied. Here, we demonstrated that quercetin decreased cell growth and induce apoptosis in HepG2 cells via suppression of Sp1 using 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, 4',6-diamidino-2-phenylindole (DAPI) staining, Annexin V, and Western blot analysis, an effect that was dose- and time-dependent manner. Treatment of HepG2 cells with quercetin reduced cell growth and induced apoptosis, followed by regulation of Sp1 and Sp1 regulatory protein. Taken together, the results suggest that quercetin can induce apoptotic cell death by regulating cell cycle and suppressing antiapoptotic proteins. Therefore, quercetin may be useful for cancer prevention. Drug Dev Res 76 : 9-16, 2015. © 2015 Wiley Periodicals, Inc.

  16. Activation of apoptosis by ethyl acetate fraction of ethanol extract of Dianthus superbus in HepG2 cell line.

    PubMed

    Yu, Jian-Qing; Yin, Yan; Lei, Jia-Chuan; Zhang, Xiu-Qiao; Chen, Wei; Ding, Cheng-Li; Wu, Shan; He, Xiao-Yu; Liu, Yan-Wen; Zou, Guo-Lin

    2012-02-01

    Dianthus superbus L. is commonly used as a traditional Chinese medicine. We recently showed that ethyl acetate fraction (EE-DS) from ethanol extract of D. superbus exhibited the strongest antioxidant and cytotoxic activities. In this study, we examined apoptosis of HepG2 cells induced by EE-DS, and the mechanism underlying apoptosis was also investigated. Treatment of HepG2 cells with EE-DS (20-80 μg/ml) for 48 h led to a significant dose-dependent increase in the percentage of cells in sub-G1 phase by analysis of the content of DNA in cells, and a large number of apoptotic bodies containing nuclear fragments were observed in cells treated with 80 μg/ml of EE-DS for 24 h by using Hoechst 33258 staining. These data show that EE-DS can induce apoptosis of HepG2 cells. Immunoblot analysis showed that EE-DS significantly suppressed the expressions of Bcl-2 and NF-κB. Treatment of cells with EE-DS (80 μg/ml) for 48 h resulted in significant increase of cytochrome c in the cytosol, which indicated cytochrome c release from mitochondria. Activation of caspase-9 and -3 were also determined when the cells treated with EE-DS. The results suggest that apoptosis of HepG2 cells induced by EE-DS could be through the mitochondrial intrinsic pathway. High performance liquid chromatography (HPLC) data showed that the composition of EE-DS is complicated. Further studies are needed to find the effective constituents of EE-DS.

  17. Realgar quantum dots induce apoptosis and necrosis in HepG2 cells through endoplasmic reticulum stress

    PubMed Central

    QIN, YU; WANG, HUAN; LIU, ZHENG-YUN; LIU, JIE; WU, JIN-ZHU

    2015-01-01

    Realgar (As4S4) has been used in traditional Chinese medicines for treatment of malignancies. However, the poor water solubility of realgar limits its clinical application. To overcome this problem, realgar quantum dots (RQDs; 5.48±1.09 nm) were prepared by a photoluminescence method. The mean particle size was characterized by high-resolution transmission electron microscopy and scanning electron microscopy. Our recent studies revealed that the RQDs were effective against tumor growth in tumor-bearing mice without producing apparent toxicity. The present study investigated their anticancer effects and mechanisms in human hepatocellular carcinoma (HepG2) cells. The HepG2 cells and human normal liver (L02) cells were used to determine the cytotoxicity of RQDs. The portion of apoptotic and dead cells were measured by flow cytometry with Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Apoptosis-related proteins and genes were examined by western blot analysis and reverse transcription-quantitative polymerase chain reaction, and the mitochondrial membrane potential was assayed by confocal microscope with JC-1 as a probe. RQDs exhibited cytotoxicity in a concentration-dependent manner and HepG2 cells were more sensitive compared with normal L02 cells. At 15 µg/ml, 20% of the cells were apoptotic, while 60% of the cells were necrotic at 30 µg/ml. The anti-apoptosis protein Bcl-2 was dose-dependently decreased, while pro-apoptotic protein Bax was increased. There was a loss of mitochondrial membrane potential and expression of the stress genes C/EBP-homologous protein 10 and glucose-regulated protein 78 was increased by RQDs. RQDs were effective in the inhibition of HepG2 cell proliferation and this effect was due to induction of apoptosis and necrosis through endoplasmic reticulum stress. PMID:26405541

  18. Comparative cytotoxicity of dolomite nanoparticles in human larynx HEp2 and liver HepG2 cells.

    PubMed

    Ahamed, Maqusood; Alhadlaq, Hisham A; Ahmad, Javed; Siddiqui, Maqsood A; Khan, Shams T; Musarrat, Javed; Al-Khedhairy, Abdulaziz A

    2015-06-01

    Dolomite is a natural mineral of great industrial and commercial importance. With the advent of nanotechnology, natural minerals including dolomite in the form of nanoparticles (NPs) are being utilized in various applications to improve the quality of products. However, safety or toxicity information of dolomite NPs is largely lacking. This study evaluated the cytotoxicity of dolomite NPs in two widely used in vitro cell culture models: human airway epithelial (HEp2) and human liver (HepG2) cells. Concentration-dependent decreased cell viability and damaged cell membrane integrity revealed the cytotoxicity of dolomite NPs. We further observed that dolomite NPs induce oxidative stress in a concentration-dependent manner, as indicated by depletion of glutathione and induction of reactive oxygen species (ROS) and lipid peroxidation. Quantitative real-time PCR data demonstrated that the mRNA level of tumor suppressor gene p53 and apoptotic genes (bax, CASP3 and CASP9) were up-regulated whereas the anti-apoptotic gene bcl-2 was down-regulated in HEp2 and HepG2 cells exposed to dolomite NPs. Moreover, the activity of apoptotic enzymes (caspase-3 and caspase-9) was also higher in both kinds of cells treated with dolomite NPs. It is also worth mentioning that HEp2 cells seem to be marginally more susceptible to dolomite NPs exposure than HepG2 cells. Cytotoxicity induced by dolomite NPs was efficiently prevented by N-acetyl cysteine treatment, which suggests that oxidative stress is primarily responsible for the cytotoxicity of dolomite NPs in both HEp2 and HepG2 cells. Toxicity mechanisms of dolomite NPs warrant further investigations at the in vivo level.

  19. Knockdown of Decoy Receptor 3 Impairs Growth and Invasiveness of Hepatocellular Carcinoma Cell Line of HepG2

    PubMed Central

    Zhou, Xiao-Na; Li, Guang-Ming; Xu, Ying-Chen; Zhao, Tuan-Jie; Wu, Ji-Xiang

    2016-01-01

    Background: Decoy receptor 3 (DcR3) binds to Fas ligand (FasL) and inhibits FasL-induced apoptosis. The receptor is overexpressed in hepatocellular carcinoma (HCC), and it is associated with the growth and metastatic spread of tumors. DcR3 holds promises as a new target for the treatment of HCC, but little is known regarding the molecular mechanisms underlying the oncogenic properties of DcR3. The present work, therefore, examined the role of DcR3 in regulating the growth and invasive property of liver cancer cell HepG2. Methods: HepG2 cells were stably transfected with lentivirus-based short hairpin RNA vector targeting DcR3. After the knockdown of DcR3 was confirmed, cell proliferation, clone formation, ability of migrating across transwell membrane, and wound healing were assessed in vitro. Matrix metalloproteinase-9 (MMP 9) and vascular epithelial growth factor (VEGF)-C and D expressions of the DcR3 knockdown were also studied. Comparisons between multiple groups were done using one-way analysis of variance (ANOVA), while pairwise comparisons were performed using Student's t test. P < 0.05 was regarded statistically significant. Results: DcR3 was overexpressed in HepG2 compared to other HCC cell lines and normal hepatocyte Lo-2. Stable knockdown of DcR3 slowed down the growth of HepG2 (P < 0.05) and reduced the number of clones formed by 50% compared to those without DcR3 knockdown (P < 0.05). The knockdown also reduced the migration of HepG2 across transwell matrix membrane by five folds compared to the control (P < 0.05) and suppressed the closure of scratch wound (P < 0.05). In addition, the messenger RNA levels of MMP 9, VEGF-C, and VEGF-D were significantly suppressed by DcR3 knockdown by 90% when compared with the mock control (P < 0.05). Conclusions: Loss of DcR3 impaired the growth and invasive property of HCC cell line of HepG2. Targeting DcR3 may be a potential therapeutic approach for the treatment of HCC. PMID:27779171

  20. Apoptosis induction by silica nanoparticles mediated through reactive oxygen species in human liver cell line HepG2

    SciTech Connect

    Ahmad, Javed; Ahamed, Maqusood; Akhtar, Mohd Javed; Alrokayan, Salman A.; Siddiqui, Maqsood A.; Musarrat, Javed; Al-Khedhairy, Abdulaziz A.

    2012-03-01

    Silica nanoparticles are increasingly utilized in various applications including agriculture and medicine. In vivo studies have shown that liver is one of the primary target organ of silica nanoparticles. However, possible mechanisms of hepatotoxicity caused by silica nanoparticles still remain unclear. In this study, we explored the reactive oxygen species (ROS) mediated apoptosis induced by well-characterized 14 nm silica nanoparticles in human liver cell line HepG2. Silica nanoparticles (25–200 μg/ml) induced a dose-dependent cytotoxicity in HepG2 cells. Silica nanoparticles were also found to induce oxidative stress in dose-dependent manner indicated by induction of ROS and lipid peroxidation and depletion of glutathione (GSH). Quantitative real-time PCR and immunoblotting results showed that both the mRNA and protein expressions of cell cycle checkpoint gene p53 and apoptotic genes (bax and caspase-3) were up-regulated while the anti-apoptotic gene bcl-2 was down-regulated in silica nanoparticles treated cells. Moreover, co-treatment of ROS scavenger vitamin C significantly attenuated the modulation of apoptotic markers along with the preservation of cell viability caused by silica nanoparticles. Our data demonstrated that silica nanoparticles induced apoptosis in human liver cells, which is ROS mediated and regulated through p53, bax/bcl-2 and caspase pathways. This study suggests that toxicity mechanisms of silica nanoparticles should be further investigated at in vivo level. -- Highlights: ► We explored the mechanisms of toxicity caused by silica NPs in human liver HepG2 cells. ► Silica NPs induced a dose-dependent cytotoxicity in HepG2 cells. ► Silica NPs induced ROS generation and oxidative stress in a dose-dependent manner. ► Silica NPs were also modulated apoptosis markers both at mRNA and protein levels. ► ROS mediated apoptosis induced by silica NPs was preserved by vitamin C.

  1. Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report

    NASA Technical Reports Server (NTRS)

    Khaoustov, V. I.; Risin, D.; Pellis, N. R.; Yoffe, B.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

  2. Antiproliferative effects of crocin in HepG2 cells by telomerase inhibition and hTERT down-regulation.

    PubMed

    Noureini, Sakineh Kazemi; Wink, Michael

    2012-01-01

    Crocin, the main pigment of Crocus sativus L., has been shown to have antiproliferative effects on cancer cells, but the involved mechanisms are only poor understood. This study focused on probable effect of crocin on the immortality of hepatic cancer cells. Cytotoxicity of crocin (IC50 3 mg/ml) in hepatocarcinoma HepG2 cells was determined after 48 h by neutral red uptake assay and MTT test. Immortality was investigated through quantification of relative telomerase activity with a quantitative real-time PCR-based telomerase repeat amplification protocol (qTRAP). Telomerase activity in 0.5 μg protein extract of HepG2 cells treated with 3 mg/ml crocin was reduced to about 51% as compared to untreated control cells. Two mechanisms of inhibition, i.e. interaction of crocin with telomeric quadruplex sequences and down regulation of hTERT expression, were examined using FRET analysis to measure melting temperature of a synthetic telomeric oligonucleotide in the presence of crocin and quantitative real-time RT-PCR, respectively. No significant changes were observed in the Tm telomeric oligonucleotides, while the relative expression level of the catalytic subunit of telomerase (hTERT) gene showed a 60% decrease as compared to untreated control cells. In conclusion, telomerase activity of HepG2 cells decreases after treatment with crocin, which is probably caused by down-regulation of the expression of the catalytic subunit of the enzyme.

  3. Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report.

    PubMed

    Khaoustov, V I; Risin, D; Pellis, N R; Yoffe, B

    2001-02-01

    Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

  4. Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report

    NASA Technical Reports Server (NTRS)

    Khaoustov, V. I.; Risin, D.; Pellis, N. R.; Yoffe, B.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

  5. Validation of weak biological effects by round robin experiments: cytotoxicity/biocompatibility of SiO2 and polymer nanoparticles in HepG2 cells.

    PubMed

    Landgraf, Lisa; Nordmeyer, Daniel; Schmiel, Peter; Gao, Qi; Ritz, Sandra; S Gebauer, Julia; Graß, Stefan; Diabaté, Silvia; Treuel, Lennart; Graf, Christina; Rühl, Eckart; Landfester, Katharina; Mailänder, Volker; Weiss, Carsten; Zellner, Reinhard; Hilger, Ingrid

    2017-06-28

    All over the world, different types of nanomaterials with a diversified spectrum of applications are designed and developed, especially in the field of nanomedicine. The great variety of nanoparticles (NPs), in vitro test systems and cell lines led to a vast amount of publications with conflicting data. To identify the decisive principles of these variabilities, we conducted an intercomparison study of collaborating laboratories within the German DFG Priority Program SPP1313, using well-defined experimental parameters and well-characterized NPs. The participants analyzed the in vitro biocompatibility of silica and polymer NPs on human hepatoma HepG2 cells. Nanoparticle mediated effects on cell metabolism, internalization, and inflammation were measured. All laboratories showed that both nanoparticle formulations were internalized and had a low cytotoxicity profile. Interestingly, small variations in nanoparticle preparation, cell handling and the type of culture slide influenced the nanoparticle stability and the outcomes of cell assays. The round robin test demonstrated the importance of the use of clearly defined and characterized NPs and parameters for reproducible results across laboratories. Comparative analyses of in vitro screening methods performed in multiple laboratories are absolutely essential to establish robust standard operation procedure as a prerequisite for sound hazard assessment of nanomaterials.

  6. Mangiferin, a Dietary Xanthone Protects Against Mercury-Induced Toxicity in HepG2 Cells

    PubMed Central

    Agarwala, Sobhika; Rao, B. Nageshwar; Mudholkar, Kaivalya; Bhuwania, Ridhirama; Rao, B. S. Satish

    2012-01-01

    Mercury is one of the noxious heavy metal environmental toxicants and is a cause of concern for human exposure. Mangiferin (MGN), a glucosylxanthone found in Mangifera indica, reported to have a wide range of pharmacological properties. The objective of this study was to evaluate the cytoprotective potential of MGN, against mercury chloride (HgCl2) induced toxicity in HepG2 cell line. The cytoprotective effect of MGN on HgCl2 induced toxicity was assessed by colony formation assay, while antiapoptotic effect by fluorescence microscopy, flow cytometric DNA analysis, and DNA fragmentation pattern assays. Further, the cytoprotective effect of MGN against HgCl2 toxicity was assessed by using biochemical parameters like reduced glutathione (GSH), glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) by spectrophotometrically, mitochondrial membrane potential by flowcytometry and the changes in reactive oxygen species levels by DCFH-DA spectrofluoremetric analysis. A significant increase in the surviving fraction was observed with 50 µM of MGN administered two hours prior to various concentrations of HgCl2. Further, pretreatment of MGN significantly decreased the percentage of HgCl2 induced apoptotic cells. Similarly, the levels of ROS generated by the HgCl2 treatment were inhibited significantly (P < 0.01) by MGN. MGN also significantly (P < 0.01) inhibited the HgCl2 induced decrease in GSH, GST, SOD, and CAT levels at all the post incubation intervals. Our study demonstrated the cytoprotective potential of MGN, which may be attributed to quenching of the ROS generated in the cells due to oxidative stress induced by HgCl2, restoration of mitochondrial membrane potential and normalization of cellular antioxidant levels. PMID:20629087

  7. Anti-tumor effects of bemiparin in HepG2 and MIA PaCa-2 cells.

    PubMed

    Alur, İhsan; Dodurga, Yavuz; Seçme, Mücahit; Elmas, Levent; Bağcı, Gülseren; Gökşin, İbrahim; Avcı, Çığır Biray

    2016-07-10

    Recent researches have demonstrated improved survival in oncologic patients treated with low molecular weight heparins (LMWHs) which are anticoagulant drugs. We evaluated "second generation" LMWH bemiparin and its in vitro anti-tumor effects on HepG2 hepatocellular carcinoma and MIA PaCa-2 cancer cells. The aim of the study is to investigate anti-cancer mechanism of bemiparin in HepG2 and Mia-Paca-2 cancer cells. Cytotoxic effects of bemiparin were determined by XTT assay. IC50 dose of bemiparin was found to be 200 IU/mL in the 48th hour in the MiaPaCa-2 cell line and 50 IU/mL in the 48th hour in the HepG2 cell line. CCND1 (cyclin D1), CDK4, CDK6, p21, p16, p53, caspase-3, caspase-9, caspase-8, Bcl-2, BID, DR4, DR5, FADD, TRADD, Bax, gene mRNA expressions were evaluated by Real-time PCR. Real-time PCR analysis showed that CCND1 expression was reduced in HepG2 dose the group cells when compared with the control group cells and p53, caspase-3, caspase p21, caspase-8 and expressions were increased in the dose group cells when compared with the control group cells. CCND1, CDK4 and CDK6 expressions were reduced in MIA PaCa-2 dose group cells when compared with the control group cells and p53 expression was increased in the dose group cells when compared with the control group cells. Other expressions of genes were found statistically insignificant both of cell lines. It was found that bemiparin in HepG2 and MIA PaCa-2 cells suppressed invasion, migration, and colony formation by using matrigel invasion chamber, and colony formation assay, respectively. In conclusion, it is thought that bemiparin indicates anti-tumor activity by affecting cell cycle arrest, apoptosis, invasion, migration, and colony formation on cancer cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Alantolactone Induces Apoptosis in HepG2 Cells through GSH Depletion, Inhibition of STAT3 Activation, and Mitochondrial Dysfunction

    PubMed Central

    Khan, Muhammad; Li, Ting; Ahmad Khan, Muhammad Khalil; Rasul, Azhar; Nawaz, Faisal; Sun, Meiyan; Zheng, Yongchen; Ma, Tonghui

    2013-01-01

    Signal transducer and activator of transcription 3 (STAT3) constitutively expresses in human liver cancer cells and has been implicated in apoptosis resistance and tumorigenesis. Alantolactone, a sesquiterpene lactone, has been shown to possess anticancer activities in various cancer cell lines. In our previous report, we showed that alantolactone induced apoptosis in U87 glioblastoma cells via GSH depletion and ROS generation. However, the molecular mechanism of GSH depletion remained unexplored. The present study was conducted to envisage the molecular mechanism of alantolactone-induced apoptosis in HepG2 cells by focusing on the molecular mechanism of GSH depletion and its effect on STAT3 activation. We found that alantolactone induced apoptosis in HepG2 cells in a dose-dependent manner. This alantolactone-induced apoptosis was found to be associated with GSH depletion, inhibition of STAT3 activation, ROS generation, mitochondrial transmembrane potential dissipation, and increased Bax/Bcl-2 ratio and caspase-3 activation. This alantolactone-induced apoptosis and GSH depletion were effectively inhibited or abrogated by a thiol antioxidant, N-acetyl-L-cysteine (NAC). The data demonstrate clearly that intracellular GSH plays a central role in alantolactone-induced apoptosis in HepG2 cells. Thus, alantolactone may become a lead chemotherapeutic candidate for the treatment of liver cancer. PMID:23533997

  9. N-acetyl-cysteine protects against DNA damage associated with lead toxicity in HepG2 cells.

    PubMed

    Yedjou, Clement G; Tchounwou, Christine K; Haile, Samuel; Edwards, Falicia; Tchounwou, Paul B

    2010-01-01

    Lead toxicity has been associated with its ability to interact and damage DNA. However, its molecular mechanisms of action are not fully understood. In vitro studies in our laboratory indicated that lead nitrate (PbNO3) induces cytotoxicity and oxidative stress to human liver carcinoma (HepG2) cells in a dose-dependent manner. In this research, we hypothesized that n-acetyl-cysteine (NAC), a known antioxidant compound, affords protection against lead-induced cell death associated with genotoxic damage. To test this hypothesis, HepG2 cells were treated either with a physiologic dose of NAC, NAC plus PbNO3, or PbNO3 alone, followed by incubation in humidified 5% CO2 incubator at 37 degrees C for 48 hr. The cell viability was determined by trypan blue exclusion test. The degree of DNA damage was detected by micro gel electrophoresis (comet) assay. Our results showed that lead exposure induces a substantial cytotoxicity as well as a significant genotoxicity to HepG2 cells. However, co-treatment with a physiologic dose (500 microM) of NAC slightly increases cell viability, and significantly reduced (P < .05) the degree of DNA damage. Hence, NAC treatment may be a promising therapeutic candidate for chemoprevention against lead toxicity, based on its ability to scavenge free radicals.

  10. Transcriptional down regulation of hTERT and senescence induction in HepG2 cells by chelidonine

    PubMed Central

    Kazemi Noureini, Sakineh; Wink, Michael

    2009-01-01

    AIM: To investigate the potential effects of chelidonine, the main alkaloid of Chelidonium majus, on telomerase activity and its regulation in HepG2 cells. METHODS: Cytotoxicity of chelidonine for HepG2 cells was determined by neutral red assay. A modified polymerase chain reaction (PCR)-based telomerase repeat amplification protocol was used to estimate relative telomerase activity in chelidonine-treated cells in comparison with the untreated control cells. Relative expression level of the catalytic subunit of telomerase (hTERT) gene and P-glycoprotein (pgp) were estimated using semi-quantitative real-time reverse transcription-PCR (RT-PCR). Cell senescence in treated cells was demonstrated using a β-galactosidase test. RESULTS: Cytotoxicity of chelidonine in HepG2 cells was not dose-dependent and tended to reach plateau immediately after the living cells were reduced in number to slightly higher than 50%. However, 12 μmol/L concentration of chelidonine was considered as LD50, where the maximal attainable effects were realized. Real-time RT-PCR data showed that the expression of pgp increased three-fold in chelidonine treated HepG2 cells in comparison with the untreated controls. Morphologically, treated HepG2 cells showed apoptotic features after 24 h and a small fraction of cells appeared with single blister cell death. The relative expression level of Bcl-2 dropped to less than 50% of control cells at a sub-apoptotic concentration of chelidonine and subsequently increased to higher than 120% at LD50. Telomerase activity was reduced considerably after administration of very low doses of chelidonine, whereas higher concentrations of chelidonine did not remarkably enhance the effect. Real-time RT-PCR experiments indicated a drastic decrease in expression level of hTERT subunit of telomerase under treatment with chelidonine. Repeated treatment of cells with very low doses of chelidonine caused a decline in growth rate by 4 wk and many of the cells appeared to be

  11. Effects of Nano-CeO₂ with Different Nanocrystal Morphologies on Cytotoxicity in HepG2 Cells.

    PubMed

    Wang, Lili; Ai, Wenchao; Zhai, Yanwu; Li, Haishan; Zhou, Kebin; Chen, Huiming

    2015-09-02

    Cerium oxide nanoparticles (nano-CeO₂) have been reported to cause damage and apoptosis in human primary hepatocytes. Here, we compared the toxicity of three types of nano-CeO₂ with different nanocrystal morphologies (cube-, octahedron-, and rod-like crystals) in human hepatocellular carcinoma cells (HepG2). The cells were treated with the nano-CeO₂ at various concentrations (6.25, 12.5, 25, 50, 100 μg/mL). The crystal structure, size and morphology of nano-CeO₂ were investigated by X-ray diffractometry and transmission electron microscopy. The specific surface area was detected using the Brunauer, Emmet and Teller method. The cellular morphological and internal structure were observed by microscopy; apoptotic alterations were measured using flow cytometry; nuclear DNA, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and glutathione (GSH) in HepG2 cells were measured using high content screening technology. The scavenging ability of hydroxyl free radicals and the redox properties of the nano-CeO₂ were measured by square-wave voltammetry and temperature-programmed-reduction methods. All three types of nano-CeO₂ entered the HepG2 cells, localized in the lysosome and cytoplasm, altered cellular shape, and caused cytotoxicity. The nano-CeO₂ with smaller specific surface areas induced more apoptosis, caused an increase in MMP, ROS and GSH, and lowered the cell's ability to scavenge hydroxyl free radicals and antioxidants. In this work, our data demonstrated that compared with cube-like and octahedron-like nano-CeO₂, the rod-like nano-CeO₂ has lowest toxicity to HepG2 cells owing to its larger specific surface areas.

  12. Hepatitis C Virus Genotype 4 Replication in the Hepatocellular Carcinoma Cell Line HepG2/C3A

    PubMed Central

    Shier, Medhat K.; El-Wetidy, Mohammad S.; Ali, Hebatallah H.; Al-Qattan, Mohammad M.

    2016-01-01

    Background/Aims: The lack of a reliable cell culture system allowing persistent in vitro hepatitis C virus (HCV) propagation is still restraining the search for novel antiviral strategies. HepG2 cells transfection with HCV allows for viral replication. However, the replication is weak presumably because of HepG2 lack of miRNA-122, which is essential for viral replication. Other agents such as polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) have been shown to increase the efficiency of infection with other viruses. This study included comparison of HCV genotype 4 5′UTR and core RNA levels and HCV core protein expression at different time intervals in the absence or presence of PEG and/or DMSO postinfection. Materials and Methods: We used serum with native HCV particles in infecting HepG2 cells in vitro. HCV replication was assessed by reverse transcriptase polymerase chain reaction for detection of HCV RNA and immunofluorescence and flow cytometry for detection of HCV core protein. Results: HCV 5′UTR and core RNA expression was evident at different time intervals after viral infection, especially after cells were treated with PEG. HCV core protein was also evident at different time intervals using both immunofluorescence and flow cytometry. PEG, not DMSO, has increased the HCV core protein expression in the treated cells, similar to its effect on viral RNA expression. Conclusions: These expression profiles suggest that the current model of cultured HepG2 cells allows the study of HCV genotype 4 replication and different stages of the viral life cycle. PMID:27184644

  13. Quercetin modulates Nrf2 and glutathione-related defenses in HepG2 cells: Involvement of p38.

    PubMed

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

    2012-01-25

    Dietary flavonoid quercetin has been suggested as a cancer chemopreventive agent, but the mechanisms of action remain unclear. This study investigated the influence of quercetin on p38-MAPK and the potential regulation of the nuclear transcription factor erythroid-2p45-related factor (Nrf2) and the cellular antioxidant/detoxifying defense system related to glutathione (GSH) by p38 in HepG2 cells. Incubation of HepG2 cells with quercetin at a range of concentrations (5-50μM) for 4 or 18h induced a differential effect on the modulation of p38 and Nrf2 in HepG2 cells, 50μM quercetin showed the highest activation of p38 at 4h of treatment and values of p38 similar to those of control cells after 18 h of incubation, together with the inhibition of Nrf2 at both incubation times. Quercetin (50μM) induced a time-dependent activation of p38, which was in concert with a transient stimulation of Nrf2 to provoke its inhibition afterward. Quercetin also increased GSH content, mRNA levels of glutamylcysteine-synthetase (GCS) and expression and/or activity of glutathione-peroxidase, glutathione-reductase and GCS after 4h of incubation, and glutathione-S-transferase after 18h of exposure. Further studies with the p38 specific inhibitor SB203580 showed that the p38 blockage restored the inhibited Nrf2 transcription factor and the enzymatic expression and activity of antioxidant/detoxificant enzymes after 4h exposure. In conclusion, p38-MAPK is involved in the mechanisms of the cell response to quercetin through the modulation of Nrf2 and glutathione-related enzymes in HepG2 cells.

  14. Pfaffosidic Fraction from Hebanthe paniculata Induces Cell Cycle Arrest and Caspase-3-Induced Apoptosis in HepG2 Cells

    PubMed Central

    da Silva, Tereza Cristina; Cogliati, Bruno; Latorre, Andréia Oliveira; Akisue, Gokithi; Nagamine, Márcia Kazumi; Haraguchi, Mitsue; Hansen, Daiane; Sanches, Daniel Soares; Dagli, Maria Lúcia Zaidan

    2015-01-01

    Hebanthe paniculata roots (formerly Pfaffia paniculata and popularly known as Brazilian ginseng) show antineoplastic, chemopreventive, and antiproliferative properties. Functional properties of these roots and their extracts are usually attributed to the pfaffosidic fraction, which is composed mainly by pfaffosides A–F. However, the therapeutic potential of this fraction in cancer cells is not yet entirely understood. This study aimed to analyze the antitumoral effects of the purified pfaffosidic fraction or saponinic fraction on the human hepatocellular carcinoma HepG2 cell line. Cellular viability, proliferation, and apoptosis were evaluated, respectively, by MTT assay, BrdU incorporation, activated caspase-3 immunocytochemistry, and DNA fragmentation assay. Cell cycle was analyzed by flow cytometry and the cell cycle-related proteins were analyzed by quantitative PCR and Western blot. The cells exposed to pfaffosidic fraction had reduced viability and cellular growth, induced G2/M at 48 h or S at 72 h arrest, and increased sub-G1 cell population via cyclin E downregulation, p27KIP1 overexpression, and caspase-3-induced apoptosis, without affecting the DNA integrity. Antitumoral effects of pfaffosidic fraction from H. paniculata in HepG2 cells originated by multimechanisms of action might be associated with cell cycle arrest in the S phase, by CDK2 and cyclin E downregulation and p27KIP1 overexpression, besides induction of apoptosis through caspase-3 activation. PMID:26075002

  15. The role of oxidative stress in citreoviridin-induced DNA damage in human liver-derived HepG2 cells.

    PubMed

    Bai, Yuntao; Jiang, Li-Ping; Liu, Xiao-Fang; Wang, Dong; Yang, Guang; Geng, Cheng-Yan; Li, Qiujuan; Zhong, Lai-Fu; Sun, Qinghua; Chen, Min

    2015-05-01

    We hypothesize that citreoviridin (CIT) induces DNA damage in human liver-derived HepG2 cells through an oxidative stress mechanism and that N-acetyl-l-cysteine (NAC) protects against CIT-induced DNA damage in HepG2 cells. CIT-induced DNA damage in HepG2 cells was evaluated by alkaline single-cell gel electrophoresis assay. To elucidate the genotoxicity mechanisms, the level of oxidative DNA damage was tested by immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG); the intracellular generation of reactive oxygen species (ROS) and reduced glutathione (GSH) were examined; mitochondrial membrane potential and lysosomal membranes' permeability were detected; furthermore, protective effects of NAC on CIT-induced ROS formation and CIT-induced DNA damage were evaluated in HepG2 cells. A significant dose-dependent increment in DNA migration was observed at tested concentrations (2.50-10.00 µM) of CIT. The levels of ROS, 8-OHdG formation were increased by CIT, and significant depletion of GSH in HepG2 cells was induced by CIT. Destabilization of lysosome and mitochondria was also observed in cells treated with CIT. In addition, NAC significantly decreased CIT-induced ROS formation and CIT-induced DNA damage in HepG2 cells. The data indicate that CIT induces DNA damage in HepG2 cells, most likely through oxidative stress mechanisms; that NAC protects against DNA damage induced by CIT in HepG2 cells; and that depolarization of mitochondria and lysosomal protease leakage may play a role in CIT-induced DNA damage in HepG2 cells. © 2014 The Authors. Published by Wiley Periodicals Inc.

  16. A comparative toxicity evaluation of Escherichia coli-targeted ssDNA and chlorine in HepG2 cells.

    PubMed

    Kaushik, Rajni; Balasubramanian, Rajasekhar

    2014-01-01

    In this study, a comparative assessment of the effectiveness of ssDNA and chlorine as disinfectants for treating water contaminated with Escherichia coli (E. coli) was investigated on the basis of cytotoxicity and genotoxicity. The gene targets addressed for the ssDNA based inhibition method were marA (multiple antibiotic resistance) and groL (essential gene Hsp60) in E. coli. Based on the maximum log reduction in E. coli cell numbers when compared to no ssDNA control, groL-1 was chosen as the optimized ssDNA for gene silencing-based inactivation. For toxicity assessment, HepG2 cells were exposed to extracts corresponding to concentrations of 0.2, 1, 5, 25 and 50 mL water/mL medium of chlorine doped water and 1, 10, 100, 300 nM of ssDNA. Compared with ssDNA, HepG2 cells exposed to extracts of chlorine doped water for 24 h showed higher cytotoxicity, caspase 3/7 levels, DNA damage, micronuclei frequency, and decreased cell viability. Water doped with chlorine was found to be more toxic than that by ssDNA when exposed to HepG2 cells. The results of this study provide a scientific basis for comparative evaluation of new and conventional disinfection methods by taking into consideration the outcome of cytotoxicity and genotoxicity assessments.

  17. Chenodeoxycholic acid increases the induction of CYP1A1 in HepG2 and H4IIE cells

    PubMed Central

    IBRAHIM, ZEIN SHABAN

    2015-01-01

    Bile acids are considered to promote carcinogenesis. Cytochrome P450 1A1 (CYP1A1) plays a critical role in the biotransformation of drugs and procarcinogens. This study aimed to investigate the ability of bile acids to modulate CYP1A1 expression. Treatment of HepG2 cells with chenodeoxycholic acid (CDCA) and Sudan III (S.III) upregulated CYP1A1 transcriptional activity in HepG2 cells and CYP1A1 mRNA expression in H4IIE cells. Pretreatment of the HepG2 and H4IIE cells with CDCA upregulated the S.III-induced CYP1A transcriptional activity and mRNA expression. The CDCA-induced enhancement of CYP1A1 was not abolished by the p38 inhibitor SB203580. However, exposure of the cells to the mitogen-activated protein kinase kinase (MEK)1/2 inhibitor PD98059 suppressed the CDCA-induced enhancement of CYP1A1. These results show the ability of CDCA to upregulate CYP1A1 transcription and expression, which may explain the hepatocarcinogenesis-inducing effect of cholestasis. The CDCA-induced upregulation of CYP1A1 most probably proceeded through MEK1/2 activation, indicating that this may be a therapeutic target to prevent the cancer-promoting effects of excessive amounts of bile acids. PMID:26640583

  18. Protective effects of the extracts of Barringtonia racemosa shoots against oxidative damage in HepG2 cells

    PubMed Central

    Kong, Kin Weng; Mat-Junit, Sarni; Aminudin, Norhaniza; Hassan, Fouad Abdulrahman; Ismail, Amin

    2016-01-01

    Barringtonia racemosa is a tropical plant with medicinal values. In this study, the ability of the water extracts of the leaf (BLE) and stem (BSE) from the shoots to protect HepG2 cells against oxidative damage was studied. Five major polyphenolic compounds consisting of gallic acid, ellagic acid, protocatechuic acid, quercetin and kaempferol were identified using HPLC-DAD and ESI-MS. Cell viability assay revealed that BLE and BSE were non-cytotoxic (cell viabilities >80%) at concentration less than 250 µg/ml and 500 µg/ml, respectively. BLE and BSE improved cellular antioxidant status measured by FRAP assay and protected HepG2 cells against H2O2-induced cytotoxicity. The extracts also inhibited lipid peroxidation in HepG2 cells as well as the production of reactive oxygen species. BLE and BSE could also suppress the activities of superoxide dismutase and catalase during oxidative stress. The shoots of B. racemosa can be an alternative bioactive ingredient in the prevention of oxidative damage. PMID:26839752

  19. Encapsulation of honokiol into self-assembled pectin nanoparticles for drug delivery to HepG2 cells.

    PubMed

    Zhang, Yuxia; Chen, Tong; Yuan, Pei; Tian, Rui; Hu, Wenjing; Tang, Yalan; Jia, Yuntao; Zhang, Liangke

    2015-11-20

    Self-assembled pectin nanoparticles was prepared and evaluated for delivering the hydrophobic drug, honokiol (HK), to HepG2 cells. These hydrophobic drug-loaded nanoparticles were developed without using any surfactant and organic solvent. Hydroxypropyl-β-cyclodextrin (HCD) was used to fabricate an inclusion complex with HK (HKHCD) to increase the solubility of the drug and thus facilitate its encapsulation and dispersion in the pectin nanoparticles. Investigation of the in vitro release indicated that the drug-loaded nanoparticles exhibited a higher drug release rate than free honokiol and an effective sustained-release. Cytotoxicity, cell apoptosis and cellular uptake studies further confirmed that the pectin nanoparticles with galactose residues generated higher cytotoxicity than free honokiol on HepG2 cells which highly expressed asialoglycoprotein receptors (ASGR). Nevertheless, these findings were not observed in ASGR-negative A549 cells under similar condition. Therefore, pectin nanoparticles demonstrated a specific active targeting ability to ASGR-positive HepG2 cells and could be used as a potential drug carrier for treatment of liver-related tumors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Fucoidan induces apoptosis of HepG2 cells by down-regulating p-Stat3.

    PubMed

    Roshan, Sadia; Liu, Yun-yi; Banafa, Amal; Chen, Hui-jie; Li, Ke-xiu; Yang, Guang-xiao; He, Guang-yuan; Chen, Ming-jie

    2014-06-01

    Fucoidan is one of the main bioactive components of polysaccharides. The current study was focused on the anti-tumor effects of fucoidan on human heptoma cell line HepG2 and the possible mechanisms. Fucoidan treatment resulted in cell cycle arrest and apoptosis of HepG2 cells in a dose-dependent manner detected by MTT assay, flow cytometry and fluorescent microscopy. The results of flow cytometric analysis revealed that fucoidan induced G2/M arrest in the cell cycle progression. Hoechst 33258 and Annexin V/PI staining results showed that the apoptotic cell number was increased, which was associated with a dose-dependent up-regulation of Bax and down-regulation of Bcl-2 and p-Stat3. In parallel, the up-regulation of p53 and the increase in reactive oxygen species were also observed, which may play important roles in the inhibition of HepG2 growth by fucoidan. In the meantime, Cyclin B1 and CDK1 were down-regulated by fucoidan treatment. Down-regulation of p-Stat3 by fucoidan resulted in apoptosis and an increase in ROS in response to fucoidan exposure. We therefore concluded that fucoidan induces apoptosis through the down-regulation of p-Stat3. These results suggest that fucoidan may be used as a novel anti-cancer agent for hepatocarcinoma.

  1. Protective effects of the extracts of Barringtonia racemosa shoots against oxidative damage in HepG2 cells.

    PubMed

    Kong, Kin Weng; Mat-Junit, Sarni; Aminudin, Norhaniza; Hassan, Fouad Abdulrahman; Ismail, Amin; Abdul Aziz, Azlina

    2016-01-01

    Barringtonia racemosa is a tropical plant with medicinal values. In this study, the ability of the water extracts of the leaf (BLE) and stem (BSE) from the shoots to protect HepG2 cells against oxidative damage was studied. Five major polyphenolic compounds consisting of gallic acid, ellagic acid, protocatechuic acid, quercetin and kaempferol were identified using HPLC-DAD and ESI-MS. Cell viability assay revealed that BLE and BSE were non-cytotoxic (cell viabilities >80%) at concentration less than 250 µg/ml and 500 µg/ml, respectively. BLE and BSE improved cellular antioxidant status measured by FRAP assay and protected HepG2 cells against H2O2-induced cytotoxicity. The extracts also inhibited lipid peroxidation in HepG2 cells as well as the production of reactive oxygen species. BLE and BSE could also suppress the activities of superoxide dismutase and catalase during oxidative stress. The shoots of B. racemosa can be an alternative bioactive ingredient in the prevention of oxidative damage.

  2. Lipid Accumulation in HepG2 Cells Is Attenuated by Strawberry Extract through AMPK Activation.

    PubMed

    Forbes-Hernández, Tamara Y; Giampieri, Francesca; Gasparrini, Massimiliano; Afrin, Sadia; Mazzoni, Luca; Cordero, Mario D; Mezzetti, Bruno; Quiles, José L; Battino, Maurizio

    2017-06-16

    Regulation of lipid metabolism is essential for treatment and prevention of several chronic diseases such as obesity, diabetes, and cardiovascular diseases, which are responsible for most deaths worldwide. It has been demonstrated that the AMP-activated protein kinase (AMPK) has a direct impact on lipid metabolism by modulating several downstream-signaling components. The main objective of the present work was to evaluate the in vitro effect of a methanolic strawberry extract on AMPK and its possible repercussion on lipid metabolism in human hepatocellular carcinoma cel