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Sample records for hepg2 hepatoma cells

  1. Radiation induced bystander effect on hepatoma HepG2 cells under hypoxia condition

    International Nuclear Information System (INIS)

    Zhang Jianghong; Jin Yizun; Shao Chunlin; Prise KM

    2009-01-01

    Objective: To investigate radiation induced bystander effect and its mechanism on hepatoma HepG2 cells under hypoxia condition. Methods: Non-irradiated bystander hepatoma cells were co-cultured with irradiated cells or treated with the conditioned medium (CM) from irradiated cells, then micronuclei (MN) were measured for both irradiated cells and bystander cells. Results: The MN yield of irradiated HepG2 cells under hypoxic condition was significantly lower than that under normoxia, the oxygen enhancement ratio of HepG2 cells of MN was 1.6. For both hypoxic and normoxic condition, the MN yield of bystander cells were obviously enhanced to a similar high level after co-culturing with irradiated cells or with CM treatment, and it also correlated with the irradiation dose. When the hypoxic HepG2 cells were treated with either DMSO, a scavenger of reactive oxygen species (ROS), or aminoguanidine, an iNOS inhibitor, the yield of bystander MN was partly diminished, and the reducing rate of DMSO was 42.2%-46.7%, the reducing rate of aminoguanidine was 42% . Conclusion: ROS, NO and their downstream signal factors are involved in the radiation induced bystander effect of hypoxic HepG2 cells. (authors)

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

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

    2018-03-01

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

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

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

    2014-12-02

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

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

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    Li, Shuai; Guo, Lianyi

    2018-01-01

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

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

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    Gabriel Yarmush

    2016-01-01

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

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

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    Peng Liu

    2018-05-01

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

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

    International Nuclear Information System (INIS)

    Kuo, H.-C.; Lee, H.-J.; Hu, C.-C.; Shun, H.-I; Tseng, T.-H.

    2006-01-01

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

  8. Radiosensitization by inhibiting survivin in human hepatoma HepG2 cells to high-LET radiation

    International Nuclear Information System (INIS)

    Jin Xiaodong; Li Qiang; Wu Qingfeng; Li Ping; Gong Li; Hao Jifang; Dai Zhongying; Matsumoto, Yoshitaka; Furusawa, Yoshiya

    2011-01-01

    In this study, whether survivin plays a direct role in mediating high-linear energy transfer (LET) radiation resistance in human hepatoma cells was investigated. Small interfering RNA (siRNA) targeting survivin mRNA was designed and transfected into human hepatoma HepG2 cells. Real-time polymerase chain reaction (PCR) and western blotting analyses revealed that survivin expression in HepG2 cells decreased at both transcriptional and post-transcriptional levels after treatment with survivin-specific siRNA. Caspase-3 activity was determined with a microplate reader assay as well. Following exposure to high-LET carbon ions, a reduced clonogenic survival effect, increased apoptotic rates and caspase-3 activity were observed in the cells treated with the siRNA compared to those untreated with the siRNA. The cells with transfection of the survivin-specific siRNA also increased the level of G 2 /M arrest. These results suggest that survivin definitely plays a role in mediating the resistance of HepG2 cells to high-LET radiation and depressing survivin expression might be useful to improve the therapeutic efficacy of heavy ions for radioresistant solid tumors. (author)

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

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

    2018-02-01

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

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

    International Nuclear Information System (INIS)

    Lenich, C.M.

    1985-01-01

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

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

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    Shi Lixin

    2011-01-01

    Full Text Available Abstract 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.

  12. microRNA-mediated resistance to hypoglycemia in the HepG2 human hepatoma cell line

    International Nuclear Information System (INIS)

    Ueki, Satomi; Murakami, Yuko; Yamada, Shoji; Kimura, Masaki; Saito, Yoshimasa; Saito, Hidetsugu

    2016-01-01

    It is generally accepted that the energy resources of cancer cells rely on anaerobic metabolism or the glycolytic system, even if they have sufficient oxygen. This is known as the Warburg effect. The cells skillfully survive under hypoglycemic conditions when their circumstances change, which probably at least partly involves microRNA (miRNA)-mediated regulation. To determine how cancer cells exploit miRNA-mediated epigenetic mechanisms to survive in hypoglycemic conditions, we used DNA microarray analysis to comprehensively and simultaneously compare the expression of miRNAs and mRNAs in the HepG2 human hepatoma cell line and in cultured normal human hepatocytes. The hypoglycemic condition decreased the expression of miRNA-17-5p and -20a-5p in hepatoma cells and consequently upregulated the expression of their target gene p21. These regulations were also confirmed by using antisense inhibitors of these miRNAs. In addition to this change, the hypoglycemic condition led to upregulated expression of heat shock proteins and increased resistance to caspase-3-induced apoptosis. However, we could not identify miRNA-mediated regulations, despite using comprehensive detection. Several interesting genes were also found to be upregulated in the hypoglycemic condition by the microarray analysis, probably because of responding to this cellular stress. These results suggest that cancer cells skillfully survive in hypoglycemic conditions, which frequently occur in malignancies, and that some of the gene regulation of this process is manipulated by miRNAs. The online version of this article (doi:10.1186/s12885-016-2762-7) contains supplementary material, which is available to authorized users

  13. Effects of PARP-1 inhibitors AG-014699 and AZD2281 on proliferation and apoptosis of human hepatoma cell line HepG2

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    DU Senrong

    2015-06-01

    Full Text Available ObjectiveTo observe the inhibitory and pro-apoptotic effects of two poly(ADP-ribose polymerase (PARP-1 inhibitors, AG-014699 and AZD2281, on human hepatoma HepG2 cells and preliminarily explore the mechanism by which AG-014699 induces HepG2 cell apoptosis, and to provide a new therapeutic target for hepatoma. MethodsThe effects of different concentrations of AG-014699 and AZD2281 on HepG2 cell proliferation were determined by MTT assay. The cell apoptosis rate was measured by flow cytometry. The expression levels of caspase-3 and caspase-8 were measured by Western Blot. Inter-group comparison was made by t test. ResultsBoth AG-014699 and AZD2281 suppressed HepG2 cell proliferation in a time- and dose-dependent manner. However, the sensitivity of HepG2 cells to the two PARP-1 inhibitors was different. The half-maximal inhibitory concentrations of AG-014699 and AZD2281 at 48 h determined by MTT assay were about 20 μmol/L and 400 μmol/L, respectively. Flow cytometry and Western blot were not used to evaluate the apoptosis of HepG2 cells exposed to AZD2281 to which these cells were not sensitive. HepG2 cell apoptosis could be induced by 10, 30, and 50 μmol/L AG-014699, and the highest apoptosis rate at 48 h was significantly higher than that of the control group (3100%±2.13% vs 09%±0013%, P<0.01. Compared with those in the control group, the protein levels of caspase-3 and caspase-8 in HepG2 cells after 48-h exposure to 30, and 50 μmol/L AG-014699 increased. ConclusionThe two PARP-1 inhibitors AG-014699 and AZD2281 can inhibit the proliferation of HepG2 cells, which showed different sensitivities to the two inhibitors. AG-014699 can induce HepG2 cell apoptosis by up-regulating the protein expression of caspase-3 and caspase-8.

  14. Tyramine-O-sulfate is produced and secreted by human hepatoma cells, line HepG2

    International Nuclear Information System (INIS)

    Liu, M.C.; Yu, S.; Suiko, M.

    1987-01-01

    Human hepatoma cells, line HepG2, were metabolically labeled with [ 35 S]sulfate. The spent medium separated following 24 hr labeling was subjected to ultrafiltration using an Amicon Centricon unit. The filtrate obtained was analyzed by a two-dimensional separation procedure combining high-voltage electrophoresis and thin-layer chromatography. The autoradiograph taken from the cellulose thin-layer plate following the analysis revealed the presence of tyramine-O-[ 35 ]sulfate in addition to tyrosine-O-[ 35 ]sulfate. Using adenosine, 3'-phosphate, 5'-phospho[ 35 S]sulfate as the sulfate donor, it was shown that tyramine was actively sulfated to form tyramine-O-[ 35 S]sulfate as catalyzed by the sulfotransferase(s) present in dog liver homogenate. Attempts to decarboxylate tyrosine-O-sulfate to tyramine-O-sulfate using intrinsic p-tyrosine decarboxylase present in dog liver homogenate, however, were unsuccessful. Employing purified Streptococcus faecalis tyrosine decarboxylase, it was shown that L-tyrosine was actively decarboxylated to tyramine, whereas tyrosine-O-sulfate could not serve as a substrate

  15. Cytotoxicity and apoptotic effects of tea polyphenol-loaded chitosan nanoparticles on human hepatoma HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Jin [Key Laboratory of Tea Biochemistry and Biotechnology of Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei 230036 (China); College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Li, Feng [College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Fang, Yong; Yang, Wenjian [College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210023 (China); An, Xinxin; Zhao, Liyan; Xin, Zhihong; Cao, Lin [College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Hu, Qiuhui, E-mail: qiuhuihu@njau.edu.cn [College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210023 (China)

    2014-03-01

    Tea polyphenols have strong antioxidant and antitumor activities. However, these health benefits are limited due to their poor in vivo stability and low bioavailability. Chitosan nanoparticles as delivery systems may provide an alternative approach for enhancing bioavailability of poorly absorbed drugs. In this study, tea polyphenol-loaded chitosan nanoparticles have been prepared using two different chitosan biomaterials, and their antitumor effects were evaluated in HepG2 cells, including cell cytotoxicity comparison, cell morphology analysis, cell apoptosis and cell cycle detection. The results indicated that the tea polyphenol-loaded chitosan nanoparticles showed a branch shape and heterogeneous distribution in prepared suspension. MTT assay suggested that tea polyphenol-loaded chitosan nanoparticles could inhibit the proliferation of HepG2 cells, and the cytotoxicity rates were increased gradually and appeared an obvious dose-dependent relationship. Transmission electron microscope images showed that the HepG2 cells treated with tea polyphenol-loaded chitosan nanoparticles exhibited some typical apoptotic features, such as microvilli disappearance, margination of nuclear chromatin, intracytoplasmic vacuoles and the mitochondrial swelling. In addition, the tea polyphenol-loaded chitosan nanoparticles had relatively weak inhibitory effects on HepG2 cancer cells compared with tea polyphenols. Tea polyphenols not only induced cancer cell apoptosis, but also promoted their necrosis. However, tea polyphenol-loaded chitosan nanoparticles exhibited their antitumor effects mainly through inducing cell apoptosis. Our results revealed that the inhibition effects of tea polyphenol-loaded chitosan nanoparticles on tumor cells probably depended on their controlled drug release and effective cell delivery. The chitosan nanoparticles themselves as the delivery carrier showed limited antitumor effects compared with their encapsulated drugs. - Highlights: • Tea polyphenol

  16. Cytotoxicity and apoptotic effects of tea polyphenol-loaded chitosan nanoparticles on human hepatoma HepG2 cells

    International Nuclear Information System (INIS)

    Liang, Jin; Li, Feng; Fang, Yong; Yang, Wenjian; An, Xinxin; Zhao, Liyan; Xin, Zhihong; Cao, Lin; Hu, Qiuhui

    2014-01-01

    Tea polyphenols have strong antioxidant and antitumor activities. However, these health benefits are limited due to their poor in vivo stability and low bioavailability. Chitosan nanoparticles as delivery systems may provide an alternative approach for enhancing bioavailability of poorly absorbed drugs. In this study, tea polyphenol-loaded chitosan nanoparticles have been prepared using two different chitosan biomaterials, and their antitumor effects were evaluated in HepG2 cells, including cell cytotoxicity comparison, cell morphology analysis, cell apoptosis and cell cycle detection. The results indicated that the tea polyphenol-loaded chitosan nanoparticles showed a branch shape and heterogeneous distribution in prepared suspension. MTT assay suggested that tea polyphenol-loaded chitosan nanoparticles could inhibit the proliferation of HepG2 cells, and the cytotoxicity rates were increased gradually and appeared an obvious dose-dependent relationship. Transmission electron microscope images showed that the HepG2 cells treated with tea polyphenol-loaded chitosan nanoparticles exhibited some typical apoptotic features, such as microvilli disappearance, margination of nuclear chromatin, intracytoplasmic vacuoles and the mitochondrial swelling. In addition, the tea polyphenol-loaded chitosan nanoparticles had relatively weak inhibitory effects on HepG2 cancer cells compared with tea polyphenols. Tea polyphenols not only induced cancer cell apoptosis, but also promoted their necrosis. However, tea polyphenol-loaded chitosan nanoparticles exhibited their antitumor effects mainly through inducing cell apoptosis. Our results revealed that the inhibition effects of tea polyphenol-loaded chitosan nanoparticles on tumor cells probably depended on their controlled drug release and effective cell delivery. The chitosan nanoparticles themselves as the delivery carrier showed limited antitumor effects compared with their encapsulated drugs. - Highlights: • Tea polyphenol

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

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    Haider Raza

    Full Text Available 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.

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

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

    1999-09-15

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

  19. Cytotoxicity and apoptotic effects of tea polyphenol-loaded chitosan nanoparticles on human hepatoma HepG2 cells.

    Science.gov (United States)

    Liang, Jin; Li, Feng; Fang, Yong; Yang, Wenjian; An, Xinxin; Zhao, Liyan; Xin, Zhihong; Cao, Lin; Hu, Qiuhui

    2014-03-01

    Tea polyphenols have strong antioxidant and antitumor activities. However, these health benefits are limited due to their poor in vivo stability and low bioavailability. Chitosan nanoparticles as delivery systems may provide an alternative approach for enhancing bioavailability of poorly absorbed drugs. In this study, tea polyphenol-loaded chitosan nanoparticles have been prepared using two different chitosan biomaterials, and their antitumor effects were evaluated in HepG2 cells, including cell cytotoxicity comparison, cell morphology analysis, cell apoptosis and cell cycle detection. The results indicated that the tea polyphenol-loaded chitosan nanoparticles showed a branch shape and heterogeneous distribution in prepared suspension. MTT assay suggested that tea polyphenol-loaded chitosan nanoparticles could inhibit the proliferation of HepG2 cells, and the cytotoxicity rates were increased gradually and appeared an obvious dose-dependent relationship. Transmission electron microscope images showed that the HepG2 cells treated with tea polyphenol-loaded chitosan nanoparticles exhibited some typical apoptotic features, such as microvilli disappearance, margination of nuclear chromatin, intracytoplasmic vacuoles and the mitochondrial swelling. In addition, the tea polyphenol-loaded chitosan nanoparticles had relatively weak inhibitory effects on HepG2 cancer cells compared with tea polyphenols. Tea polyphenols not only induced cancer cell apoptosis, but also promoted their necrosis. However, tea polyphenol-loaded chitosan nanoparticles exhibited their antitumor effects mainly through inducing cell apoptosis. Our results revealed that the inhibition effects of tea polyphenol-loaded chitosan nanoparticles on tumor cells probably depended on their controlled drug release and effective cell delivery. The chitosan nanoparticles themselves as the delivery carrier showed limited antitumor effects compared with their encapsulated drugs. Copyright © 2013. Published by

  20. GEP100/Arf6 is required for epidermal growth factor-induced ERK/Rac1 signaling and cell migration in human hepatoma HepG2 cells.

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    ZhenZhen Hu

    Full Text Available BACKGROUND: Epidermal growth factor (EGF signaling is implicated in the invasion and metastasis of hepatoma cells. However, the signaling pathways for EGF-induced motility of hepatoma cells remain undefined. METHODOLOGY/PRINCIPAL FINDINGS: We found that EGF dose-dependently stimulated the migration of human hepatoma cells HepG2, with the maximal effect at 10 ng/mL. Additionally, EGF increased Arf6 activity, and ectopic expression of Arf6 T27N, a dominant negative Arf6 mutant, largely abolish EGF-induced cell migration. Blocking GEP100 with GEP100 siRNA or GEP100-△PH, a pleckstrin homology (PH domain deletion mutant of GEP100, blocked EGF-induced Arf6 activity and cell migration. EGF also increased ERK and Rac1 activity. Ectopic expression GEP100 siRNA, GEP100-△PH, or Arf6-T27N suppressed EGF-induced ERK and Rac1 activity. Furthermore, blocking ERK signaling with its inhibitor U0126 remarkably inhibited both EGF-induced Rac1 activation as well as cell migration, and ectopic expression of inactive mutant form of Rac1 (Rac1-T17N also largely abolished EGF-induced cell migration. CONCLUSIONS/SIGNIFICANCE: Taken together, this study highlights the function of the PH domain of GEP100 and its regulated Arf6/ERK/Rac1 signaling cascade in EGF-induced hepatoma cell migration. These findings could provide a rationale for designing new therapy based on inhibition of hepatoma metastasis.

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

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    Xiao-pei Tong

    2017-01-01

    Full Text Available 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.

  2. Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism

    Directory of Open Access Journals (Sweden)

    Hesham M. Korashy

    2012-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  5. Over-expression and siRNA of a novel environmental lipopolysaccharide-responding gene on the cell cycle of the human hepatoma-derived cell line HepG2

    International Nuclear Information System (INIS)

    Du Kejun; Chai Yubo; Hou Lichao; Chang Wenhui; Chen Suming; Luo Wenjing; Cai Tongjian; Zhang Xiaonan; Chen Nanchun; Chen Yaoming; Chen Jingyuan

    2008-01-01

    Lipopolysaccharide (LPS) is the toxic determinant for Gram-negative bacterium infection. The individual response to LPS was related to its gene background. It is necessary to identify new molecules and signaling transduction pathways about LPS. The present study was undertaken to evaluate the effects of a novel environmental lipopolysaccharide-responding (Elrg) gene on the regulation of proliferation and cell cycle of the hepatoma-derived cell line, HepG2. By means of RT-PCR, the new molecule of Elrg was generated from a human dental pulp cell cDNA library. Expression level of Elrg in HepG2 cells was remarkably upgraded by the irritation of LPS. Localization of Elrg in HepG2 cells was positioned mainly in cytoplasm. HepG2 cells were markedly arrested in the G1 phase by over-expressing Elrg. The percentage of HepG2 cells in G1 phase partly decreased after Elrg-siRNA. In conclusion, Elrg is probably correlative with LPS responding. Elrg is probably a new protein in cytoplasm which plays an important role in regulating cell cycle. The results will deepen our understanding about the potential effects of Elrg on the human hepatoma-derived cell line HepG2

  6. Analysis of changes in energy and redox states in HepG2 hepatoma and C6 glioma cells upon exposure to cadmium

    International Nuclear Information System (INIS)

    Yang, M.S.; Yu, L.C.; Gupta, R.C.

    2004-01-01

    The energy and redox states of the HepG2 hepatoma and the C6 glioma cells were studied by quantifying the levels of ATP, ADP, AMP, GSH, and GSSG. These values were used to calculate the energy charge potential (ECP = [ATP + 0.5ADP]/TAN), total adenosine nucleotides (TAN = ATP + ADP + AMP), total glutathione (TG = [GSH + GSSG]/TAN), and the redox state (GSH/GSSG ratio). For comparison between cell types, the level of each energy metabolite (ATP, ADP, and AMP) was normalized against TAN of the respective cell. The results showed that ATP:ADP:AMP ratio was 0.76:0.11:0.13 for the HepG2 cells and 0.80:0.11:0.09 for the C6 glioma cells. ECP was 0.81 ± 0.01 and 0.85 ± 0.01 for the HepG2 and the C6 glioma cells, respectively. GSH/GSSG ratio was 2.66 ± 0.16 and 3.63 ± 0.48 for HepG2 and C6 glioma cells, respectively. TG was 3.2 ± 0.54 for the HepG2 cells and 2.43 ± 0.18 for the C6 glioma cells, indicating that the level of total glutathione is more than two to three times higher than the total energy metabolites in these cell lines. Following a 3-h incubation in medium containing different concentrations of Cd, there was a dose-dependent decrease in cell viability. The 3-h LC 50 for the HepG2 cells was 0.5 mM and that for the C6 glioma cells was 0.4 mM. Cellular TAN decreased with a decrease in cell viability. Upon careful analysis of the energy state, there was a significant increase in relative amount of ATP and decrease in ADP and AMP in both cells as Cd concentration increased from 0 to 0.1, 0.2, and 0.6 mM. However, ECP in both cell lines increased, which indicated that the level of high energy phosphate was adequate. There was also a significant increase in TG and a significant decrease in GSH/GSSG in the C6 glioma cells when cells were exposed to as low as 0.1 mM Cd, which suggested that the cellular redox state was compromised. The HepG2 cells, on the other hand, showed no significant change in both TG and GSH/GSSG level until Cd concentration reached 0.6 m

  7. Salmonella typhimurium strain SL7207 induces apoptosis and inhibits the growth of HepG2 hepatoma cells in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Baowei Li

    2012-12-01

    Full Text Available Salmonella typhimurium is probably most extensively studied tumor-targeting bacteria and SL7207 is one of its attenuated strains. SL7207 was first made for bacterial vaccine development and its therapeutic efficacy and safety for hepatocellular carcinoma has not been characterized. In this study, the inhibitory ability of SL7207-lux on human hepatoma HepG2 cells was tested in vitro and in vivo. A bacterial luminescent gene cluster (lux CDABE was transfected into SL7207 to better monitor the invasion of the bacteria. The results show that SL7207-lux can rapidly enter HepG2 cells and localize in the cytoplasm. This invasion represses cell proliferation and induces apoptosis. In vivo real-time invasion studies showed that the bacteria gradually accumulate in the tumor. This enrichment was confirmed by anatomic observation at 5 days after inoculation. About 40% of tumor growth was inhibited by SL7207-lux at 34 days post-treatment without significant loss of body weight. The area of necrosis of tumor tissue was clearly increased in the treated group. Bacterial quantification showed that the number of colony-forming units per gram of bacteria within tumor tissue was approximately 1000-fold higher than that of liver and spleen. These data suggest that attenuated S. typhimurium strain SL7207 has potential for the treatment of cancers.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

    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.

  9. 3-Nitrobenzanthrone (3-NBA) induced micronucleus formation and DNA damage in human hepatoma (HepG2) cells.

    Science.gov (United States)

    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.

  10. Basil extract inhibits the sulfotransferase mediated formation of DNA adducts of the procarcinogen 1′-hydroxyestragole by rat and human liver S9 homogenates and in HepG2 human hepatoma cells

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Punt, A.; Delatour, T.; Rietjens, I.M.C.M.

    2008-01-01

    The effects of a basil extract on the sulfation and concomitant DNA adduct formation of the proximate carcinogen 1′-hydroxyestragole were studied using rat and human liver S9 homogenates and the human hepatoma cell line HepG2. Basil was chosen since it contains the procarcinogen estragole that can

  11. Basil extract inhibits the sulfotransferase mediated formation of DNA adducts of the procarcinogen 1'-hydroxyestragole by rat and human liver S9 homogenates and in HepG2 human hepatoma cells

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Punt, A.; Delatour, T.; Rietjens, I.M.C.M.

    2008-01-01

    The effects of a basil extract on the sulfation and concomitant DNA adduct formation of the proximate carcinogen 1¿-hydroxyestragole were studied using rat and human liver S9 homogenates and the human hepatoma cell line HepG2. Basil was chosen since it contains the procarcinogen estragole that can

  12. Genotoxic potential of montmorillonite clay mineral and alteration in the expression of genes involved in toxicity mechanisms in the human hepatoma cell line HepG2

    Energy Technology Data Exchange (ETDEWEB)

    Maisanaba, Sara, E-mail: saramh@us.es [Area of Toxicology, Faculty of Pharmacy, University of Sevilla, Profesor García González no. 2, 41012 Seville (Spain); Hercog, Klara; Filipic, Metka [National Institute of Biology, Department for Genetic Toxicology and Cancer Biology, Vecna pot 111, 1000 Ljubljana (Slovenia); Jos, Ángeles [Area of Toxicology, Faculty of Pharmacy, University of Sevilla, Profesor García González no. 2, 41012 Seville (Spain); Zegura, Bojana [National Institute of Biology, Department for Genetic Toxicology and Cancer Biology, Vecna pot 111, 1000 Ljubljana (Slovenia)

    2016-03-05

    Highlights: • Cloisite{sup ®}Na{sup +} has a wide range of well-documented and novel applications. • Cloisite{sup ®}Na{sup +} induces micronucleus, but not nuclear bridges or nuclear buds in HepG2 cells. • Cloisite{sup ®}Na{sup +} induces changes in the gene expression. • Gene alteration is presented mainly after 24 h of exposure to Cloisite{sup ®}Na{sup +}. - Abstract: Montmorillonite, also known as Cloisite{sup ®}Na{sup +} (CNa{sup +}), 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{sup +} 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{sup +} (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{sup +} on the expression of several genes involved in toxicity mechanisms using the real-time quantitative PCR. The results showed that CNa{sup +} 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{sup +} is potentially genotoxic. Therefore further studies that will elucidate the molecular mechanisms involved in toxic activity of CNa{sup +} are needed for hazard identification and human safety assessment.

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

    Directory of Open Access Journals (Sweden)

    Liu X

    2016-07-01

    Full Text Available Xi Liu,1–4 Yan Liu,1–4 Pengcheng Zhang,1–4 Xiaodong Jin,1–3 Xiaogang Zheng,1–4 Fei Ye,1–4 Weiqiang Chen,1–3 Qiang Li1–3 1Institute of Modern Physics, 2Key Laboratory of Heavy Ion Radiation Biology and Medicine, Chinese Academy of Sciences, 3Key Laboratory of Basic Research on Heavy Ion Radiation Application in Medicine, Gansu Province, Lanzhou, 4School of Life Science, University of Chinese Academy of Sciences, Beijing, People’s Republic of China Abstract: 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. Keywords: AuNPs, radiation enhancement, synergistic effect, human hepatoma cells, hydroxyl radical production

  14. Genotoxic potential of montmorillonite clay mineral and alteration in the expression of genes involved in toxicity mechanisms in the human hepatoma cell line HepG2.

    Science.gov (United States)

    Maisanaba, Sara; Hercog, Klara; Filipic, Metka; Jos, Ángeles; Zegura, Bojana

    2016-03-05

    Montmorillonite, also known as Cloisite(®)Na(+) (CNa(+)), is a natural clay with a wide range of well-documented and novel applications, such as pharmaceutical products or food packaging. Although considered a low toxic product, the expected increased exposure to CNa(+) arises concern on the potential consequences on human and environmental health especially as its genotoxicity has scarcely been investigated so far. Thus, we investigated, for the first time, the influence of non-cytotoxic concentrations of CNa(+) (15.65, 31.25 and 62.5 μg/mL) on genomic instability of human hepatoma cell line (HepG2) by determining the formation of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) with the Cytokinesis block micronucleus cytome assay. Further on we studied the influence of CNa(+) on the expression of several genes involved in toxicity mechanisms using the real-time quantitative PCR. The results showed that CNa(+) increased the number of MNi, while the numbers of NBUDs and NPBs were not affected. In addition it deregulated genes in all the groups studied, mainly after longer time of exposure. These findings provide the evidence that CNa(+) is potentially genotoxic. Therefore further studies that will elucidate the molecular mechanisms involved in toxic activity of CNa(+) are needed for hazard identification and human safety assessment. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  18. Oroxylin A regulates glucose metabolism in response to hypoxic stress with the involvement of Hypoxia-inducible factor-1 in human hepatoma HepG2 cells.

    Science.gov (United States)

    Dai, Qinsheng; Yin, Qian; Wei, Libin; Zhou, Yuxin; Qiao, Chen; Guo, Yongjian; Wang, Xiaotang; Ma, Shiping; Lu, Na

    2016-08-01

    Metabolic alteration in cancer cells is one of the most conspicuous characteristics that distinguish cancer cells from normal cells. In this study, we investigated the influence and signaling ways of oroxylin A affecting cancer cell energy metabolism under hypoxia. The data showed that oroxylin A remarkably reduced the generation of lactate and glucose uptake under hypoxia in HepG2 cells. Moreover, oroxylin A inhibited HIF-1α expression and its stability. The downstream targets (PDK1, LDHA, and HK II), as well as their mRNA levels were also suppressed by oroxylin A under hypoxia. The silencing or the overexpression of HIF-1α assays suggested that HIF-1α is required for metabolic effect of oroxylin A in HepG2 cells during hypoxia. Furthermore, oroxylin A could reduce the expression of complex III in mitochondrial respiratory chain, and then decrease the accumulation of ROS at moderate concentrations (0-50 µM) under hypoxia, which was benefit for its inhibition on glycolytic activity by decreasing ROS-mediated HIF-1 expression. Besides, oroxylin A didn't cause the loss of MMP under hypoxia and had no obvious effects on the expression of OXPHOS complexes, suggesting that oroxylin A did not affect mitochondrial mass at the moderate stress of oroxylin A. The suppressive effect of oroxylin A on glycolysis led to a significantly repress of ATP generation, for ATP generation mostly depends on glycolysis in HepG2 cells. This study revealed a new aspect of glucose metabolism regulation of oroxylin A under hypoxia, which may contribute to its new anticancer mechanism. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  19. Bioluminescence-based cytotoxicity assay for simultaneous evaluation of cell viability and membrane damage in human hepatoma HepG2 cells.

    Science.gov (United States)

    Uno, Katsuhiro; Murotomi, Kazutoshi; Kazuki, Yasuhiro; Oshimura, Mitsuo; Nakajima, Yoshihiro

    2018-05-01

    We have developed a bioluminescence-based non-destructive cytotoxicity assay in which cell viability and membrane damage are simultaneously evaluated using Emerald luciferase (ELuc) and endoplasmic reticulum (ER)-targeted copepod luciferase (GLuc-KDEL), respectively, by using multi-integrase mouse artificial chromosome (MI-MAC) vector. We have demonstrated that the time-dependent concentration response curves of ELuc luminescence intensity and WST-1 assay, and GLuc-KDEL luminescence intensity and lactate dehydrogenase (LDH) activity in the culture medium accompanied by cytotoxicity show good agreement in toxicant-treated ELuc- and GLuc-KDEL-expressing HepG2 stable cell lines. We have clarified that the increase of GLuc-KDEL luminescence intensity in the culture medium reflects the type of cell death, including necrosis and late apoptosis, but not early apoptosis. We have also uncovered a strong correlation between GLuc-KDEL luminescence intensity in the culture medium and the extracellular release of high mobility group box 1 (HMGB1), a representative damage-associated molecular pattern (DAMP) molecule. The bioluminescence measurement assay using ELuc and GLuc-KDEL developed in this study can simultaneously monitor cell viability and membrane damage, respectively, and the increase of GLuc-KDEL luminescence intensity in the culture medium accompanied by the increase of cytotoxicity is an index of necrosis and late apoptosis associated with the extracellular release of DAMP molecules. Copyright © 2018 John Wiley & Sons, Ltd.

  20. Differences in TCDD-elicited gene expression profiles in human HepG2, mouse Hepa1c1c7 and rat H4IIE hepatoma cells

    Directory of Open Access Journals (Sweden)

    Burgoon Lyle D

    2011-04-01

    Full Text Available Abstract Background 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD is an environmental contaminant that elicits a broad spectrum of toxic effects in a species-specific manner. Current risk assessment practices routinely extrapolate results from in vivo and in vitro rodent models to assess human risk. In order to further investigate the species-specific responses elicited by TCDD, temporal gene expression responses in human HepG2, mouse Hepa1c1c7 and rat H4IIE cells were compared. Results Microarray analysis identified a core set of conserved gene expression responses across species consistent with the role of AhR in mediating adaptive metabolic responses. However, significant species-specific as well as species-divergent responses were identified. Computational analysis of the regulatory regions of species-specific and -divergent responses suggests that dioxin response elements (DREs are involved. These results are consistent with in vivo rat vs. mouse species-specific differential gene expression, and more comprehensive comparative DRE searches. Conclusions Comparative analysis of human HepG2, mouse Hepa1c1c7 and rat H4IIE TCDD-elicited gene expression responses is consistent with in vivo rat-mouse comparative gene expression studies, and more comprehensive comparative DRE searches, suggesting that AhR-mediated gene expression is species-specific.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  3. Estrogen receptor α and aryl hydrocarbon receptor cross-talk in a transfected hepatoma cell line (HepG2 exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

    Directory of Open Access Journals (Sweden)

    Manuela Göttel

    2014-01-01

    Full Text Available The prototype dioxin congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD is known to exert anti-estrogenic effects via activation of the aryl hydrocarbon receptor (AhR by interfering with the regulation of oestrogen homeostasis and the estrogen receptor α (ERα signalling pathway. The AhR/ER cross-talk is considered to play a crucial role in TCDD- and E2-dependent mechanisms of carcinogenesis, though the concerted mechanism of action in the liver is not yet elucidated. The present study investigated TCDD's impact on the transcriptional cross-talk between AhR and ERα and its modulation by 17β-estradiol (E2 in the human hepatoma cell line HepG2, which is AhR-responsive but ERα-negative. Transient transfection assays with co-transfection of hERα and supplementation of receptor antagonists showed anti-estrogenic action of TCDD via down-regulation of E2-induced ERα signaling. In contrast, enhancement of AhR signaling dependent on ERα was observed providing evidence for increased cytochrome P450 (CYP induction to promote E2 metabolism. However, relative mRNA levels of major E2-metabolizing CYP1A1 and 1B1 and the main E2-detoxifying catechol-O-methyltransferase were not affected by the co-treatments. This study provides new evidence of a TCDD-activated AhR-mediated molecular AhR/ERα cross-talk mechanism at transcriptional level via indirect inhibition of ERα and enhanced transcriptional activity of AhR in HepG2 cells.

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

    Directory of Open Access Journals (Sweden)

    Haider Raza

    Full Text Available 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.

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

    Science.gov (United States)

    Yamaguchi, Masayoshi; Murata, Tomiyasu

    2018-04-05

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Tee, Thiam-Tsui, E-mail: thiamtsu@yahoo.com [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Cheah, Yew-Hoong [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, Jalan Pahang, Kuala Lumpur (Malaysia); Meenakshii, Nallappan [Biology Department, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Mohd Sharom, Mohd Yusof; Azimahtol Hawariah, Lope Pihie [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia)

    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.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    Highlights: ► We isolated xanthorrhizol, a sesquiterpenoid compound from Curcuma xanthorrhiza. ► Xanthorrhizol induced apoptosis in HepG2 cells as observed using SEM. ► Apoptosis in xanthorrhizol-treated HepG2 cells involved Bcl-2 family proteins. ► DNA fragmentation was observed in xanthorrhizol-treated HepG2 cells. ► 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 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.

  8. Mannose 6-phosphate-independent targeting of cathepsin D to lysosomes in HepG2 cells

    NARCIS (Netherlands)

    Rijnboutt, S.; Kal, A. J.; Geuze, H. J.; Aerts, H.; Strous, G. J.

    1991-01-01

    We have studied the role of N-linked oligosaccharides and proteolytic processing on the targeting of cathepsin D to the lysosomes in the human hepatoma cell line HepG2. In the presence of tunicamycin cathepsin D was synthesized as an unglycosylated 43-kDa proenzyme which was proteolytically

  9. Isolation and establishment of radiotolerant hepatoma cell subline

    International Nuclear Information System (INIS)

    Jin Wensen; Kong Zhaolu; Zhang Jianghong; Shen Zhifen; Tong Shungao; Ji Huajun; Jin Yizun

    2009-01-01

    Objective: To induce and isolate the monoclonal cell subline, in order to establish the experimental model for further investigating biologic characteristics in radiotolerant hepatoma cells. Methods: HepG2 cells were irradiated by γ-rays at the dose of 2 Gy each time with the total absorbed dose of 60 Gy. After monoclonal cell being selected and extensively cultured, the cell subline was named as HepG2/R60. Furthermore, HepG2/R60 cells were identified by observing the changes of morphology, ultrastructure, growth characteristics and radiosensitivity. The levels of radioresistant correlative gene mRNA in HepG2/R60 cells after exposure to 2 Gy irradiation, were also detected by RT-PCR, and then compared with parental HepG2 cells. Results: HepG2/R60 cell subline was successfully established by fractionated irradiation at 2 Gy. HepG2/R60 cells displayed higher irregularity, the clearer appearance and dissociation of cell junctions compared with parental HepG2 cells. Ultrastnictural investigations through transmission electron microscopy (TEM) showed that there was an increase of microvillus on the surfaces of HepG2/R60 cells with plenty of rough endo-plasmic reticulum, abundance of mitochondria and viable Golgi complex. Further observation found that the growth of HepG2/R60 cells was slower and its population doubling time (PDT) prolonged arrived at 34.9 h. Moreover, the radiosensitivity of HepG2/R60 cells was lower than that of parental HepG2 cells. Additionally, the levels of radioresistance correlative genes were increased in HepG2/R60 cells by 2 Gy irradiaiton Conclusions: Radiotolerant cell subline - HepG2/R60 was successfully isolated and established by fractionated irradiation. (authors)

  10. Investigating free radical generation in HepG2 cells using immuno-spin trapping.

    Science.gov (United States)

    Horinouchi, Yuya; Summers, Fiona A; Ehrenshaft, Marilyn; Kawazoe, Kazuyoshi; Tsuchiya, Koichiro; Tamaki, Toshiaki; Mason, Ronald P

    2014-10-01

    Oxidative stress can induce the generation of free radicals, which are believed to play an important role in both physiological and pathological processes and a number of diseases such as cancer. Therefore, it is important to identify chemicals which are capable of inducing oxidative stress. In this study, we evaluated the ability of four environmental chemicals, aniline, nitrosobenzene (NB), N,N-dimethylaniline (DMA) 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 (LDH) assays and morphological changes were observed using phase contrast microscopy. Free radicals were detected by immuno-spin trapping (IST) in in-cell western experiments or in confocal microscopy experiments to determine the subcellular localization of free radical generation. DMNA induced free radical generation, LDH release and morphological changes in HepG2 cells whereas aniline, NB and DMA 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 upon subsequent incubation with DMNA, whereas preincubation with apocynin and dimethyl sulfoxide did not. These results suggest that DMNA induces oxidative stress and that reactive oxygen species, metals and free radical generation play a critical role in DMNA-induced cytotoxicity. Copyright © 2014. Published by Elsevier Inc.

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

    International Nuclear Information System (INIS)

    Alia, Mario; Ramos, Sonia; Mateos, Raquel; Granado-Serrano, Ana Belen; Bravo, Laura; Goya, Luis

    2006-01-01

    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 μM quercetin completely prevented lactate dehydrogenase leakage from the cells. Pretreatment for 2 or 20 h with all doses of quercetin (0.1-10 μM) 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 μM and for 20 h with 5 μM 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

  12. Biodistribution and SPECT imaging of 99Tcm labeling NGR peptide in nude mice bearing human HePG2 hepatoma

    International Nuclear Information System (INIS)

    Ma Wenhui; Wang Jing; Yang Weidong; Li Guiyu; Ma Xiaowei; Wang Zhe

    2012-01-01

    A peptide containing the Asn-Gly-Arg (NGR) sequence was radiolabeled by 99 Tc m and its radiochemical characteristics, biodistribution and SPECT imaging in nude mice bearing human HePG2 hepatoma were evaluated. 99 Tc m -NGR was prepared directly with a labeling yield higher than 90%, and the radiochemical purity (RCP) higher than 95%. Nude mice bearing human HePG2 hepatoma were randomly divided into 6 groups with 3 mice in each group. The control group mice were blocked by injecting 100 μg unlabeled NGR 0.5 h before 99 Tc m -NGR injection. The mice were sacrificed at 1, 2, 4, 8, 12 h after caudal intravenous injection of 7.4 MBq 99 Tc m -NGR. The uptakes of kidney and liver were very high. Tumor uptake was (2.52±0.62)% ID/g at 1 h, with the highest uptake of (7.26±2.71) %ID/g. At 12 h, the uptake was still (3.93±1.93) %ID/g. In comparison, the uptake of the blocked control group was (1.29±0.85) %ID/g. The SPECT static images of 3 mice and the tumor/muscle (T/NT) value were obtained. The highest T/NT value was 3.25 at 4 h. The xenografted tumor became visible at 1 h and the clearest image of the tumor was observed at 12 h. Results from this work shows that 99 Tc m -NGR can be efficiently prepared, can favorably target tumor angiogenesis, and should be a potential probe in tumor therapy. (authors)

  13. PPARγ activates ABCA1 gene transcription but reduces the level of ABCA1 protein in HepG2 cells

    International Nuclear Information System (INIS)

    Mogilenko, Denis A.; Shavva, Vladimir S.; Dizhe, Ella B.; Orlov, Sergey V.; Perevozchikov, Andrej P.

    2010-01-01

    Research highlights: → PPARγ activates ABCA1 gene expression but decreases ABCA1 protein content in human hepatoma cell line HepG2. → Treatment of HepG2 cells with PPARγ agonist GW1929 leads to dissociation of LXRβ from ABCA1-LXRβ complex. → Inhibition of protein kinases MEK1/2 abolishes PPARγ-mediated dissociation of LXRβ from ABCA1/LXRβ complex. → Activation of PPARγ leads to increasing of the level of LXRβ associated with LXRE within ABCA1 gene promoter. -- Abstract: Synthesis of ABCA1 protein in liver is necessary for high-density lipoproteins (HDL) formation in mammals. Nuclear receptor PPARγ is known as activator of ABCA1 expression, but details of PPARγ-mediated regulation of ABCA1 at both transcriptional and post-transcriptional levels in hepatocytes have not still been well elucidated. In this study we have shown, that PPARγ activates ABCA1 gene transcription in human hepatoma cells HepG2 through increasing of LXRβ binding with promoter region of ABCA1 gene. Treatment of HepG2 cells with PPARγ agonist GW1929 leads to dissociation of LXRβ from ABCA1/LXRβ complex and to nuclear translocation of this nuclear receptor resulting in reduction of ABCA1 protein level 24 h after treatment. Inhibition of protein kinases MEK1/2 abolishes PPARγ-mediated dissociation of LXRβ from ABCA1/LXRβ complex, but does not block PPARγ-dependent down-regulation of ABCA1 protein in HepG2 cells. These data suggest that PPARγ may be important for regulation of the level of hepatic ABCA1 protein and indicate the new interplays between PPARγ, LXRβ and MEK1/2 in regulation of ABCA1 mRNA and protein expression.

  14. Cellular trafficking of thymosin beta-4 in HEPG2 cells following serum starvation.

    Directory of Open Access Journals (Sweden)

    Giuseppina Pichiri

    Full Text Available Thymosin beta-4 (Tβ4 is an ubiquitous multi-functional regenerative peptide, related to many critical biological processes, with a dynamic and flexible conformation which may influence its functions and its subcellular distribution. For these reasons, the intracellular localization and trafficking of Tβ4 is still not completely defined and is still under investigation in in vivo as well as in vitro studies. In the current study we used HepG2 cells, a human hepatoma cell line; cells growing in normal conditions with fetal bovine serum expressed high levels of Tβ4, restricted to the cytoplasm until 72 h. At 84 h, a diffuse Tβ4 cytoplasmic immunostaining shifted to a focal perinuclear and nuclear reactivity. In the absence of serum, nuclear reactivity was localized in small granules, evenly dispersed throughout the entire nuclear envelop, and was observed as earlier as at 48 h. Cytoplasmic immunostaining for Tβ4 in HepG2 cells under starvation appeared significantly lower at 48 h and decreased progressively at 72 and at 84 h. At these time points, the decrease in cytoplasmic staining was associated with a progressive increase in nuclear reactivity, suggesting a possible translocation of the peptide from the cytoplasm to the nuclear membrane. The normal immunocytochemical pattern was restored when culture cells submitted to starvation for 84 h received a new complete medium for 48 h. Mass spectrometry analysis, performed on the nuclear and cytosolic fractions of HepG2 growing with and without serum, showed that Tβ4 was detectable only in the cytosolic and not in the intranuclear fraction. These data suggest that Tβ4 is able to translocate from different cytoplasmic domains to the nuclear membrane and back, based on different stress conditions within the cell. The punctuate pattern of nuclear Tβ4 immunostaining associated with Tβ4 absence in the nucleoplasm suggest that this peptide might be localized in the nuclear pores, where it could

  15. Actin filaments and microtubules are involved in different membrane traffic pathways that transport sphingolipids to the apical surface of polarized HepG2 cells

    NARCIS (Netherlands)

    Zegers, MMP; Zaal, KJM; van Ijzendoorn, SCD; Klappe, K; Hoekstra, D

    In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and

  16. Effects of the radiolysis products of sennoside A on HepG2 and PC-3 cell

    International Nuclear Information System (INIS)

    Kim, Dong Ho; Jo, Min Ho

    2016-01-01

    Radiolysis of sennoside A was carried out by gamma irradiation and the anti-cancer activities of the radiolysis product were evaluated. An aqueous solution of sennoside A was exposed to 0.5-3 kGy of gamma irradiation and the radiolysis products were analyzed by HPLC. A fraction of radiolysis product (RLF) of sennoside A was isolated and the RLF was presumed as a rhein-8-β-D-glucoside. The anticancer effect of the RLF was compared with the sennoside and rhein using a in vitro assay system of human prostate cancer cells (PC-3) and human hepatoma HepG2 cells. The cell viability of PC-3 and HepG2 cell was significantly decreased to 12.4±1.2% and 32.4±2.1%, respectively, by the treatment of 0.6 μM of RLF. The sennoside A (range from 0 to 25 μM) had no cytotoxic effect on PC-3 and HepG2 cells, while the rhein had the effect on HepG2 cells with a LD_5_0 at 80 μM

  17. Effects of the radiolysis products of sennoside A on HepG2 and PC-3 cell

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dong Ho; Jo, Min Ho [Research Division for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2016-11-15

    Radiolysis of sennoside A was carried out by gamma irradiation and the anti-cancer activities of the radiolysis product were evaluated. An aqueous solution of sennoside A was exposed to 0.5-3 kGy of gamma irradiation and the radiolysis products were analyzed by HPLC. A fraction of radiolysis product (RLF) of sennoside A was isolated and the RLF was presumed as a rhein-8-β-D-glucoside. The anticancer effect of the RLF was compared with the sennoside and rhein using a in vitro assay system of human prostate cancer cells (PC-3) and human hepatoma HepG2 cells. The cell viability of PC-3 and HepG2 cell was significantly decreased to 12.4±1.2% and 32.4±2.1%, respectively, by the treatment of 0.6 μM of RLF. The sennoside A (range from 0 to 25 μM) had no cytotoxic effect on PC-3 and HepG2 cells, while the rhein had the effect on HepG2 cells with a LD{sub 50} at 80 μM.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Hugo Miguel Santos

    2013-12-01

    Full Text Available 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.

  20. α-Tocopherol modulates the low density lipoprotein receptor of human HepG2 cells

    Directory of Open Access Journals (Sweden)

    Bottema Cynthia DK

    2003-05-01

    Full Text Available Abstract The aim of this study was to determine the effects of vitamin E (α-tocopherol on the low density lipoprotein (LDL receptor, a cell surface protein which plays an important role in controlling blood cholesterol. Human HepG2 hepatoma cells were incubated for 24 hours with increasing amounts of α, δ, or γ-tocopherol. The LDL receptor binding activity, protein and mRNA, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA reductase mRNA, cell cholesterol and cell lathosterol were measured. The effect of α-tocopherol was biphasic. Up to a concentration of 50 μM, α-tocopherol progressively increased LDL receptor binding activity, protein and mRNA to maximum levels 2, 4 and 6-fold higher than control, respectively. The HMG-CoA reductase mRNA and the cell lathosterol concentration, indices of cholesterol synthesis, were also increased by 40% over control by treatment with 50 μM α-tocopherol. The cell cholesterol concentration was decreased by 20% compared to control at 50 μM α-tocopherol. However, at α-tocopherol concentrations higher than 50 μM, the LDL receptor binding activity, protein and mRNA, the HMG-CoA reductase mRNA and the cell lathosterol and cholesterol concentrations all returned to control levels. The biphasic effect on the LDL receptor was specific for α-tocopherol in that δ and γ-tocopherol suppressed LDL receptor binding activity, protein and mRNA at all concentrations tested despite the cells incorporating similar amounts of the three homologues. In conclusion, α-tocopherol, exhibits a specific, concentration-dependent and biphasic "up then down" effect on the LDL receptor of HepG2 cells which appears to be at the level of gene transcription. Cholesterol synthesis appears to be similarly affected and the cell cholesterol concentration may mediate these effects.

  1. Time- and concentration-dependent effects of resveratrol in HL-60 and HepG2 cells

    DEFF Research Database (Denmark)

    Stervbo, Ulrik; Vang, Ole; Bonnesen, Christine

    2006-01-01

    Resveratrol, a phytochemical present in grapes, has been demonstrated to inhibit tumourigenesis in animal models. However, the specific mechanism by which resveratrol exerts its anticarcinogenic effect has yet to be elucidated. In the present study, the inhibitory effects of resveratrol on cell...... proliferation and apoptosis were evaluated in the human leukaemia cell line HL-60 and the human hepatoma derived cell line HepG2. We found that after a 2 h incubation period, resveratrol inhibited DNA synthesis in a concentration-dependent manner. The IC50 value was 15 μM in both HL-60 and HepG2 cells. When...... the time of treatment was extended, an increase in IC50 value was observed; for example, at 24 h the IC50 value was 30 μM for HL-60 cells and 60 μM for HepG2 cells. Flow cytometry revealed that cells accumulated in different phases of the cell cycle depending on the resveratrol concentration. Furthermore...

  2. silver nanoparticles on liver cancer cells (HepG2

    Directory of Open Access Journals (Sweden)

    Ahmed I. El-Batal

    2018-01-01

    Full Text Available This study demonstrates a novel approach for the synthesis of silver nanoparticles (AgNPs against human liver cancer cell line (HepG2 using prodigiosin pigment isolated from Serratia marcescens. It further investigates the influence of various parameters such as initial pH, temperature, silver nitrate (AgNO 3 concentration, and prodigiosin concentration on stability and optical properties of synthesized prodigiosin AgNPs. Highly stable, spherical prodigiosin-conjugated AgNPs were synthesized with a mean diameter of 9.98 nm using a rapid one-step method. The cytotoxic activity investigated in the present study indicated that prodigiosin and prodigiosin-conjugated AgNPs possessed a strong cytotoxic potency against human liver cancer. The In silico molecular docking results of prodigiosin and prodigiosin-conjugated AgNPs are congruent with the In vitro studies and these AgNPs can be considered as good inhibitors of mitogen-activated protein kinase 1 (MEK kinases. The study opened the possibility of using prodigiosin-conjugated AgNPs to increase the efficiency of liver cancer treatment.

  3. Stimulation of LDL receptor activity in Hep-G2 cells by a serum factor(s)

    International Nuclear Information System (INIS)

    Ellsworth, J.L.; Brown, C.; Cooper, A.D.

    1988-01-01

    The regulation of low-density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep-G2 by serum components was examined. Incubation of dense monolayers of Hep-G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time-dependent increase in LDL receptor activity. Uptake and degradation of 125I-LDL was stimulated two- to four-fold, as compared with that of Hep-G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125I-LDL uptake plus degradation increased from 0.2 microgram/mg cell protein/4 h to 0.8 microgram/mg cell protein/4 h. In addition, a two-fold increase in cell surface binding of 125I-LDL to Hep-G2 cells was observed when binding was measured at 4 degrees C. There was no change in the apparent Kd. The stimulation of LDL receptor activity was suppressed in a concentration-dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125I-asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five-fold, as measured by [3H]thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; [14C]acetate incorporation into unesterified and esterified cholesterol was increased approximately five-fold. Incubation of Hep-G2 cells with high-density lipoproteins (200 micrograms protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125I-LDL. Stimulation of LDL receptor activity was dependent on a heat-stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G-75 column

  4. 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

    International Nuclear Information System (INIS)

    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

    2006-01-01

    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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    Highlights: ► VCC-1 is hypothesized to be associated with carcinogenesis. ► Levels of VCC-1 are increased significantly in HCC. ► Over-expression of VCC-1 could promotes cellular proliferation rate. ► Over-expression of VCC-1 inhibit the cisplatin-provoked apoptosis in HepG2 cells. ► 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.

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Oxidative stress induced lipid accumulation via SREBP1c activation in HepG2 cells

    International Nuclear Information System (INIS)

    Sekiya, Mika; Hiraishi, Ako; Touyama, Maiko; Sakamoto, Kazuichi

    2008-01-01

    SREBP1c (sterol regulatory element-binding protein 1c) is a metabolic-syndrome-associated transcription factor that controls fatty acid biosynthesis under glucose/insulin stimulation. Oxidative stress increases lipid accumulation, which promotes the generation of reactive oxygen species (ROS). However, we know little about the role of oxidative stress in fatty acid biosynthesis. To clarify the action of oxidative stress in lipid accumulation via SREBP1c, we examined SREBP1c activity in H 2 O 2 -treated mammalian cells. We introduced a luciferase reporter plasmid carrying the SREBP1c-binding site into HepG2 or COS-7 cells. With increasing H 2 O 2 dose, SREBP1c transcriptional activity increased in HepG2 cells but declined in COS-7 cells. RT-PCR analysis revealed that mRNA expression of SREBP1c gene or of SREBP1c-regulated genes rose H 2 O 2 dose-dependently in HepG2 cells but dropped in COS-7 cells. Lipid accumulation and levels of the nuclear form of SREBP1c increased in H 2 O 2 -stimulated HepG2 cells. ROS may stimulate lipid accumulation in HepG2 cells via SREBP1c activation

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    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)

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

    Energy Technology Data Exchange (ETDEWEB)

    Rathinaraj, Pierson [Auckland University of Technology, Institute of Biomedical Technologies (New Zealand); Lee, Kyubae; Choi, Yuri; Park, Soo-Young [Kyungpook National University, School of Applied Chemical Engineering, Graduate School (Korea, Republic of); Kwon, Oh Hyeong [Kumoh National Institute of Technology, Department of Polymer Science and Engineering (Korea, Republic of); Kang, Inn-Kyu, E-mail: ikkang@knu.ac.kr [Kyungpook National University, School of Applied Chemical Engineering, Graduate School (Korea, Republic of)

    2015-07-15

    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)

  10. ATM phosphorylation in HepG2 cells following continuous low dose-rate irradiation

    International Nuclear Information System (INIS)

    Mei Quelin; Du Duanming; Chen Zaizhong; Liu Pengcheng; Yang Jianyong; Li Yanhao

    2008-01-01

    Objective: To investigate the change of ATM phosphorylation in HepG2 cells following a continuous low dose-rate irradiation. Methods: Cells were persistently exposed to low dose-rate (8.28 cGy/h) irradiation. Indirect immunofluorescence and Western blot were used to detect the expression of ATM phosphorylated proteins. Colony forming assay was used to observe the effect of a low dose-rate irradiation on HepG2 cell survival. Results: After 30 min of low dose-rate irradiation, the phosphorylation of ATM occurred. After 6 h persistent irradiation, the expression of ATM phosphorylated protein reached the peak value, then gradually decreased. After ATM phosphorylation was inhibited with Wortmannin, the surviving fraction of HepG2 cells was lower than that of the irradiation alone group at each time point (P<0.05). Conclusions: Continuous low dose-rate irradiation attenuated ATM phosphorylation, suggesting that continuous low dose-rate irradiation has a potential effect for increasing the radiosensitivity of HepG2 cells. (authors)

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  13. Preparation of three-dimensional macroporous chitosan-gelatin B microspheres and HepG2-cell culture.

    Science.gov (United States)

    Huang, Fang; Cui, Long; Peng, Cheng-Hong; Wu, Xu-Bo; Han, Bao-San; Dong, Ya-Dong

    2016-12-01

    Chitosan-gelatin B microspheres with an open, interconnected, highly macroporous (100-200 µm) structure were prepared via a three-step protocol combining freeze-drying with an electrostatic and ionic cross-linking method. Saturated tripolyphosphate ethanol solution (85% ethanol) was chosen as the crosslinking agent to prevent destruction of the porous structure and to improve the biostability of the chitosan-gelatin B microspheres, with N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide/N-hydroxysuccinimide as a second crosslinking agent to react with gelatin A and fixed chitosan-gelatin B microspheres to attain improved biocompatibility. Water absorption of the three-dimensional macroporous chitosan-gelatin B microspheres (3D-P-CGMs) was 12.84, with a porosity of 85.45%. In vitro lysozyme degradation after 1, 3, 5, 7, 10, 14, and 21 days showed improved biodegradation in the 3D-P-CGMs. The morphology of human hepatoma cell lines (HepG2 cells) cultured on the 3D-P-CGMs was spherical, unlike that of cells cultured under traditional two-dimensional conditions. Scanning electron microscopy and paraffin sections were used to confirm the porous structure of the 3D-P-CGMs. HepG2 cells were able to migrate inside through the pore. Cell proliferation and levels of albumin and lactate dehydrogenase suggested that the 3D-P-CGMs could provide a larger specific surface area and an appropriate microenvironment for cell growth and survival. Hence, the 3D-P-CGMs are eminently suitable as macroporous scaffolds for cell cultures in tissue engineering and cell carrier studies. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  14. Impaired mitochondrial function in HepG2 cells treated with hydroxy-cobalamin[c-lactam]: A cell model for idiosyncratic toxicity

    International Nuclear Information System (INIS)

    Haegler, Patrizia; Grünig, David; Berger, Benjamin; Krähenbühl, Stephan; Bouitbir, Jamal

    2015-01-01

    The vitamin B12 analog hydroxy-cobalamin[c-lactam] (HCCL) impairs mitochondrial protein synthesis and the function of the electron transport chain. Our goal was to establish an in vitro model for mitochondrial dysfunction in human hepatoma cells (HepG2), which can be used to investigate hepatotoxicity of idiosyncratic mitochondrial toxicants. For that, HepG2 cells were treated with HCCL, which inhibits the function of methylmalonyl-CoA mutase and impairs mitochondrial protein synthesis. Secondary, cells were incubated with propionate that served as source of propionyl-CoA, a percursor of methylmalonyl-CoA. Dose-finding experiments were conducted to evaluate the optimal dose and treatment time of HCCL and propionate for experiments on mitochondrial function. 50 μM HCCL was cytotoxic after exposure of HepG2 cells for 2 d and 10 and 50 μM HCCL enhanced the cytotoxicity of 100 or 1000 μM propionate. Co-treatment with HCCL (10 μM) and propionate (1000 μM) dissipated the mitochondrial membrane potential and impaired the activity of enzyme complex IV of the electron transport chain. Treatment with HCCL decreased the mRNA content of mitochondrially encoded proteins, whereas the mtDNA content remained unchanged. We observed mitochondrial ROS accumulation and decreased mitochondrial SOD2 expression. Moreover, electron microscopy showed mitochondrial swelling. Finally, HepG2 cells pretreated with a non-cytotoxic combination of HCCL (10 μM) and propionate (100 μM) were more sensitive to the mitochondrial toxicants dronedarone, benzbromarone, and ketoconazole than untreated cells. In conclusion, we established and characterized a cell model, which could be used for testing drugs with idiosyncratic mitochondrial toxicity

  15. Hyperglycemia and anthocyanin inhibit quercetin metabolism in HepG2 cells

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    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. © 2016 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    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.

  19. Metformin affects the features of a human hepatocellular cell line (HepG2) by regulating macrophage polarization in a co-culture microenviroment.

    Science.gov (United States)

    Chen, Miaojiao; Zhang, Jingjing; Hu, Fang; Liu, Shiping; Zhou, Zhiguang

    2015-11-01

    cells. Notch signalling pathway is a potentially important mechanism in the regulation of metformin on macrophage polarization and the subsequent change of hepatoma cells. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  20. HepG2 human hepatocarcinomas cells sensitization by endogenous porphyrins

    Science.gov (United States)

    Vonarx-Coinsmann, Veronique; Foultier, Marie-Therese; de Brito, Leonor X.; Morlet, Laurent; Patrice, Thierry

    1995-03-01

    We assessed the ability of the human hepatocarcinoma cell line HepG2 to synthesize PpIX in vitro from exogenous ALA and analyzed ALA-induced toxicity and phototoxicity on this cell line. ALA induced a slight dose-dependent dark toxicity, with 79 and 66% cell survival respectively for ALA 50 and 100 mg/ml after 3-h incubation. Whereas the same treatment followed by laser irradiation (l equals 632 nm, 25 J/sq cm) induced dose-dependent phototoxicity, with 54 and 19% cell survival 24 h after PDT. Whatever the incubation time with ALA, a 3-h delay before light exposure was found optimal to reach a maximal phototoxicity. Photoproducts induced by porphyrin light irradiation absorbed light in the red spectral region at longer wavelengths than did the original porphyrins. The possible enhancement of PDT effects after ALA HepG2 cell incubation was investigated by irradiating cells successively with red light (l equals 632 nm) and light (l equals 650 nm). Total fluence was kept constant at 25 J/sq cm. Phototoxicity was lower when cells were irradiated for increased periods of l equals 650 nm light than with l equals 632 nm light alone. Any photoproducts involved had either a short life or were poorly photoreactive. HepG2 cells, synthesizing enzymes and precursors of endogenous porphyrin synthesis, represent a good in vitro model for experiments using ALA-PpIX-PDT.

  1. Surface Grafted Glycopolymer Brushes to Enhance Selective Adhesion of HepG2 Cells

    DEFF Research Database (Denmark)

    Chernyy, Sergey; Jensen, Bettina Elisabeth Brøgger; Shimizu, Kyoko

    2013-01-01

    on the polymerization kinetics of 2-lactobionamidoethyl methacrylate) (LAMA) monomer on thermally oxidized silicon wafer. Both monolayer and multilayered aminosilane precursor layers have been prepared followed by reaction with 2-bromoisobutyrylbromide to form the ATRP initiator layer. It is inferred from the kinetic...... studies that the rate of termination is low on a multilayered initiator layer compared to a disordered monolayer structure. However both initiator types results in similar graft densities. Furthermore, it is shown that thick comb-like poly(LAMA) brushes can be constructed by initiating a second ATRP...... process on a previously formed poly(LAMA) brushes. The morphology of human hepatocellular carcinoma cancer cells (HepG2) on the comb-like poly(LAMA) brush layer has been studied. The fluorescent images of the HepG2 cells on the glycopolymer brush surface display distinct protrusions that extend outside...

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

    Science.gov (United States)

    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.

  3. Aglycemia keeps mitochondrial oxidative phosphorylation under hypoxic conditions in HepG2 cells

    Czech Academy of Sciences Publication Activity Database

    Plecitá-Hlavatá, Lydie; Ježek, Jan; Ježek, Petr

    2015-01-01

    Roč. 47, č. 6 (2015), s. 467-476 ISSN 0145-479X R&D Projects: GA ČR(CZ) GAP302/10/0346; GA ČR(CZ) GA13-02033S; GA MŠk(CZ) LH11055 Institutional support: RVO:67985823 Keywords : cancer mitochondria * non-canonical response to hypoxia * hypoxia-inducible factor * glutaminolysis * HepG2 cell s Subject RIV: ED - Physiology Impact factor: 2.080, year: 2015

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

    Science.gov (United States)

    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.

  5. MicroRNA-122 mimic transfection contributes to apoptosis in HepG2 cells.

    Science.gov (United States)

    Huang, Hongyan; Zhu, Yueyong; Li, Shaoyang

    2015-11-01

    There is currently a requirement for effective treatment strategies for human hepatocellular carcinoma (HCC), a leading cause of cancer‑associated mortality. MicroRNA-122 (miR-122), a repressor of the endogenous apoptosis regulator Bcl‑w, is frequently downregulated in HCC. Thus, it is hypothesized that the activation of miR‑122 may induce selective hepatocellular apoptosis via caspase activation in a model of HCC. In the present study, an miR‑122 mimic transfection was performed in HepG2 cells, and used to investigate the role and therapeutic potential of miR‑122 in the regulation of HCC‑derived cell lines. The apoptotic rates of HepG2 cells were significantly increased following miR‑122 mimic transfection. Reverse transcription‑polymerase chain reaction analysis revealed that Bcl‑w mRNA was significantly reduced, while the mRNA levels of caspase‑9 and caspase‑3 were markedly increased. The immunocytochemistry results supported the mRNA trends. Collectively, the present results suggest that endogenous miR‑122 contributes to HepG2 apoptosis and that transfection of mimic miR‑122 normalizes apoptotic levels in a model of HCC.

  6. Effects of low priming dose irradiation on cell cycle arrest of HepG2 cells caused by high dose irradiation

    International Nuclear Information System (INIS)

    Xia Jingguang; Jin Xiaodong; Chinese Academy of Sciences, Beijing; Li Wenjian; Wang Jufang; Guo Chuanling; Gao Qingxiang

    2005-01-01

    Human hepatoma cells hepG2 were irradiated twice by 60 Co γ-rays with a priming dose of 5 cGy and a higher dose of 3 Gy performed 4h or 8h after the low dose irradiation. Effects of the priming dose irradiation on cell cycle arrest caused by high dose were examined with flow cytometry. Cells in G 2 /M phase accumulated temporarily after the 5 cGy irradiation, and proliferation of tumor cells was promoted significantly by the low dose irradiation. After the 3 Gy irradiation, G 2 phase arrest occurred, and S phase delayed temporally. In comparison with 3 kGy irradiation only, the priming dose delivered 4h prior to the high dose irradiation facilitated accumulation of hepG2 cells in G 2 /M phase, whereas the priming dose delivered 8h prior to the high dose irradiation helped the cells to overcome G 2 arrest. It was concluded that effects of the priming dose treatment on cell cycle arrest caused by high dose irradiation were dependent on time interval between the two irradiations. (authors)

  7. Specific binding of tubeimoside-2 with proteins in hepatocarcinoma HepG2 cells: investigation by molecular spectroscopy

    Science.gov (United States)

    Yang, Sun; Shi-Sheng, Sun; Ying-Yong, Zhao; Jun, Fan

    2012-07-01

    In this study, we compared different binding interactions of TBMS2 with proteins both in hepatocarcinoma HepG2 cells and in normal embryo hepatic L02 cells by using fluorescence, absorption, and CD spectroscopy. The fluorescence data revealed that the fluorescence intensity of proteins in the HepG2 and L02 cells decreased in the presence of TBMS2 by 30.79% and 12.01%, respectively. Binding constants and thermodynamic parameters were obtained for systems of TBMS2 with the two kinds of cell proteins. The results indicated that HepG2 cell proteins had a higher TBMS2 binding activity than those in the L02 cells. Analysis of the TBMS2 cytotoxic activities showed that TBMS2 could selectively induce apoptosis of HepG2 cells by binding to them, while its apoptotic effect on L02 cells was relatively weaker.

  8. Cytotoxic effects of the synthetic oestrogens and androgens on Balb/c 3T3 and HepG2 cells

    Directory of Open Access Journals (Sweden)

    Minta Maria

    2014-12-01

    Full Text Available The aim of the study was to test and compare the cytotoxic potential of two synthetic oestrogens: diethylstilboestrol (DES and ethinyloestradiol (EE2 and two androgens: testosterone propionate (TP and trenbolone (TREN on two cell lines. The fibroblast cell line Balb/c 3T3 and the hepatoma cell line HepG2 were selected. To get more insight into the mode of toxic action, four methods were used, which evaluated different biochemical endpoints: mitochondrial activity (3-(4,5-dimethylthiazol-2-yl- 2,5-diphenyltetrazolium bromide reduction assay, lysosomal activity (neutral red uptake assay, total protein content, and lactate dehydrogenase release. Cytotoxicity was assessed after 24, 48, and 72 h exposure to eight concentrations ranging from 0.78 to 100 μg/mL. Concentration- and time- dependent effects were observed. Depending on the line and assay used, half maximal effective concentration after 72 h (EC50-72h values ranged as follows: DES 1-13.7 μg/mL (Balb/c 3T3 and 3.7-5.2 μg/mL (HepG2; EE2 2.1-14.3 μg/mL (Balb/c 3T3 and 1.8-7.8 μg/mL (HepG2; TP-14.9-17.5 μg/mL (Balb/c 3T3, and 63.9- 100 μg/mL (HepG2; and TREN 11.3-31.4 μg/mL (Balb/c 3T3 and 12.5-59.4 μg/mL (HepG2. The results revealed that oestrogens were more toxic than androgens and the most affected endpoint was mitochondrial activity. In contrast to oestrogens, for which EC50-72h values were similar in both lines and by all assays used, Balb/c 3T3 cells were more sensitive than HepG2 cells to TP.

  9. Differential genomic effects on signaling pathways by two different CeO2 nanoparticles in HepG2 cells

    Data.gov (United States)

    U.S. Environmental Protection Agency — Differential genomic effects on signaling pathways by two different CeO2 nanoparticles in HepG2 cells. This dataset is associated with the following publication:...

  10. Linoleic acid-menthyl ester reduces the secretion of apolipoprotein B100 in HepG2 cells.

    Science.gov (United States)

    Inoue, Nao; Yamano, Naomi; Sakata, Kotaro; Arao, Keisuke; Kobayashi, Takashi; Nagao, Toshihiro; Shimada, Yuji; Nagao, Koji; Yanagita, Teruyoshi

    2009-01-01

    The effect of linoleic acid-menthyl ester (LAME) on lipid metabolism were assessed in HepG2 cells. It is well known that high level of apolipoprotein (apo) B100 in the serum is risk for atherosclerosis. Although linoleic acid (LA) treatment and LA plus L-mentol treatment increased apo B100 secretion, LAME treatment significantly decreased apo B100 secretion in HepG2 cells compared with control medium. The hypolipidemic effect of LAME was attributable to the suppression of triglyceride synthesis in HepG2 cells. It is also known that the risk of coronary heart disease is negatively related to the concentration of serum apo A-1. In the present study, LAME treatment increased apo A-1 secretion as compared with LA treatment in HepG2 cells. These results suggest that mentyl-esterification of fatty acids may be beneficial in anti-atherogenic dietary therapy.

  11. Effect of Toxicants on Fatty Acid Metabolism in HepG2 Cells

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    David Grünig

    2018-04-01

    Full Text Available Impairment of hepatic fatty acid metabolism can lead to liver steatosis and injury. Testing drugs for interference with hepatic fatty acid metabolism is therefore important. To find out whether HepG2 cells are suitable for this purpose, we investigated the effect of three established fatty acid metabolism inhibitors and of three test compounds on triglyceride accumulation, palmitate metabolism, the acylcarnitine pool and dicarboxylic acid accumulation in the cell supernatant and on ApoB-100 excretion in HepG2 cells. The three established inhibitors [etomoxir, methylenecyclopropylacetic acid (MCPA, and 4-bromocrotonic acid (4-BCA] depleted mitochondrial ATP at lower concentrations than cytotoxicity occurred, suggesting mitochondrial toxicity. They inhibited palmitate metabolism at similar or lower concentrations than ATP depletion, and 4-BCA was associated with cellular fat accumulation. They caused specific changes in the acylcarnitine pattern and etomoxir an increase of thapsic (C18 dicarboxylic acid in the cell supernatant, and did not interfere with ApoB-100 excretion (marker of VLDL export. The three test compounds (amiodarone, tamoxifen, and the cannabinoid WIN 55,212-2 depleted the cellular ATP content at lower concentrations than cytotoxicity occurred. They all caused cellular fat accumulation and inhibited palmitate metabolism at similar or higher concentrations than ATP depletion. They suppressed medium-chain acylcarnitines in the cell supernatant and amiodarone and tamoxifen impaired thapsic acid production. Tamoxifen and WIN 55,212-2 decreased cellular ApoB-100 excretion. In conclusion, the established inhibitors of fatty acid metabolism caused the expected effects in HepG2 cells. HepG cells proved to be useful for the detection of drug-associated toxicities on hepatocellular fatty acid metabolism.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Thaís de Oliveira Fernandes

    2015-01-01

    Full Text Available 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.

  14. Enhancing cisplatin delivery to hepatocellular carcinoma HepG2 cells using dual sensitive smart nanocomposite.

    Science.gov (United States)

    Salimi, Farzaneh; Dilmaghani, Karim Akbari; Alizadeh, Effat; Akbarzadeh, Abolfazl; Davaran, Soodabeh

    2017-07-07

    Targeted entrance and accumulation of higher doses of drugs into malignant cells could help in intensification of tumor specific cytotoxicity. A dual-responsive nanogel, poly(N-isopropylacrylamide)-co-poly(N,N-(dimethylamino)ethyl methacrylate) [P(NIPAM-co-DMA)] containing N-isopropylacrylamide (NIPAM) as thermoresponsive monomer and N,N-(dimethylamino)ethyl methacrylate (DMA) as pH-responsive monomer and methylene-bis-acrylamide (MBA) as cross-linking agent, was synthesized by free radical emulsion polymerization. Cisplatin along with magnetic Fe 3 O 4 nanoparticles (MNPs) was loaded into the nanogel by physically embedding the magnetic nanoparticles into hydrogel matrix after gelation to obtain drug-loaded magnetic nanocomposite [P(NIPAM-co-DMA)/Fe 3 O 4 ]. Drug loading efficiencies and drug release profiles of cisplatin-loaded P(NIPAM-co-DMA) nanogel and P(NIPAM-co-DMA)/Fe 3 O 4 nanocomposite were evaluated in vitro for controlled drug delivery in different temperature and pH conditions. Finally, the anticancer activity of P(NIPAM-co-DMA)/Fe 3 O 4 nanocomposite on human liver HepG2 cells was evaluated. Nanogel and nanocomposite showed significantly higher (p < .05) cisplatin release at 40 °C compared to 37 °C and at pH 5.7 compared to pH 7.4, demonstrating their temperature and pH sensitivity, respectively. The cytotoxicity assay of drug free nanogel on HepG2 cell line indicated that the nanogel is biocompatible and suitable as drug carrier. Moreover, MTT assay revealed that the cisplatin-loaded nanocomposite represented significant superior cytotoxicity (p < .05) to HepG2 cells as compared with free cisplatin.

  15. Involvement of enniatins-induced cytotoxicity in human HepG2 cells.

    Science.gov (United States)

    Juan-García, Ana; Manyes, Lara; Ruiz, María-José; Font, Guillermina

    2013-04-12

    Enniatins (ENNs) are mycotoxins found in Fusarium fungi and they appear in nature as mixtures of cyclic depsipeptides. The ability to form ionophores in the cell membrane is related to their cytotoxicity. Changes in ion distribution between inner and outer phases of the mitochondria affect to their metabolism, proton gradient, and chemiosmotic coupling, so a mitochondrial toxicity analysis of enniatins is highly recommended because they host the homeostasis required for cellular survival. Two ENNs, ENN A and ENN B on hepatocarcinoma cells (HepG2) at 1.5 and 3 μM and three exposure times (24, 48 and 72 h) were studied. Flow cytometry was used to examine their effects on cell proliferation, to characterize at which phase of the cell cycle progression the cells were blocked and to study the role of the mitochondrial in ENNs-induced apoptosis. In conclusion, apoptosis induction on HepG2 cells allowed to compare cytotoxic effects caused by both ENNs, A and B. It is reported the possible mechanism observed in MMP changes, cell cycle analysis and apoptosis/necrosis, identifying ENN B more toxic than ENN A. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  16. Exogenous FABP4 induces endoplasmic reticulum stress in HepG2 liver cells.

    Science.gov (United States)

    Bosquet, Alba; Guaita-Esteruelas, Sandra; Saavedra, Paula; Rodríguez-Calvo, Ricardo; Heras, Mercedes; Girona, Josefa; Masana, Lluís

    2016-06-01

    Fatty acid binding protein 4 (FABP4) is an intracellular fatty acid (FA) carrier protein that is, in part, secreted into circulation. Circulating FABP4 levels are increased in obesity, diabetes and other insulin resistance (IR) diseases. FAs contribute to IR by promoting endoplasmic reticulum stress (ER stress) and altering the insulin signaling pathway. The effect of FABP4 on ER stress in the liver is not known. The aim of this study was to investigate whether exogenous FABP4 (eFABP4) is involved in the lipid-induced ER stress in the liver. HepG2 cells were cultured with eFABP4 (40 ng/ml) with or without linoleic acid (LA, 200 μM) for 18 h. The expression of ER stress-related markers was determined by Western blotting (ATF6, EIF2α, IRE1 and ubiquitin) and real-time PCR (ATF6, CHOP, EIF2α and IRE1). Apoptosis was studied by flow cytometry using Annexin V-FITC and propidium iodide staining. eFABP4 increased the ER stress markers ATF6 and IRE1 in HepG2 cells. This effect led to insulin resistance mediated by changes in AKT and JNK phosphorylation. Furthermore, eFABP4 significantly induced both apoptosis, as assessed by flow cytometry, and CHOP expression, without affecting necrosis and ubiquitination. The presence of LA increased the ER stress response induced by eFABP4. eFABP4, per se, induces ER stress and potentiates the effect of LA in HepG2 cells, suggesting that FABP4 could be a link between obesity-associated metabolic abnormalities and hepatic IR mechanisms. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  17. Taurine reduces the secretion of apolipoprotein B100 and lipids in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Nagao Koji

    2008-10-01

    Full Text Available Abstract Background Higher concentrations of serum lipids and apolipoprotein B100 (apoB are major individual risk factors of atherosclerosis and coronary heart disease. Therefore ameliorative effects of food components against the diseases are being paid attention in the affluent countries. The present study was undertaken to investigate the effect of taurine on apoB secretion and lipid metabolism in human liver model HepG2 cells. Results The results demonstrated that an addition of taurine to the culture media reduces triacylglycerol (TG-mass in the cells and the medium. Similarly, cellular cholesterol-mass was decreased. Taurine inhibited the incorporation of [14C] oleate into cellular and medium TG, suggesting the inhibition of TG synthesis. In addition, taurine reduced the synthesis of cellular cholesterol ester and its secretion, suggesting the inhibition of acyl-coenzyme A:cholesterol acyltransferase activity. Furthermore, taurine reduced the secretion of apoB, which is a major protein component of very low-density lipoprotein. Conclusion This is a first report to demonstrate that taurine inhibits the secretion of apoB from HepG2 cells.

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

    Science.gov (United States)

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

    2017-10-01

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

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

    International Nuclear Information System (INIS)

    Wu Hai; Cai Ping; Clemens, Dahn L.; Jerrells, Thomas R.; Ansari, G.A. Shakeel; Kaphalia, Bhupendra S.

    2006-01-01

    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

  20. Proanthocyanidins modulate microRNA expression in human HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Anna Arola-Arnal

    Full Text Available Mi(croRNAs are small non-coding RNAs of 18-25 nucleotides in length that modulate gene expression at the post-transcriptional level. These RNAs have been shown to be involved in a several biological processes, human diseases and metabolic disorders. Proanthocyanidins, which are the most abundant polyphenol class in the human diet, have positive health effects on a variety of metabolic disorders such as inflammation, obesity, diabetes and insulin resistance. The present study aimed to evaluate whether proanthocyanidin-rich natural extracts modulate miRNA expression. Using microarray analysis and Q-PCR, we investigated miRNA expression in HepG2 cells treated with proanthocyanidins. Our results showed that when HepG2 cells were treated with grape seed proanthocyanidin extract (GSPE, cocoa proanthocyanidin extract (CPE or pure epigallocatechin gallate isolated from green tea (EGCG, fifteen, six and five differentially expressed miRNAs, respectively, were identified out of 904 mRNAs. Specifically, miR-30b* was downregulated by the three treatments, and treatment with GSPE or CPE upregulated miR-1224-3p, miR-197 and miR-532-3p. Therefore, these results provide evidence of the capacity of dietary proanthocyanidins to influence microRNA expression, suggesting a new mechanism of action of proanthocyanidins.

  1. Flow cytometry based micronucleus assay for evaluation of genotoxic potential of 2-ACBs in hepatic cells HepG2

    International Nuclear Information System (INIS)

    Barbezan, Angélica B.; Santos, Carla J.B.; Carvalho, Luma R.; Vieira, Daniel P.; Villavicêncio, Anna L.C.H.; Santelli, Glaucia M.M.

    2017-01-01

    Food irradiation is approved for use in more than 60 countries for applications and purposes in a wide variety of foods, being an effective and safe method for preservation and long-term storage. 2-Alkylcyclobutanones (2-ACBs) are the only known radiolytic products generated from foods that contain fatty acids (Triglycerides) when irradiated. The acids analyzed in this study are palmitic and stearic, which when irradiated form 2-Dodecylcyclobutanones (2-dDCB) and 2-Tetradecylcyclobutanone (2-tDCB). Part of the 2-ACBs ingested is excreted through feces and part is deposited in adipose tissues. In vitro studies so far have been only in colon cells. The work used a human hepatoma cell line (HepG2) since the accumulation of fat in this organ is quite common. Micronucleus test was selected to evaluate possible genotoxic effects of 2-dDCB and 2-tDCB compounds when exposed to high concentrations (447, 1422 and 2235 μM) for 4 and 24 hours. Tests were performed in quadriplicates using flow cytometric analysis. None detectable genotoxic damage was observed after 4 hours of exposure to the compounds, and cytotoxic effects were only significant at the highest concentration (2235 μM) of 2-dDCB. After 24 hours of exposure, slight genotoxic damage was observed at all concentrations evaluated, and cytotoxic effects were only present when exposed to compound 2-tDCB. Although there is a genotoxic and cytotoxic effect in some of the situations tested, the two compounds predominantly induced proliferation reduction effects of this hepatic tumor cell line. (author)

  2. Flow cytometry based micronucleus assay for evaluation of genotoxic potential of 2-ACBs in hepatic cells HepG2

    Energy Technology Data Exchange (ETDEWEB)

    Barbezan, Angélica B.; Santos, Carla J.B.; Carvalho, Luma R.; Vieira, Daniel P.; Villavicêncio, Anna L.C.H., E-mail: abarbezan@ipen.br [Instituto de Pesquisas Energéticas e Nucleares (IPEN/CNEN-SP), São Paulo, SP (Brazil); Santelli, Glaucia M.M. [Universidade de São Paulo (USP), SP (Brazil). Departamento de Biologia Celular e do Desenvolvimento

    2017-07-01

    Food irradiation is approved for use in more than 60 countries for applications and purposes in a wide variety of foods, being an effective and safe method for preservation and long-term storage. 2-Alkylcyclobutanones (2-ACBs) are the only known radiolytic products generated from foods that contain fatty acids (Triglycerides) when irradiated. The acids analyzed in this study are palmitic and stearic, which when irradiated form 2-Dodecylcyclobutanones (2-dDCB) and 2-Tetradecylcyclobutanone (2-tDCB). Part of the 2-ACBs ingested is excreted through feces and part is deposited in adipose tissues. In vitro studies so far have been only in colon cells. The work used a human hepatoma cell line (HepG2) since the accumulation of fat in this organ is quite common. Micronucleus test was selected to evaluate possible genotoxic effects of 2-dDCB and 2-tDCB compounds when exposed to high concentrations (447, 1422 and 2235 μM) for 4 and 24 hours. Tests were performed in quadriplicates using flow cytometric analysis. None detectable genotoxic damage was observed after 4 hours of exposure to the compounds, and cytotoxic effects were only significant at the highest concentration (2235 μM) of 2-dDCB. After 24 hours of exposure, slight genotoxic damage was observed at all concentrations evaluated, and cytotoxic effects were only present when exposed to compound 2-tDCB. Although there is a genotoxic and cytotoxic effect in some of the situations tested, the two compounds predominantly induced proliferation reduction effects of this hepatic tumor cell line. (author)

  3. HBV X Protein induces overexpression of HERV-W env through NF-κB in HepG2 cells.

    Science.gov (United States)

    Liu, Cong; Liu, Lijuan; Wang, Xiuling; Liu, Youyi; Wang, Miao; Zhu, Fan

    2017-12-01

    Human endogenous retrovirus W family (HERV-W) envelope (env) at chromosome 7 is highly expressed in the placenta and possesses fusogenic activity in trophoblast development. HERV-W env has been found to be overexpressed in some cancers and immune diseases. Viral transactivators can induce the overexpression of HERV-W env in human cell lines. Hepatitis B virus X protein (HBx) is believed to be a multifunctional oncogenic protein. Here, we reported that HBx could increase the promoter activity of HERV-W env and upregulate the mRNA levels of non-spliced and spliced HERV-W env and also its protein in human hepatoma HepG2 cells. Interestingly, we found that the inhibition of nuclear factor κB (NF-κB) using shRNA targeting NF-κB/p65 or PDTC (an inhibitor of NF-κB) could attenuate the upregulation of HERV-W env induced by HBx. These suggested that HBx might upregulate the expression of HERV-W env through NF-κB in HepG2 cells. This study might provide a new insight in HBV-associated liver diseases including HCC.

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

    Science.gov (United States)

    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.

  5. Extracellular visfatin activates gluconeogenesis in HepG2 cells through the classical PKA/CREB-dependent pathway.

    Science.gov (United States)

    Choi, Y J; Choi, S-E; Ha, E S; Kang, Y; Han, S J; Kim, D J; Lee, K W; Kim, H J

    2014-04-01

    Adipokines reportedly affect hepatic gluconeogenesis, and the adipokine visfatin is known to be related to insulin resistance and type 2 diabetes. However, whether visfatin contributes to hepatic gluconeogenesis remains unclear. Visfatin, also known as nicotinamide phosphoribosyltransferase (NAMPT), modulates sirtuin1 (SIRT1) through the regulation of nicotinamide adenine dinucleotide (NAD). Therefore, we investigated the effect of extracellular visfatin on glucose production in HepG2 cells, and evaluated whether extracellular visfatin affects hepatic gluconeogenesis via an NAD+-SIRT1-dependent pathway. Treatment with visfatin significantly increased glucose production and the mRNA expression and protein levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in HepG2 cells in a time- and concentration-dependent manner. Knockdown of SIRT1 had no remarkable effect on the induction of gluconeogenesis by visfatin. Subsequently, we evaluated if extracellular visfatin stimulates the production of gluconeogenic enzymes through the classical protein kinase A (PKA)/cyclic AMP-responsive element (CRE)-binding protein (CREB)-dependent process. The phosphorylation of CREB and PKA increased significantly in HepG2 cells treated with visfatin. Additionally, knockdown of CREB and PKA inhibited visfatin-induced gluconeogenesis in HepG2 cells. In summary, extracellular visfatin modulates glucose production in HepG2 cells through the PKA/CREB pathway, rather than via SIRT1 signaling. © Georg Thieme Verlag KG Stuttgart · New York.

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

    Directory of Open Access Journals (Sweden)

    Jung Dae Lim

    2015-02-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  10. Silencing of cytosolic NADP+-dependent isocitrate dehydrogenase gene enhances ethanol-induced toxicity in HepG2 cells.

    Science.gov (United States)

    Yang, Eun Sun; Lee, Su-Min; Park, Jeen-Woo

    2010-07-01

    It has been shown that acute and chronic alcohol administrations increase the production of reactive oxygen species, lower cellular antioxidant levels and enhance oxidative stress in many tissues. We recently reported that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) functions as an antioxidant enzyme by supplying NADPH to the cytosol. Upon exposure to ethanol, IDPc was susceptible to the loss of its enzyme activity in HepG2 cells. Transfection of HepG2 cells with an IDPc small interfering RNA noticeably downregulated IDPc and enhanced the cells' vulnerability to ethanol-induced cytotoxicity. Our results suggest that suppressing the expression of IDPc enhances ethanol-induced toxicity in HepG2 cells by further disruption of the cellular redox status.

  11. Antioxidative and cytoprotective effects of andrographolide against CCl4-induced hepatotoxicity in HepG2 cells.

    Science.gov (United States)

    Krithika, R; Verma, R J; Shrivastav, P S

    2013-05-01

    This article describes antioxidative and cytoprotective property of andrographolide, a major active component of the plant Andrographis paniculata (A. paniculata). High yields (2.7%) of andrographolide was isolated from the aerial parts of this plant via silica column chromatography. The purity of the compound was determined by high-performance thin-layer chromatography (HPTLC) and reversed phase high-performance liquid chromatography (HPLC) analysis. The structure was elucidated using techniques such as UV-visible spectrophotometry, elemental analysis, Fourier transform infrared (FT-IR), (1)H nuclear magnetic resonance ((1)H NMR), (13)C nuclear magnetic resonance ((13)C NMR) and mass spectral analysis and the data obtained were comparable with reported results. It was observed that andrographolide exhibited significant antioxidative property (IC50 = 3.2 µg/ml) by its ability to scavenge a stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) as compared to known antioxidants like ascorbic acid, butylated hydroxy toluene (BHT) and the plant extract. The cytoprotective role of andrographolide against carbon tetrachloride (CCl4) toxicity in human hepatoma HepG2 cell line was assessed using trypan blue exclusion test, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, by estimation of various leakage enzymes and by measuring the glutathione levels. The recovery obtained for andrographolide treatment in the presence of CCl4 was two-fold compared to A. paniculata extract for all other related biochemical parameters investigated. The results of the study indicate that andrographolide is a potent inhibitor of CCl4-mediated lipid peroxidation.

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

    International Nuclear Information System (INIS)

    Piret, Jean-Pascal; Vankoningsloo, Sebastien; Noel, Florence; Saout, Christelle; Toussaint, Olivier; Mendoza, Jorge Mejia; Lucas, Stephane

    2011-01-01

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 (P2,3-DPG level, phosphatidylserine externalization, and extra-erythrocytic free Hb were significantly altered by hyperthermia plus high concentrations of cisplatin (100 and 200 μg/ml) (P0.05). 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.

  16. Antiproliferative effects of cinobufacini on human hepatocellular carcinoma HepG2 cells detected by atomic force microscopy

    Science.gov (United States)

    Wu, Qing; Lin, Wei-Dong; Liao, Guan-Qun; Zhang, Li-Guo; Wen, Shun-Qian; Lin, Jia-Ying

    2015-01-01

    AIM: To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action. METHODS: HepG2 cells were treated with different concentrations of cinobufacini. Cell viability was measured by methylthiazolyl tetrazolium (MTT) assay. Cell cycle distribution was analyzed by flow cytometry (FCM). Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope. Changes in morphology and ultrastructure of cells were detected by atomic force microscopy (AFM) at the nanoscale level. RESULTS: MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dose-dependent manner. With the concentration of cinobufacini increasing from 0 to 0.10 mg/mL, the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1% (P deep pores in the cell membrane, with larger particles and a rougher cell surface. CONCLUSION: Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity. PMID:25624718

  17. Relationship between P53 and bystander effect induced by radiated hepatoma cells

    International Nuclear Information System (INIS)

    Zhao Meijia; Shen Bo; Yuan Dexiao; Cheng Honghong; Shao Chunlin

    2009-01-01

    The role of p53 in bystander responses on normal liver cells were investigated by co-culturing irradiated hepatoma cells with non-irradiated bystander Chang liver cells. It was found that radiosensitivity of the hepatoma cells was relative to p53. HepG2 cells with wtp53 had the highest radiosensitivity followed by PLC/PRF/5 cells with mtp53 and Hep3B cells with null-p53. The induction of bystander micronucleus(MN) was observed only in the Chang liver cells that had been co-cultured with HepG2 cells but not co-cultured with PLC/PRF/5 or Hep3B. Also, this bystander MN was relative to the irradiation dose and the cell co-culture rime. When the hepatoma cells were treated with pifithrin-α, a p53 inhibitor, their radiosensitivities were reduced, and the bystander effect was diminished. The results indicate that p53 could regulate not only the radiosensitivity but also the bystander response. (authors)

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

    International Nuclear Information System (INIS)

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

    2008-01-01

    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

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

    Directory of Open Access Journals (Sweden)

    Mohd Fadzelly Abu Bakar

    2015-01-01

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Laura Paesano

    2017-04-01

    Full Text Available 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.

  1. Curcumin induced nanoscale CD44 molecular redistribution and antigen-antibody interaction on HepG2 cell surface

    Energy Technology Data Exchange (ETDEWEB)

    Wang Mu [Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China); Ruan Yuxia [Department of Ophthalmology, The First Affiliated Hospital, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China); Xing Xiaobo; Chen Qian; Peng, Yuan [Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China); Cai Jiye, E-mail: tjycai@jnu.edu.cn [Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China)

    2011-07-04

    Graphical abstract: Highlights: > In this study, we investigate the changes of CD44 expression and distribution on HepG2 cells after curcumin treatment. > We find curcumin is able to change the morphology and ultrastructure of HepG2 cells. > Curcumin can reduce the expression of CD44 molecules and induce the nanoscale molecular redistribution on cell surface. > The binding force between CD44-modified AFM tip and the HepG2 cell surface decreases after curcumin-treatment. - Abstract: The cell surface glycoprotein CD44 was implicated in the progression, metastasis and apoptosis of certain human tumors. In this study, we used atomic force microscope (AFM) to monitor the effect of curcumin on human hepatocellular carcinoma (HepG2) cell surface nanoscale structure. High-resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with curcumin. The membrane average roughness increased (10.88 {+-} 4.62 nm to 129.70 {+-} 43.72 nm) and the expression of CD44 decreased (99.79 {+-} 0.16% to 75.14 {+-} 8.37%). Laser scanning confocal microscope (LSCM) imaging showed that CD44 molecules were located on the cell membrane. The florescence intensity in control group was weaker than that in curcumin treated cells. Most of the binding forces between CD44 antibodies and untreated HepG2 cell membrane were around 120-220 pN. After being incubated with curcumin, the major forces focused on 70-150 pN (10 {mu}M curcumin-treated) and 50-120 pN (20 {mu}M curcumin-treated). These results suggested that, as result of nanoscale molecular redistribution, changes of the cell surface were in response to external treatment of curcumin. The combination of AFM and LSCM could be a powerful method to detect the distribution of cell surface molecules and interactions between molecules and their ligands.

  2. Nickel oxide nanoparticles exert cytotoxicity via oxidative stress and induce apoptotic response in human liver cells (HepG2).

    Science.gov (United States)

    Ahamed, Maqusood; Ali, Daoud; Alhadlaq, Hisham A; Akhtar, Mohd Javed

    2013-11-01

    Increasing use of nickel oxide nanoparticles (NiO NPs) necessitates an improved understanding of their potential impact on human health. Previously, toxic effects of NiO NPs have been investigated, mainly on airway cells. However, information on effect of NiO NPs on human liver cells is largely lacking. In this study, we investigated the reactive oxygen species (ROS) mediated cytotoxicity and induction of apoptotic response in human liver cells (HepG2) due to NiO NPs exposure. Prepared NiO NPs were crystalline and spherical shaped with an average diameter of 44 nm. NiO NPs induced cytotoxicity (cell death) and ROS generation in HepG2 cells in dose-dependent manner. Further, ROS scavenger vitamin C reduced cell death drastically caused by NiO NPs exposure indicating that oxidative stress plays an important role in NiO NPs toxicity. Micronuclei induction, chromatin condensation and DNA damage in HepG2 cells treated with NiO NPs suggest that NiO NPs induced cell death via apoptotic pathway. Quantitative real-time PCR analysis showed that following the exposure of HepG2 cells to NiO NPs, the expression level of mRNA of apoptotic genes (bax and caspase-3) were up-regulated whereas the expression level of anti-apoptotic gene bcl-2 was down-regulated. Moreover, activity of caspase-3 enzyme was also higher in NiO NPs treated cells. To the best of our knowledge this is the first report demonstrating that NiO NPs caused cytotoxicity via ROS and induced apoptosis in HepG2 cells, which is likely to be mediated through bax/bcl-2 pathway. This work warrants careful assessment of Ni NPs before their commercial and industrial applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Cytotoxicity assessments of Portulaca oleracea and Petroselinum sativum seed extracts on human hepatocellular carcinoma cells (HepG2).

    Science.gov (United States)

    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.

  4. The Growth Suppressing Effects of Girinimbine on Hepg2 Involve Induction of Apoptosis and Cell Cycle Arrest

    Directory of Open Access Journals (Sweden)

    Tang Sook Wah

    2011-08-01

    Full Text Available Murraya koenigii is an edible herb widely used in folk medicine. Here we report that girinimbine, a carbazole alkaloid isolated from this plant, inhibited the growth and induced apoptosis in human hepatocellular carcinoma, HepG2 cells. The MTT and LDH assay results showed that girinimbine decreased cell viability and increased cytotoxicity in a dose-and time-dependent manner selectively. Girinimbine-treated HepG2 cells showed typical morphological features of apoptosis, as observed from normal inverted microscopy and Hoechst 33342 assay. Furthermore, girinimbine treatment resulted in DNA fragmentation and elevated levels of caspase-3 in HepG2 cells. Girinimbine treatment also displayed a time-dependent accumulation of the Sub-G0/G1 peak (hypodiploid and caused G0/G1-phase arrest. Together, these results demonstrated for the first time that girinimbine could effectively induce programmed cell death in HepG2 cells and suggests the importance of conducting further investigations in preclinical human hepatocellular carcinoma models, especially on in vivo efficacy, to promote girinimbine for use as an anticancer agent against hepatocellular carcinoma.

  5. Necrosis of HepG2 cancer cells induced by the vibration of magnetic particles

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Biran [Laboratoire de Physique de la Matière Condensée (LPMC), CNRS UMR 7336, Université de Nice Sophia Antipolis, Parc Valrose, 06108 Nice (France); Institut de Chimie de Nice, UMR 7272, Université de Nice Sophia Antipolis, CNRS, 28 Avenue de Valrose, F-06100 Nice (France); Bienvenu, Céline [Institut de Chimie de Nice, UMR 7272, Université de Nice Sophia Antipolis, CNRS, 28 Avenue de Valrose, F-06100 Nice (France); Mendez-Garza, Juan; Lançon, Pascal; Madeira, Alexandra [Laboratoire de Physique de la Matière Condensée (LPMC), CNRS UMR 7336, Université de Nice Sophia Antipolis, Parc Valrose, 06108 Nice (France); Vierling, Pierre [Institut de Chimie de Nice, UMR 7272, Université de Nice Sophia Antipolis, CNRS, 28 Avenue de Valrose, F-06100 Nice (France); Di Giorgio, Christophe, E-mail: christophe.di-giorgio@unice.fr [Institut de Chimie de Nice, UMR 7272, Université de Nice Sophia Antipolis, CNRS, 28 Avenue de Valrose, F-06100 Nice (France); Bossis, Georges, E-mail: bossis@unice.fr [Laboratoire de Physique de la Matière Condensée (LPMC), CNRS UMR 7336, Université de Nice Sophia Antipolis, Parc Valrose, 06108 Nice (France)

    2013-10-15

    Experiments of magnetolysis, i.e., destruction of cells induced with magnetic particles (MPs) submitted to the application of a magnetic field, were conducted on HepG2 cancer cells. We herein demonstrate the usefulness of combining anisotropic MPs with an alternative magnetic field in magnetolysis. Thus, the application of an alternative magnetic field of low frequency (a few Hertz) in the presence of anisotropic, submicronic particles allowed the destruction of cancer cells “in vitro”. We also show that a constant magnetic field is far less efficient than an oscillating one. Moreover, we demonstrate that, at equal particle volume, it is much more efficient to utilize spindle shaped particles rather than spherical ones. In order to get deeper insight into the mechanism of magnetolysis experiments, we performed a study by AFM, which strongly supports that the magnetic field induces the formation of clusters of particles becoming then large enough todamage cell membranes. - Highlights: • Magnetic force was applied on cancer cells through magnetic particles. • The penetration depth was predicted, both for spherical and ellipsoidal particles. • Alternative force was shown to damage the cells contrary to static force. • The effect of indentation of magnetic particles was compared to the one of AFM tips. • The damage was attributed to the formation of clusters of particles.

  6. The role of the vascular endothelial growth factor/vascular endothelial growth factor receptors axis mediated angiogenesis in curcumin-loaded nanostructured lipid carriers induced human HepG2 cells apoptosis

    Directory of Open Access Journals (Sweden)

    Fengling Wang

    2015-01-01

    Full Text Available Background: Curcumin (diferuloylmethane, the active constituent of turmeric extract has potent anti-cancer properties have been demonstrated in hepatocellular carcinoma (HCC. However, its underlying molecular mechanism of therapeutic effects remains unclear. Vascular endothelial growth factor (VEGF and its receptors (VEGFRs have crucial roles in tumor angiogenesis. Purpose: The goal of this study was to investigate the role of the VEGF/VEGFRs mediated angiogenesis during the proliferation and apoptosis of human HepG2 hepatoma cell line and the effect of curcumin-loaded nanostructured lipid carriers (Cur-NLC. Materials and Methods: The proliferation of HepG2 cells was determined by methyl thiazolyl tetrazolium after exposure to Cur-NLC and native curcumin. Apoptosis was quantified by flow cytometry with annexin V-fluorescein isothiocyanate and propidium iodide staining. Cellular internalization of Cur-NLC was observed by fluorescent microscope. The level of VEGF was detected by enzyme-linked immunosorbent assay kits. The expression of VEGFRs was quantified by Western blotting. Results: Cur-NLC was more effective in inhibiting the proliferation and enhancing the apoptosis of HepG2 cells than native curcumin. Fluorescent microscope analysis showed that HepG2 cells internalized Cur-NLC more effectively than native curcumin. Furthermore, Cur-NLC down-regulated the level of VEGF and the expression of VEGFR-2, but had a slight effect on VEGFR-1. Conclusion: These results clearly demonstrated that Cur-NLC was more effective in anti-cancer activity than the free form of curcumin. These studies demonstrate for the 1 st time that Cur-NLC exerts an antitumor effect on HepG2 cells by modulating VEGF/VEGFRs signaling pathway.

  7. Dihydrotestosterone regulating apolipoprotein M expression mediates via protein kinase C in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Yi-zhou Ye

    2012-12-01

    Full Text Available Abstract 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

  8. Galactosylated poly(ε-caprolactone) membrane promoted liver-specific functions of HepG2 cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yan, E-mail: zhang_yan@ecust.edu.cn [The Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 (China); Zhang, Yi [The Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 (China); Chen, Min; Zhou, Yan [The State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai, 200237 (China); Lang, Meidong, E-mail: mdlang@ecust.edu.cn [The Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 (China)

    2014-08-01

    The lack of pendant functional groups on the PCL backbone has been a great challenge for surface bioactivation of poly(ε-caprolactone) (PCL). In the present study, covalently galactosylated PCL (GPCL) was developed through coupling between the amino-functionalized PCL (NPCL) and the lactobionic acid (LA) and its potential application in maintenance of physiological functions of HepG2 cells was further evaluated. The structure and properties of GPCL were explored by {sup 1}H NMR, FT-IR, GPC and DSC. Moreover, the incorporation of galactose ligands onto GPCL membranes not only promoted higher wettability, but also radically changed surface morphology in comparison with PCL and NPCL according to the contact angle measurement and atomic force microscopy. When HepG2 cells were seeded onto these membranes, the cells on GPCL membranes showed more pronounced cell adhesion and tended to form aggregates during the initial adhesion stage and then progressively grew into multi-layer structures compared to those without galactose ligands by the observation with fluorescence microscope and scanning electron microscopy. Furthermore, live–dead assay and functional tests demonstrated that HepG2 cells on GPCL membranes had superior viability and maintained better liver-specific functions. Collectively, GPCL has great potential for hepatic tissue engineering scaffolds. - Graphical abstract: The specific recognition between the galactose ligands on the galactosylated poly(ε-caprolactone) membrane and the ASGPR on the HepG2 cell surface. The galactosylated poly(ε-caprolactone) membranes improved the cell-matrix interaction. The galactosylated functionalized PCL scaffold is a potential candidate for liver tissue engineering. - Highlights: • The specific recognition between the galactose ligands on the galactosylated poly(ε-caprolactone) membrane and the ASGPR on the HepG2 cell surface. • The galactosylated poly(ε-caprolactone) membranes improved the cell-matrix interaction.

  9. Up-regulation of P-glycoprotein expression by catalase via JNK activation in HepG2 cells.

    Science.gov (United States)

    Li, Lin; Xu, Jianfeng; Min, Taishan; Huang, Weida

    2006-01-01

    Overexpression of the MDR1 gene is one of the reasons for multidrug resistance (MDR). Some studies suggested that antioxidants could down-regulate MDR1 expression as a possible cancer treatment. In this report, we try to determine the effects of antioxidants (catalase or N-acetylcysteine [NAC]) on the regulation of intrinsic MDR1 overexpression in HepG2 cells. Adding catalase or N-acetylcysteine to the HepG2 culture led to a significant increase of MDR1 mRNA and P-glycoprotein drug transporter activity. After catalase or NAC treatment, a reduced intracellular reactive oxygen species (ROS) was observed. The JNK inhibitor SP600125 abolished the positive effects of catalase on drug transporter activity in a dose-dependent manner. Furthermore, the up-regulation of P-glycoprotein functions by catalase was only observed in HepG2 cells but not in other cell lines tested (MCF-7, A549, A431). These data suggested that catalase can up-regulate P-glycoprotein expression in HepG2 cells via reducing intracellular ROS, and JNK may mediate this process.

  10. Biochemical Effects of six Ti02 and four Ce02 Nanomaterials in HepG2 cells

    Science.gov (United States)

    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

    Science.gov (United States)

    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) made b...

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

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Yi, E-mail: yi.luo@pfizer.com; 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

  13. Ginseng (Panax quinquefolius and Licorice (Glycyrrhiza uralensis Root Extract Combinations Increase Hepatocarcinoma Cell (Hep-G2 Viability

    Directory of Open Access Journals (Sweden)

    David G. Popovich

    2011-01-01

    Full Text Available The combined cytoactive effects of American ginseng (Panax quinquefolius and licorice (Glycyrrhiza uralensis root extracts were investigated in a hepatocarcinoma cell line (Hep-G2. An isobolographic analysis was utilized to express the possibility of synergistic, additive or antagonistic interaction between the two extracts. Both ginseng and licorice roots are widely utilized in traditional Chinese medicine preparations to treat a variety of ailments. However, the effect of the herbs in combination is currently unknown in cultured Hep-G2 cells. Ginseng (GE and licorice (LE extracts were both able to reduce cell viability. The LC50 values, after 72 h, were found to be 0.64 ± 0.02 mg/mL (GE and 0.53 ± 0.02 mg/mL (LE. An isobologram was plotted, which included five theoretical LC50s calculated, based on the fixed fraction method of combination ginseng to licorice extracts to establish a line of additivity. All combinations of GE to LE (1/5, 1/3, 1/2, 2/3, 4/5 produced an effect on Hep-G2 cell viability but they were all found to be antagonistic. The LC50 of fractions 1/3, 1/2, 2/3 were 23%, 21% and 18% above the theoretical LC50. Lactate dehydrogenase release indicated that as the proportion of GE to LE increased beyond 50%, the influence on membrane permeability increased. Cell-cycle analysis showed a slight but significant arrest at the G1 phase of cell cycle for LE. Both GE and LE reduced Hep-G2 viability independently; however, the combinations of both extracts were found to have an antagonistic effect on cell viability and increased cultured Hep-G2 survival.

  14. Surface ligand dependent toxicity of zinc oxide nanoparticles in HepG2 cell model

    International Nuclear Information System (INIS)

    Bartczak, D; Baradez, M-O; Merson, S; Goenaga-Infante, H; Marshall, D

    2013-01-01

    Physicochemical properties of nanoparticles (NP) strongly affect their influence on cell behaviour, but can be significantly distorted by interactions with the proteins present in biological solutions. In this study we show how different surface functionalities of zinc oxide (ZnO) NP lead to changes in the size distribution and dissolution of the NP in serum containing cell culture media and how this impacts on NP toxicity. NPs capped with weakly bound large proteins undergo substantial transformations due to the exchange of the original surface ligands to the components of the cell culture media. Conversely, NP capped with a tight monolayer of small organic molecules or with covalently conjugated proteins show significantly higher stability. These differences in ligand exchange also affect the toxicity of the NP to the HepG2 liver cell model, with the NP capped with small organic molecules being more toxic than those capped with large proteins. This study highlights the importance of characterising NPs in biological media and the effect the media has during in-vitro analysis.

  15. Kaempferol induces apoptosis in HepG2 cells via activation of the endoplasmic reticulum stress pathway.

    Science.gov (United States)

    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.

  16. Nitric oxide and TGF-β1 inhibit HNF-4α function in HEPG2 cells

    International Nuclear Information System (INIS)

    Lucas, Susana de; Lopez-Alcorocho, Juan Manuel; Bartolome, Javier; Carreno, Vicente

    2004-01-01

    This study analyzes if the profibrogenic factors nitric oxide and transforming growth factor-β1 (TGF-β1) affect hepatocyte nuclear factor-4α (HNF-4α) function. For this purpose, HepG2 cells were treated with TGF-β1 or with a nitric oxide donor to determine mRNA levels of coagulation factor VII and HNF-4α. Treatment effect on factor VII gene promoter was assessed by chloramphenicol acetyl-transferase assays in cells transfected with the pFVII-CAT plasmid. HNF-4α binding and protein levels were determined by gel shift assays and Western blot. TGF-β1 and nitric oxide downregulated factor VII mRNA levels by inhibiting its gene promoter activity. This inhibition is caused by a decrease in the DNA binding of HNF-4α. TGF-β1 induces degradation of HNF-4α in the proteasome while nitric oxide provokes nitrosylation of cysteine residues in this factor. TGF-β1 and nitric oxide inhibit HNF-4α activity. These findings may explain the loss of liver functions that occurs during fibrosis progression

  17. BAG3-dependent noncanonical autophagy induced by proteasome inhibition in HepG2 cells.

    Science.gov (United States)

    Liu, Bao-Qin; Du, Zhen-Xian; Zong, Zhi-Hong; Li, Chao; Li, Ning; Zhang, Qiang; Kong, De-Hui; Wang, Hua-Qin

    2013-06-01

    Emerging lines of evidence have shown that blockade of ubiquitin-proteasome system (UPS) activates autophagy. The molecular players that regulate the relationship between them remain to be elucidated. Bcl-2 associated athanogene 3 (BAG3) is a member of the BAG co-chaperone family that regulates the ATPase activity of heat shock protein 70 (HSP70) chaperone family. Studies on BAG3 have demonstrated that it plays multiple roles in physiological and pathological processes, including antiapoptotic activity, signal transduction, regulatory role in virus infection, cell adhesion and migration. Recent studies have attracted much attention on its role in initiation of autophagy. The current study, for the first time, demonstrates that proteasome inhibitors elicit noncanonical autophagy, which was not suppressed by inhibitors of class III phosphatidylinositol 3-kinase (PtdIns3K) or shRNA against Beclin 1 (BECN1). In addition, we demonstrate that BAG3 is ascribed to activation of autophagy elicited by proteasome inhibitors and MAPK8/9/10 (also known as JNK1/2/3 respectively) activation is also implicated via upregulation of BAG3. Moreover, we found that noncanonical autophagy mediated by BAG3 suppresses responsiveness of HepG2 cells to proteasome inhibitors.

  18. Effects of elaidic acid in a HepG2-SF liver cell model

    DEFF Research Database (Denmark)

    Hansen, Toke Peter Krogager

    Det primære mål for dette Ph.D-studie var at identificere potentielle proteinbiomarkører for menneskelig indtagelse af elaidinsyre, hvilken er den mest almindelige transfedtsyre i fødevarer. En serum fri HepG2 celle model (HepG-SF) blev inkuberet i syv dage med elaidinsyre eller med andre...... human blodplasma. Det sekundære mål for Ph.D-studiet var at undersøge årsagerne til den specifikke cellulære respons for elaidinsyre. Det blev observeret at inkubation med elaidinsyre resulterede i at 28 % af de esterficerede fedtsyrerne i fosfolipid-fraktionen var elaidinsyre, hvilket indikerer en...... vigtige modulatorer af SREBPs fundet reguleret på en sådan måde at det teoretisk ville betyder en reduceret aktivering af SREBPs, hvis dette er tilfældet kunne det tyde på en afkobling af kolesterol sensor systemet og syntesen af lipider. Denne afkobling kan måske forklare de negative helbreds effekter...

  19. Terpenoids from Curcuma wenyujin increased glucose consumption on HepG2 cells.

    Science.gov (United States)

    Zhou, Chang-Xin; Zhang, Li-Sha; Chen, Fei-Fei; Wu, Hao-Shu; Mo, Jian-Xia; Gan, Li-She

    2017-09-01

    Thirty four terpenoids, including two new cadinane-type sesquiterpenoids containing conjugated aromatic-ketone moieties, curcujinone A (1) and curcujinone B (2), were isolated from 95% ethanol extract of the root tubers of Curcuma wenyujin. Their structures were determined by spectroscopic methods, especially 2D NMR and HRMS techniques. The relative and absolute configurations of 1 and 2 were identified by quantum chemical DFT and TDDFT calculations of the 13 C NMR chemical shifts, ECD spectra, and specific optical rotations. All compounds and extracts were evaluated for their anti-diabetic activities with a glucose consumption model on HepG2 Cells. The petroleum fraction CWP (10μg/mL) and compounds curcumenol (4), 7α,11α-epoxy-5β-hydroxy-9-guaiaen-8-one (5), curdione (17), (1S, 4S, 5S 10S)-germacrone (18), zederone (20), a mixture of curcumanolide A (25) and curcumanolide B (26), gajutsulactone B (27), and wenyujinin C (30) showed promising activities with over 45% increasing of glucose consumption at 10μM. Copyright © 2017. Published by Elsevier B.V.

  20. Cholesterol-lowing effect of taurine in HepG2 cell.

    Science.gov (United States)

    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.

  1. Evaluation of anti-hepatocarcinoma capacity of puerarin nanosuspensions against human HepG2 cells

    Science.gov (United States)

    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.

  2. STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma membrane, ER, and ERC

    DEFF Research Database (Denmark)

    Garbarino, J.; Pan, M. H.; Chin, H. F.

    2012-01-01

    small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT...... synthesized cholesteryl esters. Furthermore, D4 KD cells exhibited a reduced rate of sterol transport to the endocytic recycling compartment after cholesterol repletion. Although these cells displayed normal endocytic trafficking in cholesterol-poor and replete conditions, cell surface low density lipoprotein...... membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well. -Garbarino, J., M. Pan, H.F. Chin, F.W. Lund, F.R. Maxfield, and J.L. Breslow. STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma...

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    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 (TiO 2 ) 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 TiO 2 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 TiO 2 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.

  4. Activation of apoptosis by ethyl acetate fraction of ethanol extract of Dianthus superbus in HepG2 cell line.

    Science.gov (United States)

    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. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Assessment of the predictive capacity of the optimized in vitro comet assay using HepG2 cells.

    Science.gov (United States)

    Hong, Yoon-Hee; Jeon, Hye Lyun; Ko, Kyung Yuk; Kim, Joohwan; Yi, Jung-Sun; Ahn, Ilyoung; Kim, Tae Sung; Lee, Jong Kwon

    2018-03-01

    Evaluation of DNA damage is critical during the development of new drugs because it is closely associated with genotoxicity and carcinogenicity. The in vivo comet assay to assess DNA damage is globally harmonized as OECD TG 489. However, a comet test guideline that evaluates DNA damage without sacrificing animals does not yet exist. The goal of this study was to select an appropriate cell line for optimization of the in vitro comet assay to assess DNA damage. We then evaluated the predictivity of the in vitro comet assay using the selected cell line. In addition, the effect of adding S9 was evaluated using 12 test chemicals. For cell line selection, HepG2, Chinese hamster lung (CHL/IU), and TK6 cell lines were evaluated. We employed a method for the in vitro comet assay based on that for the in vivo comet assay. The most appropriate cell line was determined by% tail DNA increase after performing in vitro comet assays with 6 test chemicals. The predictivity of the in vitro comet assay using the selected cell line was measured with 10 test chemicals (8 genotoxins and 2 non-genotoxic chemicals). The HepG2 cell line was found to be the most appropriate, and in vitro comet assays using HepG2 cells exhibited a high accuracy of 90% (9/10). This study suggests that HepG2 is an optimal cell line for the in vitro comet assay to assess DNA damage. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. In vitro investigations of Cynara scolymus L. extract on cell physiology of HepG2 liver cells

    Directory of Open Access Journals (Sweden)

    Gesine Löhr

    2009-06-01

    Full Text Available The objective of this study was the investigation of a potential influence of artichoke leaf extract (ALE on the cell physiology and gene expression of phase I/II enzymes of human liver cells HepG2 and investigation on potential cell protective effects against ethanol-induced cell toxicity against HepG2 cells. Cell biological assays under in vitro conditions using HepG2 liver cells and investigation of mitochondrial activity (MTT test, proliferation assay (BrdU incorporation ELISA, LDH as toxicity marker, gene expression analysis by RT-PCR and enzyme activity of glutationtransferase. Artichocke extract, containing 27% caffeoylquinic acids and 7% flavonoids induced mitochondrial activity, proliferation and total protein content under in vitro conditions in human liver cells HepG2. These effects could not be correlated to the well-known artichoke secondary compounds cynarin, caffeic acid, chlorogenic acid, luteolin and luteolin-7-O-glucoside. The flavones luteolin and luteolin-7-O-glucoside had inhibitory effects at 100 µg/mL level on HepG2 cells, with luteolin being a significant stronger inhibitor compared to the respective glucoside. Artichoke leaf extract had minor stimulating effect on gene expression of CYP1A2, while CYP3A4, GGT, GPX2, GSR and GST were slightly inhibited. GST inhibition under in vitro conditions was also shown by quantification of GST enzyme activity. Induction of gene expression of CYP1A2 was shown to be supraadditive after simultaneous application of ethanol plus artichoke extract. Artichoke leaf extract exhibited cell protective effects against ethanol-induced toxicity within cotreatment under in vitro conditions. Also H2O2 damage was significantly inhibited by simultaneous artichoke incubation. Pre- and posttreatments did not exert protective effects. DMSO-induced toxicity was significantly reduced by pre-, post- and cotreatment with artichoke extract and especially with luteolin-7-O-glucoside, indicating a direct

  7. HepG2 cells develop signs of riboflavin deficiency within four days of culture in riboflavin-deficient medium*

    OpenAIRE

    Werner, Ricarda; Manthey, Karoline C.; Griffin, Jacob B.; Zempleni, Janos

    2005-01-01

    Flavin mononucleotide and flavin adenine dinucleotide are essential coenzymes in redox reactions. For example, flavin adenine dinucleotide is a coenzyme for both glutathione reductase and enzymes that mediate the oxidative folding of secretory proteins. Here we investigated short-term effects of moderately riboflavin-deficient culture medium on flavin-related responses in HepG2 hepatocarcinoma cells. Cells were cultured in riboflavin-deficient (3.1 nmol/L) medium for up to six days; controls ...

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

    Directory of Open Access Journals (Sweden)

    Li Y

    2016-12-01

    Full Text Available Yinghua Li,1,* Min Guo,1,* Zhengfang Lin,1 Mingqi Zhao,1 Misi Xiao,1 Changbing Wang,1 Tiantian Xu,1 Tianfeng Chen,2 Bing Zhu1 1Center Laboratory, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, 2Department of Chemistry, Jinan University, Guangzhou, People’s Republic of China *These authors contributed equally to this work Abstract: 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. Keywords: silver nanoparticles, polyethylenimine, paclitaxel, reactive oxygen species, apoptosis

  9. Diosgenin Induces Apoptosis in HepG2 Cells through Generation of Reactive Oxygen Species and Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Dae Sung Kim

    2012-01-01

    Full Text Available Diosgenin, a naturally occurring steroid saponin found abundantly in legumes and yams, is a precursor of various synthetic steroidal drugs. Diosgenin is studied for the mechanism of its action in apoptotic pathway in human hepatocellular carcinoma cells. Based on DAPI staining, diosgenin-treated cells manifested nuclear shrinkage, condensation, and fragmentation. Treatment of HepG2 cells with 40 μM diosgenin resulted in activation of the caspase-3, -8, -9 and cleavage of poly-ADP-ribose polymerase (PARP and the release of cytochrome c. In the upstream, diosgenin increased the expression of Bax, decreased the expression of Bid and Bcl-2, and augmented the Bax/Bcl-2 ratio. Diosgenin-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of JNK, p38 MAPK and apoptosis signal-regulating kinase (ASK-1, as well as generation of the ROS. NAC administration, a scavenger of ROS, reversed diosgene-induced cell death. These results suggest that diosgenin-induced apoptosis in HepG2 cells through Bcl-2 protein family-mediated mitochndria/caspase-3-dependent pathway. Also, diosgenin strongly generated ROS and this oxidative stress might induce apoptosis through activation of ASK1, which are critical upstream signals for JNK/p38 MAPK activation in HepG2 cancer cells.

  10. Protective effects of the extracts of Barringtonia racemosa shoots against oxidative damage in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Kin Weng Kong

    2016-01-01

    Full Text Available 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.

  11. In HepG2 cells, coexisting carnitine deficiency masks important indicators of marginal biotin deficiency.

    Science.gov (United States)

    Bogusiewicz, Anna; Boysen, Gunnar; Mock, Donald M

    2015-01-01

    A large number of birth defects are related to nutrient deficiencies; concern that biotin deficiency is teratogenic in humans is reasonable. Surprisingly, studies indicate that increased urinary 3-hydroxyisovalerylcarnitine (3HIAc), a previously validated marker of biotin deficiency, is not a valid biomarker in pregnancy. In this study we hypothesized that coexisting carnitine deficiency can prevent the increase in 3HIAc due to biotin deficiency. We used a 2-factor nutrient depletion design to induce isolated and combined biotin and carnitine deficiency in HepG2 cells and then repleted cells with carnitine. To elucidate the metabolic pathogenesis, we quantitated intracellular and extracellular free carnitine, acylcarnitines, and acylcarnitine ratios using liquid chromatography-tandem mass spectrometry. Relative to biotin-sufficient, carnitine-sufficient cells, intracellular acetylcarnitine increased by 90%, propionylcarnitine more than doubled, and 3HIAc increased by >10-fold in biotin-deficient, carnitine-sufficient (BDCS) cells, consistent with a defensive mechanism in which biotin-deficient cells transesterify the acyl-coenzyme A (acyl-CoA) substrates of the biotin-dependent carboxylases to the related acylcarnitines. Likewise, in BDCS cells, the ratio of acetylcarnitine to malonylcarnitine and the ratio of propionylcarnitine to methylmalonylcarnitine both more than tripled, and the ratio of 3HIAc to 3-methylglutarylcarnitine (MGc) increased by >10-fold. In biotin-deficient, carnitine-deficient (BDCD) cells, the 3 substrate-derived acylcarnitines changed little, but the substrate:product ratios were masked to a lesser extent. Moreover, carnitine repletion unmasked biotin deficiency in BDCD cells as shown by increases in acetylcarnitine, propionylcarnitine, and 3HIAc (each increased by >50-fold). Likewise, ratios of acetylcarnitine:malonylcarnitine, propionylcarnitine:methylmalonylcarnitine, and 3HIAc:MGc all increased by >8-fold. Our findings provide strong

  12. Effects of Nano-CeO2 with Different Nanocrystal Morphologies on Cytotoxicity in HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Lili Wang

    2015-09-01

    Full Text Available 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.

  13. Curcumin induced nanoscale CD44 molecular redistribution and antigen-antibody interaction on HepG2 cell surface

    International Nuclear Information System (INIS)

    Wang Mu; Ruan Yuxia; Xing Xiaobo; Chen Qian; Peng, Yuan; Cai Jiye

    2011-01-01

    Graphical abstract: Highlights: → In this study, we investigate the changes of CD44 expression and distribution on HepG2 cells after curcumin treatment. → We find curcumin is able to change the morphology and ultrastructure of HepG2 cells. → Curcumin can reduce the expression of CD44 molecules and induce the nanoscale molecular redistribution on cell surface. → The binding force between CD44-modified AFM tip and the HepG2 cell surface decreases after curcumin-treatment. - Abstract: The cell surface glycoprotein CD44 was implicated in the progression, metastasis and apoptosis of certain human tumors. In this study, we used atomic force microscope (AFM) to monitor the effect of curcumin on human hepatocellular carcinoma (HepG2) cell surface nanoscale structure. High-resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with curcumin. The membrane average roughness increased (10.88 ± 4.62 nm to 129.70 ± 43.72 nm) and the expression of CD44 decreased (99.79 ± 0.16% to 75.14 ± 8.37%). Laser scanning confocal microscope (LSCM) imaging showed that CD44 molecules were located on the cell membrane. The florescence intensity in control group was weaker than that in curcumin treated cells. Most of the binding forces between CD44 antibodies and untreated HepG2 cell membrane were around 120-220 pN. After being incubated with curcumin, the major forces focused on 70-150 pN (10 μM curcumin-treated) and 50-120 pN (20 μM curcumin-treated). These results suggested that, as result of nanoscale molecular redistribution, changes of the cell surface were in response to external treatment of curcumin. The combination of AFM and LSCM could be a powerful method to detect the distribution of cell surface molecules and interactions between molecules and their ligands.

  14. Biphasic Estradiol-induced AKT Phosphorylation Is Modulated by PTEN via MAP Kinase in HepG2 Cells

    Science.gov (United States)

    Marino, Maria; Acconcia, Filippo; Trentalance, Anna

    2003-01-01

    We reported previously in HepG2 cells that estradiol induces cell cycle progression throughout the G1–S transition by the parallel stimulation of both PKC-α and ERK signaling molecules. The analysis of the cyclin D1 gene expression showed that only the MAP kinase pathway was involved. Here, the presence of rapid/nongenomic, estradiol-regulated, PI3K/AKT signal transduction pathway, its modulation by the levels of the tumor suppressor PTEN, its cross-talk with the ERK pathway, and its involvement in DNA synthesis and cyclin D1 gene promoter activity have all been studied in HepG2 cells. 17β-Estradiol induced the rapid and biphasic phosphorylation of AKT. These phosphorylations were independent of each other, being the first wave of activation independent of the estrogen receptor (ER), whereas the second was dependent on ER. Both activations were dependent on PI3K activity; furthermore, the ERK pathway modulated AKT phosphorylation by acting on the PTEN levels. The results showed that the PI3K pathway, as well as ER, were strongly involved in both G1–S progression and cyclin D1 promoter activity by acting on its proximal region (-254 base pairs). These data indicate that in HepG2 cells, different rapid/nongenomic estradiol-induced signal transduction pathways modulate the multiple steps of G1–S phase transition. PMID:12808053

  15. Ethanol Extract of Dianthus chinensis L. Induces Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells In Vitro

    Science.gov (United States)

    Nho, Kyoung Jin; Chun, Jin Mi; Kim, Ho Kyoung

    2012-01-01

    Dianthus chinensis L. is used to treat various diseases including cancer; however, the molecular mechanism by which the ethanol extract of Dianthus chinensis L. (EDCL) induces apoptosis is unknown. In this study, the apoptotic effects of EDCL were investigated in human HepG2 hepatocellular carcinoma cells. Treatment with EDCL significantly inhibited cell growth in a concentration- and time-dependent manner by inducing apoptosis. This induction was associated with chromatin condensation, activation of caspases, and cleavage of poly (ADP-ribose) polymerase protein. However, apoptosis induced by EDCL was attenuated by caspase inhibitor, indicating an important role for caspases in EDCL responses. Furthermore, EDCL did not alter the expression of bax in HepG2 cells but did selectively downregulate the expression of bcl-2 and bcl-xl, resulting in an increase in the ratio of bax:bcl-2 and bax:bcl-xl. These results support a mechanism whereby EDCL induces apoptosis through the mitochondrial pathway and caspase activation in HepG2 cells. PMID:22645629

  16. 9-cis-retinoic acid increases apolipoprotein AI secretion and mRNA expression in HepG2 cells.

    Science.gov (United States)

    Haghpassand, M; Moberly, J B

    1995-10-01

    HepG2 cells were studied as a model for regulation of hepatic apolipoprotein AI (apo AI) secretion and gene expression by 9-cis-retinoic acid. HepG2 cells cultured on plastic dishes were exposed to 9-cis-retinoic acid (9-cis-RA) for 48 h with a complete media change at 24 h. Apo AI mass in cultured media was determined by ELISA, by quantitative immunoblotting and by steady-state 35S-methionine labeling. Messenger RNA levels were determined by RNase protection using probes for apo AI and the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (G3PDH). 9-cis-RA increased secretion of apo AI by 52% at doses of 10 and 1 microM (6.3 +/- 0.6 vs. 4.2 +/- 0.3; P G3PDH mRNA was slightly decreased (14%, P < 0.05). Thus, 9-cis-RA stimulates apo AI expression in HepG2 cells, suggesting a role for retinoids in activating endogenous apo AI gene expression.

  17. Ku70 acetylation and modulation of c-Myc/ATF4/CHOP signaling axis by SIRT1 inhibition lead to sensitization of HepG2 cells to TRAIL through induction of DR5 and down-regulation of c-FLIP

    DEFF Research Database (Denmark)

    Kim, Mi-Ju; Hong, Kyung-Soo; Kim, Hak-Bong

    2013-01-01

    In this study, we investigated the role of c-Myc/ATF4/CHOP signaling pathway in sensitization of human hepatoma HepG2 cells to TRAIL. Knockdown of SIRT1 or treatment with SIRT1 inhibitor caused the up-regulation of DR5 and down-regulation of c-FLIP through modulation of c-Myc/ATF4/CHOP pathway, a...

  18. Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death

    International Nuclear Information System (INIS)

    Sun, Hengwen; Yang, Shana; Li, Jianhua; Zhang, Yajie; Gao, Dongsheng; Zhao, Shenting

    2016-01-01

    Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The aim of radiotherapy is to eradicate cancer cells with ionizing radiation. Except for the caspase-dependent mechanism, several lines of evidence demonstrated that caspase-independent mechanism is directly involved in the cell death responding to irradiation. For this reason, defining the contribution of caspase-independent molecular mechanisms represents the main goal in radiotherapy. In this study, we focused on the role of apoptosis-inducing factor (AIF), the caspase-independent molecular, in ionizing radiation induced hepatocellular carcinoma cell line (HepG2) cell death. We found that ionizing radiation has no function on AIF expression in HepG2 cells, but could induce AIF release from the mitochondria and translocate into nuclei. Inhibition of AIF could reduce ionizing radiation induced HepG2 cell death. These studies strongly support a direct relationship between AIF nuclear translocation and radiation induced cell death. What's more, AIF nuclear translocation is caspase-independent manner, but not caspase-dependent manner, in this process. These new findings add a further attractive point of investigation to better define the complex interplay between caspase-independent cell death and radiation therapy. - Highlights: • AIF nuclear translocation is involved in ionizing radiation induced hepatocellular carcinoma cell line HepG2 cell death. • AIF mediated cell death induced by ionizing radiation is caspase-independent. • Caspase-independent pathway is involved in ionzing radiation induced HepG2 cell death.

  19. The 3-D Culture and In Vivo Growth of the Human Hepatocellular Carcinoma Cell Line HepG2 in a Self-Assembling Peptide Nanofiber Scaffold

    International Nuclear Information System (INIS)

    Wu, M.; Yang, Z.; Liu, Y.; Liu, B.; Zhao, X.

    2010-01-01

    We report the use of the RADA16-I scaffold to mimic the ECM microenvironment and support tumor cell adherence and survival. Cellular morphology, proliferation, adhesion ability, and in vivo tumor formation were studied in the human hepatocellular carcinoma cell line HepG2 in the 3-D RADA16-I scaffold. No significant differences in HepG2 cell proliferation, adhesion, and albumin secretion were observed in the peptide scaffold compared to collagen I. Furthermore, the HepG2 cells pre cultured in the peptide scaffold showed a higher proliferation rate and formed significantly bigger tumors when compared to cells grown on a traditional 2D monolayer, suggesting that the 3-D RADA16-I scaffold can mimic the tumor microenvironment and promote a malignant phenotype in HepG2 cells. Our results indicate that the RADA16-I scaffold can serve as an ideal model for tumorigenesis, growth, local invasion, and metastasis.

  20. Body weight management effect of burdock (Arctium lappa L.) root is associated with the activation of AMP-activated protein kinase in human HepG2 cells.

    Science.gov (United States)

    Kuo, Daih-Huang; Hung, Ming-Chi; Hung, Chao-Ming; Liu, Li-Min; Chen, Fu-An; Shieh, Po-Chuen; Ho, Chi-Tang; Way, Tzong-Der

    2012-10-01

    Burdock (Arcticum lappa L.) root is used in folk medicine and also as a vegetable in Asian countries. In the present study, burdock root treatment significantly reduced body weight in rats. To evaluate the bioactive compounds, we successively extracted the burdock root with ethanol (AL-1), and fractionated it with n-hexane (AL-2), ethyl acetate (AL-3), n-butanol (AL-4), and water (AL-5). Among these fractions, AL-2 contained components with the most effective hypolipidemic potential in human hepatoma HepG2 cells. AL-2 decreased the expression of fatty acid synthase (FASN) and inhibited the activity of acetyl-coenzyme A carboxylase (ACC) by stimulating AMP-activated protein kinase (AMPK) through the LKB1 pathway. Three active compounds were identified from the AL-2, namely α-linolenic acid, methyl α-linolenate, and methyl oleate. These results suggest that burdock root is expected to be useful for body weight management. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Activation of farnesoid X receptor increases the expression of cytokine inducible SH2-containing protein in HepG2 cells.

    Science.gov (United States)

    Xu, Zhizhen; Huang, Gang; Gong, Wei; Zhao, Yuanyin; Zhou, Peng; Zeng, Yijun; He, Fengtian

    2012-11-01

    Cytokine inducible SH2-containing protein (CISH), which negatively regulates cytokine signaling by inhibiting JAK2/STAT5 activity, is regarded as a therapeutic target for inflammatory diseases. Farnesoid X receptor (FXR), a ligand-activated transcription factor, has been proposed to play a protective function in the inflammatory responses. However, the role of FXR in modulation of CISH expression is unknown. In the present study, we for the first time identified that in human hepatoma cell line HepG2 the activation of FXR by the natural agonist chenodeoxycholic acid (CDCA) and the synthetic specific agonist GW4064 upregulated CISH at both transcriptional and translational levels, and inhibited interleukin (IL)6-induced STAT5 activation. Moreover, the in vivo experiment demonstrated that gavaging mice with CDCA increased CISH expression and reduced basal STAT5 phosphorylation in liver tissues. Reporter assay showed that FXR agonists enhanced the transcriptional activity of CISH promoter. These data suggest that FXR may serve as a novel molecular target for manipulating CISH expression in hepatocytes. FXR-mediated upregulation of CISH may play an important role in the homeostasis of cytokine signal networks and be beneficial to control cytokine-associated inflammatory diseases.

  2. BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity

    International Nuclear Information System (INIS)

    Lu, Zheng; Ping, Liang; JianBo, Zhou; XiaoBing, Huang; Yu, Wen; Zheng, Wang; Jing, Li

    2012-01-01

    The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG 2 cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG 2 cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41% in HepG 2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G 1 phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+) BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms

  3. Identification, purification and partial characterisation of an oligonucleotide receptor in membranes of HepG2 cells

    OpenAIRE

    Diesbach, Philippe de; Berens, Catherine; N’Kuli, Francisca; Monsigny, Michel; Sonveaux, Etienne; Wattiez, Ruddy; Courtoy, Pierre J.

    2000-01-01

    The low and unpredictable uptake and cytosolic transfer of oligonucleotides (ODN) is a major reason for their limited benefit. Improving the ODN potential for therapy and research requires a better understanding of their receptor-mediated endocytosis. We have undertaken to identify a membrane ODN receptor on HepG2 cells by ligand blotting of cell extracts with [(125)I]ODN and by photolabelling of living cells with a [(125)I]ODN-benzophenone conjugate. A major band at 66 kDa was identified by ...

  4. Hepatoprotective potential of Lavandula coronopifolia extracts against ethanol induced oxidative stress-mediated cytotoxicity in HepG2 cells.

    Science.gov (United States)

    Farshori, Nida Nayyar; Al-Sheddi, Ebtsam S; Al-Oqail, Mai M; Hassan, Wafaa H B; Al-Khedhairy, Abdulaziz A; Musarrat, Javed; Siddiqui, Maqsood A

    2015-08-01

    The present investigations were carried out to study the protective potential of four extracts (namely petroleum ether extract (LCR), chloroform extract (LCM), ethyl acetate extract (LCE), and alcoholic extract (LCL)) of Lavandula coronopifolia on oxidative stress-mediated cell death induced by ethanol, a known hepatotoxin in human hapatocellular carcinoma (HepG2) cells. Cells were pretreated with LCR, LCM, LCE, and LCL extracts (10-50 μg/ml) of L. coronopifolia for 24 h and then ethanol was added and incubated further for 24 h. After the exposure, cell viability using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and neutral red uptake assays and morphological changes in HepG2 cells were studied. Pretreatment with various extracts of L. coronpifolia was found to be significantly effective in countering the cytotoxic responses of ethanol. Antioxidant properties of these L. coronopifolia extracts against reactive oxygen species (ROS) generation, lipid peroxidation (LPO), and glutathione (GSH) levels induced by ethanol were investigated. Results show that pretreatment with these extracts for 24 h significantly inhibited ROS generation and LPO induced and increased the GSH levels reduced by ethanol. The data from the study suggests that LCR, LCM, LCE, and LCL extracts of L. coronopifolia showed hepatoprotective activity against ethanol-induced damage in HepG2 cells. However, a comparative study revealed that the LCE extract was found to be the most effective and LCL the least effective. The hepatoprotective effects observed in the study could be associated with the antioxidant properties of these extracts of L. coronopifolia. © The Author(s) 2013.

  5. Gallic acid reduces cell growth by induction of apoptosis and reduction of IL-8 in HepG2 cells.

    Science.gov (United States)

    Lima, Kelly Goulart; Krause, Gabriele Catyana; Schuster, Aline Daniele; Catarina, Anderson Velasque; Basso, Bruno Souza; De Mesquita, Fernanda Cristina; Pedrazza, Leonardo; Marczak, Elisa Simon; Martha, Bianca Andrade; Nunes, Fernanda Bordignon; Chiela, Eduardo Cremonese Filippi; Jaeger, Natália; Thomé, Marcos Paulo; Haute, Gabriela Viegas; Dias, Henrique Bregolin; Donadio, Márcio Vinícius Fagundes; De Oliveira, Jarbas Rodrigues

    2016-12-01

    Hepatocellular carcinoma is the most prevalent primary liver tumor and is among the top ten cancer that affect the world population. Its development is related, in most cases, to the existence of chronic liver injury, such as in cirrhosis. The knowledge about the correlation between chronic inflammation and cancer has driven new researches with anti-inflammatory agents that have potential for the development of antitumor drugs. Gallic acid is a phenolic acid found in many natural products and have shown anti-inflammatory, anti-tumor, anti-mutagenic and antioxidant actions. The purpose of this study was to investigate the effect of gallic acid on acute and chronic cell proliferation and inflammatory parameters of hepatocellular carcinoma cells (HepG2), as well as to investigate the mechanisms involved. Results showed that the gallic acid decreased the proliferation of HepG2 cells in a dose-dependent manner (Trypan blue exclusion assay), without causing necrosis (LDH assay). We observed a significant increase in the percentage of small and regular nuclei (Nuclear Morphometric Analysis assay), a significant induction of apoptosis by Annexin V-FITC and PI assay and no interference with the cell cycle using the FITC BrdU Flow Kit. We observed a significant reduction in the levels of IL-8 and increased levels of IL-10 and IL-12 (Cytometric Bead Array Human Inflammation Assay). Furthermore, gallic acid caused no cancer cells regrowth at a long term (Cumulative Population Doubling assay). According to these results, gallic acid showed a strong potential as an anti-tumor agent in hepatocellular carcinoma cells. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  6. [Production effect comparison of SEPP and GPx between HepG2 and Hela cells with different selenocompounds].

    Science.gov (United States)

    Wang, Qin; Gao, Lina; Han, Feng; Lu, Jiaxi; Liu, Yiqun; Sun, Licui; Huang, Zhenwu

    2016-03-01

    To compare the effect of several selenocompounds on the productions of SEPP and GPx in HepG2 and Hela cells. The cultured HepG2 and Hela cells were divided into the control, Na2SeO3, SeMet and MeSeCys groups. After adding the selected selenocompounds (with the respective concentration 0.01 and 0.1 μmol/L), the experimental groups were then incubated for 48 h and 72 h. Finally, the cell culture supernatants and homogenates were collected for the SEPP and GPx concentrations detection by a double-antibody sandwich enyme-linked immuno-sorbent-assay (ELISA). The SEPP and GPx concentrations in Hela cells treated with 0.1 μmol/L SeMet and MeSeCys were significantly higher than that in the control group (P cell treated with 0.1 μmol/L selenocompounds were significantly higher than that in Hela cells (P cells are more beneficial to the production of selenoproteins than Hela cells.

  7. Increased glucose metabolism and alpha-glucosidase inhibition in Cordyceps militaris water extract-treated HepG2 cells

    Science.gov (United States)

    Kim, Dae Jung; Kang, Yun Hwan; Kim, Kyoung Kon; Kim, Tae Woo; Park, Jae Bong

    2017-01-01

    BACKGROUND/OBJECTIVES Recent living condition improvements, changes in dietary habits, and reductions in physical activity are contributing to an increase in metabolic syndrome symptoms including diabetes and obesity. Through such societal developments, humankind is continuously exposed to metabolic diseases such as diabetes, and the number of the victims is increasing. This study investigated Cordyceps militaris water extract (CMW)-induced glucose uptake in HepG2 cells and the effect of CMW treatment on glucose metabolism. MATERIALS/METHODS Colorimetric assay kits were used to determine the glucokinase (GK) and pyruvate dehydrogenase (PDH) activities, glucose uptake, and glycogen content. Either RT-PCR or western blot analysis was performed for quantitation of glucose transporter 2 (GLUT2), hepatocyte nuclear factor 1 alpha (HNF-1α), phosphatidylinositol 3-kinase (PI3k), protein kinase B (Akt), phosphorylated AMP-activated protein kinase (pAMPK), phosphoenolpyruvate carboxykinase, GK, PDH, and glycogen synthase kinase 3 beta (GSK-3β) expression levels. The α-glucosidase inhibitory activities of acarbose and CMW were evaluated by absorbance measurement. RESULTS CMW induced glucose uptake in HepG2 cells by increasing GLUT2 through HNF-1α expression stimulation. Glucose in the cells increased the CMW-induced phosphorylation of AMPK. In turn, glycolysis was stimulated, and glyconeogenesis was inhibited. Furthermore, by studying the mechanism of action of PI3k, Akt, and GSK-3β, and measuring glycogen content, the study confirmed that the glucose was stored in the liver as glycogen. Finally, CMW resulted in a higher level of α-glucosidase inhibitory activity than that from acarbose. CONCLUSION CMW induced the uptake of glucose into HepG2 cells, as well, it induced metabolism of the absorbed glucose. It is concluded that CMW is a candidate or potential use in diabetes prevention and treatment. PMID:28584574

  8. Fucosterol activates the insulin signaling pathway in insulin resistant HepG2 cells via inhibiting PTP1B.

    Science.gov (United States)

    Jung, Hyun Ah; Bhakta, Himanshu Kumar; Min, Byung-Sun; Choi, Jae Sue

    2016-10-01

    Insulin resistance is a characteristic feature of type 2 diabetes mellitus (T2DM) and is characterized by defects in insulin signaling. This study investigated the modulatory effects of fucosterol on the insulin signaling pathway in insulin-resistant HepG2 cells by inhibiting protein tyrosine phosphatase 1B (PTP1B). In addition, molecular docking simulation studies were performed to predict binding energies, the specific binding site of fucosterol to PTP1B, and to identify interacting residues using Autodock 4.2 software. Glucose uptake was determined using a fluorescent D-glucose analogue and the glucose tracer 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose, and the signaling pathway was detected by Western blot analysis. We found that fucosterol enhanced insulin-provoked glucose uptake and conjointly decreased PTP1B expression level in insulin-resistant HepG2 cells. Moreover, fucosterol significantly reduced insulin-stimulated serine (Ser307) phosphorylation of insulin receptor substrate 1 (IRS1) and increased phosphorylation of Akt, phosphatidylinositol-3-kinase, and extracellular signal- regulated kinase 1 at concentrations of 12.5, 25, and 50 µM in insulin-resistant HepG2 cells. Fucosterol inhibited caspase-3 activation and nuclear factor kappa B in insulin-resistant hepatocytes. These results suggest that fucosterol stimulates glucose uptake and improves insulin resistance by downregulating expression of PTP1B and activating the insulin signaling pathway. Thus, fucosterol has potential for development as an anti-diabetic agent.

  9. Polyethyleneimine-coated quantum dots for miRNA delivery and its enhanced suppression in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Liang G

    2016-11-01

    Full Text Available Gaofeng Liang,1 Yang Li,1 Wenpo Feng,1 Xinshuai Wang,2 Aihua Jing,1 Jinghua Li,1 Kaiwang Ma1 1Department of Biomedical Engineering, School of Medical Technology & Engineering, 2Department of Oncology, The First Affiliated Hospital, Henan University of Science & Technology, Luoyang, People’s Republic of China Abstract: Quantum dots (QDs have been intensively investigated for bioimaging, drug delivery, and labeling probes because of their unique optical properties. In this study, CdSe/ZnS QDs-based nonviral vectors with the dual functions of delivering miR-26a plasmid and bioimaging were formulated by capping the surface of CdSe/ZnS QDs with polyethyleneimine (PEI. The PEI-coated QDs were capable of condensing miR-26a expression vector into nanocomplexes that can emit strong red luminescence when loaded with CdSe/ZnS QDs. Further results showed that PEI-modified nanoparticles (NPs could transfect miR-26a plasmid into HepG2 cells in vitro. Meanwhile, imaging of living cells could be achieved based on the CdSe/ZnS QDs. Further study suggested that miR-26a transfection up-regulated miR-26a expression, induced cycle arrest, and triggered proliferation inhibition in HepG2 cells. The results indicated that PEI-coated QD NPs possess the capability of bioimaging and gene delivery and could be a promising vehicle with the engineering of QD NPs for gene therapy in the future. Keywords: miR-26a, PEI/QDs, HepG2, gene delivery, bioimaging

  10. ANTIPROLIFERATIVE AND APOPTOTIC EFFECTS OF THE ESSENTIAL OIL OF ORIGANUM ONITES AND CARVACROL ON HEP-G2 CELLS

    Directory of Open Access Journals (Sweden)

    Özlem TOMSUK

    2011-08-01

    Full Text Available The essential oil Origanum onites L. and its phenolic constituent carvacrol were examined for their cytotoxic and apoptotic effects in a human hepatocellular carcinoma cells Hep-G2. WST-1 and neutral red uptake assays were performed to determine the inhibitory effects of the oil and carvacrol on the growth of the cells. Possible induction of apoptosis by Origanum oil and carvacrol was further investigated by acridine orange/ethidium bromide (AO/EB staining. Results showed that the Ori- ganum oil and carvacrol was significantly cytotoxic and induced apoptosis in Hep-G2 cells. IC₅₀ value of essential oil and carvacrol was found about 0,009% (v/v and 500 μM, respectively. After incuba- tion of the cells with Origanum oil and carvacrol, characteristics of apoptotic morphology such as chromatin condensation, shrinkage of the cells and cytoplasmic blebbing was observed. In conclusion, both essential oil and its major constituent carvacrol significantly exhibited cytotoxic and apoptotic activities in hepatocellular carcinoma cells, indicating its potential for use as an anticancer agent.

  11. Hypocholesterolemic mechanism of phenolics-enriched extract from Moringa oleifera leaves in HepG2 cell lines

    Directory of Open Access Journals (Sweden)

    Peera Tabboon

    2016-04-01

    Full Text Available Previous studies have demonstrated the hypolipidemic activity of Moringa oleifera (MO leaves via lowering serum levels of cholesterol, but the mechanism of action is unknown. In this study, we demonstrated the hypocholesterolemic mechanism of a phenolics-enriched extract of Moringa oleifera leaf (PMO in HepG2 cells. When compared to the control treatment, PMO significantly decreased total intracellular cholesterol, inhibited the activity of HMG CoA reductase in a dosedependent manner and enhanced LDL receptor binding activity. Moreover, PMO also significantly increased the genetic expressions of HMG CoA reductase and LDL receptor.

  12. Effect of human mesenchymal stem cells on the growth of HepG2 and Hela cells.

    Science.gov (United States)

    Long, Xiaohui; Matsumoto, Rena; Yang, Pengyuan; Uemura, Toshimasa

    2013-01-01

    Human mesenchymal stem cells (hMSCs) accumulate at carcinomas and have a great impact on cancer cell's behavior. Here we demonstrated that hMSCs could display both the promotional and inhibitive effects on growth of HepG2 and Hela cells by using the conditioned media, indirect co-culture, and cell-to-cell co-culture. Cell growth was increased following the addition of lower proportion of hMSCs while decreased by treatment of higher proportion of hMSCs. We also established a novel noninvasive label way by using internalizing quantum dots (i-QDs) for study of cell-cell contact in the co-culture, which was effective and sensitive for both tracking and distinguishing different cells population without the disturbance of cells. Furthermore, we investigated the role of hMSCs in regulation of cell growth and showed that mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways were involved in hMSC-mediated cell inhibition and proliferation. Our findings suggested that hMSCs regulated cancer cell function by providing a suitable environment, and the discovery from the study would provide some clues for development of effective strategy for hMSC-based cancer therapies.

  13. Copper excess in liver HepG2 cells interferes with apoptosis and lipid metabolic signaling at the protein level.

    Science.gov (United States)

    Liu, Yu; Yang, Huarong; Song, Zhi; Gu, Shaojuan

    2014-12-01

    Copper is an essential trace element that serves as an important catalytic cofactor for cuproenzymes, carrying out major biological functions in growth and development. Although Wilson's disease (WD) is unquestionably caused by mutations in the ATP7B gene and subsequent copper overload, the precise role of copper in inducing pathological changes remains poorly understood. Our study aimed to explore, in HepG2 cells exposed to copper, the cell viability and apoptotic cells was tested by MTT and Hoechst 33342 stainning respectively, and the signaling pathways involved in oxidative stress response, apoptosis and lipid metabolism were determined by real time RT-PCR and Western blot analysis. The results demonstrate dose- and time-dependent cell viability and apoptosis in HepG2 cells following treatment with 10 μM, 200 μM and 500 μM of copper sulfate for 8 and 24 h. Copper overload significantly induced the expression of HSPA1A (heat shock 70 kDa protein 1A), an oxidative stress-responsive signal gene, and BAG3 (BCL2 associated athanogene3), an anti-apoptotic gene, while expression of HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase), a lipid biosynthesis and lipid metabolism gene, was inhibited. These findings provide new insights into possible mechanisms accounting for the development of liver apoptosis and steatosis in the early stages of Wilson's disease.

  14. Effervescent Granules Prepared Using Eucommia ulmoides Oliv. and Moso Bamboo Leaves: Hypoglycemic Activity in HepG2 Cells

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    Xiang-Zhou Li

    2016-01-01

    Full Text Available Eucommia ulmoides Oliv. (E. ulmoides Oliv. and moso bamboo (Phyllostachys pubescens leaves are used as folk medicines in central-western China to treat diabetes. To investigate the hypoglycemic activity of the effervescent granules prepared using E. ulmoides Oliv. and moso bamboo leaves (EBEG in HepG2 cells, EBEG were prepared with 5% of each of polysaccharides and chlorogenic acids from moso bamboo and E. ulmoides Oliv. leaves, respectively. HepG2 cells cultured in a high-glucose medium were classified into different groups. The results displayed EBEG-treated cells showed better glucose utilization than the negative controls; thus, the hypoglycemic effect of EBEG was much greater than that of granules prepared using either component alone, thereby indicating that this effect was due to a synergistic action of the components. Further, glucose consumption levels in the cells treated with EBEG (156.35% at 200 μg/mL and the positive controls (metformin, 162.29%; insulin, 161.52% were similar. Thus, EBEG exhibited good potential for use as a natural antidiabetic agent. The hypoglycemic effect of EBEG could be due to the synergistic action of polysaccharides from the moso bamboo leaves and chlorogenic acids from E. ulmoides Oliv. leaves via the inhibition of alpha-glucosidase and glucose-6-phosphate displacement enzyme.

  15. Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG2 cells

    International Nuclear Information System (INIS)

    Guo Dongping; Li Xiaoyu; Sun, Ping; Tang Yibo; Chen Xiuying; Chen Qi; Fan Leming; Zang Bin; Shao Lizheng; Li Xiaorong

    2006-01-01

    Ultrasound-targeted microbubble destruction had been employed in gene delivery and promised great potential. Liver has unique features that make it attractive for gene therapy. However, it poses formidable obstacles to hepatocyte-specific gene delivery. This study was designed to test the efficiency of therapeutic gene transfer and expression mediated by ultrasound/microbubble strategy in HepG 2 cell line. Air-filled albumin microbubbles were prepared and mixed with plasmid DNA encoding low density lipoprotein receptor (LDLR) and green fluorescent protein. The mixture of the DNA and microbubbles was administer to cultured HepG 2 cells under variable ultrasound conditions. Transfection rate of the transferred gene and cell viability were assessed by FACS analysis, confocal laser scanning microscopy, Western blot analysis and Trypan blue staining. The result demonstrated that microbubbles with ultrasound irradiation can significantly elevate exogenous LDLR gene expression and the expressed LDLRs were functional and active to uptake their ligands. We conclude that ultrasound-targeted microbubble destruction has the potential to promote safe and efficient LDLR gene transfer into hepatocytes. With further refinement, it may represent an effective nonviral avenue of gene therapy for liver-involved genetic diseases

  16. [Inhibitory effect of Biejiajian pills on HepG2 cell xenograft growth and expression of β-catenin and Tbx3 in nude mice].

    Science.gov (United States)

    Wen, Bin; Sun, Hai-Tao; He, Song-Qi; LA, Lei; An, Hai-Yan; Pang, Jie

    2016-02-01

    To explore the molecular mechanism by which Biejiajian pills inhibit hepatocellular carcinoma in a nude mouse model bearing HepG2 cell xenograft. The inhibitory effect of Biejiajian pills on the growth of HepG2 cell xenograft in nude mice was observed. Immunohistochemical method was used to examine proliferating cell nuclear antigen (PCNA) expression in HepG2 cell xenograft, and TUNEL method was employed to detect the cell apoptosis; the expression levels of β-catenin and Tbx3 were measured by Western blotting. Biejiajian pills significantly suppressed the growth of HepG2 cell xenograft in nude mice. The tumor-bearing mice treated with a high and a moderate dose of Biejiajian pills showed significantly increased apoptosis rate of the tumor cells [(22.9±1.220)% and (14.7±0.50)%, respectively] compared with the control group [(5.5±0.90)%, Ppills significantly decreased the expressions of PNCA, β-catenin, and Tbx3 in the cell xenograft (Ppills can inhibit the growth of HepG2 cell xenograft in nude mice and promote tumor cell apoptosis possibly by inhibiting PNCA expression and the Wnt/β-catenin signaling pathway.

  17. Sagunja-Tang Improves Lipid Related Disease in a Postmenopausal Rat Model and HepG2 Cells

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    Hiroe Go

    2015-01-01

    Full Text Available The present study was conducted to investigate the effect of Sagunja-tang on the lipid related disease in a rat model of menopausal hyperlipidemia and lipid accumulation in methyl-β-cyclodextrin-induced HepG2 cells. In in vivo study using menopausal hyperlipidemia rats, Sagunja-tang reduced retroperitoneal and perirenal fat, serum lipids, atherogenic index, cardiac risk factor, media thickness, and nonalcoholic steatohepatitis score, when compared to menopausal hyperlipidemia control rats. In HepG2 cells, Sagunja-tang significantly decreased the lipid accumulation, total cholesterol levels, and low-density/very-low-density lipoprotein levels. Moreover, Sagunja-tang reversed the methyl-β-cyclodextrin-induced decrease in the protein levels of critical molecule involved in cholesterol synthesis, sterol regulatory element binding protein-2, and low-density lipoprotein receptor and inhibited protein levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase as well as activity. Phosphorylation level of AMP-activated protein kinase was stimulated by Sagunja-tang. These results suggest that Sagunja-tang has effect on inhibiting hepatic lipid accumulation through regulation of cholesterol synthesis and AMPK activity in vitro. These observations support the idea that Sagunja-tang is bioavailable both in vivo and in vitro and could be developed as a preventive and therapeutic agent of hyperlipidemia in postmenopausal females.

  18. Activation of human stearoyl-coenzyme A desaturase 1 contributes to the lipogenic effect of PXR in HepG2 cells.

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    Jun Zhang

    Full Text Available The pregnane X receptor (PXR was previously known as a xenobiotic receptor. Several recent studies suggested that PXR also played an important role in lipid homeostasis but the underlying mechanism remains to be clearly defined. In this study, we found that rifampicin, an agonist of human PXR, induced lipid accumulation in HepG2 cells. Lipid analysis showed the total cholesterol level increased. However, the free cholesterol and triglyceride levels were not changed. Treatment of HepG2 cells with rifampicin induced the expression of the free fatty acid transporter CD36 and ABCG1, as well as several lipogenic enzymes, including stearoyl-CoA desaturase-1 (SCD1, long chain free fatty acid elongase (FAE, and lecithin-cholesterol acyltransferase (LCAT, while the expression of acyl:cholesterol acetyltransferase(ACAT1 was not affected. Moreover, in PXR over-expressing HepG2 cells (HepG2-PXR, the SCD1 expression was significantly higher than in HepG2-Vector cells, even in the absence of rifampicin. Down-regulation of PXR by shRNA abolished the rifampicin-induced SCD1 gene expression in HepG2 cells. Promoter analysis showed that the human SCD1 gene promoter is activated by PXR and a novel DR-7 type PXR response element (PXRE response element was located at -338 bp of the SCD1 gene promoter. Taken together, these results indicated that PXR activation promoted lipid synthesis in HepG2 cells and SCD1 is a novel PXR target gene.

  19. Electrokinetic gated injection-based microfluidic system for quantitative analysis of hydrogen peroxide in individual HepG2 cells.

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    Zhang, Xinyuan; Li, Qingling; Chen, Zhenzhen; Li, Hongmin; Xu, Kehua; Zhang, Lisheng; Tang, Bo

    2011-03-21

    A microfluidic system to determine hydrogen peroxide (H(2)O(2)) in individual HepG2 cells based on the electrokinetic gated injection was developed for the first time. A home-synthesized fluorescent probe, bis(p-methylbenzenesulfonate)dichlorofluorescein (FS), was employed to label intracellular H(2)O(2) in the intact cells. On a simple cross microchip, multiple single-cell operations, including single cell injection, cytolysis, electrophoresis separation and detection of H(2)O(2), were automatically carried out within 60 s using the electrokinetic gated injection and laser-induced fluorescence detection (LIFD). The performance of the method was evaluated under the optimal conditions. The linear calibration curve was over a range of 4.39-610 amol (R(2)=0.9994). The detection limit was 0.55 amol or 9.0×10(-10) M (S/N=3). The relative standard deviations (RSDs, n=6) of migration time and peak area were 1.4% and 4.8%, respectively. With the use of this method, the average content of H(2)O(2) in single HepG2 cells was found to be 16.09±9.84 amol (n=15). Separation efficiencies in excess of 17,000 theoretical plates for the cells were achieved. These results demonstrated that the efficient integration and automation of these single-cell operations enabled the sensitive, reproducible, and quantitative examination of intracellular H(2)O(2) at single-cell level. Owing to the advantages of simple microchip structure, controllable single-cell manipulation and ease in building, this platform provides a universal way to automatically determine other intracellular constituents within single cells. This journal is © The Royal Society of Chemistry 2011

  20. Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death

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    Sun, Hengwen [Department of Radiation, Cancer Center of Guangdong General Hospital (Guangdong Academy of Medical Science), Guangzhou, 510080, Guangdong (China); Yang, Shana; Li, Jianhua [Department of Physiology, Guangzhou Medical University, Guangzhou, 510182, Guangdong (China); Zhang, Yajie [Department of Pathology, Guangzhou Medical University, Guangzhou, 510182, Guangdong (China); Gao, Dongsheng [Department of Oncology, Guangdong Medical College Affiliated Pengpai Memorial Hospital, Hai Feng, 516400, Gungdong (China); Zhao, Shenting, E-mail: zhaoshenting@126.com [Department of Physiology, Guangzhou Medical University, Guangzhou, 510182, Guangdong (China)

    2016-03-25

    Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The aim of radiotherapy is to eradicate cancer cells with ionizing radiation. Except for the caspase-dependent mechanism, several lines of evidence demonstrated that caspase-independent mechanism is directly involved in the cell death responding to irradiation. For this reason, defining the contribution of caspase-independent molecular mechanisms represents the main goal in radiotherapy. In this study, we focused on the role of apoptosis-inducing factor (AIF), the caspase-independent molecular, in ionizing radiation induced hepatocellular carcinoma cell line (HepG2) cell death. We found that ionizing radiation has no function on AIF expression in HepG2 cells, but could induce AIF release from the mitochondria and translocate into nuclei. Inhibition of AIF could reduce ionizing radiation induced HepG2 cell death. These studies strongly support a direct relationship between AIF nuclear translocation and radiation induced cell death. What's more, AIF nuclear translocation is caspase-independent manner, but not caspase-dependent manner, in this process. These new findings add a further attractive point of investigation to better define the complex interplay between caspase-independent cell death and radiation therapy. - Highlights: • AIF nuclear translocation is involved in ionizing radiation induced hepatocellular carcinoma cell line HepG2 cell death. • AIF mediated cell death induced by ionizing radiation is caspase-independent. • Caspase-independent pathway is involved in ionzing radiation induced HepG2 cell death.

  1. A three-dimensional in vitro HepG2 cells liver spheroid model for genotoxicity studies.

    Science.gov (United States)

    Shah, Ume-Kulsoom; Mallia, Jefferson de Oliveira; Singh, Neenu; Chapman, Katherine E; Doak, Shareen H; Jenkins, Gareth J S

    2018-01-01

    The liver's role in metabolism of chemicals makes it an appropriate tissue for toxicity testing. Current testing protocols, such as animal testing and two-dimensional liver cell systems, offer limited resemblance to in vivo liver cell behaviour, in terms of gene expression profiles and metabolic competence; thus, they do not always accurately predict human toxicology. In vitro three-dimensional liver cell models offer an attractive alternative. This study reports on the development of a 3D liver model, using HepG2 cells, by a hanging-drop technique, with a focus on evaluating spheroid growth characteristics and suitability for genotoxicity testing. The cytokinesis-blocked micronucleus assay protocol was adapted to enable micronucleus (MN) detection in the 3D spheroid models. This involved evaluating the difference between hanging vs non-hanging drop positions for dosing of the test agents and comparison of automated Metafer scoring with manual scoring for MN detection in HepG2 spheroids. The initial seeding density, used for all experiments, was 5000 cells/20 μl drop hanging spheroids, harvested on day 4, with >75% cell viability. Albumin secretion (7.8 g/l) and both CYP1A1 and CYP1A2 gene expression were highest in the 3D environment at day 4. Exposure to metabolically activated genotoxicants for 24 h resulted in a 6-fold increase in CYP1A1 enzyme activity (3 μM B[a]P) and a 30-fold increase in CYP1A2 enzyme activity (5 μM PhIP) in 3D hanging spheroids. MN inductions in response to B[a]P or PhIP were 2-fold and 3-fold, respectively, and were greater in 3D hanging spheroids than in 2D format, showing that hanging spheroids are more sensitive to genotoxic agents. HepG2 hanging-drop spheroids are an exciting new alternative system for genotoxicity studies, due to their improved structural and physiological properties, relative to 2D cultures. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Ursodeoxycholic acid inhibits overexpression of P-glycoprotein induced by doxorubicin in HepG2 cells.

    Science.gov (United States)

    Komori, Yuki; Arisawa, Sakiko; Takai, Miho; Yokoyama, Kunihiro; Honda, Minako; Hayashi, Kazuhiko; Ishigami, Masatoshi; Katano, Yoshiaki; Goto, Hidemi; Ueyama, Jun; Ishikawa, Tetsuya; Wakusawa, Shinya

    2014-02-05

    The hepatoprotective action of ursodeoxycholic acid (UDCA) was previously suggested to be partially dependent on its antioxidative effect. Doxorubicin (DOX) and reactive oxygen species have also been implicated in the overexpression of P-glycoprotein (P-gp), which is encoded by the MDR1 gene and causes antitumor multidrug resistance. In the present study, we assessed the effects of UDCA on the expression of MDR1 mRNA, P-gp, and intracellular reactive oxygen species levels in DOX-treated HepG2 cells and compared them to those of other bile acids. DOX-induced increases in reactive oxygen species levels and the expression of MDR1 mRNA were inhibited by N-acetylcysteine, an antioxidant, and the DOX-induced increase in reactive oxygen species levels and DOX-induced overexpression of MDR1 mRNA and P-gp were inhibited by UDCA. Cells treated with UDCA showed improved rhodamine 123 uptake, which was decreased in cells treated with DOX alone. Moreover, cells exposed to DOX for 24h combined with UDCA accumulated more DOX than that of cells treated with DOX alone. Thus, UDCA may have inhibited the overexpression of P-gp by suppressing DOX-induced reactive oxygen species production. Chenodeoxycholic acid (CDCA) also exhibited these effects, whereas deoxycholic acid and litocholic acid were ineffective. In conclusion, UDCA and CDCA had an inhibitory effect on the induction of P-gp expression and reactive oxygen species by DOX in HepG2 cells. The administration of UDCA may be beneficial due to its ability to prevent the overexpression of reactive oxygen species and acquisition of multidrug resistance in hepatocellular carcinoma cells. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Comparison of gene expression profiles of HepG2 cells exposed to Crambescins C1 and A1

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    María R. Sánchez

    2014-06-01

    Full Text Available Crambescins are guanidine alkaloids firstly isolated in the early 90s from the encrusting Mediterranean sponge Crambe crambe (Schmidt, 1862 (Bondu et al., 2012, Laville et al., 2009, Berlinck et al., 1990. C. crambe derivatives are divided in two families named crambescins and crambescidins (Gerlinck et al., 1992. Although data on the bioactivity of these compounds is scarce, crambescidins have recognized cytotoxic, antifungal, antioxidant, antimicrobial and antiviral activities (Buscema and Van de Vyver, 1985, Jares-Erijman., 1998, Olszewski et al., 2004, Lazaro et al., 2006, Suna et al., 2007, AOKI et al., 2004. Recently we have carefully evaluated the cytotoxic activity of C816 over several human tumor cell types and characterized some of the cellular mechanisms responsible of the anti-proliferative effect of this compound on human liver-derived tumor cells (Rubiolo et al., 2013. Taking this into account, and to better understand the mechanism of action of crambescins and their potential as therapeutic agents, we made a comparative gene expression profiling of HepG2 cells after crambescin C1 (C1 and crambescin A1 (CA1 exposures. Results have shown that C1 induces genes involved in sterol and glucose metabolisms and metabolism involving growth factors. It also down regulates genes mainly involved in cell cycle control, DNA replication, recombination and repair, and drug metabolism. Flow cytometry assays revealed that C1 produces a G0/G1 arrest in HepG2 cell cycle progression. CA1 also down-regulates genes involved in cell cycle regulation, DNA recombination and pathways related to tumor cells proliferation with lower potency when compared to C1.

  4. Mercury-Induced Externalization of Phosphatidylserine and Caspase 3 Activation in Human Liver Carcinoma (HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Paul B. Tchounwou

    2006-03-01

    Full Text Available Apoptosis arises from the active initiation and propagation of a series of highly orchestrated specific biochemical events leading to the demise of the cell. It is a normal physiological process, which occurs during embryonic development as well as in the maintenance of tissue homeostasis. Diverse groups of molecules are involved in the apoptosis pathway and it functions as a mechanism to eliminate unwanted or irreparably damaged cells. However, inappropriate induction of apoptosis by environmental agents has broad ranging pathologic implications and has been associated with several diseases including cancer. The toxicity of several heavy metals such as mercury has been attributed to their high affinity to sulfhydryl groups of proteins and enzymes, and their ability to disrupt cell cycle progression and/or apoptosis in various tissues. The aim of this study was to assess the potential for mercury to induce early and late-stage apoptosis in human liver carcinoma (HepG2 cells. The Annexin-V and Caspase 3 assays were performed by flow cytometric analysis to determine the extent of phosphatidylserine externalization and Caspase 3 activation in mercury-treated HepG2 cells. Cells were exposed to mercury for 10 and 48 hours respectively at doses of 0, 1, 2, and 3 μg/mL based on previous cytotoxicity results in our laboratory indicating an LD50 of 3.5 ± 0.6 μg/mL for mercury in HepG2 cells. The study data indicated a dose response relationship between mercury exposure and the degree of early and late-stage apoptosis in HepG2 cells. The percentages of cells undergoing early apoptosis were 0.03 ± 0.03%, 5.19 ± 0.04%, 6.36 ± 0.04%, and 8.84 ± 0.02% for 0, 1, 2, and 3 μg/mL of mercury respectively, indicating a gradual increase in apoptotic cells with increasing doses of mercury. The percentages of Caspase 3 positive cells undergoing late apoptosis were 3.58 ± 0.03%, 17.06 ± 0

  5. Synergistic effect of intervention of glypican-3 gene transcription combined with antitumor drugs in inhibiting hepatoma cell proliferation

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    YANG Jie

    2016-12-01

    Full Text Available ObjectiveTo investigate the inhibitory effect of intervention of glypican-3 (GPC3 gene transcription combined with antitumor drugs on hepatoma cell proliferation. MethodsFour types of GPC3-shRNA plasmids were established and transfected into HepG2 hepatoma cells. Quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression of GPC3 to analyze its association with hepatoma cell proliferation and apoptosis. The independent samples t-test was used for comparison of continuous data between any two groups, and a one-way analysis of variance was used for comparison between multiple groups. ResultsAmong these four plasmids, shRNA1 had a transfection efficiency of >85% in the transfection of HepG2 cells and a silence efficiency of 89.3% at the mRNA level, and the protein expression of GPC3 was significantly inhibited(P<0.01). At 72 hours, the GPC3-shRNA1 co-intervention group had an HepG2 cell inhibition rate of 71.1%, significantly different from that in the negative group (t=18.092, P<0.001, an inhibition rate of migration of 89.1%, significantly lower than that in the negative group (t=8.326, P<0.001, and inhibition rates of HepG2 cell movement and invasion of 53.6% and 60.1%, which were significantly different from those in the negative group (t=52.400 and 48.245, both P<0.001. The GPC3-shRNA1 co-intervention group had a β-catenin mRNA inhibition rate of 46.9% and a Gli1 mRNA upregulation rate of 7.4%, significantly different from those in the negative group (t=30.108 and -3.551, P<0.001 and P=0.009. At 24 hours, 10 μmol/L sorafenib combined with shRNA1 had an inhibition rate of tumor cells of 52.6% and 100 μmol/L sorafenib combined with shRNA1 had an inhibition rate of tumor cells of 79.5%, which were significantly different from that in the control group (t=23.314 and 50.352, both P<0.001. The half-maximal inhibitory concentrations of sorafenib, rapamycin, and erlotinib for HepG2 were 4.67±1

  6. OSBP-related protein 8 (ORP8) interacts with Homo sapiens sperm associated antigen 5 (SPAG5) and mediates oxysterol interference of HepG2 cell cycle

    International Nuclear Information System (INIS)

    Zhong, Wenbin; Zhou, You; Li, Jiwei; Mysore, Raghavendra; Luo, Wei; Li, Shiqian; Chang, Mau-Sun; Olkkonen, Vesa M.; Yan, Daoguang

    2014-01-01

    We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum/nuclear envelope oxysterol-binding protein implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, a yeast two-hybrid screen identified Homo sapiens sperm associated antigen 5 (SPAG5)/Astrin as interaction partner of ORP8. The putative interaction was further confirmed by pull-down and co-immunoprecipitation assays. ORP8 did not colocalize with kinetochore-associated SPAG5 in mitotic HepG2 or HuH7 cells, but overexpressed ORP8 was capable of recruiting SPAG5 onto endoplasmic reticulum membranes in interphase cells. In our experiments, 25-hydroxycholesterol (25OHC) retarded the HepG2 cell cycle, causing accumulation in G2/M phase; ORP8 overexpression resulted in the same phenotype. Importantly, ORP8 knock-down dramatically inhibited the oxysterol effect on HepG2 cell cycle, suggesting a mediating role of ORP8. Furthermore, knock-down of SPAG5 significantly reduced the effects of both ORP8 overexpression and 25OHC on the cell cycle, placing SPAG5 downstream of the two cell-cycle interfering factors. Taken together, the present results suggest that ORP8 may via SPAG5 mediate oxysterol interference of the HepG2 cell cycle. - Highlights: • The oxysterol-binding protein ORP8 was found to interact with the mitotic regulator SPAG5/Astrin. • Treatment of HepG2 cells with 25-hydroxycholesterol caused cell cycle retardation in G2/M. • ORP8 overexpression caused a similar G2/M accumulation, and ORP8 knock-down reversed the 25-hydroxycholesterol effect. • Reduction of cellular of SPAG5/Astrin reversed the cell cycle effects of both 25-hydroxycholesterol and ORP8 overexpression. • Our results suggest that ORP8 mediates via SPAG5/Astrin the oxysterol interference of HepG2 cell cycle

  7. OSBP-related protein 8 (ORP8) interacts with Homo sapiens sperm associated antigen 5 (SPAG5) and mediates oxysterol interference of HepG2 cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Zhong, Wenbin [Department of Biotechnology, Jinan University, Guangzhou 510632 (China); Zhou, You [Minerva Foundation Institute for Medical Research, Helsinki (Finland); Li, Jiwei [Department of Biotechnology, Jinan University, Guangzhou 510632 (China); Mysore, Raghavendra [Minerva Foundation Institute for Medical Research, Helsinki (Finland); Luo, Wei; Li, Shiqian [Department of Biotechnology, Jinan University, Guangzhou 510632 (China); Chang, Mau-Sun [Institute of Biochemical Sciences, National Taiwan University, No. 1, Taipei, Taiwan (China); Olkkonen, Vesa M. [Minerva Foundation Institute for Medical Research, Helsinki (Finland); Yan, Daoguang, E-mail: tydg@jnu.edu.cn [Department of Biotechnology, Jinan University, Guangzhou 510632 (China)

    2014-04-01

    We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum/nuclear envelope oxysterol-binding protein implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, a yeast two-hybrid screen identified Homo sapiens sperm associated antigen 5 (SPAG5)/Astrin as interaction partner of ORP8. The putative interaction was further confirmed by pull-down and co-immunoprecipitation assays. ORP8 did not colocalize with kinetochore-associated SPAG5 in mitotic HepG2 or HuH7 cells, but overexpressed ORP8 was capable of recruiting SPAG5 onto endoplasmic reticulum membranes in interphase cells. In our experiments, 25-hydroxycholesterol (25OHC) retarded the HepG2 cell cycle, causing accumulation in G2/M phase; ORP8 overexpression resulted in the same phenotype. Importantly, ORP8 knock-down dramatically inhibited the oxysterol effect on HepG2 cell cycle, suggesting a mediating role of ORP8. Furthermore, knock-down of SPAG5 significantly reduced the effects of both ORP8 overexpression and 25OHC on the cell cycle, placing SPAG5 downstream of the two cell-cycle interfering factors. Taken together, the present results suggest that ORP8 may via SPAG5 mediate oxysterol interference of the HepG2 cell cycle. - Highlights: • The oxysterol-binding protein ORP8 was found to interact with the mitotic regulator SPAG5/Astrin. • Treatment of HepG2 cells with 25-hydroxycholesterol caused cell cycle retardation in G2/M. • ORP8 overexpression caused a similar G2/M accumulation, and ORP8 knock-down reversed the 25-hydroxycholesterol effect. • Reduction of cellular of SPAG5/Astrin reversed the cell cycle effects of both 25-hydroxycholesterol and ORP8 overexpression. • Our results suggest that ORP8 mediates via SPAG5/Astrin the oxysterol interference of HepG2 cell cycle.

  8. MicroRNA expression in the vildagliptin-treated two- and three-dimensional HepG2 cells.

    Science.gov (United States)

    Yamashita, Yasunari; Asakura, Mitsutoshi; Mitsugi, Ryo; Fujii, Hideaki; Nagai, Kenichiro; Atsuda, Koichiro; Itoh, Tomoo; Fujiwara, Ryoichi

    2016-06-01

    Vildagliptin is an inhibitor of dipeptidyl peptidase-4 that is used for the treatment of type 2 diabetes mellitus. While vildagliptin can induce hepatic dysfunction in humans, the molecular mechanism has not been determined yet. Recent studies indicated that certain types of microRNA (miRNA) were linking to the development of drug-induced hepatotoxicity. In the present study, therefore, we identified hepatic miRNAs that were highly induced or reduced by the vildagliptin treatment in mice. MiR-222 and miR-877, toxicity-associated miRNAs, were induced 31- and 53-fold, respectively, by vildagliptin in the liver. While a number of miRNAs were significantly regulated by the orally treated vildagliptin in vivo, such regulation was not observed in the vildagliptin-treated HepG2 cells. In addition to the regular two-dimensional (2D) culture, we carried out the three-dimensional (3D) culturing of HepG2 cells. In the 3D-HepG2 cells, a significant reduction of miR-222 was observed compared to the expression level in 2D-HepG2 cells. A slight induction of miR-222 by vildagliptin was observed in the 3D-HepG2 cells, although miR-877 was not induced by vildagliptin even in the 3D-HepG2 cells. Further investigations are needed to overcome the discrepancy in the responsiveness of the miRNA expressions to vildagliptin between in vivo and in vitro. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  9. Comparative cytotoxic response of nickel ferrite nanoparticles in human liver HepG2 and breast MFC-7 cancer cells.

    Science.gov (United States)

    Ahamed, Maqusood; Akhtar, Mohd Javed; Alhadlaq, Hisham A; Khan, M A Majeed; Alrokayan, Salman A

    2015-09-01

    Nickel ferrite nanoparticles (NPs) have received much attention for their potential applications in biomedical fields such as magnetic resonance imaging, drug delivery and cancer hyperthermia. However, little is known about the toxicity of nickel ferrite NPs at the cellular and molecular levels. In this study, we investigated the cytotoxic responses of nickel ferrite NPs in two different types of human cells (i.e., liver HepG2 and breast MCF-7). Nickel ferrite NPs induced dose-dependent cytotoxicity in both types of cells, which was demonstrated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) assays. Nickel ferrite NPs were also found to induce oxidative stress, which was evident by the depletion of glutathione and the induction of reactive oxygen species (ROS) and lipid peroxidation. The mitochondrial membrane potential due to nickel ferrite NP exposure was also observed. The mRNA levels for the tumor suppressor gene p53 and the apoptotic genes bax, CASP3 and CASP9 were up-regulated, while the anti-apoptotic gene bcl-2 was down-regulated following nickel ferrite NP exposure. Furthermore, the activities of apoptotic enzymes (caspase-3 and caspase-9) were also higher in both types of cells treated with nickel ferrite NPs. Cytotoxicity induced by nickel ferrite was efficiently prevented by N-acetyl cysteine (ROS scavenger) treatment, which suggested that oxidative stress might be one of the possible mechanisms of nickel ferrite NP toxicity. We also observed that MCF-7 cells were slightly more susceptible to nickel ferrite NP exposure than HepG2 cells. This study warrants further investigation to explore the potential mechanisms of different cytotoxic responses of nickel ferrite NPs in different cell lines. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. ML-7 amplifies the quinocetone-induced cell death through akt and MAPK-mediated apoptosis on HepG2 cell line.

    Science.gov (United States)

    Zhou, Yan; Zhang, Shen; Deng, Sijun; Dai, Chongshan; Tang, Shusheng; Yang, Xiayun; Li, Daowen; Zhao, Kena; Xiao, Xilong

    2016-01-01

    The study aims at evaluating the combination of the quinocetone and the ML-7 in preclinical hepatocellular carcinoma models. To this end, the effect of quinocetone and ML-7 on apoptosis induction and signaling pathways was analyzed on HepG2 cell lines. Here, we report that ML-7, in a nontoxic concentration, sensitized the HepG2 cells to quinocetone-induced cytotoxicity. Also, ML-7 profoundly enhances quinocetone-induced apoptosis in HepG2 cell line. Mechanistic investigations revealed that ML-7 and quinocetone act in concert to trigger the cleavage of caspase-8 as well as Bax/Bcl-2 ratio up-regulation and subsequent cleavage of Bid, capsases-9 and -3. Importantly, ML-7 weakened the quinocetone-induced Akt pathway activation, but strengthened the phosphorylation of p-38, ERK and JNK. Further treatment of Akt activator and p-38 inhibitor almost completely abolished the ML-7/quinocetone-induced apoptosis. In contrast, the ERK and JNK inhibitor aggravated the ML-7/quinocetone-induced apoptosis, indicating that the synergism critically depended on p-38 phosphorylation and HepG2 cells provoke Akt, ERK and JNK signaling pathways to against apoptosis. In conclusion, the rational combination of quinocetone and ML-7 presents a promising approach to trigger apoptosis in hepatocellular carcinoma, which warrants further investigation.

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

    Directory of Open Access Journals (Sweden)

    Chen Lei

    2011-06-01

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

  12. Protective Effect of Curcumin against Ionizing Radiation (IR)-induced Cytotoxicity and Genotoxicity in HepG2 Cells

    International Nuclear Information System (INIS)

    Chung, Dong Min; Nasir Uddin, S. M.; Ryu, Tae Ho; Kang, Mi Young; Kim, Jin Kyu

    2013-01-01

    Ionizing radiation (IR) has many practical applications such as medicine, foods, agricultures, industries, and research laboratories. However, the increasing use of radiation is associated with radiation accidents threatening human health. It is well known that exposure to IR gives rise to genomic alterations, mutagenesis, and cell death. IR is absorbed directly by DNA, leading to various DNA damages (single or double-strand breaks, base damage, and DNA-DNA or DNA-protein cross-linkages) in many living organisms. Therefore, the development of effective and nontoxic radioprotective agents is of considerable interest. Curcumin (C 12 H 20 O 6 , structure is the major yellow component of Curcuma longa with biological activities (antioxidant, anti-proliferative and anti-inflammatory properties). It has been widely used as food and medicine for a long time. The aim of our present study is to investigate the protective effects of curcumin against IR-induced cytotoxicity and genotoxicity in cultured HepG2 cells

  13. Structure of Sphingolipids From Sea Cucumber Cucumaria frondosa and Structure-Specific Cytotoxicity Against Human HepG2 Cells.

    Science.gov (United States)

    Jia, Zicai; Song, Yu; Tao, Suyuan; Cong, Peixu; Wang, Xiaoxu; Xue, Changhu; Xu, Jie

    2016-03-01

    To investigate the relationship between structure and activity, three glucocerebroside series (CFC-1, CFC-2 and CFC-3), ceramides (CF-Cer) and long-chain bases (CF-LCB) of sea cucumber Cucumaria frondosa (C. frondosa) were isolated and evaluated in HepG2 cells. The molecular species of CFC-1, CFC-2 and CFC-3 and CF-Cer were identified using reversed-phase liquid chromatography with heated electrospray ionization coupled to high-resolution mass spectrometry (RPLC-HESI-HRMS), and determined on the basis of chemical and spectroscopic evidence: For the three glucocerebroside series, fatty acids (FA) were mainly saturated (18:0 and 22:0), monounsaturated (22:1, 23:1 and 24:1) and 2-hydroxyl FA (2-HFA) (23:1 h and 24:1 h), the structure of long-chain bases (LCB) were dihydroxy (d17:1, d18:1 and d18:2) and trihydroxy (t16:0 and t17:0), and the glycosylation was glucose; For CF-Cer, FA were primarily saturated (17:0) and monounsaturated (16:1 and 19:1), the structure of LCB were dihydroxy (d17:1 and d18:1), and trihydroxy (t16:0). The results of cell experiment indicated that all of three glucocerebroside series, CF-Cer and CF-LCB exhibited an inhibitory effects on cell proliferation. Moreover, CFC-3 was most effective in three glucocerebrosides to HepG-2 cell viability. The inhibition effect of CF-LCB was the strongest, and the inhibition effect of CF-Cer was much stronger than glucocerebrosides.

  14. HepG2 cells biospecific extraction and HPLC-ESI-MS analysis for screening potential antiatherosclerotic active components in Bupeuri radix.

    Science.gov (United States)

    Liu, Shuqiang; Tan, Zhibin; Li, Pingting; Gao, Xiaoling; Zeng, Yuaner; Wang, Shuling

    2016-03-20

    HepG2 cells biospecific extraction method and high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) analysis was proposed for screening of potential antiatherosclerotic active components in Bupeuri radix, a well-known Traditional Chinese Medicine (TCM). The hypothesis suggested that when cells are incubated together with the extracts of TCM, the potential bioactive components in the TCM should selectively combine with the receptor or channel of HepG2 cells, then the eluate which contained biospecific component binding to HepG2 cells was identified using HPLC-ESI-MS analysis. The potential bioactive components of Bupeuri radix were investigated using the proposed approach. Five compounds in the saikosaponins of Bupeuri radix were detected as these components selectively combined with HepG2 cells, among these compounds, two potentially bioactive compounds namely saikosaponin b1 and saikosaponin b2 (SSb2) were identified by comparing with the chromatography of the standard sample and analysis of the structural clearance characterization of MS. Then SSb2 was used to assess the uptake of DiI-high density lipoprotein (HDL) in HepG2 cells for antiatherosclerotic activity. The results have showed that SSb2, with indicated concentrations (5, 15, 25, and 40 μM) could remarkably uptake dioctadecylindocarbocyanine labeled- (DiI) -HDL in HepG2 cells (Vs control group, *PESI-MS analysis is a rapid, convenient, and reliable method for screening potential bioactive components in TCM and SSb2 may be a valuable novel drug agent for the treatment of atherosclerosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Serum microRNA miR-206 is decreased in hyperthyroidism and mediates thyroid hormone regulation of lipid metabolism in HepG2 human hepatoblastoma cells.

    Science.gov (United States)

    Zheng, Yingjuan; Zhao, Chao; Zhang, Naijian; Kang, Wenqin; Lu, Rongrong; Wu, Huadong; Geng, Yingxue; Zhao, Yaping; Xu, Xiaoyan

    2018-04-01

    The actions of thyroid hormone (TH) on lipid metabolism in the liver are associated with a number of genes involved in lipogenesis and lipid metabolism; however, the underlying mechanisms through which TH impacts on lipid metabolism remain to be elucidated. The present study aimed to investigate the effects of hyperthyroidism on the serum levels of the microRNA (miR) miR‑206 and the role of miR‑206 on TH‑regulated lipid metabolism in liver cells. Serum was obtained from 12 patients diagnosed with hyperthyroidism and 10 healthy control subjects. Human hepatoblastoma (HepG2) cells were used to study the effects of triiodothyronine (T3) and miR‑206 on lipid metabolism. Expression of miR‑206 in serum and cells was determined by reverse transcription‑quantitative polymerase chain reaction analysis. Lipid accumulation in HepG2 cells was assessed with Oil Red O staining. Suppression or overexpression of miR‑206 was performed via transfection with a miR‑206 mimic or miR‑206 inhibitor. Serum miR‑206 was significantly decreased in patients with hyperthyroidism compared with euthyroid controls. Treatment of HepG2 cells with T3 led to reduced total cholesterol (TC) and triglyceride (TG) content, accompanied by reduced miR‑206 expression. Inhibition of endogenous miR‑206 expression decreased intracellular TG and TC content in HepG2 cells. By contrast, overexpression of miR‑206 in HepG2 partially prevented the reduction in TG content induced by treatment with T3. In conclusion, serum miR‑206 expression is reduced in patients with hyperthyroidism. In addition, miR‑206 is involved in T3‑mediated regulation of lipid metabolism in HepG2 cells, indicating a role for miR‑206 in thyroid hormone‑induced disorders of lipid metabolism in the liver.

  16. Studies on Cytotoxic Activity against HepG-2 Cells of Naphthoquinones from Green Walnut Husks of Juglans mandshurica Maxim.

    Science.gov (United States)

    Zhou, Yuanyuan; Yang, Bingyou; Jiang, Yanqiu; Liu, Zhaoxi; Liu, Yuxin; Wang, Xiaoli; Kuang, Haixue

    2015-08-26

    Twenty-seven naphthoquinones and their derivatives, including four new naphthalenyl glucosides and twenty-three known compounds, were isolated from green walnut husks, which came from Juglans mandshurica Maxim. The structures of four new naphthalenyl glucosides were elucidated based on extensive spectroscopic analyses. All of these compounds were evaluated for their cytotoxic activities against the growth of human cancer cells lines HepG-2 by MTT [3-(4,5-dimethylthiazo l-2-yl)-2,5 diphenyl tetrazolium bromide] assay. The results were shown that most naphthoquinones in an aglycone form exhibited better cytotoxicity in vitro than naphthalenyl glucosides with IC50 values in the range of 7.33-88.23 μM. Meanwhile, preliminary structure-activity relationships for these compounds were discussed.

  17. Hydrogen sulfide (H2S)/cystathionine γ-lyase (CSE) pathway contributes to the proliferation of hepatoma cells

    International Nuclear Information System (INIS)

    Pan, Yan; Ye, Shuang; Yuan, Dexiao; Zhang, Jianghong; Bai, Yang; Shao, Chunlin

    2014-01-01

    Highlights: • Inhibition of H 2 S/CSE pathway strongly stimulates cellular apoptosis. • Inhibition of H 2 S/CSE pathway suppresses cell growth by blocking EGFR pathway. • H 2 S/CSE pathway is critical for maintaining the proliferation of hepatoma cells. - Abstract: Hydrogen sulfide (H 2 S)/cystathionine γ-lyase (CSE) pathway has been demonstrated to play vital roles in physiology and pathophysiology. However, its role in tumor cell proliferation remains largely unclear. Here we found that CSE over-expressed in hepatoma HepG2 and PLC/PRF/5 cells. Inhibition of endogenous H 2 S/CSE pathway drastically decreased the proliferation of HepG2 and PLC/PRF/5 cells, and it also enhanced ROS production and mitochondrial disruption, pronounced DNA damage and increased apoptosis. Moreover, this increase of apoptosis was associated with the activation of p53 and p21 accompanied by a decreased ratio of Bcl-2/Bax and up-regulation of phosphorylated c-Jun N-terminal kinase (JNK) and caspase-3 activity. In addition, the negative regulation of cell proliferation by inhibition of H 2 S/CSE system correlated with the blockage of cell mitogenic and survival signal transduction of epidermal growth factor receptor (EGFR) via down-regulating the extracellular-signal-regulated kinase 1/2 (ERK1/2) activation. These results demonstrate that H 2 S/CSE and its downstream pathway contribute to the proliferation of hepatoma cells, and inhibition of this pathway strongly suppress the excessive growth of hepatoma cells by stimulating mitochondrial apoptosis and suppressing cell growth signal transduction

  18. Protection of betulin against cadmium-induced apoptosis in hepatoma cells

    International Nuclear Information System (INIS)

    Oh, Seon-Hee; Choi, Jeong-Eun; Lim, Sung-Chul

    2006-01-01

    The protective effects of betulin (BT) against cadmium (Cd)-induced cytotoxicity have been previously reported. However, the mechanisms responsible for these protective effects are unclear. Therefore, this study investigated the mechanisms responsible for the protection of BT against Cd-induced cytotoxicity in human hepatoma cell lines. The protection of BT against Cd cytotoxicity was more effective in the HepG2 than in the Hep3B cells. The protection of BT on Cd-induced cytotoxicity in the HepG2 cells appeared to be related to the inhibition of apoptosis, as determined by PI staining and DNA fragmentation analysis. The anti-apoptosis exerted by BT involved the blocking of Cd-induced reactive oxygen species (ROS) generation, the abrogation of the Cd-induced Fas upregulation, the blocking of caspase-8-dependent Bid activation, and subsequent inhibition of mitochondrial pathway. The BT pretreatment did not affect the p21 and p53 expression levels, when compared with those of the treated cells with Cd alone. BT induced the transient S phase arrest at an early stage and the G /G 1 arrest at a relatively late stage, but it did not observe the sub-G1 apoptotic peak. In the Hep3B cells, Cd did not induce ROS generation. The BT pretreatment partially inhibited the Cd-induced apoptosis, which was related with the incomplete blockage in caspase-9 or -3 activation, as well as in Bax activation. Taken together, it was found that Cd can induce apoptosis via the Fas-dependent and -independent apoptosis pathways. However, the observed protective effects of BT were clearly more sensitive to Fas-expressing HepG2 cells than to Fas-deficient Hep3B cells

  19. Multifunctional selenium nanoparticles as carriers of HSP70 siRNA to induce apoptosis of HepG2 cells

    Directory of Open Access Journals (Sweden)

    Li Y

    2016-07-01

    Full Text Available Yinghua Li,1 Zhengfang Lin,1 Mingqi Zhao,1 Tiantian Xu,1 Changbing Wang,1 Huimin Xia,1,* Hanzhong Wang,2,* Bing Zhu1,* 1Guangzhou Women and Children’s Medical Center, Guangzhou, Guangdong, 2State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People’s Republic of China *These authors contributed equally to this work Abstract: Small interfering RNA (siRNA as a new therapeutic modality holds promise for cancer treatment, but it is unable to cross cell membrane. To overcome this limitation, nanotechnology has been proposed for mediation of siRNA transfection. Selenium (Se is a vital dietary trace element for mammalian life and plays an essential role in the growth and functioning of humans. As a novel Se species, Se nanoparticles have attracted more and more attention for their higher anticancer efficacy. In the present study, siRNAs with polyethylenimine (PEI-modified Se nanoparticles (Se@PEI@siRNA have been demonstrated to enhance the apoptosis of HepG2 cells. Heat shock protein (HSP-70 is overexpressed in many types of human cancer and plays a significant role in several biological processes including the regulation of apoptosis. The objective of this study was to silence inducible HSP70 and promote the apoptosis of Se-induced HepG2 cells. Se@PEI@siRNA were successfully prepared and characterized by various microscopic methods. Se@PEI@siRNA showed satisfactory size distribution, high stability, and selectivity between cancer and normal cells. The cytotoxicity of Se@PEI@siRNA was lower for normal cells than tumor cells, indicating that these compounds may have fewer side effects. The gene-silencing efficiency of Se@PEI@siRNA was significantly much higher than Lipofectamine 2000@siRNA and resulted in a significantly reduced HSP70 mRNA and protein expression in cancer cells. When the expression of HSP70 was diminished, the function of cell protection was also removed and cancer cells became more

  20. Prostaglandin (PG) synthesis by hepatoma cells

    International Nuclear Information System (INIS)

    Cyran, J.; Lysz, T.W.; Lea, M.A.

    1987-01-01

    Proliferation of cultured HTC hepatoma cells was reported to be inhibited by indomethacin but synthesis of PG in these cells was no detected. The authors have found that omission of fetal calf serum from the medium permits detection of synthesis of 6-keto-PGF1 alpha, PFG2 alpha, PGE2 and TxB2 from labeled arachidonic acid. Two additional peaks were identified as metabolites of PGF2 alpha and PGE2 by retention times on HPLC. Indomethacin inhibited the formation of the PGs and the metabolites. When 3 H-PGE2 and 3 H-PGF2 alpha were added to the cultures, approximately 50% of the label was recovered as the PG metabolites after a 4 day incubation. Metabolism of 3 H-TxB2 was not detected. When HTC cells were grown in the presence of 100 μM flurbiprofen, a cyclooxygenase inhibitor, there was significant inhibition of both cell proliferation and 3 H-thymidine uptake. The authors data suggest that proliferation of hepatoma cells is facilitated by synthesis of PGs

  1. Portulaca oleracea Seed Oil Exerts Cytotoxic Effects on Human Liver Cancer (HepG2) and Human Lung Cancer (A-549) Cell Lines.

    Science.gov (United States)

    Al-Sheddi, Ebtesam Saad; Farshori, Nida Nayyar; Al-Oqail, Mai Mohammad; Musarrat, Javed; Al-Khedhairy, Abdulaziz Ali; Siddiqui, Maqsood Ahmed

    2015-01-01

    Portulaca oleracea (Family: Portulacaceae), is well known for its anti-inflammatory, antioxidative, anti- bacterial, and anti-tumor activities. However, cytotoxic effects of seed oil of Portulaca oleracea against human liver cancer (HepG2) and human lung cancer (A-549) cell lines have not been studied previously. Therefore, the present study was designed to investigate the cytotoxic effects of Portulaca oleracea seed oil on HepG2 and A-549 cell lines. Both cell lines were exposed to various concentrations of Portulaca oleracea seed oil for 24h. After the exposure, percentage cell viability was studied by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), neutral red uptake (NRU) assays, and cellular morphology by phase contrast inverted microscopy. The results showed a concentration-dependent significant reduction in the percentage cell viability and an alteration in the cellular morphology of HepG2 and A-549 cells. The percentage cell viability was recorded as 73%, 63%, and 54% by MTT assay and 76%, 61%, and 50% by NRU assay at 250, 500, and 1000 μg/ml, respectively in HepG2 cells. Percentage cell viability was recorded as 82%, 72%, and 64% by MTT assay and 83%, 68%, and 56% by NRU assay at 250, 500, and 1000 μg/ml, respectively in A-549 cells. The 100 μg/ml and lower concentrations were found to be non cytotoxic to A-549 cells, whereas decrease of 14% and 12% were recorded by MTT and NRU assay, respectively in HepG2 cells. Both HepG2 and A-549 cell lines exposed to 250, 500, and 1000 μg/ ml of Portulaca oleracea seed oil lost their normal morphology, cell adhesion capacity, become rounded, and appeared smaller in size. The data from this study showed that exposure to seed oil of Portulaca oleracea resulted in significant cytotoxicity and inhibition of growth of the human liver cancer (HepG2) and human lung cancer (A-549) cell lines.

  2. GENE EXPRESSION PROFILING OF HUMAN LIVER CARCINOMA (HepG2) CELLS EXPOSED TO THE MARINE TOXIN OKADAIC ACID

    Science.gov (United States)

    Fieber, Lynne A.; Greer, Justin B.; Guo, Fujiang; Crawford, Douglas C.; Rein, Kathleen S.

    2012-01-01

    The marine toxin, okadaic acid (OA) is produced by dinoflagellates of the genera Prorocentrum and Dinophysis and is the causative agent of the syndrome known as diarrheic shellfish poisoning (DSP). In addition, OA acts as both a tumor promoter, attributed to OA-induced inhibition of protein phosphatases as well as an inducer of apoptosis. To better understand the potentially divergent toxicological profile of OA, the concentration dependent cytotoxicity and alterations in gene expression on the human liver tumor cell line HepG2 upon OA exposure were determined using RNA microarrays, DNA fragmentation, and cell proliferation assays as well as determinations of cell detachment and cell death in different concentrations of OA. mRNA expression was quantified for approximately 15,000 genes. Cell attachment and proliferation were both negatively correlated with OA concentration. Detached cells displayed necrotic DNA signatures but apoptosis also was broadly observed. Data suggest that OA has a concentration dependent effect on cell cycle, which might explain the divergent effects that at low concentration OA stimulates genes involved in the cell cycle and at high concentrations it stimulates apoptosis. PMID:23172983

  3. Epoxy Stearic Acid, an Oxidative Product Derived from Oleic Acid, Induces Cytotoxicity, Oxidative Stress, and Apoptosis in HepG2 Cells.

    Science.gov (United States)

    Liu, Ying; Cheng, Yajun; Li, Jinwei; Wang, Yuanpeng; Liu, Yuanfa

    2018-05-23

    In the present study, effects of cis-9,10-epoxy stearic acid (ESA) generated by the thermal oxidation of oleic acid on HepG2 cells, including cytotoxicity, apoptosis, and oxidative stress, were investigated. Our results revealed that ESA decreased the cell viability and induced cell death. Cell cycle analysis with propidium iodide staining showed that ESA induced cell cycle arrest at the G0/G1 phase in HepG2 cells. Cell apoptosis analysis with annexin V and propidium iodide staining demonstrated that ESA induced HepG2 cell apoptotic events in a dose- and time-dependent manner; the apoptosis of cells after treated with 500 μM ESA for 12, 24, and 48 h was 32.16, 38.70, and 65.80%, respectively. Furthermore, ESA treatment to HepG2 cells resulted in an increase in reactive oxygen species and malondialdehyde (from 0.84 ± 0.02 to 8.90 ± 0.50 nmol/mg of protein) levels and a reduction in antioxidant enzyme activity, including superoxide dismutase (from 1.34 ± 0.27 to 0.10 ± 0.007 units/mg of protein), catalase (from 100.04 ± 5.05 to 20.09 ± 3.00 units/mg of protein), and glutathione peroxidase (from 120.44 ± 7.62 to 35.84 ± 5.99 milliunits/mg of protein). These findings provide critical information on the effects of ESA on HepG2 cells, particularly cytotoxicity and oxidative stress, which is important for the evaluation of the biosafety of the oxidative product of oleic acid.

  4. Existence of B/E and E receptors on Hep-G2 cells: a study using colloidal gold- and 125I-labeled lipoproteins

    International Nuclear Information System (INIS)

    Hesz, A.; Ingolic, E.; Krempler, F.; Kostner, G.M.

    1987-01-01

    The presence of specific receptors for apolipoprotein B (low-density lipoproteins) and apolipoprotein E (HDL-E) on Hep-G2 cells and human skin fibroblasts was studied by chemical methods and by electron microscopy using a differential gold labeling technique. Fibroblasts bound both types of lipoproteins to one and the same receptor (B/E receptor) as deduced from competition experiments with HDL-E and LDL. Labeled HDL-E, on the other hand, was only partially displaced by cold LDL but was completely displaced by unlabeled HDL-E. Scatchard analysis of lipoprotein binding to Hep-G2 cells revealed an approx 10 times higher binding affinity of apoE-containing lipoproteins as compared to apoB-containing ones. No differences between apoE- or apoB-containing lipoproteins with respect to the morphology of cell binding and intracellular processing were observed. The results are compatible with the concept that Hep-G2 cells possess two kinds of receptors, one specific for apoB- and apoE-containing lipoproteins (B/E receptor) and another specific for apoE only. From these studies we conclude that Hep-G2 cells may serve as a suitable model for studying the lipoprotein metabolism in the liver

  5. Gelsolin negatively regulates the activity of tumor suppressor p53 through their physical interaction in hepatocarcinoma HepG2 cells

    International Nuclear Information System (INIS)

    An, Joo-Hee; Kim, Jung-Woong; Jang, Sang-Min; Kim, Chul-Hong; Kang, Eun-Jin; Choi, Kyung-Hee

    2011-01-01

    Highlights: → The actin binding protein Gelsolin (GSN) interacts with transcription factor p53. → GSN interacts with transactivation- and DNA binding domains of p53. → GSN represses transactivity of p53 via inhibition of nuclear translocation of p53. → GSN inhibits the p53-mediated apoptosis in hepatocarcinoma HepG2 cells. -- Abstract: As a transcription factor, p53 modulates several cellular responses including cell-cycle control, apoptosis, and differentiation. In this study, we have shown that an actin regulatory protein, gelsolin (GSN), can physically interact with p53. The nuclear localization of p53 is inhibited by GSN overexpression in hepatocarcinoma HepG2 cells. Additionally, we demonstrate that GSN negatively regulates p53-dependent transcriptional activity of a reporter construct, driven by the p21-promoter. Furthermore, p53-mediated apoptosis was repressed in GSN-transfected HepG2 cells. Taken together, these results suggest that GSN binds to p53 and this interaction leads to the inhibition of p53-induced apoptosis by anchoring of p53 in the cytoplasm in HepG2 cells.

  6. Statins Prevent Dextrose-Induced Endoplasmic Reticulum Stress and Oxidative Stress in Endothelial and HepG2 Cells.

    Science.gov (United States)

    Kojanian, Hagop; Szafran-Swietlik, Anna; Onstead-Haas, Luisa M; Haas, Michael J; Mooradian, Arshag D

    Statins have favorable effects on endothelial function partly because of their capacity to reduce oxidative stress. However, antioxidant vitamins, unlike statins, are not as cardioprotective, and this paradox has been explained by failure of vitamin antioxidants to ameliorate endoplasmic reticulum (ER) stress. To determine whether statins prevent dextrose-induced ER stress in addition to their antioxidative effects, human umbilical vein endothelial cells and HepG2 hepatocytes were treated with 27.5 mM dextrose in the presence of simvastatin (lipophilic statin that is a prodrug) and pravastatin (water-soluble active drug), and oxidative stress, ER stress, and cell death were measured. Superoxide generation was measured using 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride. ER stress was measured using the placental alkaline phosphatase assay and Western blot of glucose-regulated protein 75, c-jun-N-terminal kinase, phospho-JNK, eukaryotic initiating factor 2α and phospho-eIF2α, and X-box binding protein 1 mRNA splicing. Cell viability was measured by propidium iodide staining. Superoxide anion production, ER stress, and cell death induced by 27.5 mM dextrose were inhibited by therapeutic concentrations of simvastatin and pravastatin. The salutary effects of statins on endothelial cells in reducing both ER stress and oxidative stress observed with pravastatin and the prodrug simvastatin suggest that the effects may be independent of cholesterol-lowering activity.

  7. Melittin restores PTEN expression by down-regulating HDAC2 in human hepatocelluar carcinoma HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Hui Zhang

    Full Text Available Melittin is a water-soluble toxic peptide derived from the venom of the bee. Although many studies show the anti-tumor activity of melittin in human cancer including glioma cells, the underlying mechanisms remain elusive. Here the effect of melittin on human hepatocelluar carcinoma HepG2 cell proliferation in vitro and further mechanisms was investigated. We found melittin could inhibit cell proliferation in vitro using Flow cytometry and MTT method. Besides, we discovered that melittin significantly downregulated the expressions of CyclinD1 and CDK4. Results of western Blot and Real-time PCR analysis indicated that melittin was capable to upregulate the expression of PTEN and attenuate histone deacetylase 2 (HDAC2 expression. Further studies demonstrated that knockdown of HDAC2 completely mimicked the effects of melittin on PTEN gene expression. Conversely, it was that the potential utility of melittin on PTEN expression was reversed in cells treated with a recombinant pEGFP-C2-HDAC2 plasmid. In addition, treatment with melittin caused a downregulation of Akt phosphorylation, while overexpression of HDAC2 promoted Akt phosphorylation. These findings suggested that the inhibitory of cell growth by melittin might be led by HDAC2-mediated PTEN upregulation, Akt inactivation, and inhibition of the PI3K/Akt signaling pathways.

  8. Metabolomic effects of CeO2, SiO2 and CuO metal oxide nanomaterials on HepG2 cells

    Data.gov (United States)

    U.S. Environmental Protection Agency — The data set is a matrix of cellular biochemical (metabolites) in HepG2 cells treated with various metal oxide nanomaterials composed of CeO2, SiO2 and CuO. This...

  9. TRAF1 knockdown alleviates palmitate-induced insulin resistance in HepG2 cells through NF-κB pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wanlu [Department of Pathogen Biology, Medical College, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu Province (China); Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu Province (China); Tang, Zhuqi; Zhu, Xiaohui [Department of Endocrinology, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu Province (China); Xia, Nana; Zhao, Yun; Wang, Suxin [Department of Pathogen Biology, Medical College, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu Province (China); Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu Province (China); Cui, Shiwei, E-mail: neifenmicui@163.com [Department of Endocrinology, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu Province (China); Wang, Cuifang, E-mail: binghuodinghuo@163.com [Department of Endocrinology, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu Province (China)

    2015-11-20

    High-fat diet (HFD) and inflammation are key contributors to insulin resistance (IR) and Type 2 diabetes mellitus (T2DM). With HFD, plasma free fatty acids (FFAs) can activate the nuclear factor-κB (NF-κB) in target tissues, then initiate negative crosstalk between FFAs and insulin signaling. However, the molecular link between IR and inflammation remains to be identified. We here reported that tumor necrosis factor receptor-associated factor 1 (TRAF1), an adapter in signal transduction, was involved in the onset of IR in hepatocytes. TRAF1 was significantly up-regulated in insulin-resistant liver tissues and palmitate (PA)-treated HepG2 cells. In addition, we showed that depletion of TRAF1 led to inhibition of the activity of NF-κB. Given the fact that the activation of NF-κB played a facilitating role in IR, the phosphorylation of Akt and GSK3β was also analyzed. We found that depletion of TRAF1 markedly reversed PA-induced attenuation of the phosphorylation of Akt and GSK3β in the cells. The accumulation of lipid droplets in hepatocyte and expression of two key gluconeogenic enzymes, PEPCK and G6Pase, were also determined and found to display a similar tendency with the phosphorylation of Akt and GSK3β. Glucose uptake assay indicated that knocking down TRAF1 blocked the effect of PA on the suppression of glucose uptake. These data implicated that TRAF1 knockdown might alleviate PA-induced IR in HepG2 cells through NF-κB pathway. - Highlights: • TRAF1 accelerated PA-induced IR in HepG2 cells mediated through NF-κB signaling. • Knockdown of TRAF1 alleviated PA-induced IR in HepG2 cells. • Knockdown of TRAF1 alleviated PA-induced lipid accumulation in HepG2 cells. • Knockdown of TRAF1 reversed PA-induced suppression of glucose uptake in HepG2 cells. • Knockdown of TRAF1 reversed PA-induced gluconeogenesis in HepG2 cells.

  10. TRAF1 knockdown alleviates palmitate-induced insulin resistance in HepG2 cells through NF-κB pathway

    International Nuclear Information System (INIS)

    Zhang, Wanlu; Tang, Zhuqi; Zhu, Xiaohui; Xia, Nana; Zhao, Yun; Wang, Suxin; Cui, Shiwei; Wang, Cuifang

    2015-01-01

    High-fat diet (HFD) and inflammation are key contributors to insulin resistance (IR) and Type 2 diabetes mellitus (T2DM). With HFD, plasma free fatty acids (FFAs) can activate the nuclear factor-κB (NF-κB) in target tissues, then initiate negative crosstalk between FFAs and insulin signaling. However, the molecular link between IR and inflammation remains to be identified. We here reported that tumor necrosis factor receptor-associated factor 1 (TRAF1), an adapter in signal transduction, was involved in the onset of IR in hepatocytes. TRAF1 was significantly up-regulated in insulin-resistant liver tissues and palmitate (PA)-treated HepG2 cells. In addition, we showed that depletion of TRAF1 led to inhibition of the activity of NF-κB. Given the fact that the activation of NF-κB played a facilitating role in IR, the phosphorylation of Akt and GSK3β was also analyzed. We found that depletion of TRAF1 markedly reversed PA-induced attenuation of the phosphorylation of Akt and GSK3β in the cells. The accumulation of lipid droplets in hepatocyte and expression of two key gluconeogenic enzymes, PEPCK and G6Pase, were also determined and found to display a similar tendency with the phosphorylation of Akt and GSK3β. Glucose uptake assay indicated that knocking down TRAF1 blocked the effect of PA on the suppression of glucose uptake. These data implicated that TRAF1 knockdown might alleviate PA-induced IR in HepG2 cells through NF-κB pathway. - Highlights: • TRAF1 accelerated PA-induced IR in HepG2 cells mediated through NF-κB signaling. • Knockdown of TRAF1 alleviated PA-induced IR in HepG2 cells. • Knockdown of TRAF1 alleviated PA-induced lipid accumulation in HepG2 cells. • Knockdown of TRAF1 reversed PA-induced suppression of glucose uptake in HepG2 cells. • Knockdown of TRAF1 reversed PA-induced gluconeogenesis in HepG2 cells.

  11. STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma membrane, ER, and ERC.

    Science.gov (United States)

    Garbarino, Jeanne; Pan, Meihui; Chin, Harvey F; Lund, Frederik W; Maxfield, Frederick R; Breslow, Jan L

    2012-12-01

    STARD4, a member of the evolutionarily conserved START gene family, has been implicated in the nonvesicular intracellular transport of cholesterol. However, the direction of transport and the membranes with which this protein interacts are not clear. We present studies of STARD4 function using small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT synthesized cholesteryl esters. Furthermore, D4 KD cells exhibited a reduced rate of sterol transport to the endocytic recycling compartment after cholesterol repletion. Although these cells displayed normal endocytic trafficking in cholesterol-poor and replete conditions, cell surface low density lipoprotein receptor (LDLR) levels were increased and decreased, respectively. We also observed a decrease in NPC1 protein expression, suggesting the induction of compensatory pathways to maintain cholesterol balance. These data indicate a role for STARD4 in nonvesicular transport of cholesterol from the plasma membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well.

  12. Synergy analysis reveals association between insulin signaling and desmoplakin expression in palmitate treated HepG2 cells.

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    Xuewei Wang

    Full Text Available The regulation of complex cellular activities in palmitate treated HepG2 cells, and the ensuing cytotoxic phenotype, involves cooperative interactions between genes. While previous approaches have largely focused on identifying individual target genes, elucidating interacting genes has thus far remained elusive. We applied the concept of information synergy to reconstruct a "gene-cooperativity" network for palmititate-induced cytotoxicity in liver cells. Our approach integrated gene expression data with metabolic profiles to select a subset of genes for network reconstruction. Subsequent analysis of the network revealed insulin signaling as the most significantly enriched pathway, and desmoplakin (DSP as its top neighbor. We determined that palmitate significantly reduces DSP expression, and treatment with insulin restores the lost expression of DSP. Insulin resistance is a common pathological feature of fatty liver and related ailments, whereas loss of DSP has been noted in liver carcinoma. Reduced DSP expression can lead to loss of cell-cell adhesion via desmosomes, and disrupt the keratin intermediate filament network. Our findings suggest that DSP expression may be perturbed by palmitate and, along with insulin resistance, may play a role in palmitate induced cytotoxicity, and serve as potential targets for further studies on non-alcoholic fatty liver disease (NAFLD.

  13. Galangin suppresses HepG2 cell proliferation by activating the TGF-β receptor/Smad pathway

    International Nuclear Information System (INIS)

    Wang, Yajun; Wu, Jun; Lin, Biyun; Li, Xv; Zhang, Haitao; Ding, Hang; Chen, Xiaoyi; Lan, Liubo; Luo, Hui

    2014-01-01

    Galangin can suppress hepatocellular carcinoma (HCC) cell proliferation. In this study, we demonstrated that galangin induced autophagy by activating the transforming growth factor (TGF)-β receptor/Smad pathway and increased TGF-β receptor I (RI), TGF-βRII, Smad1, Smad2, Smad3 and Smad4 levels but decreased Smad6 and Smad7 levels. Autophagy induced by galangin appears to depend on the TGF-β receptor/Smad signalling pathway because the down-regulation of Smad4 by siRNA or inhibition of TGF-β receptor activation by LY2109761 blocked galangin-induced autophagy. The down-regulation of Beclin1, autophagy-related gene (ATG) 16L, ATG12 and ATG3 restored HepG2 cell proliferation and prevented galangin-induced apoptosis. Our findings indicate a novel mechanism for galangin-induced autophagy via activation of the TGF-β receptor/Smad pathway. The induction of autophagy thus reflects the anti-proliferation effect of galangin on HCC cells

  14. Antagonism of Secreted PCSK9 Increases Low Density Lipoprotein Receptor Expression in HepG2 Cells

    Energy Technology Data Exchange (ETDEWEB)

    McNutt, Markey C.; Kwon, Hyock Joo; Chen, Chiyuan; Chen, Justin R.; Horton, Jay D.; Lagace, Thomas A.; (USMC); (UTSMC)

    2009-07-10

    PCSK9 is a secreted protein that degrades low density lipoprotein receptors (LDLRs) in liver by binding to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. It is not known whether PCSK9 causes degradation of LDLRs within the secretory pathway or following secretion and reuptake via endocytosis. Here we show that a mutation in the LDLR EGF-A domain associated with familial hypercholesterolemia, H306Y, results in increased sensitivity to exogenous PCSK9-mediated cellular degradation because of enhanced PCSK9 binding affinity. The crystal structure of the PCSK9-EGF-A(H306Y) complex shows that Tyr-306 forms a hydrogen bond with Asp-374 in PCSK9 at neutral pH, which strengthens the interaction with PCSK9. To block secreted PCSK9 activity, LDLR (H306Y) subfragments were added to the medium of HepG2 cells stably overexpressing wild-type PCSK9 or gain-of-function PCSK9 mutants associated with hypercholesterolemia (D374Y or S127R). These subfragments blocked secreted PCSK9 binding to cell surface LDLRs and resulted in the recovery of LDLR levels to those of control cells. We conclude that PCSK9 acts primarily as a secreted factor to cause LDLR degradation. These studies support the concept that pharmacological inhibition of the PCSK9-LDLR interaction extracellularly will increase hepatic LDLR expression and lower plasma low density lipoprotein levels.

  15. Preparation and Optimization Lipid Nanocapsules to Enhance the Antitumor Efficacy of Cisplatin in Hepatocellular Carcinoma HepG2 Cells.

    Science.gov (United States)

    Zhai, Qingqing; Li, Hailong; Song, Yanlin; Wu, Ruijiao; Tang, Chuanfang; Ma, Xiaodong; Liu, Zhihao; Peng, Jinyong; Zhang, Jianbin; Tang, Zeyao

    2018-04-20

    This work aimed to develop and optimize several lipid nanocapsule formulations (LNCs) to encapsulate cisplatin (CDDP) for treatment of hepatocellular carcinoma. By comparing the effect of oil/surfactant ratio, lecithin content, and oil/surfactant type on LNC characteristics, two LNCs were selected as optimal formulations: HS15-LNC (Solutol HS 15/MCT/lecithin, 54.5:42.5:3%, w/w) and EL-LNC (Cremophor EL/MCT/lecithin, 54.5:42.5:3%, w/w). Both LNCs could effectively encapsulate CDDP with the encapsulation efficiency of 73.48 and 78.84%. In vitro release study showed that both LNCs could sustain the release CDDP. Moreover, cellular uptake study showed that C6-labeled LNCs could be effectively internalized by HepG2 cells. Cellular cytotoxicity study revealed that both LNCs showed negligible cellular toxicity when their concentrations were below 313 μg/mL. Importantly, CDDP-loaded LNCs exhibited much stronger cell killing potency than free CDDP, with the IC50 values decreased from 17.93 to 3.53 and 5.16 μM after 72-h incubation. In addition, flow cytometric analysis showed that the percentage of apoptotic cells was significantly increased after treatment with LNCs. Therefore, the prepared LNC formulations exhibited promising anti-hepatocarcinoma effect, which could be beneficial to hepatocellular carcinoma therapy.

  16. Generation of Multilayered 3D Structures of HepG2 Cells Using a Bio-printing Technique.

    Science.gov (United States)

    Jeon, Hyeryeon; Kang, Kyojin; Park, Su A; Kim, Wan Doo; Paik, Seung Sam; Lee, Sang-Hun; Jeong, Jaemin; Choi, Dongho

    2017-01-15

    Chronic liver disease is a major widespread cause of death, and whole liver transplantation is the only definitive treatment for patients with end-stage liver diseases. However, many problems, including donor shortage, surgical complications and cost, hinder their usage. Recently, tissue-engineering technology provided a potential breakthrough for solving these problems. Three-dimensional (3D) printing technology has been used to mimic tissues and organs suitable for transplantation, but applications for the liver have been rare. A 3D bioprinting system was used to construct 3D printed hepatic structures using alginate. HepG2 cells were cultured on these 3D structures for 3 weeks and examined by fluorescence microscopy, histology and immunohistochemistry. The expression of liverspecific markers was quantified on days 1, 7, 14, and 21. The cells grew well on the alginate scaffold, and liver-specific gene expression increased. The cells grew more extensively in 3D culture than two-dimensional culture and exhibited better structural aspects of the liver, indicating that the 3D bioprinting method recapitulates the liver architecture. The 3D bioprinting of hepatic structures appears feasible. This technology may become a major tool and provide a bridge between basic science and the clinical challenges for regenerative medicine of the liver.

  17. Cholesterol lowering effects of mono-lactose-appended β-cyclodextrin in Niemann–Pick type C disease-like HepG2 cells

    Directory of Open Access Journals (Sweden)

    Keiichi Motoyama

    2015-11-01

    Full Text Available The Niemann–Pick type C disease (NPC is one of inherited lysosomal storage disorders, emerges the accumulation of unesterified cholesterol in endolysosomes. Currently, 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD has been applied for the treatment of NPC. HP-β-CyD improved hepatosplenomegaly in NPC patients, however, a high dose of HP-β-CyD was necessary. Therefore, the decrease in dose by actively targeted-β-CyD to hepatocytes is expected. In the present study, to deliver β-CyD selectively to hepatocytes, we newly fabricated mono-lactose-appended β-CyD (Lac-β-CyD and evaluated its cholesterol lowering effects in NPC-like HepG2 cells, cholesterol accumulated HepG2 cells induced by treatment with U18666A. Lac-β-CyD (degree of substitution of lactose (DSL 1 significantly decreased the intracellular cholesterol content in a concentration-dependent manner. TRITC-Lac-β-CyD was associated with NPC-like HepG2 cells higher than TRITC-β-CyD. In addition, TRITC-Lac-β-CyD was partially localized with endolysosomes after endocytosis. Thus, Lac-β-CyD entered NPC-like HepG2 cells via asialoglycoprotein receptor (ASGPR-mediated endocytosis and decreased the accumulation of intracellular cholesterol in NPC-like HepG2 cells. These results suggest that Lac-β-CyD may have the potential as a drug for the treatment of hepatosplenomegaly in NPC disease.

  18. Saponins, especially platycodin D, from Platycodon grandiflorum modulate hepatic lipogenesis in high-fat diet-fed rats and high glucose-exposed HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Yong Pil [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Department of Pharmaceutical Engineering, International University of Korea, Jinju (Korea, Republic of); Choi, Jae Ho; Kim, Hyung Gyun; Khanal, Tilak; Song, Gye Young [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Nam, Myoung Soo [College of Agriculture and Life Sciences, Chungnam National University, Daejeon (Korea, Republic of); Lee, Hyun-Sun [Molecular Cancer Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Chung, Young Chul; Lee, Young Chun [Division of Food Science, International University of Korea, Jinju (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon 305-764 (Korea, Republic of)

    2013-03-01

    AMP-activated protein kinase (AMPK) plays a central role in controlling hepatic lipid metabolism through modulating the downstream acetyl CoA carboxylase (ACC) and sterol regulatory element-binding protein-1c (SREBP-1c) pathway. Saponins, particularly platycodin D, from the roots of Platycodon grandiflorum (Changkil saponins, CKS) have a variety of pharmacological properties, including antioxidant and hepatoprotective properties. The aim of this study was to investigate the effects of CKS on hepatic lipogenesis and on the expression of genes involved in lipogenesis, and the mechanisms involved. CKS attenuated fat accumulation and the induction of the lipogenic genes encoding SREBP-1c and fatty acid synthase in the livers of HFD-fed rats and in steatotic HepG2 cells. Blood biochemical analyses and histopathological examinations showed that CKS prevented liver injury. CKS and platycodin D each increased the phosphorylation of AMPK and acetyl-CoA carboxylase in HFD-fed rats and HepG2 cells. The use of specific inhibitors showed that platycodin D activated AMPK via SIRT1/CaMKKβ in HepG2 cells. This study demonstrates that CKS or platycodin D alone can regulate hepatic lipogenesis via an AMPK-dependent signalling pathway. - Highlights: ► CKS attenuated fat accumulation in HFD-fed rats and in steatotic HepG2 cells. ► CKS and its major component, platycodin D, inhibited the levels of SREBP-1 and FAS. ► CKS and platycodin D increased the phosphorylation of AMPK and ACC. ► Platycodin D activated AMPK via SIRT1/CaMKKβ in HepG2 cells.

  19. An earthworm protease cleaving serum fibronectin and decreasing HBeAg in HepG2.2.15 cells

    Directory of Open Access Journals (Sweden)

    Zhao Jing

    2008-11-01

    Full Text Available Abstract Background Virus-binding activity is one of the important functions of fibronectin (FN. It has been reported that a high concentration of FN in blood improves the transmission frequency of hepatitis viruses. Therefore, to investigate a protease that hydrolyzes FN rapidly is useful to decrease the FN concentration in blood and HBV infection. So far, however, no specific protease digesting FN in serum has been reported. Methods We employed a purified earthworm protease to digest serum proteins. The rapidly cleaved protein (FN was identified by MALDI-TOF MS and western blotting. The cleavage sites were determined by N-terminus amino acid residues sequencing. The protease was orally administrated to rats to investigate whether serum FN in vivo became decreased. The serum FN was determined by western blotting and ELISA. In cytological studies, the protease was added to the medium in the culture of HepG2.2.15 cells and then HBsAg and HBeAg were determined by ELISA. Results The protease purified from earthworm Eisenia fetida was found to function as a fibronectinase (FNase. The cleavage sites on FN by the FNase were at R and K, exhibiting a trypsin alkaline serine-like function. The earthworm fibronectinase (EFNase cleaved FN at four sites, R259, R1005, K1557 and R2039, among which the digested fragments at R259, K1557 and R2039 were related to the virus-binding activity as reported. The serum FN was significantly decreased when the earthworm fibronectinase was orally administrated to rats. The ELISA results showed that the secretion of HBeAg from HepG2.2.15 cells was significantly inhibited in the presence of the FNase. Conclusion The earthworm fibronectinase (EFNase cleaves FN much faster than the other proteins in serum, showing a potential to inhibit HBV infection through its suppressing the level of HBeAg. This suggests that EFNase is probably used as one of the candidates for the therapeutic agents to treat hepatitis virus infection.

  20. Zinc protects HepG2 cells against the oxidative damage and DNA damage induced by ochratoxin A

    International Nuclear Information System (INIS)

    Zheng, Juanjuan; Zhang, Yu; Xu, Wentao; Luo, YunBo; Hao, Junran; Shen, Xiao Li; Yang, Xuan; Li, Xiaohong; Huang, Kunlun

    2013-01-01

    Oxidative stress and DNA damage are the most studied mechanisms by which ochratoxin A (OTA) induces its toxic effects, which include nephrotoxicity, hepatotoxicity, immunotoxicity and genotoxicity. Zinc, which is an essential trace element, is considered a potential antioxidant. The aim of this paper was to investigate whether zinc supplement could inhibit OTA-induced oxidative damage and DNA damage in HepG2 cells and the mechanism of inhibition. The results indicated that that exposure of OTA decreased the intracellular zinc concentration; zinc supplement significantly reduced the OTA-induced production of reactive oxygen species (ROS) and decrease in superoxide dismutase (SOD) activity but did not affect the OTA-induced decrease in the mitochondrial membrane potential (Δψ m ). Meanwhile, the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly aggravated the OTA-induced oxidative damage. This study also demonstrated that zinc helped to maintain the integrity of DNA through the reduction of OTA-induced DNA strand breaks, 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation and DNA hypomethylation. OTA increased the mRNA expression of metallothionein1-A (MT1A), metallothionein2-A (MT2A) and Cu/Zn superoxide dismutase (SOD1). Zinc supplement further enhanced the mRNA expression of MT1A and MT2A, but it had no effect on the mRNA expression of SOD1 and catalase (CAT). Zinc was for the first time proven to reduce the cytotoxicity of OTA through inhibiting the oxidative damage and DNA damage, and regulating the expression of zinc-associated genes. Thus, the addition of zinc can potentially be used to reduce the OTA toxicity of contaminated feeds. - Highlights: ► OTA decreased the intracellular zinc concentration. ► OTA induced the formation of 8-OHdG in HepG2 cells. ► It was testified for the first time that OTA induced DNA hypomethylation. ► Zinc protects against the oxidative damage and DNA damage induced by OTA in

  1. Zinc protects HepG2 cells against the oxidative damage and DNA damage induced by ochratoxin A

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Juanjuan; Zhang, Yu [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); Xu, Wentao, E-mail: xuwentaoboy@sina.com [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Luo, YunBo [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Hao, Junran [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); Shen, Xiao Li [The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Yang, Xuan [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); Li, Xiaohong [The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Huang, Kunlun, E-mail: hkl009@163.com [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China)

    2013-04-15

    Oxidative stress and DNA damage are the most studied mechanisms by which ochratoxin A (OTA) induces its toxic effects, which include nephrotoxicity, hepatotoxicity, immunotoxicity and genotoxicity. Zinc, which is an essential trace element, is considered a potential antioxidant. The aim of this paper was to investigate whether zinc supplement could inhibit OTA-induced oxidative damage and DNA damage in HepG2 cells and the mechanism of inhibition. The results indicated that that exposure of OTA decreased the intracellular zinc concentration; zinc supplement significantly reduced the OTA-induced production of reactive oxygen species (ROS) and decrease in superoxide dismutase (SOD) activity but did not affect the OTA-induced decrease in the mitochondrial membrane potential (Δψ{sub m}). Meanwhile, the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly aggravated the OTA-induced oxidative damage. This study also demonstrated that zinc helped to maintain the integrity of DNA through the reduction of OTA-induced DNA strand breaks, 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation and DNA hypomethylation. OTA increased the mRNA expression of metallothionein1-A (MT1A), metallothionein2-A (MT2A) and Cu/Zn superoxide dismutase (SOD1). Zinc supplement further enhanced the mRNA expression of MT1A and MT2A, but it had no effect on the mRNA expression of SOD1 and catalase (CAT). Zinc was for the first time proven to reduce the cytotoxicity of OTA through inhibiting the oxidative damage and DNA damage, and regulating the expression of zinc-associated genes. Thus, the addition of zinc can potentially be used to reduce the OTA toxicity of contaminated feeds. - Highlights: ► OTA decreased the intracellular zinc concentration. ► OTA induced the formation of 8-OHdG in HepG2 cells. ► It was testified for the first time that OTA induced DNA hypomethylation. ► Zinc protects against the oxidative damage and DNA damage induced by

  2. Statins Activate Human PPAR Promoter and Increase PPAR mRNA Expression and Activation in HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Makoto Seo

    2008-01-01

    Full Text Available Statins increase peroxisome proliferator-activated receptor (PPAR mRNA expression, but the mechanism of this increased PPAR production remains elusive. To examine the regulation of PPAR production, we examined the effect of 7 statins (atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin on human PPAR promoter activity, mRNA expression, nuclear protein levels, and transcriptional activity. The main results are as follows. (1 Majority of statins enhanced PPAR promoter activity in a dose-dependent manner in HepG2 cells transfected with the human PPAR promoter. This enhancement may be mediated by statin-induced HNF-4. (2 PPAR mRNA expression was increased by statin treatment. (3 The PPAR levels in nuclear fractions were increased by statin treatment. (4 Simvastatin, pravastatin, and cerivastatin markedly enhanced transcriptional activity in 293T cells cotransfected with acyl-coenzyme A oxidase promoter and PPAR/RXR expression vectors. In summary, these data demonstrate that PPAR production and activation are upregulated through the PPAR promoter activity by statin treatment.

  3. Protective Effect of Curcumin against Ionizing Radiation (IR)-induced Cytotoxicity and Genotoxicity in HepG2 Cells

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Dong Min; Nasir Uddin, S. M.; Ryu, Tae Ho; Kang, Mi Young; Kim, Jin Kyu [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2013-10-15

    Ionizing radiation (IR) has many practical applications such as medicine, foods, agricultures, industries, and research laboratories. However, the increasing use of radiation is associated with radiation accidents threatening human health. It is well known that exposure to IR gives rise to genomic alterations, mutagenesis, and cell death. IR is absorbed directly by DNA, leading to various DNA damages (single or double-strand breaks, base damage, and DNA-DNA or DNA-protein cross-linkages) in many living organisms. Therefore, the development of effective and nontoxic radioprotective agents is of considerable interest. Curcumin (C{sub 12}H{sub 20}O{sub 6}, structure is the major yellow component of Curcuma longa with biological activities (antioxidant, anti-proliferative and anti-inflammatory properties). It has been widely used as food and medicine for a long time. The aim of our present study is to investigate the protective effects of curcumin against IR-induced cytotoxicity and genotoxicity in cultured HepG2 cells.

  4. Behavior of HepG2 liver cancer cells using microfluidic-microscopy: a preliminary study

    Science.gov (United States)

    Karamahmutoglu, Hande; ćetin, Metin; Yaǧcı, Tamer; Elitaş, Meltem

    2018-02-01

    Hepatocellular carcinoma is one of the most common types of liver cancer causing death all over the world. Although early-stage liver cancer can sometimes be treated with partial hepatectomy, liver transplantation, ablation, and embolization, sorafenib treatment is the only approved systemic therapy for advanced HCC. The aim of this research is to develop tools and methods to understand the individuality of hepatocellular carcinoma. Microfluidic cell-culture platform has been developed to observe behavior of single-cells; fluorescence microscopy has been implemented to investigate phenotypic changes of cells. Our preliminary data proved high-level heterogeneity of hepatocellular carcinoma while verifying limited growth of liver cancer cell lines on the silicon wafer.

  5. Signaling dynamics of palmitate-induced ER stress responses mediated by ATF4 in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Cho Hyunju

    2013-01-01

    Full Text Available Abstract Background Palmitic acid, the most common saturated free fatty acid, has been implicated in ER (endoplasmic reticulum stress-mediated apoptosis. This lipoapotosis is dependent, in part, on the upregulation of the activating transcription factor-4 (ATF4. To better understand the mechanisms by which palmitate upregulates the expression level of ATF4, we integrated literature information on palmitate-induced ER stress signaling into a discrete dynamic model. The model provides an in silico framework that enables simulations and predictions. The model predictions were confirmed through further experiments in human hepatocellular carcinoma (HepG2 cells and the results were used to update the model and our current understanding of the signaling induced by palmitate. Results The three key things from the in silico simulation and experimental results are: 1 palmitate induces different signaling pathways (PKR (double-stranded RNA-activated protein kinase, PERK (PKR-like ER kinase, PKA (cyclic AMP (cAMP-dependent protein kinase A in a time dependent-manner, 2 both ATF4 and CREB1 (cAMP-responsive element-binding protein 1 interact with the Atf4 promoter to contribute to a prolonged accumulation of ATF4, and 3 CREB1 is involved in ER-stress induced apoptosis upon palmitate treatment, by regulating ATF4 expression and possibly Ca2+ dependent-CaM (calmodulin signaling pathway. Conclusion The in silico model helped to delineate the essential signaling pathways in palmitate-mediated apoptosis.

  6. Cytotoxicity of Triterpenes from Green Walnut Husks of Juglans mandshurica Maxim in HepG-2 Cancer Cells.

    Science.gov (United States)

    Zhou, Yuanyuan; Yang, Bingyou; Liu, Zhaoxi; Jiang, Yanqiu; Liu, Yuxin; Fu, Lei; Wang, Xiaoli; Kuang, Haixue

    2015-10-22

    Among the classes of identified natural products, triterpenoids, one of the largest families, have been studied extensively for their diverse structures and variety of biological activities, including antitumor effects. In the present study, a phytochemical study of the green walnut husks of Juglans mandshurica Maxim led to the isolation of a new dammarane triterpene, 12β, 20(R), 24(R)-trihydroxydammar-25-en-3-one (6), together with sixteen known compounds, chiefly from chloroform and ethyl acetate extracts. According to their structural characteristics, these compounds were divided into dammarane-type, oleanane- and ursane-type. Dammarane-type triterpenoids were isolated for the first time from the Juglans genus. As part of our continuing search for biologically active compounds from this plant, all of these compounds were also evaluated for their cytotoxic activities against the growth of human cancer cells lines HepG-2 by the MTT assay. The results were shown that 20(S)-protopanaxadiol, 2α,3β,23-trihydroxyolean-12-en-28-oic acid and 2α,3β,23-trihydroxyurs-12-en-28-oic acid exhibited better cytotoxicity in vitro with IC50 values of 10.32±1.13, 16.13±3.83, 15.97±2.47 μM, respectively. Preliminary structure-activity relationships for these compounds were discussed.

  7. Temporal metabolomic responses of cultured HepG2 liver cells to high fructose and high glucose exposures.

    Science.gov (United States)

    Meissen, John K; Hirahatake, Kristin M; Adams, Sean H; Fiehn, Oliver

    2015-06-01

    High fructose consumption has been implicated with deleterious effects on human health, including hyperlipidemia elicited through de novo lipogenesis. However, more global effects of fructose on cellular metabolism have not been elucidated. In order to explore the metabolic impact of fructose-containing nutrients, we applied both GC-TOF and HILIC-QTOF mass spectrometry metabolomic strategies using extracts from cultured HepG2 cells exposed to fructose, glucose, or fructose + glucose. Cellular responses were analyzed in a time-dependent manner, incubated in media containing 5.5 mM glucose + 5.0 mM fructose in comparison to controls incubated in media containing either 5.5 mM glucose or 10.5 mM glucose. Mass spectrometry identified 156 unique known metabolites and a large number of unknown compounds, which revealed metabolite changes due to both utilization of fructose and high-carbohydrate loads independent of hexose structure. Fructose was shown to be partially converted to sorbitol, and generated higher levels of fructose-1-phosphate as a precursor for glycolytic intermediates. Differentially regulated ratios of 3-phosphoglycerate to serine pathway intermediates in high fructose media indicated a diversion of carbon backbones away from energy metabolism. Additionally, high fructose conditions changed levels of complex lipids toward phosphatidylethanolamines. Patterns of acylcarnitines in response to high hexose exposure (10.5 mM glucose or glucose/fructose combination) suggested a reduction in mitochondrial beta-oxidation.

  8. Preliminary screening of some traditional Zulu medicinal plants for antineoplastic activities versus the HepG2 cell line.

    Science.gov (United States)

    Opoku, A R; Geheeb-Keller, M; Lin, J; Terblanche, S E; Hutchings, A; Chuturgoon, A; Pillay, D

    2000-11-01

    Aqueous and methanol extracts of nine traditional Zulu medicinal plants, Cissus quandrangularis L., Cyphostemma flaviflorum (Sprague) Descoings, Cyphostemma lanigerum (Harv.) Descoings ex Wild & Drum, Cyphostemma natalitium (Szyszyl.) J. v. d. Merwe, Cyphostemma sp., Rhoicissus digitata (L. F.) Gilg & Brandt, Rhoicissus rhomboidea (E. Mey. Ex harv.) Planch, Rhoicissus tomentosa (Lam.) Wild & Drum, R. tridentata (L. F.) Wild & Drum and Rhoicissus tridentata (L. F.) Wild & Drum subsp. cuneifolia (Eckl. & Zeyh.) N. R. Urton, all belonging to the Vitaceae family, were evaluated to determine their therapeutic potentials as antineoplastic agents. The antiproliferative activity in vitro against HepG2 cells was determined. Twenty-two of the twenty-seven crude plant extracts showed activities ranging from 25% to 97% inhibition of proliferation when compared with the control which showed no inhibitory activity. Higher degrees of growth inhibition were found in aqueous root extracts in comparison with the methanol extracts of the same plant parts. The results show potential antineoplastic activity, indicating some scientific validation for traditional usage. Copyright 2000 John Wiley & Sons, Ltd.

  9. Reduced mitochondrial coenzyme Q10 levels in HepG2 cells treated with high-dose simvastatin: A possible role in statin-induced hepatotoxicity?

    International Nuclear Information System (INIS)

    Tavintharan, S.; Ong, C.N.; Jeyaseelan, K.; Sivakumar, M.; Lim, S.C.; Sum, C.F.

    2007-01-01

    Lowering of low-density lipoprotein cholesterol is well achieved by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins). Statins inhibit the conversion of HMG-CoA to mevalonate, a precursor for cholesterol and coenzyme Q10 (CoQ 10 ). In HepG2 cells, simvastatin decreased mitochondrial CoQ 10 levels, and at higher concentrations was associated with a moderately higher degree of cell death, increased DNA oxidative damage and a reduction in ATP synthesis. Supplementation of CoQ 10 , reduced cell death and DNA oxidative stress, and increased ATP synthesis. It is suggested that CoQ 10 deficiency plays an important role in statin-induced hepatopathy, and that CoQ 10 supplementation protects HepG2 cells from this complication

  10. Effect of Phenolic Compounds from Elderflowers on Glucose- and Fatty Acid Uptake in Human Myotubes and HepG2-Cells

    Directory of Open Access Journals (Sweden)

    Giang Thanh Thi Ho

    2017-01-01

    Full Text Available Type 2 diabetes (T2D is manifested by progressive metabolic impairments in tissues such as skeletal muscle and liver, and these tissues become less responsive to insulin, leading to hyperglycemia. In the present study, stimulation of glucose and oleic acid uptake by elderflower extracts, constituents and metabolites were tested in vitro using the HepG2 hepatocellular liver carcinoma cell line and human skeletal muscle cells. Among the crude extracts, the 96% EtOH extract showed the highest increase in glucose and oleic acid uptake in human skeletal muscle cells and HepG2-cells. The flavonoids and phenolic acids contained therein were potent stimulators of glucose and fatty acid uptake in a dose-dependent manner. Most of the phenolic constituents and several of the metabolites showed high antioxidant activity and showed considerably higher α-amylase and α-glucosidase inhibition than acarbose. Elderflower might therefore be valuable as a functional food against diabetes.

  11. Effect of Genistein and 17-β Estradiol on the Viability and Apoptosis of Human Hepatocellular Carcinoma HepG2 cell line

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    Masumeh Sanaei

    2017-01-01

    Full Text Available Background: One of the most lethal cancers is hepatocellular carcinoma (HCC. Genistein (GE is a choice compound for treatment of certain types of cancer. Phytoestrogens are plant derivatives that bear a structural similarity to 17-β estradiol (E2 and act in a similar manner. They are a group of lipophillic plant compounds with tumorigenic and antitumorigenic effects. E2 has stimulatory and inhibitory effects on cancer cell lines. This study was designed to investigate the antiproliferative and apoptotic effects of GE and E2 on the HCC HepG2 cell line. Materials and Methods: HepG2 cells were cultured and treated with various concentrations of GE and E2 and then 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromideand flow cytometry assay were performed to determine cell viability and apoptosis. Results: GE and E2 induced apoptosis and inhibited cell growth significantly. Reduction of cell viability by 50% required 20 μM E2 for E2-treatment groups and 20 μMGE for GE-treatment groups. The percentage of the GE-treated apoptotic cells was reduced by about 35%, 42%, and 47% (P < 0.001 and that of E2-treated groups 34%, 39%, and 42% (P < 0.001 after 24, 48, and 72 h, respectively. Conclusions: Our experimental work clearly demonstrated that GE and E2 exhibited significant antiproliferative and apoptotic effects on human HCC HepG2 cells.

  12. Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells

    International Nuclear Information System (INIS)

    Capraro, Jessica; Magni, Chiara; Faoro, Franco; Maffi, Dario; Scarafoni, Alessio; Tedeschi, Gabriella; Maffioli, Elisa; Parolari, Anna; Manzoni, Cristina; Lovati, Maria Rosa; Duranti, Marcello

    2013-01-01

    Highlights: •A glycaemia-reducing lupin seed protein is internalized by HepG2 cells. •The protein accumulates in the cytosol in an intact form. •The internalized protein is multiply phosphorylated. -- Abstract: Lupin seed γ-Conglutin is a protein capable of reducing glycaemia in mammalians and increasing glucose uptake by model cells. This work investigated whether γ-Conglutin is internalised into the target cells and undergoes any covalent change during the process, as a first step to understanding its mechanism of action. To this purpose, γ-Conglutin-treated and untreated HepG2 cells were submitted to confocal and transmission electron microscopy. Immune-revelation of γ-Conglutin at various intervals revealed its accumulation inside the cytosol. In parallel, 2D-electrophoresis of the cell lysates and antibody reaction of the blotted maps showed the presence of the protein intact subunits inside the treated cells, whilest no trace of the protein was found in the control cells. However, γ-Conglutin-related spots with an unexpectedly low pI were also observed in the maps. These spots were excised, trypsin-treated and submitted to MS/MS spectrometric analysis. The presence of phosphorylated amino acids was detected. These findings, by showing that γ-Conglutin is internalised by HepG2 cells in an intact form and is modified by multiple phosphorylation, open the way to the understanding of the lupin γ-Conglutin insulin-mimetic activity

  13. Measurement of Hepatic Protein Fractional Synthetic Rate with Stable Isotope Labeling Technique in Thapsigargin Stressed HepG2 Cells

    Science.gov (United States)

    Song, Juquan; Zhang, Xiao-jun; Boehning, Darren; Brooks, Natasha C.; Herndon, David N.; Jeschke, Marc G.

    2012-01-01

    Severe burn-induced liver damage and dysfunction is associated with endoplasmic reticulum (ER) stress. ER stress has been shown to regulate global protein synthesis. In the current study, we induced ER stress in vitro and estimated the effect of ER stress on hepatic protein synthesis. The aim was two-fold: (1) to establish an in vitro model to isotopically measure hepatic protein synthesis and (2) to evaluate protein fractional synthetic rate (FSR) in response to ER stress. Human hepatocellular carcinoma cells (HepG2) were cultured in medium supplemented with stable isotopes 1,2-13C2-glycine and L-[ring-13C6]phenylalanine. ER stress was induced by exposing the cells to 100 nM of thapsigargin (TG). Cell content was collected from day 0 to 14. Alterations in cytosolic calcium were measured by calcium imaging and ER stress markers were confirmed by Western blotting. The precursor and product enrichments were detected by GC-MS analysis for FSR calculation. We found that the hepatic protein FSR were 0.97±0.02 and 0.99±0.05%/hr calculated from 1,2-13C2-glycine and L-[ring-13C6]phenylalanine, respectively. TG depleted ER calcium stores and induced ER stress by upregulating p-IRE-1 and Bip. FSR dramatically decreased to 0.68±0.03 and 0.60±0.06%/hr in the TG treatment group (pisotope tracer incorporation technique is a useful method for studying the effects of ER stress on hepatic protein synthesis. PMID:22298954

  14. Effects of three different formulae of Gamisoyosan on lipid accumulation induced by oleic acid in HepG2 cells

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    Hiroe Go

    2017-12-01

    Full Text Available Background: Gamisoyosan (GSS is an herbal formula which has been used to treat women’s diseases for several hundred years in Korea. GSS is one of the three most common prescriptions among women and is used to treat menopausal symptoms. Fatty liver disease is also common in postmenopausal women and can precede more severe diseases, such as steatohepatitis. The present study compared the effects of GSS on fatty liver using three different formulae, Dongui-Bogam (KIOM A, Korean Pharmacopeia (KIOM B and Korean National Health Insurance (KIOM C. Methods: In oleic acid-induced HepG2 fatty liver cells, cellular lipid accumulation, triglycerides and total cholesterol were measured after treatment with three GSS formulae and simvastatin as a positive control. To investigate the phytoestrogen activity of GSS, MCF-7 cells were treated with GSS, and hormone levels were quantified. Also, qualitative analysis was performed with UPLC. Results: All types of GSS decreased cellular lipid accumulation. KIOM A was slightly less effective than the other two GSS formulae. KIOM B and KIOM C decreased cellular triglycerides more effective than simvastatin, but KIOM A did not affect cellular triglycerides. Cellular total cholesterol was decreased by all GSS and simvastatin. GSS showed phytoestrogen activity in MCF-7 cells. From the UPLC analysis data, geniposide, paeoniflorin and glycyrrhizin were detected form three GSS formulae. Conclusion: These results suggest that all GSS formulae have a beneficial effect on fatty liver disease during menopause and that differences of formula have no effect on the efficacy of the prescription. Keywords: fatty liver, Gamisoyosan, menopause, phytoestrogen

  15. Antioxidant mechanism of mitochondria-targeted plastoquinone SkQ1 is suppressed in aglycemic HepG2 cells dependent on oxidative phosphorylation

    Czech Academy of Sciences Publication Activity Database

    Ježek, J.; Engstová, Hana; Ježek, Petr

    2017-01-01

    Roč. 1858, č. 9 (2017), s. 750-762 ISSN 0005-2728 R&D Projects: GA ČR(CZ) GA17-01813S Institutional support: RVO:67985823 Keywords : mitochondria-targeted antioxidant SkQ1 * mitochondrial Complex I superoxide formation * mitochondrial Complex III superoxide formation * HepG2 cell s * NAD(P)H fluorescence lifetime imaging microscopy Subject RIV: ED - Physiology OBOR OECD: Cell biology Impact factor: 4.932, year: 2016

  16. Secretion of apolipoproteins A-I and B by HepG2 cells: regulation by substrates and metabolic inhibitors.

    Science.gov (United States)

    Kempen, H J; Imbach, A P; Giller, T; Neumann, W J; Hennes, U; Nakada, N

    1995-08-01

    It was the aim of this study to i) compare the effects of glucose and other hexoses with that of oleate on secretion of apolipoproteins (apos) A-I and B by HepG2 cells, and ii) document the effect of various metabolic inhibitors on the secretion of these apos in the absence or presence of extra glucose/oleate. i) The addition of 10 mM glucose increased secretion of apoA-I and apoB, as measured by enzyme immunoassay, by about 60% when cells were incubated for 48 h in DMEM + 10% fetal calf serum. The addition of extra glucose also increased the mRNA levels for these apos. Increased radioactivity was also found in these apolipoproteins by immunoprecipitation after metabolic labeling with [35S]methionine for 48 h. However, in a pulse-chase experiment (15 min labeling, 2 h chase), glucose was found to increase apoA-I synthesis but not apoB synthesis. More labeled apoB appeared in the medium during the chase because glucose inhibited its intracellular degradation. The effect of glucose on secretion of these apos could be mimicked by fructose and mannose but not by 6-deoxyglucose, showing that the hexoses must enter the cells and be phosphorylated. In contrast, the addition of 0.5 mM oleate had a weak inhibitory effect on secretion of apoA-I whereas it increased the secretion of apoB by more than twofold. The combination of 10 mM glucose and 0.5 mM oleate had no greater effect than glucose alone on apoA-I secretion but increased apoB secretion by fourfold. ii) Inhibiting glycolysis (by glucosamine) lowered secretion of both apoA-I and apoB, while inhibiting lipogenesis (using 8-Br-cyclic AMP or 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA)) did not affect apoA-I secretion but clearly decreased that of apoB. However, the inhibitory effect of TOFA on apoB secretion was much smaller in the presence of 0.5 mM oleate instead of extra glucose. Actinomycin-D and cycloheximide strongly suppressed the stimulatory effect of glucose on secretion of both apolipoproteins

  17. Heat-modified citrus pectin induces apoptosis-like cell death and autophagy in HepG2 and A549 cancer cells.

    Science.gov (United States)

    Leclere, Lionel; Fransolet, Maude; Cote, Francois; Cambier, Pierre; Arnould, Thierry; Van Cutsem, Pierre; Michiels, Carine

    2015-01-01

    Cancer is still one of the leading causes of death worldwide, and finding new treatments remains a major challenge. Previous studies showed that modified forms of pectin, a complex polysaccharide present in the primary plant cell wall, possess anticancer properties. Nevertheless, the mechanism of action of modified pectin and the pathways involved are unclear. Here, we show that citrus pectin modified by heat treatment induced cell death in HepG2 and A549 cells. The induced cell death differs from classical apoptosis because no DNA cleavage was observed. In addition, Z-VAD-fmk, a pan-caspase inhibitor, did not influence the observed cell death in HepG2 cells but appeared to be partly protective in A549 cells, indicating that heat-modified citrus pectin might induce caspase-independent cell death. An increase in the abundance of the phosphatidylethanolamine-conjugated Light Chain 3 (LC3) protein and a decrease in p62 protein abundance were observed in both cell types when incubated in the presence of heat-modified citrus pectin. These results indicate the activation of autophagy. To our knowledge, this is the first time that autophagy has been revealed in cells incubated in the presence of a modified form of pectin. This autophagy activation appears to be protective, at least for A549 cells, because its inhibition with 3-methyladenine increased the observed modified pectin-induced cytotoxicity. This study confirms the potential of modified pectin to improve chemotherapeutic cancer treatments.

  18. Nanosilica induced dose-dependent cytotoxicity and cell type-dependent multinucleation in HepG2 and L-02 cells

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Yongbo [Capital Medical University, Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, Beijing Pediatric Research Institute, Beijing Children’s Hospital (China); Duan, Junchao; Li, Yang; Yu, Yang; Hu, Hejing; Wu, Jing; Zhang, Yannan; Li, Yanbo; CaixiaGuo; Zhou, Xianqing; Sun, Zhiwei, E-mail: zwsun@ccmu.edu.cn [Capital Medical University, School of Public Health (China)

    2016-11-15

    The prevalent exposure to nanosilica gained concerns about health effects of these particles on human beings. Although nanosilica-induced multinucleation has been confirmed previously, the underlying mechanism was still not clear; this study was to investigate the origination of multinucleated cells caused by nanosilica (62 nm) in both HepG2 and L-02 cells. Cell viability and cellular uptake was determined by MTT assay and transmission electron microscope (TEM), respectively. Giemsa staining was applied to detect multinucleation. To clarify the origination of multinucleated cells, fluorescent probes, PKH26 and PKH67, time-lapse observation were further conducted by confocal microscopy. Results indicated that nanosilica particles were internalized into cells and induced cytotoxicity in a dose-dependent manner. Quantification analysis showed that nanosilica significantly increased the rates of binucleated and multinucleated cells, which suggested mitotic catastrophe induction. Moreover, dynamic visualization verified that multinucleation resulted from cell fusion in HepG2 cells not in L-02 cells after nanosilica exposure, suggesting cell type-dependent multinucleation formation. Both multinucleation and cell fusion were involved in genetic instability, which emphasized the significance to explore the multinucleation induced by nanosilica via environmental, occupational and consumer product exposure.

  19. Heat-modified citrus pectin induces apoptosis-like cell death and autophagy in HepG2 and A549 cancer cells.

    Directory of Open Access Journals (Sweden)

    Lionel Leclere

    Full Text Available Cancer is still one of the leading causes of death worldwide, and finding new treatments remains a major challenge. Previous studies showed that modified forms of pectin, a complex polysaccharide present in the primary plant cell wall, possess anticancer properties. Nevertheless, the mechanism of action of modified pectin and the pathways involved are unclear. Here, we show that citrus pectin modified by heat treatment induced cell death in HepG2 and A549 cells. The induced cell death differs from classical apoptosis because no DNA cleavage was observed. In addition, Z-VAD-fmk, a pan-caspase inhibitor, did not influence the observed cell death in HepG2 cells but appeared to be partly protective in A549 cells, indicating that heat-modified citrus pectin might induce caspase-independent cell death. An increase in the abundance of the phosphatidylethanolamine-conjugated Light Chain 3 (LC3 protein and a decrease in p62 protein abundance were observed in both cell types when incubated in the presence of heat-modified citrus pectin. These results indicate the activation of autophagy. To our knowledge, this is the first time that autophagy has been revealed in cells incubated in the presence of a modified form of pectin. This autophagy activation appears to be protective, at least for A549 cells, because its inhibition with 3-methyladenine increased the observed modified pectin-induced cytotoxicity. This study confirms the potential of modified pectin to improve chemotherapeutic cancer treatments.

  20. N-Acetyl-L-Cysteine Affords Protection against Lead-Induced Cytotoxicity and Oxidative Stress in Human Liver Carcinoma (HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Paul B. Tchounwou

    2007-06-01

    Full Text Available Although lead exposure has declined in recent years as a result of change to lead-free gasoline, several epidemiological have pointed out that it represents a medical and public health emergency, especially in young children consuming high amounts of lead-contaminated flake paints. A previous study in our laboratory indicated that lead exposure induces cytotoxicity in human liver carcinoma cells. In the present study, we evaluated the role of oxidative stress in lead-induced toxicity, and the protective effect of the anti-oxidant n-acetyl-l-cysteine (NAC. We hypothesized that oxidative stress plays a role in lead-induced cytotoxicity, and that NAC affords protection against this adverse effect. To test this hypothesis, we performed the MTT [3-(4, 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide] assay and the trypan blue exclusion test for cell viability. We also performed the thiobarbituric acid test for lipid peroxidation. Data obtained from the MTT assay indicated that NAC significantly increased the viability of HepG2 cells in a dosedependent manner upon 48 hours of exposure. Similar trend was obtained with the trypan blue exclusion test. Data generated from the thiobarbituric acid test showed a significant (p ≤ 0.05 increase of MDA levels in lead nitrate-treated HepG2 cells compared to control cells. Interestingly, the addition of NAC to lead nitrate-treated HepG2 cells significantly decreased cellular content of reactive oxygen species (ROS, as evidenced by the decrease in lipid peroxidation byproducts. Overall, findings from this study suggest that NAC inhibits lead nitrate-induced cytotoxicity and oxidative stress in HepG2 cells. Hence, NAC may be used as a salvage therapy for lead-induced toxicity in exposed persons.

  1. Intracellular distribution and mechanisms of actions of photosensitizer Zinc(II)-phthalocyanine solubilized in Cremophor EL against human hepatocellular carcinoma HepG2 cells.

    Science.gov (United States)

    Shao, Jingwei; Dai, Yongchao; Zhao, Wenna; Xie, Jingjing; Xue, Jinping; Ye, Jianhui; Jia, Lee

    2013-03-01

    Zinc(II)-phthalocyanine (ZnPc) is a metal photosensitizer. In the present study, we formulated the poorly-soluble ZnPc in Cremophor EL solution to enhance its solubility and determined its intracellular distribution and mechanisms of action on human hepatocellular carcinoma HepG2 cells. ZnPc uptake by the cells reached a plateau by 8h. ZnPc primarily located in mitochondria, lysosome and endoplasmic reticulum. The concentration-growth inhibition curves of ZnPc on the cell lines were pharmacodynamically enhanced by 10-50 folds by irradiation. Once irradiated, ZnPc produced significant amount of reactive oxygen species (ROS), activated caspase-3 and caspase-9, arrested cell cycle mainly at G2/M stage, and decreased membrane potential (ΔΨm) of HepG2 cells. In conclusion, the present study first elucidated cellular and molecular mechanisms of ZnPc on HepG2 cells. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  2. Newly developed chitosan-silver hybrid nanoparticles: biosafety and apoptosis induction in HepG2 cells

    International Nuclear Information System (INIS)

    El-Sherbiny, Ibrahim M.; Salih, Ehab; Yassin, Abdelrahman M.; Hafez, Elsayed E.

    2016-01-01

    The present study reports the biosafety assessment, the exact molecular effects, and apoptosis induction of newly developed chitosan-silver hybrid nanoparticles (Cs–Ag NPs) in HepG2 cells. The investigated hybrid NPs were green synthesized using Cs/grape leaves aqueous extract (Cs/GLE) or Cs/GLE NPs as reducing and stabilizing agents. The successful formation of Cs/GLE NPs and Cs–Ag hybrid NPs has been confirmed by UV–Vis spectrophotometry, FTIR spectroscopy, XRD, and HRTEM. From the TEM analysis, the prepared Cs/GLE NPs are uniform and spherical with an average size of 150 nm, and the AgNPs (5–10 nm) were formed mainly on their surface. The UV–Vis spectra of Cs–Ag NPs showed a surface plasmon resonance (SPR) peak at about 450 nm confirming their formation. The synthesized Cs–Ag NPs were found to be crystalline as shown by XRD patterns with fcc phase oriented along the (111), (200), (220), and (311) planes. The cytotoxicity patterns, the antiproliferative activities, and the possible mechanisms of anticancer activity at molecular level of the newly developed Cs–Ag hybrid NPs were investigated. Cytotoxicity patterns of all the preparations demonstrated that the nontoxic treatment concentrations are ranged from 0.39 to 50 %, and many of the newly prepared Cs–Ag hybrid NPs showed high anticancer activities against HpG2 cells, and induced cellular apoptosis by downregulating BCL2 gene and upregulating P53.Graphical Abstract

  3. Newly developed chitosan-silver hybrid nanoparticles: biosafety and apoptosis induction in HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    El-Sherbiny, Ibrahim M., E-mail: ielsherbiny@Zewailcity.edu.eg; Salih, Ehab [Zewail City of Science and Technology, Center for Materials Science (Egypt); Yassin, Abdelrahman M. [Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technology Applications, Biopharmaceutical Product Research Department (Egypt); Hafez, Elsayed E. [City of Scientific Research and Technology Applications, Plant Protection and Biomolecular Diagnosis Department (Egypt)

    2016-07-15

    The present study reports the biosafety assessment, the exact molecular effects, and apoptosis induction of newly developed chitosan-silver hybrid nanoparticles (Cs–Ag NPs) in HepG2 cells. The investigated hybrid NPs were green synthesized using Cs/grape leaves aqueous extract (Cs/GLE) or Cs/GLE NPs as reducing and stabilizing agents. The successful formation of Cs/GLE NPs and Cs–Ag hybrid NPs has been confirmed by UV–Vis spectrophotometry, FTIR spectroscopy, XRD, and HRTEM. From the TEM analysis, the prepared Cs/GLE NPs are uniform and spherical with an average size of 150 nm, and the AgNPs (5–10 nm) were formed mainly on their surface. The UV–Vis spectra of Cs–Ag NPs showed a surface plasmon resonance (SPR) peak at about 450 nm confirming their formation. The synthesized Cs–Ag NPs were found to be crystalline as shown by XRD patterns with fcc phase oriented along the (111), (200), (220), and (311) planes. The cytotoxicity patterns, the antiproliferative activities, and the possible mechanisms of anticancer activity at molecular level of the newly developed Cs–Ag hybrid NPs were investigated. Cytotoxicity patterns of all the preparations demonstrated that the nontoxic treatment concentrations are ranged from 0.39 to 50 %, and many of the newly prepared Cs–Ag hybrid NPs showed high anticancer activities against HpG2 cells, and induced cellular apoptosis by downregulating BCL2 gene and upregulating P53.Graphical Abstract.

  4. An untargeted multi-technique metabolomics approach to studying intracellular metabolites of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

    Science.gov (United States)

    Ruiz-Aracama, Ainhoa; Peijnenburg, Ad; Kleinjans, Jos; Jennen, Danyel; van Delft, Joost; Hellfrisch, Caroline; Lommen, Arjen

    2011-05-20

    In vitro cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Transcriptomic and proteomic approaches have been widely applied in vitro but relatively few studies have used metabolomics. Therefore, the goal of the present study was to develop an untargeted methodology for performing reproducible metabolomics on in vitro systems. The human liver cell line HepG2, and the well-known hepatotoxic and non-genotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were used as the in vitro model system and model toxicant, respectively. The study focused on the analysis of intracellular metabolites using NMR, LC-MS and GC-MS, with emphasis on the reproducibility and repeatability of the data. State of the art pre-processing and alignment tools and multivariate statistics were used to detect significantly altered levels of metabolites after exposing HepG2 cells to TCDD. Several metabolites identified using databases, literature and LC-nanomate-Orbitrap analysis were affected by the treatment. The observed changes in metabolite levels are discussed in relation to the reported effects of TCDD. Untargeted profiling of the polar and apolar metabolites of in vitro cultured HepG2 cells is a valid approach to studying the effects of TCDD on the cell metabolome. The approach described in this research demonstrates that highly reproducible experiments and correct normalization of the datasets are essential for obtaining reliable results. The effects of TCDD on HepG2 cells reported herein are in agreement with previous studies and serve to validate the procedures used in the present work.

  5. Protection of HepG2 cells against acrolein toxicity by 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide via glutathione-mediated mechanism.

    Science.gov (United States)

    Shah, Halley; Speen, Adam M; Saunders, Christina; Brooke, Elizabeth A S; Nallasamy, Palanisamy; Zhu, Hong; Li, Y Robert; Jia, Zhenquan

    2015-10-01

    Acrolein is an environmental toxicant, mainly found in smoke released from incomplete combustion of organic matter. Several studies showed that exposure to acrolein can lead to liver damage. The mechanisms involved in acrolein-induced hepatocellular toxicity, however, are not completely understood. This study examined the cytotoxic mechanisms of acrolein on HepG2 cells. Acrolein at pathophysiological concentrations was shown to cause apoptotic cell death and an increase in levels of protein carbonyl and thiobarbituric acid reactive acid substances. Acrolein also rapidly depleted intracellular glutathione (GSH), GSH-linked glutathione-S-transferases, and aldose reductase, three critical cellular defenses that detoxify reactive aldehydes. Results further showed that depletion of cellular GSH by acrolein preceded the loss of cell viability. To further determine the role of cellular GSH in acrolein-mediated cytotoxicity, buthionine sulfoximine (BSO) was used to inhibit cellular GSH biosynthesis. It was observed that depletion of cellular GSH by BSO led to a marked potentiation of acrolein-mediated cytotoxicity in HepG2 cells. To further assess the contribution of these events to acrolein-induced cytotoxicity, triterpenoid compound 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) was used for induction of GSH. Induction of GSH by CDDO-Im afforded cytoprotection against acrolein toxicity in HepG2 cells. Furthermore, BSO significantly inhibited CDDO-Im-mediated induction in cellular GSH levels and also reversed cytoprotective effects of CDDO-Im in HepG2 cells. These results suggest that GSH is a predominant mechanism underlying acrolein-induced cytotoxicity as well as CDDO-Im-mediated cytoprotection. This study may provide understanding on the molecular action of acrolein which may be important to develop novel strategies for the prevention of acrolein-mediated toxicity. © 2014 by the Society for Experimental Biology and Medicine.

  6. Regulation of human gamma-glutamylcysteine synthetase: co-ordinate induction of the catalytic and regulatory subunits in HepG2 cells.

    Science.gov (United States)

    Galloway, D C; Blake, D G; Shepherd, A G; McLellan, L I

    1997-11-15

    We have shown that in HepG2 cells treatment with 75 microM t-butylhydroquinone (tBHQ) results in a 2.5-fold increase in glutathione concentration, as part of an adaptive response to chemical stress. In these cells the elevation in intracellular glutathione level was found to be accompanied by an increase of between 2-fold and 3-fold in the level of the 73 kDa catalytic subunit of gamma-glutamylcysteine synthetase (heavy subunit, GCSh) and the 31 kDa regulatory subunit (light subunit, GCSl). Levels of GCSh and GCSl mRNA were increased by up to 5-fold in HepG2 cells in response to tBHQ. To study the transcriptional regulation of GCSl, we subcloned 6.7 kb of the upstream region of the human GCSl gene (GLCLR) from a genomic clone isolated from a P1 lymphoblastoid cell line genomic library. HepG2 cells were transfected with GLCLR promoter reporter constructs and treated with tBHQ. This resulted in an induction of between 1.5-fold and 3.5-fold in reporter activity, indicating that transcriptional regulation of GLCLR is likely to contribute to the induction of GCSl by tBHQ in HepG2 cells. Sequence analysis of the promoter region demonstrated the presence of putative enhancer elements including AP-1 sites and an antioxidant-responsive element, which might be involved in the observed induction of the GLCLR promoter.

  7. Copper(ii) oxide nanoparticles penetrate into HepG2 cells, exert cytotoxicity via oxidative stress and induce pro-inflammatory response

    Science.gov (United States)

    Piret, Jean-Pascal; Jacques, Diane; Audinot, Jean-Nicolas; Mejia, Jorge; Boilan, Emmanuelle; Noël, Florence; Fransolet, Maude; Demazy, Catherine; Lucas, Stéphane; Saout, Christelle; Toussaint, Olivier

    2012-10-01

    The potential toxic effects of two types of copper(ii) oxide (CuO) nanoparticles (NPs) with different specific surface areas, different shapes (rod or spheric), different sizes as raw materials and similar hydrodynamic diameter in suspension were studied on human hepatocarcinoma HepG2 cells. Both CuO NPs were shown to be able to enter into HepG2 cells and induce cellular toxicity by generating reactive oxygen species. CuO NPs increased the abundance of several transcripts coding for pro-inflammatory interleukins and chemokines. Transcriptomic data, siRNA knockdown and DNA binding activities suggested that Nrf2, NF-κB and AP-1 were implicated in the response of HepG2 cells to CuO NPs. CuO NP incubation also induced activation of MAPK pathways, ERKs and JNK/SAPK, playing a major role in the activation of AP-1. In addition, cytotoxicity, inflammatory and antioxidative responses and activation of intracellular transduction pathways induced by rod-shaped CuO NPs were more important than spherical CuO NPs. Measurement of Cu2+ released in cell culture medium suggested that Cu2+ cations released from CuO NPs were involved only to a small extent in the toxicity induced by these NPs on HepG2 cells.The potential toxic effects of two types of copper(ii) oxide (CuO) nanoparticles (NPs) with different specific surface areas, different shapes (rod or spheric), different sizes as raw materials and similar hydrodynamic diameter in suspension were studied on human hepatocarcinoma HepG2 cells. Both CuO NPs were shown to be able to enter into HepG2 cells and induce cellular toxicity by generating reactive oxygen species. CuO NPs increased the abundance of several transcripts coding for pro-inflammatory interleukins and chemokines. Transcriptomic data, siRNA knockdown and DNA binding activities suggested that Nrf2, NF-κB and AP-1 were implicated in the response of HepG2 cells to CuO NPs. CuO NP incubation also induced activation of MAPK pathways, ERKs and JNK/SAPK, playing a major

  8. Poly(vinyl alcohol/gelatin Hydrogels Cultured with HepG2 Cells as a 3D Model of Hepatocellular Carcinoma: A Morphological Study

    Directory of Open Access Journals (Sweden)

    Stefania Moscato

    2015-01-01

    Full Text Available It has been demonstrated that three-dimensional (3D cell culture models represent fundamental tools for the comprehension of cellular phenomena both for normal and cancerous tissues. Indeed, the microenvironment affects the cellular behavior as well as the response to drugs. In this study, we performed a morphological analysis on a hepatocarcinoma cell line, HepG2, grown for 24 days inside a bioartificial hydrogel composed of poly(vinyl alcohol (PVA and gelatin (G to model a hepatocellular carcinoma (HCC in 3D. Morphological features of PVA/G hydrogels were investigated, resulting to mimic the trabecular structure of liver parenchyma. A histologic analysis comparing the 3D models with HepG2 cell monolayers and tumor specimens was performed. In the 3D setting, HepG2 cells were viable and formed large cellular aggregates showing different morphotypes with zonal distribution. Furthermore, β-actin and α5β1 integrin revealed a morphotype-related expression; in particular, the frontline cells were characterized by a strong immunopositivity on a side border of their membrane, thus suggesting the formation of lamellipodia-like structures apt for migration. Based on these results, we propose PVA/G hydrogels as valuable substrates to develop a long term 3D HCC model that can be used to investigate important aspects of tumor biology related to migration phenomena.

  9. Cytotoxic Activity of Origanum Vulgare L. on Hepatocellular Carcinoma cell Line HepG2 and Evaluation of its Biological Activity

    Directory of Open Access Journals (Sweden)

    Hazem S. Elshafie

    2017-08-01

    Full Text Available The potential of plant essential oils (EOs in anticancer treatment has recently received many research efforts to overcome the development of multidrug resistance and their negative side effects. The aims of the current research are to study (i the cytotoxic effect of the crude EO extracted from Origanum vulgare subsp hirtum and its main constituents (carvacrol, thymol, citral and limonene on hepatocarcinoma HepG2 and healthy human renal cells HEK293; (ii the antibacterial and phytotoxic activities of the above EO and its main constituents. Results showed that cell viability percentage of treated HepG2 by EO and its main constituents was significantly decreased when compared to untreated cells. The calculated inhibition concentration (IC50 values for HepG2 were lower than healthy renal cells, indicating the sort of selectivity of the studied substances. Citral is not potentially recommended as an anticancer therapeutic agent, since there are no significant differences between IC50 values against both tested cell lines. Results showed also that oregano EO and its main constituents have a significant antibacterial activity and a moderate phytotoxic effect. The current research verified that oregano EO and its main constituents could be potentially utilized as anticancer therapeutic agents.

  10. In HepG2 Cells, Coexisting Carnitine Deficiency Masks Important Indicators of Marginal Biotin Deficiency123

    Science.gov (United States)

    Bogusiewicz, Anna; Boysen, Gunnar; Mock, Donald M

    2015-01-01

    Background: A large number of birth defects are related to nutrient deficiencies; concern that biotin deficiency is teratogenic in humans is reasonable. Surprisingly, studies indicate that increased urinary 3-hydroxyisovalerylcarnitine (3HIAc), a previously validated marker of biotin deficiency, is not a valid biomarker in pregnancy. Objective: In this study we hypothesized that coexisting carnitine deficiency can prevent the increase in 3HIAc due to biotin deficiency. Methods: We used a 2-factor nutrient depletion design to induce isolated and combined biotin and carnitine deficiency in HepG2 cells and then repleted cells with carnitine. To elucidate the metabolic pathogenesis, we quantitated intracellular and extracellular free carnitine, acylcarnitines, and acylcarnitine ratios using liquid chromatography–tandem mass spectrometry. Results: Relative to biotin-sufficient, carnitine-sufficient cells, intracellular acetylcarnitine increased by 90%, propionylcarnitine more than doubled, and 3HIAc increased by >10-fold in biotin-deficient, carnitine-sufficient (BDCS) cells, consistent with a defensive mechanism in which biotin-deficient cells transesterify the acyl-coenzyme A (acyl-CoA) substrates of the biotin-dependent carboxylases to the related acylcarnitines. Likewise, in BDCS cells, the ratio of acetylcarnitine to malonylcarnitine and the ratio of propionylcarnitine to methylmalonylcarnitine both more than tripled, and the ratio of 3HIAc to 3-methylglutarylcarnitine (MGc) increased by >10-fold. In biotin-deficient, carnitine-deficient (BDCD) cells, the 3 substrate-derived acylcarnitines changed little, but the substrate:product ratios were masked to a lesser extent. Moreover, carnitine repletion unmasked biotin deficiency in BDCD cells as shown by increases in acetylcarnitine, propionylcarnitine, and 3HIAc (each increased by >50-fold). Likewise, ratios of acetylcarnitine:malonylcarnitine, propionylcarnitine:methylmalonylcarnitine, and 3HIAc:MGc all increased

  11. Dihydromyricetin induces mitochondria-mediated apoptosis in HepG2 cells through down-regulation of the Akt/Bad pathway.

    Science.gov (United States)

    Zhang, Zhuangwei; Zhang, Huiqin; Chen, Shiyong; Xu, Yan; Yao, Anjun; Liao, Qi; Han, Liyuan; Zou, Zuquan; Zhang, Xiaohong

    2017-02-01

    The plant flavonol dihydromyricetin (DHM) was reported to induce apoptosis in human hepatocarcinoma HepG2 cells. This study was undertaken to elucidate the underlying molecular mechanism of action of DHM. In the study, DHM down-regulated Akt expression and its phosphorylation at Ser473, up-regulated the levels of mitochondrial proapoptotic proteins Bax and Bad, and inhibited the phosphorylation of Bad at Ser136 and Ser112. It also inhibited the expression of the antiapoptotic protein Bcl-2 and enhanced the cleavage and activation of caspase-3 as well as the degradation of its downstream target poly(ADP-ribose) polymerase. Our results for the first time suggest that DHM-induced apoptosis in HepG2 cells may come about by the inhibition of the Akt/Bad signaling pathway and stimulation of the mitochondrial apoptotic pathway. Dihydromyricetin may be a promising therapeutic medication for hepatocellular carcinoma. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Baicalin and its metabolites suppresses gluconeogenesis through activation of AMPK or AKT in insulin resistant HepG-2 cells.

    Science.gov (United States)

    Wang, Tao; Jiang, Hongmei; Cao, Shijie; Chen, Qian; Cui, Mingyuan; Wang, Zhijie; Li, Dandan; Zhou, Jing; Wang, Tao; Qiu, Feng; Kang, Ning

    2017-12-01

    Scutellaria baicalensis Georgi (S. baicalensis), as a traditional Chinese herbal medicine, is an important component of several famous Chinese medicinal formulas for treating patients with diabetes mellitus. Baicalin (BG), a main bioactive component of S. baicalensis, has been reported to have antidiabetic effects. However, pharmacokinetic studies have indicated that BG has poor oral bioavailability. Therefore, it is hard to explain the pharmacological effects of BG in vivo. Interestingly, several reports show that BG is extensively metabolized in rats and humans. Therefore, we speculate that the BG metabolites might be responsible for the pharmacological effects. In this study, BG and its three metabolites (M1-M3) were examined their effects on glucose consumption in insulin resistant HepG-2 cells with a commercial glucose assay kit. Real-time PCR and western blot assay were used to confirm genes and proteins of interest, respectively. The results demonstrate that BG and its metabolites (except for M3) enhanced the glucose consumption which might be associated with inhibiting the expression of the key gluconeogenic genes, including glucose-6-phosphatase (G6Pase), phosphoenolypyruvate carboxykinase (PEPCK) and glucose transporter 2 (GLUT2). Further study found that BG and M1 could suppress hepatic gluconeogenesis via activation of the AMPK pathway, while M2 could suppress hepatic gluconeogenesis via activation of the PI3K/AKT signaling pathway. Taken together, our findings suggest that both BG and its metabolites have antihyperglycemic activities, and might be the active forms of oral doses of BG in vivo. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  13. The Nitric Oxide Prodrug JS-K Induces Ca(2+)-Mediated Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells.

    Science.gov (United States)

    Liu, Ling; Wang, Dongmei; Wang, Jiangang; Wang, Shuying

    2016-04-01

    Hepatocellular carcinoma is one of the most common and deadly forms of human malignancies. JS-K, O(2)-(2, 4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1, 2-diolate, has the ability to induce apoptosis of tumor cell lines. In the present study, JS-K inhibited the proliferation of HepG2 cells in a time- and concentration-dependent manner and significantly induced apoptosis. JS-K enhanced the ratio of Bax-to-Bcl-2, released of cytochrome c (Cyt c) from mitochondria and the activated caspase-9/3. JS-K caused an increasing cytosolic Ca(2+) and the loss of mitochondrial membrane potential. Carboxy-PTIO (a NO scavenger) and BAPTA-AM (an intracellular Ca(2+) chelator) significantly blocked an increasing cytosolic Ca(2+) in JS-K-induced HepG2 cells apoptosis, especially Carboxy-PTIO. Meanwhile, Carboxy-PTIO and BAPTA-AM treatment both attenuate JS-K-induced apoptosis through upregulation of Bcl-2, downregulation of Bax, reduction of Cyt c release from mitochondria to cytoplasm and inactivation of caspase-9/3. In summary, JS-K induced HepG2 cells apoptosis via Ca(2+)/caspase-3-mediated mitochondrial pathway. © 2015 Wiley Periodicals, Inc.

  14. Lignans from Opuntia ficus-indica seeds protect rat primary hepatocytes and HepG2 cells against ethanol-induced oxidative stress.

    Science.gov (United States)

    Kim, Jung Wha; Yang, Heejung; Kim, Hyeon Woo; Kim, Hong Pyo; Sung, Sang Hyun

    2017-01-01

    Bioactivity-guided isolation of Opuntia ficus-indica (Cactaceae) seeds against ethanol-treated primary rat hepatocytes yielded six lignan compounds. Among the isolates, furofuran lignans 4-6, significantly protected rat hepatocytes against ethanol-induced oxidative stress by reducing intracellular reactive oxygen species levels, preserving antioxidative defense enzyme activities, and maintaining the glutathione content. Moreover, 4 dose-dependently induced the heme oxygenase-1 expression in HepG2 cells.

  15. Metabolomic effects of CeO2, SiO2 and CuO metal oxide nanomaterials on HepG2 cells

    Science.gov (United States)

    To better assess potential hepatotoxicity of nanomaterials, human liver HepG2 cells were exposed for 3 days to five different CeO2 (either 30 or 100 μg/ml), 3 SiO2 based (30 μg/ml) or 1 CuO (3 μg/ml) nanomaterials with dry primary particle sizes ranging from 15 to 213 nm. Metabol...

  16. Metabolomic effects in HepG2 cells exposed to CeO2, SiO2 and CuO nanomaterials.

    Science.gov (United States)

    To better assess potential hepatotoxicity of nanomaterials, human liver HepG2 cells were exposed for three days to 5 different CeO2 (either 30 or 100 ug/ml), 3 SiO2 based (30 ug/ml) or 1 CuO (3 ug/ml) nanomaterials with dry primary particle sizes ranging from 15 to 213 nm. Metab...

  17. Isoorientin induces apoptosis through mitochondrial dysfunction and inhibition of PI3K/Akt signaling pathway in HepG2 cancer cells

    International Nuclear Information System (INIS)

    Yuan, Li; Wang, Jing; Xiao, Haifang; Xiao, Chunxia; Wang, Yutang; Liu, Xuebo

    2012-01-01

    Isoorientin (ISO) is a flavonoid compound that can be extracted from several plant species, such as Phyllostachys pubescens, Patrinia, and Drosophyllum lusitanicum; however, its biological activity remains poorly understood. The present study investigated the effects and putative mechanism of apoptosis induced by ISO in human hepatoblastoma cancer (HepG2) cells. The results showed that ISO induced cell death in a dose-dependent manner in HepG2 cells, but no toxicity in human liver cells (HL-7702) and buffalo rat liver cells (BRL-3A) treated with ISO at the indicated concentrations. ISO-induced cell death included apoptosis which characterized by the appearance of nuclear shrinkage, the cleavage of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation. ISO significantly (p < 0.01) increased the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), increased the release of cytochrome c, activated caspase-3, and enhanced intracellular levels of reactive oxygen species (ROS) and nitric oxide (NO). In addition, ISO effectively inhibited the phosphorylation of Akt and increased FoxO4 expression. The PI3K/Akt inhibitor LY294002 enhanced the apoptosis-inducing effect of ISO. However, LY294002 markedly quenched ROS and NO generation and diminished the protein expression of heme peroxidase enzyme (HO-1) and inducible nitric oxide synthase (iNOS). Furthermore, the addition of a ROS inhibitor (N-acetyl cysteine, NAC) or iNOS inhibitor (N-[3-(aminomethyl) benzyl] acetamidine, dihydrochloride, 1400W) significantly diminished the apoptosis induced by ISO and also blocked the phosphorylation of Akt. These results demonstrated for the first time that ISO induces apoptosis in HepG2 cells and indicate that this apoptosis might be mediated through mitochondrial dysfunction and PI3K/Akt signaling pathway, and has no toxicity in normal liver cells, suggesting that ISO may have good potential as a therapeutic and chemopreventive agent for liver cancer. Highlights:

  18. Structure related effects of flavonoid aglycones on cell cycle progression of HepG2 cells: Metabolic activation of fisetin and quercetin by catechol-O-methyltransferase (COMT).

    Science.gov (United States)

    Poór, Miklós; Zrínyi, Zita; Kőszegi, Tamás

    2016-10-01

    Dietary flavonoids are abundant in the Plant Kingdom and they are extensively studied because of their manifold pharmacological activities. Recent studies highlighted that cell cycle arrest plays a key role in their antiproliferative effect in different tumor cells. However, structure-activity relationship of flavonoids is poorly characterized. In our study the influence of 18 flavonoid aglycones (as well as two metabolites) on cell cycle distribution was investigated. Since flavonoids are extensively metabolized by liver cells, HepG2 tumor cell line was applied, considering the potential metabolic activation/inactivation of flavonoids. Our major observations are the followings: (1) Among the tested compounds diosmetin, fisetin, apigenin, lutelin, and quercetin provoked spectacular extent of G2/M phase cell cycle arrest. (2) Inhibition of catechol-O-methyltransferase enzyme by entacapone decreased the antiproliferative effects of fisetin and quercetin. (3) Geraldol and isorhamnetin (3'-O-methylated metabolites of fisetin and quercetin, respectively) demonstrated significantly higher antiproliferative effect on HepG2 cells compared to the parent compounds. Based on these results, O-methylated flavonoid metabolites or their chemically modified derivatives may be suitable candidates of tumor therapy in the future. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  19. SIRT1 attenuates palmitate-induced endoplasmic reticulum stress and insulin resistance in HepG2 cells via induction of oxygen-regulated protein 150

    Science.gov (United States)

    Jung, T.W.; Lee, K.T.; Lee, M.W.; Ka, K.H.

    2012-01-01

    Endoplasmic reticulum (ER) stress has been implicated in the pathology of type 2 diabetes mellitus (T2DM). Although SIRT1 has a therapeutic effect on T2DM, the mechanisms by which SIRT1 ameliorates insulin resistance (IR) remain unclear. In this study, we investigated the impact of SIRT1 on palmitate-induced ER stress in HepG2 cells and its underlying signal pathway. Treatment with resveratrol, a SIRT1 activator significantly inhibited palmitate-induced ER stress, leading to the protection against palmitate-induced ER stress and insulin resistance. Resveratrol and SIRT1 overexpression induced the expression of oxygen-regulated protein (ORP) 150 in HepG2 cells. Forkhead box O1 (FOXO1) was involved in the regulation of ORP150 expression because suppression of FOXO1 inhibited the induction of ORP150 by SIRT1. Our results indicate a novel mechanism by which SIRT1 regulates ER stress by overexpression of ORP150, and suggest that SIRT1 ameliorates palmitate-induced insulin resistance in HepG2 cells via regulation of ER stress.

  20. Active Fragment of Veronica ciliata Fisch. Attenuates t-BHP-Induced Oxidative Stress Injury in HepG2 Cells through Antioxidant and Antiapoptosis Activities

    Directory of Open Access Journals (Sweden)

    Yiran Sun

    2017-01-01

    Full Text Available Excessive amounts of reactive oxygen species (ROS in the body are a key factor in the development of hepatopathies such as hepatitis. The aim of this study was to assess the antioxidation effect in vitro and hepatoprotective activity of the active fragment of Veronica ciliata Fisch. (VCAF. Antioxidant assays (DPPH, superoxide, and hydroxyl radicals scavenging were conducted, and hepatoprotective effects through the application of tert-butyl hydroperoxide- (t-BHP- induced oxidative stress injury in HepG2 cells were evaluated. VCAF had high phenolic and flavonoid contents and strong antioxidant activity. From the perspective of hepatoprotection, VCAF exhibited a significant protective effect on t-BHP-induced HepG2 cell injury, as indicated by reductions in cytotoxicity and the levels of ROS, 8-hydroxydeoxyguanosine (8-OHdG, and protein carbonyls. Further study demonstrated that VCAF attenuated the apoptosis of t-BHP-treated HepG2 cells by suppressing the activation of caspase-3 and caspase-8. Moreover, it significantly decreased the levels of ALT and AST, increased the activities of acetyl cholinesterase (AChE, glutathione (GSH, superoxide dismutase (SOD, and catalase (CAT, and increased total antioxidative capability (T-AOC. Collectively, we concluded that VCAF may be a considerable candidate for protecting against liver injury owing to its excellent antioxidant and antiapoptosis properties.

  1. Black rice extract protected HepG2 cells from oxidative stress-induced cell death via ERK1/2 and Akt activation

    Science.gov (United States)

    Yoon, Jaemin; Ham, Hyeonmi; Sung, Jeehye; Kim, Younghwa; Choi, Youngmin; Lee, Jeom-Sig; Jeong, Heon-Sang; Lee, Junsoo

    2014-01-01

    BACKGROUND/OBJECTIVES The objective of this study was to evaluate the protective effect of black rice extract (BRE) on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. MATERIALS/METHODS Methanolic extract from black rice was evaluated for the protective effect on TBHP-induced oxidative injury in HepG2 cells. Several biomarkers that modulate cell survival and death including reactive oxygen species (ROS), caspase-3 activity, and related cellular kinases were determined. RESULTS TBHP induced cell death and apoptosis by a rapid increase in ROS generation and caspase-3 activity. Moreover, TBHP-induced oxidative stress resulted in a transient ERK1/2 activation and a sustained increase of JNK1/2 activation. While, BRE pretreatment protects the cells against oxidative stress by reducing cell death, caspase-3 activity, and ROS generation and also by preventing ERKs deactivation and the prolonged JNKs activation. Moreover, pretreatment of BRE increased the activation of ERKs and Akt which are pro-survival signal proteins. However, this effect was blunted in the presence of ERKs and Akt inhibitors. CONCLUSIONS These results suggest that activation of ERKs and Akt pathway might be involved in the cytoprotective effect of BRE against oxidative stress. Our findings provide new insights into the cytoprotective effects and its possible mechanism of black rice against oxidative stress. PMID:24741394

  2. Construction of Expression Vector for Anti-Alpha-Fetoprotein Gene and Its Inhibition Effects on Alpha-Fetoprotein Positive Hepg2 Cells

    Science.gov (United States)

    Wang, Ze; Zhang, Hui

    As research previously demonstrated, suppression of AFP expression or its biological activities might inhibit the proliferation of AFP positive human hepatocellular carcinoma cells. In this study, we constructed an anti-AFP gene vector and transfected it to HepG2 cells. RT-PCR showed AFP gene expression in the transfected cells was reduced. MTT assay suggested the proliferation of the transfected cells was also inhibited comparing with the untransfected cells. This result provides a new insight into AFP as the target for preventing and treating hepatocellular carcinoma.

  3. All-trans retinoic acid inhibits the recruitment of ARNT to DNA, resulting in the decrease of CYP1A1 mRNA expression in HepG2 cells

    International Nuclear Information System (INIS)

    Ohno, Marumi; Ikenaka, Yoshinori; Ishizuka, Mayumi

    2012-01-01

    Highlights: ► AHR and ARNT transcriptionally regulate genes related to metabolisms such as CYP1A1. ► We investigated the effect of retinoic acid (RA) on the function of AHR/ARNT. ► RA inhibited the recruitment of ARNT, not AHR, to the regulatory region of CYP1A1. ► It resulted in a reduction of constitutive expression of CYP1A1 to less than half. -- Abstract: Aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) are well-conserved transcription factors among species. However, there are a very limited number of reports on the physiological function of AHR, particularly on the regulation of AHR by endogenous compounds. We hence investigated the effects of all-trans retinoic acid (atRA) on cytochrome P450 (CYP) 1A1 gene transcription as a model of AHR-regulated transcription mechanisms in HepG2 cells, a human hepatoma cell line. Treatment with atRA significantly reduced transactivation and expression of CYP1A1 mRNA to less than half of its control value, and this inhibitory effect was mediated by RARα. The result of chromatin immunoprecipitation assay indicated that treatment with atRA at 1–100 nM drastically inhibited the recruitment of ARNT to DNA regions containing xenobiotic responsive elements. In conclusion, atRA at physiological concentrations could reduce AHR-mediated gene transcription via the inhibition of recruitment of ARNT to relevant DNA regions.

  4. Inhibition effects of 125I-triplex forming oligonucleotide to hepatoma cells

    International Nuclear Information System (INIS)

    Lv Zhongwei; Hou Min; Cai Haidong; Yuan Xueyu; Yang Yuehua; Yuan Shidong; He Junmin

    2007-01-01

    Objective: Triplex forming oligonucleotide (TFO) has been reported as a new antigene strategy. The purpose of this study was to observe the inhibition effects of 125 I-TFO on hepatoma cells and to investigate the possibility of using 125 I-TFO as an antigene radiotherapy technique for hepatocellular carcinoma (HCC) related to HBV. Methods: TFO complementary to the initiator of S gene of HBV was synthesized and labeled with 125 I. HepG2.2.15 cells, in which HBV genome was integrated, were incubated with 125 I-TFO, TFO and 125 I respectively. After incubation, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) of each group were assayed with ELISA and the survival rate of cells in each group was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) reduction assay. Results: 125 I-TFO showed a high stability with a radiolabeling rate of >93%. The radiochemical purity of labeled compound was 90.8%, 81.1% and 73.2% respectively after 12, 48 and 72 h at 37 degree C. The peak inhibition effect of 125 I-TFO on synthesizing HBsAg and HBeAg by HepG2.2.15 cells were found at 48 h after transfection, with significantly the highest inhibition rate of 45.2% for HBsAg and 74.5% for HBeAg expression among the three groups(P 125 I-TFO may inhibit the antigen expression of HBV and the growth of hepatocarcinoma cells, thus it may provide a new approach to develop gene-based radiotherapeutic pharmaceuticals for anti-HBV and HCC. (authors)

  5. Inhibition effects of {sup 125}I-triplex forming oligonucleotide to hepatoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhongwei, Lv; Min, Hou; Haidong, Cai; Xueyu, Yuan; Yuehua, Yang; Shidong, Yuan [Department of Nuclear Medicine, 10th People' s Hospital, Tongji Univ., Shanghai (China); Junmin, He

    2007-08-15

    Objective: Triplex forming oligonucleotide (TFO) has been reported as a new antigene strategy. The purpose of this study was to observe the inhibition effects of {sup 125}I-TFO on hepatoma cells and to investigate the possibility of using {sup 125}I-TFO as an antigene radiotherapy technique for hepatocellular carcinoma (HCC) related to HBV. Methods: TFO complementary to the initiator of S gene of HBV was synthesized and labeled with {sup 125}I. HepG2.2.15 cells, in which HBV genome was integrated, were incubated with {sup 125}I-TFO, TFO and {sup 125}I respectively. After incubation, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) of each group were assayed with ELISA and the survival rate of cells in each group was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) reduction assay. Results: {sup 125}I-TFO showed a high stability with a radiolabeling rate of >93%. The radiochemical purity of labeled compound was 90.8%, 81.1% and 73.2% respectively after 12, 48 and 72 h at 37 degree C. The peak inhibition effect of {sup 125}I-TFO on synthesizing HBsAg and HBeAg by HepG2.2.15 cells were found at 48 h after transfection, with significantly the highest inhibition rate of 45.2% for HBsAg and 74.5% for HBeAg expression among the three groups(P<0.01 ). As the transfection time prolonged its inhibition effects were stronger. Conclusion: {sup 125}I-TFO may inhibit the antigen expression of HBV and the growth of hepatocarcinoma cells, thus it may provide a new approach to develop gene-based radiotherapeutic pharmaceuticals for anti-HBV and HCC. (authors)

  6. NMR-based metabolomics reveals that conjugated double bond content and lipid storage efficiency in HepG2 cells are affected by fatty acid cis/trans configuration and chain length

    DEFF Research Database (Denmark)

    Najbjerg, Heidi; Young, Jette F; Bertram, Hanne Christine S.

    2011-01-01

    from conjugated double bonds (5.65, 5.94, and 6.28 ppm) in cells exposed to vaccenic acid, revealing that vaccenic acid upon uptake by the HepG2 cells is converted into a conjugated fatty acid. Upon exposure of the HepG2 cells to either butyric acid (C4:0), caproic acid (C6:0), lauric acid (C12...

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

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Fabao [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); You, Xiaona [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Chi, Xiumei [Department of Hepatology, The First Hospital, Jilin University, Changchun 130021 (China); Wang, Tao [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Niu, Junqi, E-mail: junqiniu@yahoo.com.cn [Department of Hepatology, The First Hospital, Jilin University, Changchun 130021 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2014-02-07

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

  8. Hydrogen sulfide (H{sub 2}S)/cystathionine γ-lyase (CSE) pathway contributes to the proliferation of hepatoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Yan; Ye, Shuang; Yuan, Dexiao; Zhang, Jianghong; Bai, Yang; Shao, Chunlin, E-mail: clshao@shmu.edu.cn

    2014-05-15

    Highlights: • Inhibition of H{sub 2}S/CSE pathway strongly stimulates cellular apoptosis. • Inhibition of H{sub 2}S/CSE pathway suppresses cell growth by blocking EGFR pathway. • H{sub 2}S/CSE pathway is critical for maintaining the proliferation of hepatoma cells. - Abstract: Hydrogen sulfide (H{sub 2}S)/cystathionine γ-lyase (CSE) pathway has been demonstrated to play vital roles in physiology and pathophysiology. However, its role in tumor cell proliferation remains largely unclear. Here we found that CSE over-expressed in hepatoma HepG2 and PLC/PRF/5 cells. Inhibition of endogenous H{sub 2}S/CSE pathway drastically decreased the proliferation of HepG2 and PLC/PRF/5 cells, and it also enhanced ROS production and mitochondrial disruption, pronounced DNA damage and increased apoptosis. Moreover, this increase of apoptosis was associated with the activation of p53 and p21 accompanied by a decreased ratio of Bcl-2/Bax and up-regulation of phosphorylated c-Jun N-terminal kinase (JNK) and caspase-3 activity. In addition, the negative regulation of cell proliferation by inhibition of H{sub 2}S/CSE system correlated with the blockage of cell mitogenic and survival signal transduction of epidermal growth factor receptor (EGFR) via down-regulating the extracellular-signal-regulated kinase 1/2 (ERK1/2) activation. These results demonstrate that H{sub 2}S/CSE and its downstream pathway contribute to the proliferation of hepatoma cells, and inhibition of this pathway strongly suppress the excessive growth of hepatoma cells by stimulating mitochondrial apoptosis and suppressing cell growth signal transduction.

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

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yilin; Yang, Yang; Cai, Yanyan; Liu, Fang; Liu, Yingle; Zhu, Ying [State Key Laboratory of Virology, College of Life Sciences, and Chinese-French Liver Disease Research Institute at Zhongnan Hospital, Wuhan University, Wuhan 430072 (China); Wu, Jianguo, E-mail: jwu@whu.edu.cn [State Key Laboratory of Virology, College of Life Sciences, and Chinese-French Liver Disease Research Institute at Zhongnan Hospital, Wuhan University, Wuhan 430072 (China)

    2011-12-16

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

  10. The effect of agglomeration state of silver and titanium dioxide nanoparticles on cellular response of HepG2, A549 and THP-1 cells.

    Science.gov (United States)

    Lankoff, Anna; Sandberg, Wiggo J; Wegierek-Ciuk, Aneta; Lisowska, Halina; Refsnes, Magne; Sartowska, Bożena; Schwarze, Per E; Meczynska-Wielgosz, Sylwia; Wojewodzka, Maria; Kruszewski, Marcin

    2012-02-05

    Nanoparticles (NPs) occurring in the environment rapidly agglomerate and form particles of larger diameters. The extent to which this abates the effects of NPs has not been clarified. The motivation of this study was to examine how the agglomeration/aggregation state of silver (20nm and 200nm) and titanium dioxide (21nm) nanoparticles may affect the kinetics of cellular binding/uptake and ability to induce cytotoxic responses in THP1, HepG2 and A549 cells. Cellular binding/uptake, metabolic activation and cell death were assessed by the SSC flow cytometry measurements, the MTT-test and the propidium iodide assay. The three types of particles were efficiently taken up by the cells, decreasing metabolic activation and increasing cell death in all the cell lines. The magnitude of the studied endpoints depended on the agglomeration/aggregation state of particles, their size, time-point and cell type. Among the three cell lines tested, A549 cells were the most sensitive to these particles in relation to cellular binding/uptake. HepG2 cells showed a tendency to be more sensitive in relation to metabolic activation. THP-1 cells were the most resistant to all three types of particles in relation to all endpoints tested. Our findings suggest that particle features such as size and agglomeration status as well as the type of cells may contribute to nanoparticles biological impact. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  11. Coating independent cytotoxicity of citrate- and PEG-coated silver nanoparticles on a human hepatoma cell line.

    Science.gov (United States)

    Bastos, Verónica; Ferreira-de-Oliveira, José M P; Carrola, Joana; Daniel-da-Silva, Ana L; Duarte, Iola F; Santos, Conceição; Oliveira, Helena

    2017-01-01

    The antibacterial potential of silver nanoparticles (AgNPs) resulted in their increasing incorporation into consumer, industrial and biomedical products. Therefore, human and environmental exposure to AgNPs (either as an engineered product or a contaminant) supports the emergent research on the features conferring them different toxicity profiles. In this study, 30nm AgNPs coated with citrate or poly(ethylene glycol) (PEG) were used to assess the influence of coating on the effects produced on a human hepatoma cell line (HepG2), namely in terms of viability, apoptosis, apoptotic related genes, cell cycle and cyclins gene expression. Both types of coated AgNPs decreased cell proliferation and viability with a similar toxicity profile. At the concentrations used (11 and 5μg/mL corresponding to IC50 and ~IC10 levels, respectively) the amount of cells undergoing apoptosis was not significant and the apoptotic related genes BCL2 (anti-apoptotic gene) and BAX (pro-apoptotic gene) were both downregulated. Moreover, both AgNPs affected HepG2 cell cycle progression at the higher concentration (11μg/mL) by increasing the percentage of cells in S (synthesis phase) and G2 (Gap 2 phase) phases. Considering the cell-cycle related genes, the expression of cyclin B1 and cyclin E1 genes were decreased. Thus, this work has shown that citrate- and PEG-coated AgNPs impact on HepG2 apoptotic gene expression, cell cycle dynamics and cyclin regulation in a similar way. More research is needed to determine the properties that confer AgNPs at lower toxicity, since their use has proved helpful in several industrial and biomedical contexts. Copyright © 2016. Published by Elsevier B.V.

  12. A high-throughput quantitative expression analysis of cancer-related genes in human HepG2 cells in response to limonene, a potential anticancer agent.

    Science.gov (United States)

    Hafidh, Rand R; Hussein, Saba Z; MalAllah, Mohammed Q; Abdulamir, Ahmed S; Abu Bakar, Fatimah

    2017-11-14

    Citrus bioactive compounds, as active anticancer agent, have been under focus by several studies worldwide. However, the underlying genes responsible for the anticancer potential have not been sufficiently highlighted. The current study investigated the gene expression profile of hepatocellular carcinoma, HepG2, cells after treatment with Limonene. The concentration that killed 50% of HepG2 cells was used to elucidate the genetic mechanisms of limonene anticancer activity. The apoptotic induction was detected by flow cytometry and confocal fluorescence microscope. Two of pro-apoptotic events, caspase-3 activation and phosphatidylserine translocation were manifested by confocal fluorescence microscopy. High-throughput real-time PCR was used to profile 1023 cancer-related genes in 16 different gene families related to the cancer development. In comparison to untreated cells, limonene increased the percentage of apoptotic cells up to 89.61%, by flow cytometry, and 48.2% by fluorescence microscopy. There was a significant limonene-driven differential gene expression of HepG2 cells in 15 different gene families. Limonene was shown to significantly (>2log) up-regulate and down-regulate 14 and 59 genes, respectively. The affected gene families, from most to least affected, were apoptosis induction, signal transduction, cancer genes augmentation, alteration in kinases expression, inflammation, DNA damage repair, and cell cycle proteins. The current study reveals that limonene could be a promising, cheap, and effective anticancer compound. The broad spectrum of limonene anticancer activity is interesting for anticancer drug development. Further research is needed to confirm the current findings and to examine the anticancer potential of limonene along with underlying mechanisms on different cell lines. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

    Directory of Open Access Journals (Sweden)

    Chih-Jung Yao

    2012-01-01

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

  14. Alcohol depletes coenzyme-Q10 associated with increased TNF-alpha secretion to induce cytotoxicity in HepG2 cells

    International Nuclear Information System (INIS)

    Vidyashankar, Satyakumar; Nandakumar, Krishna S.; Patki, Pralhad S.

    2012-01-01

    Highlights: ► Ethanol induced cytotoxicity in HepG2 cells in absence of lipogenesis. ► Ethanol inhibited HMG-CoA reductase activity. ► Ethanol induced HMG-CoA reductase inhibition is due to decreased cell viability. ► Incubation with mevalonate could not increase the cholesterol. ► Cytotoxicity brought about by CoQ10 depletion and increased TNF-alpha. -- Abstract: Alcohol consumption has been implicated to cause severe hepatic steatosis which is mediated by alcohol dehydrogenase (ADH) activity and CYP 450 2E1 expression. In this context, the effect of ethanol was studied for its influence on lipogenesis in HepG2 cell which is deficient of ADH and does not express CYP 450 2E1. The results showed that ethanol at 100 mM concentration caused 40% cytotoxicity at 72 h as determined by MTT assay. The incorporation of labeled [2- 14 C] acetate into triacylglycerol and phospholipid was increased by 40% and 26% respectively upon 24 h incubation, whereas incorporation of labeled [2- 14 C] acetate into cholesterol was not significantly increased. Further, ethanol inhibited HMG-CoA reductase which is a rate-limiting enzyme in the cholesterol biosynthesis. It was observed that, HMG-CoA reductase inhibition was brought about by ethanol as a consequence of decreased cell viability, since incubation of HepG2 cells with mevalonate could not increase the cholesterol content and increase the cell viability. Addition of ethanol significantly increased TNF-alpha secretion and depleted mitochondrial coenzyme-Q 10 which is detrimental for cell viability. But vitamin E (10 mM) could partially restore coenzyme-Q 10 and glutathione content with decreased TNF-alpha secretion in ethanol treated cells. Further, lipid peroxidation, glutathione peroxidase and superoxide dismutase enzyme activities remained unaffected. Ethanol decreased glutathione content while, GSH/GSSG ratio was significantly higher compared to other groups showing cellular pro-oxidant and antioxidant balance remained

  15. JS-K, a nitric oxide prodrug, induces DNA damage and apoptosis in HBV-positive hepatocellular carcinoma HepG2.2.15 cell.

    Science.gov (United States)

    Liu, Zhengyun; Li, Guangmin; Gou, Ying; Xiao, Dongyan; Luo, Guo; Saavedra, Joseph E; Liu, Jie; Wang, Huan

    2017-08-01

    Hepatocellular carcinoma (HCC) is the most important cause of cancer-related death, and 85% of HCC is caused by chronic HBV infection, the prognosis of patients and the reduction of HBV DNA levels remain unsatisfactory. JS-K, a nitric oxide-releasing diazeniumdiolates, is effective against various tumors, but little is known on its effects on HBV positive HCC. We found that JS-K reduced the expression of HBsAg and HBeAg in HBV-positive HepG2.2.15 cells. This study aimed to further examine anti-tumor effects of JS-K on HepG2.2.15 cells. The MTT assay and colony forming assay were used to study the cell growth inhibition of JS-K; scratch assay and transwell assay were performed to detect cell migration. The cell cycle was detected by flow cytometry. The immunofluorescence, flow cytometry analysis, and western blot were used to study DNA damage and cell apoptosis. JS-K inhibited HepG2.2.15 cell growth in a dose-dependent manner, suppressed cell colony formation and migration, arrested cells gather in the G2 phase. JS-K (1-20μM) increased the expression of DNA damage-associated protein phosphorylation H 2 AX (γH 2 AX), phosphorylation of checkpoint kinase 1 (p-Chk1), phosphorylation of checkpoint kinase 2 (p-Chk2), ataxia-telangiectasia mutated (ATM), phosphorylation of ataxia-telangiectasia mutated rad3-related (p-ATR) and apoptotic-associated proteins cleaved caspase-3, cleaved caspase-7, cleaved poly ADP-ribose polymerase (cleaved PARP). The study demonstrated JS-K is effective against HBV-positive HepG2.2.15 cells, the mechanisms are not only related to inhibition of HBsAg and HBeAg secretion, but also related with induction of DNA damage and apoptosis. JS-K is a promising anti-cancer candidate against HBV-positive HCC. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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

    Science.gov (United States)

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

    2008-01-01

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

  17. Eicosapentaenoic acid (EPA) induced apoptosis in HepG2 cells through ROS–Ca{sup 2+}–JNK mitochondrial pathways

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yuanyuan; Han, Lirong [Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economy Technological Development Area, Tianjin 300457 (China); Qi, Wentao [Academy of State Administration of Grain, No.11 Baiwanzhuang Avenue, Xicheng District, Beijing, 100037 (China); Cheng, Dai; Ma, Xiaolei; Hou, Lihua [Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economy Technological Development Area, Tianjin 300457 (China); Cao, Xiaohong, E-mail: caoxh@tust.edu.cn [Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economy Technological Development Area, Tianjin 300457 (China); Wang, Chunling, E-mail: wangchunling@tust.edu.cn [Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economy Technological Development Area, Tianjin 300457 (China)

    2015-01-24

    Highlights: • EPA evoked ROS formation, [Ca{sup 2+}]{sub c} accumulation, the opening of MPTP and the phosphorylation of JNK. • EPA-induced [Ca{sup 2+}]{sub c} elevation was depended on production of ROS. • EPA-induced ROS generation, [Ca{sup 2+}]{sub c} increase, and JNK activated caused MPTP opening. • The apoptosis induced by EPA was related to release of cytochrome C through the MPTP. • EPA induced HepG2 cells apoptosis through ROS–Ca{sup 2+}–JNK mitochondrial pathways. - Abstract: Eicosapentaenoic acid (EPA), a well-known dietary n−3 PUFAS, has been considered to inhibit proliferation of tumor cells. However, the molecular mechanism related to EPA-induced liver cancer cells apoptosis has not been reported. In this study, we investigated the effect of EPA on HepG2 cells proliferation and apoptosis mechanism through mitochondrial pathways. EPA inhibited proliferation of HepG2 cells in a dose-dependent manner and had no significant effect on the cell viability of humor normal liver L-02 cells. It was found that EPA initially evoked ROS formation, leading to [Ca{sup 2+}]{sub c} accumulation and the mitochondrial permeability transition pore (MPTP) opening; EPA-induced HepG2 cells apoptosis was inhibited by N-acetylcysteine (NAC, an inhibitor of ROS), 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium) and CsA (inhibitor of MPTP). The relationship between ROS production, the increase of cytoplasmic Ca and MPTP opening was detected. It seems that ROS may act as an upstream regulator of EPA-induced [Ca{sup 2+}]{sub c} generation, moreover, generation of ROS, overload of mitochondrial [Ca{sup 2+}]{sub c}, and JNK activated cause the opening of MPTP. Western blotting results showed that EPA elevated the phosphorylation status of JNK, processes associated with the ROS generation. Simultaneously, the apoptosis induced by EPA was related to release of cytochrome C from mitochondria to cytoplasm through the MPTP

  18. Concurrent acetylation of FoxO1/3a and p53 due to sirtuins inhibition elicit Bim/PUMA mediated mitochondrial dysfunction and apoptosis in berberine-treated HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Shukla, Shatrunajay [Herbal Research Section, CSIR — Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow‐226001 (India); Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi ‐110062 (India); Sharma, Ankita [Herbal Research Section, CSIR — Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow‐226001 (India); Pandey, Vivek Kumar [Herbal Research Section, CSIR — Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow‐226001 (India); Academy of Scientific and Innovative Research (India); Raisuddin, Sheikh [Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi ‐110062 (India); Kakkar, Poonam, E-mail: kakkarp59@gmail.com [Herbal Research Section, CSIR — Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow‐226001 (India); Academy of Scientific and Innovative Research (India)

    2016-01-15

    Post-translational modifications i.e. phosphorylation and acetylation are pivotal requirements for proper functioning of eukaryotic proteins. The current study aimed to decode the impact of acetylation/deacetylation of non-histone targets i.e. FoxO1/3a and p53 of sirtuins (NAD{sup +} dependent enzymes with lysine deacetylase activity) in berberine treated human hepatoma cells. Berberine (100 μM) inhibited sirtuins significantly (P < 0.05) at transcriptional level as well as at translational level. Combination of nicotinamide (sirtuin inhibitor) with berberine potentiated sirtuins inhibition and increased the expression of FoxO1/3a and phosphorylation of p53 tumor suppressor protein. As sirtuins deacetylate non-histone targets including FoxO1/3a and p53, berberine increased the acetylation load of FoxO1/3a and p53 proteins. Acetylated FoxO and p53 proteins transcriptionally activate BH3-only proteins Bim and PUMA (3.89 and 3.87 fold respectively, P<0.001), which are known as direct activator of pro-apoptotic Bcl-2 family protein Bax that culminated into mitochondria mediated activation of apoptotic cascade. Bim/PUMA knock-down showed no changes in sirtuins' expression while cytotoxicity induced by berberine and nicotinamide was curtailed up to 28.3% (P < 0.001) and it restored pro/anti apoptotic protein ratio in HepG2 cells. Sirtuins inhibition was accompanied by decline in NAD{sup +}/NADH ratio, ATP generation, enhanced ROS production and decreased mitochondrial membrane potential. TEM analysis confirmed mitochondrial deterioration and cell damage. SRT-1720 (1–10 μM), a SIRT-1 activator, when pre-treated with berberine (25 μM), reversed sirtuins expression comparable to control and significantly restored the cell viability (P < 0.05). Thus, our findings suggest that berberine mediated sirtuins inhibition resulting into FoxO1/3a and p53 acetylation followed by BH3-only protein Bim/PUMA activation may in part be responsible for mitochondria

  19. Fluoro-sorafenib (Regorafenib) effects on hepatoma cells: growth inhibition, quiescence and recovery

    Science.gov (United States)

    Carr, Brian I.; Cavallini, Aldo; Lippolis, Catia; D’Alessandro, Rosalba; Messa, Caterina; Refolo, Maria Grazia; Tafaro, Angela

    2015-01-01

    To evaluate the growth-inhibitory properties of the potent multi-kinase antagonist Regorafenib (Fluoro-Sorafenib), which was synthesized as a more potent Sorafenib, a Raf inhibitor and to determine whether similar mechanisms were involved, human hepatoma cell lines were grown in the presence or absence of Regorafanib and examined for growth inhibition. Western blots were performed for Raf targets, for apoptosis and autophagy. Regorafenib inhibited growth of human Hep3B, PLC/PRF/5 and HepG2 cells in a concentration- and time-dependent manner. Multiple signaling pathways were altered, including MAP kinases phospho-ERK and phospho-JNK and its target phospho-c-Jun. There was evidence for apoptosis by FACS, cleavage of caspases and increased Bax levels; as well as induction of autophagy, as judged by increased Beclin-1 and LC3 (II) levels. Prolonged drug exposure resulted in cell quiescence. Full growth recovery occurred after drug removal, unlike with doxorubicin chemotherapy. Regorafenib is a potent inhibitor of cell growth. Cells surviving Regorafenib treatment remain viable, but quiescent and capable of regrowth following drug removal. The reversibility of tumor cell growth suppression after drug removal may have clinical implications. PMID:22777740

  20. Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro

    International Nuclear Information System (INIS)

    Dykens, James A.; Jamieson, Joseph; Marroquin, Lisa; Nadanaciva, Sashi; Billis, Puja A.; Will, Yvonne

    2008-01-01

    As a class, the biguanides induce lactic acidosis, a hallmark of mitochondrial impairment. To assess potential mitochondrial impairment, we evaluated the effects of metformin, buformin and phenformin on: 1) viability of HepG2 cells grown in galactose, 2) respiration by isolated mitochondria, 3) metabolic poise of HepG2 and primary human hepatocytes, 4) activities of immunocaptured respiratory complexes, and 5) mitochondrial membrane potential and redox status in primary human hepatocytes. Phenformin was the most cytotoxic of the three with buformin showing moderate toxicity, and metformin toxicity only at mM concentrations. Importantly, HepG2 cells grown in galactose are markedly more susceptible to biguanide toxicity compared to cells grown in glucose, indicating mitochondrial toxicity as a primary mode of action. The same rank order of potency was observed for isolated mitochondrial respiration where preincubation (40 min) exacerbated respiratory impairment, and was required to reveal inhibition by metformin, suggesting intramitochondrial bio-accumulation. Metabolic profiling of intact cells corroborated respiratory inhibition, but also revealed compensatory increases in lactate production from accelerated glycolysis. High (mM) concentrations of the drugs were needed to inhibit immunocaptured respiratory complexes, supporting the contention that bioaccumulation is involved. The same rank order was found when monitoring mitochondrial membrane potential, ROS production, and glutathione levels in primary human hepatocytes. In toto, these data indicate that biguanide-induced lactic acidosis can be attributed to acceleration of glycolysis in response to mitochondrial impairment. Indeed, the desired clinical outcome, viz., decreased blood glucose, could be due to increased glucose uptake and glycolytic flux in response to drug-induced mitochondrial dysfunction

  1. Up-Regulation of CYP2C19 Expression by BuChang NaoXinTong via PXR Activation in HepG2 Cells.

    Directory of Open Access Journals (Sweden)

    Hong Sun

    Full Text Available Cytochrome P450 2C19 (CYP2C19 is an important drug-metabolizing enzyme (DME, which is responsible for the biotransformation of several kinds of drugs such as proton pump inhibitors, platelet aggregation inhibitors and antidepressants. Previous studies showed that Buchang NaoXinTong capsules (NXT increased the CYP2C19 metabolic activity in vitro and enhanced the antiplatelet effect of clopidogrel in vivo. However, the underlying molecular mechanism remained unclear. In the present study, we examined whether Pregnane X receptor (PXR plays a role in NXT-mediated regulation of CYP2C19 expression.We applied luciferase assays, real-time quantitative PCR (qPCR, Western blotting and cell-based analysis of metabolic activity experiments to investigate the NXT regulatory effects on the CYP2C19 promoter activity, the mRNA/ protein expression and the metabolic activity.Our results demonstrated that NXT significantly increased the CYP2C19 promoter activity when co-transfected with PXR in HepG2 cells. Mutations in PXR responsive element abolished the NXT inductive effects on the CYP2C19 promoter transcription. Additionally, NXT incubation (150 and 250μg/mL also markedly up-regulated endogenous CYP2C19 mRNA and protein levels in PXR-transfected HepG2 cells. Correspondingly, NXT leaded to a significant enhancement of the CYP2C19 catalytic activity in PXR-transfected HepG2 cells.In summary, this is the first study to suggest that NXT could induce CYP2C19 expression via PXR activation.

  2. Inhibition of Cholesterol Synthesis in HepG2 Cells by GINST-Decreasing HMG-CoA Reductase Expression Via AMP-Activated Protein Kinase.

    Science.gov (United States)

    Han, Joon-Seung; Sung, Jong Hwan; Lee, Seung Kwon

    2017-11-01

    GINST, a hydrolyzed ginseng extract, has been reported to have antidiabetic effects and to reduce hyperglycemia and hyperlipidemia. Hypercholesterolemia is caused by diet or genetic factors and can lead to atherosclerosis and coronary heart disease. Thus, the purpose of this study is to determine whether GINST and the ginsenoside metabolite, IH-901 (compound K), reduce cholesterol synthesis in HepG2 cells and the signal transduction pathways involved. Concentrations of cholesterol were measured by using an enzymatic method. Expression levels of sterol regulatory element-binding protein 2 (SREBP2), HMG-CoA reductase (HMGCR), peroxisome proliferators-activated receptor γ (PPARγ), CCAAT/enhancer-binding proteins α (C/EBPα), GAPDH, and phosphorylation of AMP-activated protein kinase α (AMPKα), protein kinase B (PKB, also known as Akt), and mechanistic target of rapamycin complex 1 (mTORC1) were measured using western blot. Total cholesterol concentration decreased after GINST treatment for 24 and 48 h. Expression of HMGCR decreased more with GINST than with the inhibitors, U18666A and atorvastatin, after 48 h in a dose-dependent manner. Phosphorylation of AMPKα increased 2.5x by GINST after 360 min of treatment, and phosphorylation of Akt decreased after 120 and 360 min. We separated compound K from GINST extracts flash chromatography. Compound K decreased cholesterol synthesis in HepG2 cells at 24 and 48 h. Therefore, we conclude that GINST inhibits cholesterol synthesis in HepG2 cells by decreasing HMGCR expression via AMPKα activation. GINST, a hydrolyzed ginseng extract, can inhibit cholesterol synthesis in liver cells via activation of AMPKα. IH-901 (compound K), which is the main component with bioactivity in GINST, also has anticholesterol effects. Thus, we suggest that GINST can be used to reduce hypercholesterolemia. © 2017 Institute of Food Technologists®.

  3. Transmission of HCV to a chimpanzee using virus particles produced in an RNA-transfected HepG2 cell culture.

    Science.gov (United States)

    Dash, S; Kalkeri, G; McClure, H M; Garry, R F; Clejan, S; Thung, S N; Murthy, K K

    2001-10-01

    It was demonstrated previously that HepG2 cells produce negative strand RNA and virus-like particles after transfection with RNA transcribed from a full-length hepatitis C virus (HCV) cDNA clone [Dash et al. (1997) American Journal of Pathology, 151:363-373]. To determine in vivo infectivity of these in vitro synthesized viral particles, a chimpanzee was inoculated intravenously with HCV derived from HepG2 cells. The infected chimpanzee was examined serially for elevation of liver enzymes, for the presence of HCV RNA in the serum by reverse transcription nested polymerase chain reaction (RT-PCR), anti-HCV antibodies in the serum, and inflammation in the liver. The chimpanzee developed elevated levels of liver enzymes after the second week, but the levels fluctuated over a 10-week period. HCV RNA was detected in the serum of the chimpanzee at the second, seventh and ninth weeks after inoculation, and remained positive up to 25 weeks. Liver biopsies at Weeks 18 and 19 revealed of mild inflammation. Nucleotide sequence analysis of HCV recovered from the infected chimpanzee at the second and ninth weeks showed 100% sequence homology with the clone used for transfection studies. Serum anti-HCV antibodies were not detected by EIA during the 25 weeks follow-up period. These results suggest that intravenous administration of the virus-like particles derived from RNA-transfected HepG2 cells are infectious, and therefore, the pMO9.6-T7 clone is an infectious clone. These results provide new information that in vitro synthesized HCV particles produced from full-length HCV clone can cause infection in a chimpanzee. This study will facilitate the use of innovative approaches to the study of assembly of HCV particles and mechanisms of virus infectivity in cell culture. Copyright 2001 Wiley-Liss, Inc.

  4. A ginseng saponin metabolite-induced apoptosis in HepG2 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and Bid cleavage

    International Nuclear Information System (INIS)

    Oh, Seon-Hee; Lee, Byung-Hoon

    2004-01-01

    20-O-(β-D-Glucopyranosyl)-20(S)-protopanaxadiol (IH901), an intestinal bacterial metabolite of ginseng saponin formed from ginsenosides Rb1, Rb2, and Rc, is suggested to be a potential chemopreventive agent. Here, we show that IH901 induces apoptosis in human hepatoblastoma HepG2 cells. IH901 led to an early activation of procaspase-3 (12 h posttreatment), and the activation of caspase-8 became evident only later (18 h posttreatment). Caspase activation was a necessary requirement for apoptosis because caspase inhibitors significantly inhibited cell death by IH901. Treatment of HepG2 cells with IH901 also induced the cleavage of cytosolic factors such as Bid and Bax and translocation of truncated Bid (tBid) to mitochondria. A time-dependent release of cytochrome c from mitochondria was observed, which was accompanied by activation of caspase-9. A broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), and a specific inhibitor for caspase-8, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone (zIETD-fmk), abrogated Bid processing and translocation, and caspase-3 activation. Cytochrome c release was inhibited by zVAD-fmk, however, the inhibition by zIETD-fmk was not complete. The activation of caspase-8 was inhibited not only by zIETD-fmk but also by zVAD-fmk. The results, together with the kinetic change of caspase activation, indicate that activation of caspase-8 occurred downstream of caspase-3 and -9. Our data suggest that the activation of caspase-8 after early caspase-3 activation might act as an amplification loop necessary for successful apoptosis. Primary hepatocytes isolated from normal Sprague-Dawley rats were not affected by IH901 (0-60 μM). The very low toxicity in normal hepatocytes and high activity in hepatoblastoma HepG2 cells suggest that IH901 is a promising experimental cancer chemopreventive agent

  5. UHPLC-ESI-MS Analysis of Purified Flavonoids Fraction from Stem of Dendrobium denneaum Paxt. and Its Preliminary Study in Inducing Apoptosis of HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Chunhua Zhou

    2018-01-01

    Full Text Available Dendrobium denneaum paxt., which has been widely used for health prevention in traditional Chinese medicine (TCM, is one of the most popular tonic herbs in China. In order to analyze its flavonoids, characterization and antitumor activity of crude extract and flavonoids rich fractions from D. denneaum paxt. were investigated. Flavonoids extracted from D. denneaum paxt. were clearly enriched in fraction II after determining the total flavonoids content; there were 15 characteristic peaks which have been detected; ultra-high performance liquid chromatography-electrospray ionization/mass spectrometry (UHPLC-ESI-MS/MS was applied for structural elucidation of compounds. 13 characteristic peaks including flavonoid-O-glycosides and flavonoid-C-glycosides were determined or preliminarily characterized through comparing retention times and UV and MS spectra with standard compounds or documented literature. The antitumor activity of fraction II on human liver cancer cells HepG2 was investigated. MTT assay method was used to test the antiproliferation activity and to confirm the appropriate treatment concentration as well as inducing time. The morphological changes of the apoptosis cells after being induced by fraction II were observed by a Hoechst reagent and the apoptosis rate was tested by flow cytometry. The results showed that fraction II can inhibit HepG2 cells from proliferation in a dose-dependent and time-dependent manner. The apoptosis experiments indicated that fraction II can significantly induce apoptosis in HepG2 cells in a concentration over 50 μg/mL for 48 h and the most effective level was 150 μg/mL for 48 h.

  6. Protective Effects of Maillard Reaction Products of Whey Protein Concentrate against Oxidative Stress through an Nrf2-Dependent Pathway in HepG2 Cells.

    Science.gov (United States)

    Pyo, Min Cheol; Yang, Sung-Yong; Chun, Su-Hyun; Oh, Nam Su; Lee, Kwang-Won

    2016-09-01

    Whey protein concentrate (WPC), which contains α-lactalbumin and β-lactoglobulin, is utilized widely in the food industry. The Maillard reaction is a complex reaction that produces Maillard reaction products (MRPs), which are associated with the formation of antioxidant compounds. In this study, the hepatoprotection activity of MRPs of WPC against oxidative stress through the nuclear factor-E2-related factor 2 (Nrf2)-dependent antioxidant pathway in HepG2 cells was examined. Glucose-whey protein concentrate conjugate (Glc-WPC) was obtained from Maillard reaction between WPC and glucose. The fluorescence intensity of Glc-WPC increased after 7 d compared to native WPC, and resulted in loss of 48% of the free amino groups of WPC. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of Glc-WPC showed the presence of a high-molecular-weight portion. Treatment of HepG2 cells with Glc-WPC increased cell viability in the presence of oxidative stress, inhibited the generation of intracellular reactive oxygen species by tert-butyl hydroperoxide (t-BHP), and increased the glutathione level. Nrf2 translocation and Nrf2, reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H)-quinone oxidoreductase 1 (NOQ1), heme oxygenase-1 (HO-1), glutamate-L-cysteine ligase (GCL)M and GCLC mRNA levels were increased by Glc-WPC. Also, Glc-WPC increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK). The results of this study demonstrate that Glc-WPC activates the Nrf2-dependent pathway through the phosphorylation of ERK1/2 and JNK in HepG2 cells, and induces production of antioxidant enzymes and phase II enzymes.

  7. Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells.

    Science.gov (United States)

    Capraro, Jessica; Magni, Chiara; Faoro, Franco; Maffi, Dario; Scarafoni, Alessio; Tedeschi, Gabriella; Maffioli, Elisa; Parolari, Anna; Manzoni, Cristina; Lovati, Maria Rosa; Duranti, Marcello

    2013-08-09

    Lupin seed γ-Conglutin is a protein capable of reducing glycaemia in mammalians and increasing glucose uptake by model cells. This work investigated whether γ-Conglutin is internalised into the target cells and undergoes any covalent change during the process, as a first step to understanding its mechanism of action. To this purpose, γ-Conglutin-treated and untreated HepG2 cells were submitted to confocal and transmission electron microscopy. Immune-revelation of γ-Conglutin at various intervals revealed its accumulation inside the cytosol. In parallel, 2D-electrophoresis of the cell lysates and antibody reaction of the blotted maps showed the presence of the protein intact subunits inside the treated cells, whilest no trace of the protein was found in the control cells. However, γ-Conglutin-related spots with an unexpectedly low pI were also observed in the maps. These spots were excised, trypsin-treated and submitted to MS/MS spectrometric analysis. The presence of phosphorylated amino acids was detected. These findings, by showing that γ-Conglutin is internalised by HepG2 cells in an intact form and is modified by multiple phosphorylation, open the way to the understanding of the lupin γ-Conglutin insulin-mimetic activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. The preparation of a radionuclide labeled peptide {sup 125}I-WH16 and its characters of binding to a human liver cancer cell line HepG2 in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Sha, Luo; Xiaohua, Zhu; Hua, Wu [Department of Nuclear Medicine, Tongji Hospital, Tongji Medical College, Huazhong Univ. of Science and Technolgoy, Wuhan (China); Bing, Jia; Jing, Du; Fan, Wang

    2004-12-15

    Objective: To investigate the binding characters of a radionuclide labeled peptide {sup 125}I-WH16 which is affinitive to hepatocarcinoma cells in order to explore the potential feasibility of this artificially synthesized peptide to be a targeting reagent in diagnosis and therapy of liver cancer. Methods: 1) WH16 was labeled with Na{sup 125}I using Iodogen method, then isolated and identified with HPLC; 2)a. The tests of cell number (or time of incubation)- to-binding counts between {sup 125}I-WH16 and HepG2 cells were carried out in order to obtain better conditions for next in vitro tests; b. The average binding counts of {sup 125}I-WH16 treated HepG2 cells and L02 cells were compared in order to inspect the binding specificity between {sup 125}I-WH16 and HepG2 cells; c. A test of saturation of binding between {sup 125}I-WH16 and HepG2 cells was carried out in order to investigate the binding affinity between {sup 125}I-WH16 and HepG2 cells. Results: 1) The labeling rate of {sup 125}I was 50%. The specific activity of {sup 125}I-WH16 was 8.21x10{sup 15} Bq/mol. The radiochemical purity was 95% and the remnant radiochemical purity after a storage for 24 h at -20 degree C was 95%. The radioactive concentration was 6.64 x 10{sup 9} Bq/ L; 2) a. The binding of {sup 125}I-WH16 to HepG2 cells was cell number dependent and the optimal time of incubation was 3 h; b. There were obvious differences between HepG2 cells and L02 cells in binding with {sup 125}I-WH16; c. The binding of {sup 125}I-WH16 to HepG2 cells showed saturability. Scatchard plotting suggested that HepG2 cells contained only one type of WH16 receptors. The concentrations of Kd and Bmax were (1.42 {+-} 0.28) nmol/L and (12.15 {+-} 0.63) pmol/L, respectively. Hill modulus from Hill plotting was 1.03, which was close to 1 and suggesting that one receptor may bind only one ligand molecule. Conclusions: The present study indicates that the preparation of {sup 125}I-WH16 is stable and has good specificity and

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Wei, E-mail: detachedy@yahoo.com.cn [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China); Sun, Ting [Brain and Nerve Research Laboratory, The First Affiliated Hospital, Soochow University, Suzhou (China); Cao, Jianping; Liu, Fenju [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China); Tian, Ye [Department of Radiotherapy and Oncology, The Second Affiliated Hospital, Soochow University, Suzhou (China); Zhu, Wei [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China)

    2012-05-01

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

  11. Anticancer Effects of 1,3-Dihydroxy-2-Methylanthraquinone and the Ethyl Acetate Fraction of Hedyotis Diffusa Willd against HepG2 Carcinoma Cells Mediated via Apoptosis.

    Directory of Open Access Journals (Sweden)

    Yun-Lan Li

    Full Text Available Hedyotis Diffusa Willd, used in Traditional Chinese Medicine, is a treatment for various diseases including cancer, owing to its mild effectiveness and low toxicity. The aim of this study was to identify the main anticancer components in Hedyotis Diffusa Willd, and explore mechanisms underlying their activity. Hedyotis Diffusa Willd was extracted and fractionated using ethyl acetate to obtain the H-Ethyl acetate fraction, which showed higher anticancer activity than the other fractions obtained against HepG2 cells with sulforhodamine B assays. The active component of the H-Ethyl acetate fraction was identified to be 1,3-dihydroxy-2-methylanthraquinone (DMQ with much high inhibitory rate up to 48.9 ± 3.3% and selectivity rate up to 9.4 ± 4.5 folds (p<0.01 at 125 μmol/L. HepG2 cells treated with the fraction and DMQ visualized morphologically using light and fluorescence microscopy. Annexin V--fluorescein isothiocyanate / propidium iodide staining flow cytometry, DNA ladder and cell cycle distribution assays. Mechanistic studies showed up-regulation of caspase-3, -8, and -9 proteases activities (p<0.001, indicating involvement of mitochondrial apoptotic and death receptor pathways. Further studies revealed that reactive oxygen species in DMQ and the fraction treated HepG2 cells increased (p<0.01 while mitochondrial membrane potential reduced significantly (p<0.001 compared to the control by flow cytometry assays. Western blot analysis showed that Bax, p53, Fas, FasL, p21 and cytoplasmic cytochrome C were up-regulated (p<0.01, while Bcl-2, mitochondrial cytochrome C, cyclin E and CDK 2 were down-regulated dose-dependently (p<0.01. The reverse transcriptase-polymerase chain reaction showed that mRNA expressions of p53 and Bax increased (p<0.001 while that of Bcl-2 decreased (p<0.001. Pre-treatment with caspase-8 inhibitor Z-IETD-FMK, or caspase-9 inhibitor Z-LEHD-FMK, attenuated the growth-inhibitory and apoptosis-inducing effects of DMQ and the

  12. Palmitic acid suppresses apolipoprotein M gene expression via the pathway of PPAR{sub β/δ} in HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Guanghua; Shi, Yuanping; Zhang, Jun; Mu, Qinfeng; Qin, Li; Zheng, Lu; Feng, Yuehua [Comprehensive Laboratory, The Third Affiliated Hospital of Soochow University, Changzhou 213003 (China); Berggren-Söderlund, Maria; Nilsson-Ehle, Peter [Division of Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, S-221 85 Lund (Sweden); Zhang, Xiaoying, E-mail: zhangxy6689996@163.com [Department of Cardiothoracic Surgery, The Third Affiliated Hospital of Soochow University, Changzhou 213003 (China); Xu, Ning, E-mail: ning.xu@med.lu.se [Division of Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, S-221 85 Lund (Sweden)

    2014-02-28

    Highlights: • Palmitic acid significantly inhibited APOM gene expression in HepG2 cells. • Palmitic acid could obviously increase PPARB/D mRNA levels in HepG2 cells. • PPAR{sub β/δ} antagonist, GSK3787, had no effect on APOM expression. • GSK3787 could reverse the palmitic acid-induced down-regulation of APOM expression. • Palmitic acid induced suppression of APOM expression is mediated via the PPAR{sub β/δ} pathway. - Abstract: It has been demonstrated that apolipoprotein M (APOM) is a vasculoprotective constituent of high density lipoprotein (HDL), which could be related to the anti-atherosclerotic property of HDL. Investigation of regulation of APOM expression is of important for further exploring its pathophysiological function in vivo. Our previous studies indicated that expression of APOM could be regulated by platelet activating factor (PAF), transforming growth factors (TGF), insulin-like growth factor (IGF), leptin, hyperglycemia and etc., in vivo and/or in vitro. In the present study, we demonstrated that palmitic acid could significantly inhibit APOM gene expression in HepG2 cells. Further study indicated neither PI-3 kinase (PI3K) inhibitor LY294002 nor protein kinase C (PKC) inhibitor GFX could abolish palmitic acid induced down-regulation of APOM expression. In contrast, the peroxisome proliferator-activated receptor beta/delta (PPAR{sub β/δ}) antagonist GSK3787 could totally reverse the palmitic acid-induced down-regulation of APOM expression, which clearly demonstrates that down-regulation of APOM expression induced by palmitic acid is mediated via the PPAR{sub β/δ} pathway.

  13. Antihyperglycemia and Antihyperlipidemia Effect of Protoberberine Alkaloids From Rhizoma Coptidis in HepG2 Cell and Diabetic KK-Ay Mice.

    Science.gov (United States)

    Ma, Hang; Hu, Yinran; Zou, Zongyao; Feng, Min; Ye, Xiaoli; Li, Xuegang

    2016-06-01

    Preclinical Research Rhizoma Coptidis (RC), the root of Coptis chinensis Franch, a species in the genus Coptis (family Ranunculaceae), has been commonly prescribed for the treatment of diabetes in Chinese traditional herbal medicine applications. The present study is focused on the assessment of the antihyperglycemia and antidiabetic hyperlipidemia effect of five protoberberine alkaloids, berberine (BBR), coptisine (COP), palmatine (PAL), epiberberine (EPI), and jatrorrhizine (JAT), separated from R. Coptidis in hepatocellular carcinoma HepG2 cells and diabetic KK-Ay mice. Protoberberine alkaloids are effective in modulating hyperglycemia and hyperlipidemia. After adding BBR and COP to culture medium, glucose consumption of HepG2 cells was increased. In KK-Ay mice assays, suppressed fasting blood glucose level and ameliorated glucose tolerance were observed after BBR/COP administration. After treated with berberine and coptisine, in the same dose of 5 µg/mL, the glucose consumption of HepG2 cells were promoted and, respectively, reached 96.1% and 17.6%. Body weight, food consumption, water intake, and urinary output of KK-Ay mice were reduced after treated with EPI. Serum total cholesterol and triglyceride of mice were decreased after treated with palmatine and jatrorrhizine. Serum high-density lipoprotein cholesterol of mice was increased after palmatine, jatrorrhizine, and berberine administrated. Moreover, hepatomegaly was attenuated in JTR-treated mice. Suggested that these protoberberine alkaloids from R. Coptidis have potential curative effect for diabetes. Drug Dev Res 77 : 163-170, 2016.   © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. ApoB-100 secretion by HepG2 cells is regulated by the rate of triglyceride biosynthesis but not by intracellular lipid pools.

    Science.gov (United States)

    Benoist, F; Grand-Perret, T

    1996-10-01

    Triglycerides (TGs), cholesteryl esters (CEs), cholesterol, and phosphatidylcholine have been independently proposed as playing regulatory roles in apoB-100 secretion; the results depended on the cellular model used. In this study, we reinvestigate the role of lipids in apoB-100 production in HepG2 cells and in particular, we clarify the respective roles of intracellular mass and the biosynthesis of lipids in the regulation of apoB-100 production. In a first set of experiments, the pool size of cholesterol, CEs, and TGs was modulated by a 3-day treatment with either lipid precursors or inhibitors of enzymes involved in lipid synthesis. We used simvastatin (a hydroxymethylglutaryl coenzyme A reductase inhibitor), 58-035 (an acyl coenzyme A cholesterol acyltransferase inhibitor), 5-tetradecyloxy-2-furancarboxylic acid (TOFA, an inhibitor of fatty acid synthesis), and oleic acid. The secretion rate of apoB-100 was not affected by the large modulation of lipid mass induced by these various pre-treatments. In a second set of experiments, the same lipid modulators were added during a 4-hour labeling period. Simvastatin and 58-035 inhibited cholesterol and CE synthesis without affecting apoB-100 secretion. By contrast, treatment of HepG2 cells with TOFA resulted in the inhibition of TG synthesis and apoB-100 secretion. This effect was highly specific for apoB-100 and was reversed by adding oleic acid, which stimulated both TG synthesis and apoB-100 secretion. Moreover, a combination of oleic acid and 58-035 inhibited CE biosynthesis and increased both TG synthesis and apoB-100 secretion. These results show that in HepG2 cells TG biosynthesis regulates apoB-100 secretion, whereas the rate of cholesterol or CE biosynthesis has no effect.

  15. Effect of diphenyl ether herbicides and oxadiazon on porphyrin biosynthesis in mouse liver, rat primary hepatocyte culture and HepG2 cells.

    Science.gov (United States)

    Krijt, J; van Holsteijn, I; Hassing, I; Vokurka, M; Blaauboer, B J

    1993-01-01

    The effects of the herbicides fomesafen, oxyfluorfen, oxadiazon and fluazifop-butyl on porphyrin accumulation in mouse liver, rat primary hepatocyte culture and HepG2 cells were investigated. Ten days of herbicide feeding (0.25% in the diet) increased the liver porphyrins in male C57B1/6J mice from 1.4 +/- 0.6 to 4.8 +/- 2.1 (fomesafen) 16.9 +2- 2.9 (oxyfluorfen) and 25.9 +/- 3.1 (oxadiazon) nmol/g wet weight, respectively. Fluazifop-butyl had no effect on liver porphyrin metabolism. Fomesafen, oxyfluorfen and oxadiazon increased the cellular porphyrin content of rat hepatocytes after 24 h of incubation (control, 3.2 pmol/mg protein, fomesafen, oxyfluorfen and oxadiazon at 0.125 mM concentration 51.5, 54.3 and 44.0 pmol/mg protein, respectively). The porphyrin content of HepG2 cells increased from 1.6 to 18.2, 10.6 and 9.2 pmol/mg protein after 24 h incubation with the three herbicides. Fluazifop-butyl increased hepatic cytochrome P450 levels and ethoxy- and pentoxyresorufin O-dealkylase (EROD and PROD) activity, oxyfluorfen increased PROD activity. Peroxisomal palmitoyl CoA oxidation increased after fomesafen and fluazifop treatment to about 500% of control values both in mouse liver and rat hepatocytes. Both rat hepatocytes and HepG2 cells can be used as a test system for the porphyrogenic potential of photobleaching herbicides.

  16. Protective Effects of Black Rice Extracts on Oxidative Stress Induced by tert-Butyl Hydroperoxide in HepG2 Cells

    Science.gov (United States)

    Lee, Seon-Mi; Choi, Youngmin; Sung, Jeehye; Kim, Younghwa; Jeong, Heon-Sang; Lee, Junsoo

    2014-01-01

    Black rice contains many biologically active compounds. The aim of this study was to investigate the protective effects of black rice extracts (whole grain extract, WGE and rice bran extract, RBE) on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. Cellular reactive oxygen species (ROS), antioxidant enzyme activities, malondialdehyde (MDA) and glutathione (GSH) concentrations were evaluated as biomarkers of cellular oxidative status. Cells pretreated with 50 and 100 μg/mL of WGE or RBE were more resistant to oxidative stress in a dose-dependent manner. The highest WGE and BRE concentrations enhanced GSH concentrations and modulated antioxidant enzyme activities (glutathione reductase, glutathione-S-transferase, catalase, and superoxide dismutase) compared to TBHP-treated cells. Cells treated with RBE showed higher protective effect compared to cells treated with WGE against oxidative insult. Black rice extracts attenuated oxidative insult by inhibiting cellular ROS and MDA increase and by modulating antioxidant enzyme activities in HepG2 cells. PMID:25580401

  17. Aryl hydrocarbon receptor activation and CYP1A induction by cooked food-derived carcinogenic heterocyclic amines in human HepG2 cell lines.

    Science.gov (United States)

    Sekimoto, Masashi; Sumi, Haruna; Hosaka, Takuomi; Umemura, Takashi; Nishikawa, Akiyoshi; Degawa, Masakuni

    2016-11-01

    The ability of nine cooked food-derived heterocyclic aromatic amines (HCAs), such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-6-methylpyrido[12-a:3',2'-d]imidazole (Glu-P-1), 2-amino-pyrido[12-a:3',2'-d]imidazole hydrochloride (Glu-P-2), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridine (PhIP), to activate human aryl hydrocarbon receptor (hAhR) was examined using a HepG2-A10 cell line, which has previously established from human hepatocarcinoma-derived HepG2 cells for use in hAhR-based luciferase reporter gene assays. Trp-P-1, Trp-P-2, AαC, MeAαC, IQ and MeIQx showed a definite ability to induce not only luciferase (hAhR activation) in HepG2-A10 cells but also cytochrome P450 (CYP)1A1/1A2 mRNAs in HepG2 cells, while such the ability of Glu-P-1, Glu-P-2, and PhIP was very low. In addition, all the HCAs examined, especially MeAαC and MeIQx, had a definite capacity for inhibiting the activity of ethoxyresorfin O-deethylase (CYP1As, especially CYP1A1). The present findings demonstrate that all the HCAs examined have the ability to activate hAhR and its target genes, and further confirm that these HCAs become good substrates for human CYP1A subfamily enzyme(s). Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2005-10-15

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

  19. Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

    International Nuclear Information System (INIS)

    Atienzar, Franck A.; Novik, Eric I.; Gerets, Helga H.; Parekh, Amit; Delatour, Claude; Cardenas, Alvaro; MacDonald, James; Yarmush, Martin L.; Dhalluin, Stéphane

    2014-01-01

    Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine model along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes

  20. Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

    Energy Technology Data Exchange (ETDEWEB)

    Atienzar, Franck A., E-mail: franck.atienzar@ucb.com [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); Novik, Eric I. [H mu rel Corporation, 675 U.S. Highway 1, North Brunswick, NJ 08902 (United States); Gerets, Helga H. [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); Parekh, Amit [H mu rel Corporation, 675 U.S. Highway 1, North Brunswick, NJ 08902 (United States); Delatour, Claude; Cardenas, Alvaro [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); MacDonald, James [Chrysalis Pharma Consulting, LLC, 385 Route 24, Suite 1G, Chester, NJ 07930 (United States); Yarmush, Martin L. [Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854 (United States); Dhalluin, Stéphane [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium)

    2014-02-15

    Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine model along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes.

  1. Trichloroethylene toxicity in a human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Thevenin, E.; McMillian, J. [Medical Univ. of Charleston South Carolina, SC (United States)

    1994-12-31

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

  2. Antiproliferative Effect of the Isoquinoline Alkaloid Papaverine in Hepatocarcinoma HepG-2 Cells — Inhibition of Telomerase and Induction of Senescence

    Directory of Open Access Journals (Sweden)

    Sakineh Kazemi Noureini

    2014-08-01

    Full Text Available Cancer cells are often immortal through up-regulation of the hTERT gene, which encodes the catalytic subunit of a special reverse transcriptase to overcome end-replication problem of chromosomes. This study demonstrates that papaverine, an isoquinoline alkaloid from the Papaveraceae, can overcome telomerase dependent immortality of HepG-2 cells that was used as a model of hepatocarcinoma. Although this alkaloid does not directly interact with telomeric sequences, papaverine inhibits telomerase through down-regulation of hTERT, which was analysed using thermal FRET and qRT-PCR, respectively. The IC50 values for the reduction of both telomerase activity and hTERT expression was 60 µM, while IC50 for cytotoxicity was 120 µM. Repeated treatments of the cells with very low non-toxic concentrations of papaverine resulted in growth arrest and strong reduction of population doublings after 40 days. This treatment induced senescent morphology in HepG-2 cells, which was evaluated by beta-galactosidase staining. Altogether, papaverine can be regarded as a promising model compound for drug design targeting cancer development.

  3. Strawberry (cv. Romina Methanolic Extract and Anthocyanin-Enriched Fraction Improve Lipid Profile and Antioxidant Status in HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Tamara Y. Forbes-Hernández

    2017-05-01

    Full Text Available Dyslipidemia and oxidation of low density lipoproteins (LDL are recognized as critical factors in the development of atherosclerosis. Healthy dietary patterns, with abundant fruit and vegetable consumption, may prevent the onset of these risk factors due to the presence of phytochemical compounds. Strawberries are known for their high content of polyphenols; among them, flavonoids are the major constituents, and it is presumed that they are responsible for the biological activity of the fruit. Nevertheless, there are only a few studies that actually evaluate the effects of different fractions isolated from strawberries. In order to assess the effects of two different strawberry extracts (whole methanolic extract/anthocyanin-enriched fraction on the lipid profile and antioxidant status in human hepatocellular carcinoma (HepG2 cells, the triglycerides and LDL-cholesterol content, lipid peroxidation, intracellular reactive oxygen species (ROS content and antioxidant enzymes’ activity on cell lysates were determined. Results demonstrated that both strawberry extracts not only improved the lipid metabolism by decreasing triglycerides and LDL-cholesterol contents, but also improved the redox state of HepG2 cells by modulating thiobarbituric acid-reactive substances production, antioxidant enzyme activity and ROS generation. The observed effects were more pronounced for the anthocyanin-enriched fraction.

  4. Identification of the C-terminal domain of Daxx acts as a potential regulator of intracellular cholesterol synthesis in HepG2 cells

    International Nuclear Information System (INIS)

    Sun, Shaowei; Wen, Juan; Qiu, Fei; Yin, Yufang; Xu, Guina; Li, Tianping; Nie, Juan; Xiong, Guozuo; Zhang, Caiping; Liao, Duangfang; Chen, Jianxiong; Tuo, Qinhui

    2016-01-01

    Daxx is a highly conserved nuclear transcriptional factor, which has been implicated in many nuclear processes including transcription and cell cycle regulation. Our previous study demonstrated Daxx also plays a role in regulation of intracellular cholesterol content. Daxx contains several domains that are essential for interaction with a growing number of proteins. To delineate the underlying mechanism of hypocholesterolemic activity of Daxx, we constructed a set of plasmids which can be used to overexpress different fragments of Daxx and transfected to HepG2 cells. We found that the C- terminal region Daxx626–740 clearly reduced intracellular cholesterol levels and inhibited the expression of SREBPs and SCAP. In GST pull-down experiments and Double immunofluorescence assays, Daxx626–740 was demonstrated to bind directly to androgen receptor (AR). Our findings suggest that the interaction of Daxx626-740 and AR abolishes the AR-mediated activation of SCAP/SREBPs pathway, which suppresses the de novo cholesterol synthesis. Thus, C-terminal domain of Daxx acts as a potential regulator of intracellular cholesterol content in HepG2 cells. - Highlights: • Daxx C-terminal domain reduces cholesterol levels. • Daxx C-terminal domain binds directly to AR. • The interaction of Daxx C-terminal domain and AR suppresses cholesterol synthesis.

  5. Casein Glycomacropeptide Hydrolysates Exert Cytoprotective Effect against Cellular Oxidative Stress by Up-Regulating HO-1 Expression in HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Tiange Li

    2017-01-01

    Full Text Available Oxidative stress is considered as an important mediator in the progression of metabolic disorders. The objective of this study was to investigate the potential hepatoprotective effects and mechanisms of bovine casein glycomacropeptide hydrolysates (GHP on hydrogen peroxide (H2O2-induced oxidative damage in HepG2 cells. Results showed that GHP significantly blocked H2O2-induced intracellular reactive oxygen species (ROS generation and cell viability reduction in a dose-dependent manner. Further, GHP concentration-dependently induced heme oxygenase-1 (HO-1 expression and increased nuclear factor-erythroid 2-related factor 2 (Nrf2 nuclear translocation. Moreover, pretreatment of GHP increased the activation of p38 mitogen-activated protein kinase (p38 MAPK and extracellular signal-regulated protein kinase 1/2 (ERK1/2, which were shown to contribute to Nrf2-mediated HO-1 expression. Taken together, GHP protected HepG2 cells from oxidative stress by activation of Nrf2 and HO-1 via p38 MAPK and ERK1/2 signaling pathways. Our findings indicate that bovine casein glycomacropeptide hydrolysates might be a potential ingredient in the treatment of oxidative stress-related disorders and further studies are needed to investigate the protective effects in vivo.

  6. Engineered exosome-mediated delivery of functionally active miR-26a and its enhanced suppression effect in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Liang G

    2018-01-01

    Full Text Available Gaofeng Liang,1,2,* Shu Kan,2,* Yanliang Zhu,3 Shuying Feng,1 Wenpo Feng,1 Shegan Gao1,4 1Medical College, Henan University of Science and Technology, Luoyang, China; 2Department of Biomedical Engineering, University of California Berkeley, California, CA, USA; 3State Key laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 4Henan Key Laboratory of Cancer Epigenetics, The First Affiliated Hospital of Henan University of Science and Technology, Luoyang, China *These authors contributed equally to this work Introduction: Exosomes are closed-membrane nanovesicles that are secreted by a variety of cells and exist in most body fluids. Recent studies have demonstrated the potential of exosomes as natural vehicles that target delivery of functional small RNA and chemotherapeutics to diseased cells. Methods: In this study, we introduce a new approach for the targeted delivery of exosomes loaded with functional miR-26a to scavenger receptor class B type 1-expressing liver cancer cells. The tumor cell-targeting function of these engineered exosomes was introduced by expressing in 293T cell hosts, the gene fusion between the transmembrane protein of CD63 and a sequence from Apo-A1. The exosomes harvested from these 293T cells were loaded with miR-26a via electroporation. Results: The engineered exosomes were shown to bind selectively to HepG2 cells via the scavenger receptor class B type 1–Apo-A1 complex and then internalized by receptor-mediated endocytosis. The release of miR-26a in exosome-treated HepG2 cells upregulated miR-26a expression and decreased the rates of cell migration and proliferation. We also presented evidence that suggest cell growth was inhibited by miR-26a-mediated decreases in the amounts of key proteins that regulate the cell cycle. Conclusion: Our gene delivery strategy can be adapted to treat a broad spectrum of cancers by expressing proteins on the surface of mi

  7. Protective effect of polysaccharide from maca (Lepidium meyenii) on Hep-G2 cells and alcoholic liver oxidative injury in mice.

    Science.gov (United States)

    Zhang, Lijun; Zhao, Qingsheng; Wang, Liwei; Zhao, Mingxia; Zhao, Bing

    2017-06-01

    To study the characterization and hepatoprotective activity of polysaccharide from maca (Lepidium meyenii), the main polysaccharide from maca (MP-1) was obtained by DEAE-52 cellulose column. The average molecular weight of MP-1 was 1067.3kDa and the polysaccharide purity was 91.63%. In order to assess the antioxidant activities of MP-1, four kinds of methods were used, including scavenging hydroxyl radical, DPPH, superoxide anion radical, and FRAP, and the results indicated high antioxidant activities. Furthermore, hepatoprotective activity of MP-1 was studied both in vitro and vivo. In vitro, the alcohol induced Hep-G2 cells model was established to evaluate the protective effect of MP-1, which demonstrated MP-1 can alleviate alcohol damage in Hep-G2 cells. In vivo, the Institute of Cancer Researcch (ICR) mice were used to evaluate hepatoprotecive effects of MP-1 on alcoholic liver disease (ALD). Supplement with MP-1 supressed the triglyceride level both in serum and in hepatic tissue. In addition, MP-1 ameliorated serous transaminases increase induced by alcohol, including aspartate transaminase, alanine aminotransferase, and γ-glutamyl transpeptidase. Moreover, MP-1 also dramatically increased the superoxide dismutase, glutathione peroxidase, and glutathione s-transferase levels in alcoholic mice. Meantime, histopathologic results MP-1 lighten inflammation induced by alcohol. These results indicate that MP-1 possesses hepatoprotective activity against hepatic injury induced by alcohol. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Effect of Cudrania tricuspidata and Kaempferol in Endoplasmic Reticulum Stress-Induced Inflammation and Hepatic Insulin Resistance in HepG2 Cells.

    Science.gov (United States)

    Kim, Ok-Kyung; Jun, Woojin; Lee, Jeongmin

    2016-01-21

    In this study, we quantitated kaempferol in water extract from Cudrania tricuspidata leaves (CTL) and investigated its effects on endoplasmic reticulum (ER) stress-induced inflammation and insulin resistance in HepG2 cells. The concentration of kaempferol in the CTL was 5.07 ± 0.08 mg/g. The HepG2 cells were treated with 300 µg/mL of CTL, 500 µg/mL of CTL, 1.5 µg/mL of kaempferol or 2.5 µg/mL of kaempferol, followed immediately by stimulation with 100 nM of thapsigargin for ER stress induction for 24 h. There was a marked increase in the activation of the ER stress and inflammation response in the thapsigargin-stimulated control group. The CTL treatment interrupted the ER stress response and ER stress-induced inflammation. Kaempferol partially inhibited the ER stress response and inflammation. There was a significant increase in serine phosphorylation of insulin receptor substrate (IRS)-1 and the expression of C/EBPα and gluconeogenic genes in the thapsigargin-stimulated control group compared to the normal control. Both CTL and kaempferol suppressed serine phosphorylation of IRS-1, and the treatments did not interrupt the C/EBPα/gluconeogenic gene pathway. These results suggest that kaempferol might be the active compound of CTL and that it might protect against ER stress-induced inflammation and hyperglycemia.

  9. Studies on Anti-Hepatoma Effect of Gan-Ai-Xiao Decoction | Yuan ...

    African Journals Online (AJOL)

    Purpose: To explore the anti-hepatoma effect of Gan-Ai-Xiao Decoction (GAXD), a folk remedy. Methods: High performance liquid chromatography (HPLC) was used to identify the major chemical components of GAXD ethanol extract (EE). The cytotoxic effect of GAXD EE against HepG2 cells was measured by methyl ...

  10. Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit

    Energy Technology Data Exchange (ETDEWEB)

    Boylan, Joan M. [Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, RI (United States); Salomon, Arthur R. [Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI (United States); Department of Chemistry, Brown University, Providence, RI (United States); Tantravahi, Umadevi [Division of Genetics, Department of Pathology, Brown University and Women and Infants Hospital, Providence, RI (United States); Gruppuso, Philip A., E-mail: philip_gruppuso@brown.edu [Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, RI (United States); Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI (United States)

    2015-07-15

    Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders. - Highlights: • Lentiviral-transduced shRNA was used to generate a stable knockdown of PP6 in HepG2 cells. • Cells adapted to reduced PP6; cell proliferation was unaffected, and cell survival was normal. • However, PP6 knockdown was associated with a transition to a tetraploid state. • Genomic profiling showed downregulated anti-inflammatory signaling and DNA replication. • Phosphoproteomic profiling showed changes in proteins associated with DNA replication and repair.

  11. Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit

    International Nuclear Information System (INIS)

    Boylan, Joan M.; Salomon, Arthur R.; Tantravahi, Umadevi; Gruppuso, Philip A.

    2015-01-01

    Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders. - Highlights: • Lentiviral-transduced shRNA was used to generate a stable knockdown of PP6 in HepG2 cells. • Cells adapted to reduced PP6; cell proliferation was unaffected, and cell survival was normal. • However, PP6 knockdown was associated with a transition to a tetraploid state. • Genomic profiling showed downregulated anti-inflammatory signaling and DNA replication. • Phosphoproteomic profiling showed changes in proteins associated with DNA replication and repair

  12. Preparation of curcumin microemulsions with food-grade soybean oil/lecithin and their cytotoxicity on the HepG2 cell line.

    Science.gov (United States)

    Lin, Chuan-Chuan; Lin, Hung-Yin; Chi, Ming-Hung; Shen, Chin-Min; Chen, Hwan-Wen; Yang, Wen-Jen; Lee, Mei-Hwa

    2014-07-01

    The choice of surfactants and cosurfactants for preparation of oral formulation in microemulsions is limited. In this report, a curcumin-encapsulated phospholipids-based microemulsion (ME) using food-grade ingredients soybean oil and soybean lecithin to replace ethyl oleate and purified lecithin from our previous study was established and compared. The results indicated soybean oil is superior to ethyl oleate as the oil phase in curcumin microemulsion, as proven by the broadened microemulsion region with increasing range of surfactant/soybean oil ratio (approx. 1:1-12:1). Further preparation of two formula with different particle sizes of formula A (30nm) and B (80nm) exhibited differential effects on the cytotoxicity of hepatocellular HepG2 cell lines. At 15μM of concentration, curcumin-ME in formula A with smaller particle size resulted in the lowest viability (approx. 5%), which might be explained by increasing intake of curcumin, as observed by fluorescence microscopy. In addition, the cytotoxic effect of curcumin-ME is exclusively prominent on HepG2, not on HEK293, which showed over 80% of viability at 15μM. The results from this study might provide an innovative applied technique in the area of nutraceuticals and functional foods. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. SirT1 confers hypoxia-induced radioresistance via the modulation of c-Myc stabilization on hepatoma cells

    International Nuclear Information System (INIS)

    Xie Yuexia; Zhang Jianghong; Shao Chunlin; Xu Yanwu

    2012-01-01

    Intratumoral hypoxia is an important contributory factor to tumor cell resistance to radiotherapy. SirT1, a nicotinamide adenine dinucleotide (NAD + )-dependent histone/protein deacetylase, has been linked to the decrease of radiation-induced DNA damage and seems to be critical for cancer therapy. The purpose of this study was to investigate the role of SirT1 in hypoxia-induced radiation response on hepatoma cells. It was found that the administration with resveratrol, a putative SirT1 activator, enhanced the resistance of HepG2 cells against radiation-induced DNA damage of MN formation under hypoxia condition; while nicotinamide, a well-known SirT1 inhibitor, sensitized this radiation damage. Nevertheless, pretreatment of cells with 10058-F4, a specific inhibitor of c-Myc, almost eliminated the nicotinamide-induced radiosensitive effect. Further studies revealed that resveratrol inhibited c-Myc protein accumulation via up-regulation of SirT1 expression and deacetylase activity, and this loss of c-Myc protein was abolished by inhibiting its degradation in the presence of MG132, a potent inhibitor of proteasome. In contrast, nicotinamide attenuated c-Myc protein degradation induced by radiation under hypoxia through inhibition of SirT1 deacetylase activity. Our findings suggest that SirT1 could serve as a novel potent target of radiation-induced DNA damage and thus as a potential strategy to advance the efficiency of radiation therapy in hepatoma entities. (author)

  14. Influence of TiO{sub 2} nanoparticles on cellular antioxidant defense and its involvement in genotoxicity in HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Petkovic, Jana; Zegura, Bojana; Filipic, Metka, E-mail: metka.filipic@nib.si [Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, SI-1000 Ljubljana (Slovenia)

    2011-07-06

    We investigated the effects of two types of TiO{sub 2} nanoparticles (<25 nm anatase, TiO{sub 2}-An; <100 nm rutile, TiO{sub 2}-Ru) on cellular antioxidant defense in HepG2 cells. We previously showed that in HepG2 cells, TiO{sub 2} nanoparticles are not toxic, although they induce oxidative DNA damage, production of intracellular reactive oxygen species, and up-regulation of mRNA expression of DNA-damage-responsive genes (p53, p21, gadd45{alpha} and mdm2). In the present study, we measured changes in mRNA expression of several antioxidant enzymes: catalase, superoxide dismutase, glutathione peroxidase, nitric oxide synthase, glutathione reductase and glutamate-cysteine ligase. As reduced glutathione has a central role in cellular antioxidant defense, we determined the effects of TiO{sub 2} nanoparticles on changes in the intracellular glutathione content. To confirm a role for glutathione in protection against TiO{sub 2}-nanoparticle-induced DNA damage, we compared the extent of TiO{sub 2}-nanoparticle-induced DNA damage in HepG2 cells that were glutathione depleted with buthionine-(S,R)-sulfoximine pretreatment and in nonglutathione-depleted cells. Our data show that both types of TiO{sub 2} nanoparticles up-regulate mRNA expression of oxidative-stress-related genes, with TiO{sub 2}-Ru being a stronger inducer than TiO{sub 2}-An. Both types of TiO{sub 2} nanoparticles also induce dose-dependent increases in intracellular glutathione levels, and in glutathione-depleted cells, TiO{sub 2}-nanoparticle-induced DNA damage was significantly greater than in nonglutathione-depleted cells. Interestingly, the glutathione content and the extent of DNA damage were significantly higher in TiO{sub 2}-An- than TiO{sub 2}-Ru-exposed cells. Thus, we show that TiO{sub 2} nanoparticles cause activation of cellular antioxidant processes, and that intracellular glutathione has a critical role in defense against this TiO{sub 2}-nanoparticle-induced DNA damage.

  15. Lower concentrations of blueberry polyphenolic-rich extract differentially alter HepG2 cell proliferation and expression of genes related to cell-cycle, oxidation and epigenetic machinery

    Science.gov (United States)

    In vitro cancer models have been used to study the effect of relatively high concentrations (>200 ug/ml) of phenolic plant extracts upon cell proliferation. In this study we report that the treatment of human hepatocarcinoma HepG2 cells with lower concentrations of blueberry phenolic extract (6.5-10...

  16. All-trans retinoic acid inhibits the recruitment of ARNT to DNA, resulting in the decrease of CYP1A1 mRNA expression in HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Ohno, Marumi; Ikenaka, Yoshinori [Laboratory of Toxicology, Graduate School of Veterinary Medicine, Hokkaido University, N18 W9, Kita-ku, Sapporo 060-0818 (Japan); Ishizuka, Mayumi, E-mail: ishizum@vetmed.hokudai.ac.jp [Laboratory of Toxicology, Graduate School of Veterinary Medicine, Hokkaido University, N18 W9, Kita-ku, Sapporo 060-0818 (Japan)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer AHR and ARNT transcriptionally regulate genes related to metabolisms such as CYP1A1. Black-Right-Pointing-Pointer We investigated the effect of retinoic acid (RA) on the function of AHR/ARNT. Black-Right-Pointing-Pointer RA inhibited the recruitment of ARNT, not AHR, to the regulatory region of CYP1A1. Black-Right-Pointing-Pointer It resulted in a reduction of constitutive expression of CYP1A1 to less than half. -- Abstract: Aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) are well-conserved transcription factors among species. However, there are a very limited number of reports on the physiological function of AHR, particularly on the regulation of AHR by endogenous compounds. We hence investigated the effects of all-trans retinoic acid (atRA) on cytochrome P450 (CYP) 1A1 gene transcription as a model of AHR-regulated transcription mechanisms in HepG2 cells, a human hepatoma cell line. Treatment with atRA significantly reduced transactivation and expression of CYP1A1 mRNA to less than half of its control value, and this inhibitory effect was mediated by RAR{alpha}. The result of chromatin immunoprecipitation assay indicated that treatment with atRA at 1-100 nM drastically inhibited the recruitment of ARNT to DNA regions containing xenobiotic responsive elements. In conclusion, atRA at physiological concentrations could reduce AHR-mediated gene transcription via the inhibition of recruitment of ARNT to relevant DNA regions.

  17. Effect of PEG-PDLLA polymeric nanovesicles loaded with doxorubicin and hematoporphyrin monomethyl ether on human hepatocellular carcinoma HepG2 cells in vitro

    Directory of Open Access Journals (Sweden)

    Xiang GH

    2013-12-01

    Full Text Available Guang-Hua Xiang,1,2,* Guo-Bin Hong,2,3,* Yong Wang,2 Du Cheng,2 Jing-Xing Zhou,1 Xin-Tao Shuai21Department of Radiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China; 2PCFM Laboratory of Ministry of Education, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou, People's Republic of China; 3Department of Radiology, Fifth Affiliated Hospital, Sun Yat-Sen University, Zhuhai, People's Republic of China*These two authors contributed equally to this workObjective: To evaluate the cytotoxicity of poly(ethylene glycol-block-poly(D,L-lactic acid (PEG-PDLLA nanovesicles loaded with doxorubicin (DOX and the photosensitizer hematoporphyrin monomethyl ether (HMME on human hepatocellular carcinoma HepG2 cells and to investigate potential apoptotic mechanisms.Methods: PEG-PDLLA nanovesicles were simultaneously loaded with DOX and HMME (PEG-PDLLA-DOX-HMME, and PEG-PDLLA nanovesicles were loaded with DOX (PEG-PDLLA-DOX, HMME (PEG-PDLLA-HMME, or the PEG-PDLLA nanovesicle alone as controls. The cytotoxicity of PEG-PDLLA-DOX-HMME, PEG-PDLLA-DOX, PEG-PDLLA-HMME, and PEG-PDLLA against HepG2 cells was measured, and the cellular reactive oxygen species, percentage of cells with mitochondrial membrane potential depolarization, and apoptotic rate following treatment were determined.Results: Four nanovesicles (PEG-PDLLA-DOX-HMME, PEG-PDLLA-DOX, PEG-PDLLA-HMME, and PEG-PDLLA were synthesized, and mean particle sizes were 175±18 nm, 154±3 nm, 196±2 nm, and 147±15 nm, respectively. PEG-PDLLA-DOX-HMME was more cytotoxic than PEG-PDLLA-DOX, PEG-PDLLA-HMME, and PEG-PDLLA. PEG-PDLLA-HMME-treated cells had the highest mean fluorescence intensity, followed by PEG-PDLLA-DOX-HMME-treated cells, whereas PEG-PDLLA-DOX- and PEG-PDLLA-treated cells had a similar fluorescence intensity. Mitochondrial membrane potential depolarization was observed in 54.2%, 59.4%, 13.8%, and 14.8% of the cells treated with

  18. Recent advances in live cell imaging of hepatoma cells

    Science.gov (United States)

    2014-01-01

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

  19. Morin impedes Yap nuclear translocation and fosters apoptosis through suppression of Wnt/β-catenin and NF-κB signaling in Mst1 overexpressed HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Perumal, NaveenKumar [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu (India); Perumal, MadanKumar [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu (India); Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390 (United States); Kannan, Anbarasu [Department of Cellular and Molecular Biology, The University of Texas Health Science Center, Tyler, Texas (United States); Subramani, Kumar [Centre for Biotechnology, Anna University, Chennai 600025, Tamil Nadu (India); Halagowder, Devaraj [Department of Zoology, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu (India); Sivasithamparam, NiranjaliDevaraj, E-mail: profniranjali@gmail.com [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu (India)

    2017-06-15

    Recent clinical and experimental evidences strongly acclaim Yes-associated protein (Yap), a key oncogenic driver in liver carcinogenesis, as a therapeutic target. Of the known multiple schemes to inhibit Yap activity, activation of Mammalian Sterile 20-like Kinase 1 (Mst1), an upstream regulator of Yap, appears to be a promising one. In this study, we hypothesize that morin, a bioflavonoid, mediates its anti-cancer effect through the activation of Mst1/hippo signaling in liver cancer cells. To test this hypothesis, both full length Mst1 (F-Mst1) and kinase active N-terminal Mst1 (N-Mst1)-overexpressed HepG2 cells were used. Exposure of F-Mst1 overexpressed HepG2 cells to morin activated Mst1 by caspase-3 cleavage and thereby inhibited Yap nuclear translocation and fostered apoptosis. Morin suppressed NF-κB p65 and Wnt/β-catenin signaling through Mst1 activation via cleavage and phosphorylation, leading to cell death. Annexin-V/PI staining further confirmed the induction of apoptosis in morin treated F-Mst1 overexpressed cells. The present study shows that morin targets cell survival molecules such as NF-κB p65 and β-catenin through activation of hippo signaling. Therefore, morin could be considered as a potential anti-cancer agent against liver cancer. - Highlights: • Morin induced cytotoxicity in cultured HepG2 cells. • Morin activated hippo pathway via Mst1 activation in transfected HepG2 cells. • Morin suppressed Wnt/β-catenin signaling and induced G0/G1 cell cycle arrest. • Morin inhibited NF-κB signaling through Mst1 activation in transfected HepG2 cells. • Morin potentiates apoptosis through Mst1-JNK-caspase mediated mechanism in HepG2 cells.

  20. Morin impedes Yap nuclear translocation and fosters apoptosis through suppression of Wnt/β-catenin and NF-κB signaling in Mst1 overexpressed HepG2 cells

    International Nuclear Information System (INIS)

    Perumal, NaveenKumar; Perumal, MadanKumar; Kannan, Anbarasu; Subramani, Kumar; Halagowder, Devaraj; Sivasithamparam, NiranjaliDevaraj

    2017-01-01

    Recent clinical and experimental evidences strongly acclaim Yes-associated protein (Yap), a key oncogenic driver in liver carcinogenesis, as a therapeutic target. Of the known multiple schemes to inhibit Yap activity, activation of Mammalian Sterile 20-like Kinase 1 (Mst1), an upstream regulator of Yap, appears to be a promising one. In this study, we hypothesize that morin, a bioflavonoid, mediates its anti-cancer effect through the activation of Mst1/hippo signaling in liver cancer cells. To test this hypothesis, both full length Mst1 (F-Mst1) and kinase active N-terminal Mst1 (N-Mst1)-overexpressed HepG2 cells were used. Exposure of F-Mst1 overexpressed HepG2 cells to morin activated Mst1 by caspase-3 cleavage and thereby inhibited Yap nuclear translocation and fostered apoptosis. Morin suppressed NF-κB p65 and Wnt/β-catenin signaling through Mst1 activation via cleavage and phosphorylation, leading to cell death. Annexin-V/PI staining further confirmed the induction of apoptosis in morin treated F-Mst1 overexpressed cells. The present study shows that morin targets cell survival molecules such as NF-κB p65 and β-catenin through activation of hippo signaling. Therefore, morin could be considered as a potential anti-cancer agent against liver cancer. - Highlights: • Morin induced cytotoxicity in cultured HepG2 cells. • Morin activated hippo pathway via Mst1 activation in transfected HepG2 cells. • Morin suppressed Wnt/β-catenin signaling and induced G0/G1 cell cycle arrest. • Morin inhibited NF-κB signaling through Mst1 activation in transfected HepG2 cells. • Morin potentiates apoptosis through Mst1-JNK-caspase mediated mechanism in HepG2 cells.

  1. The effects of herbal composition Gambigyeongsinhwan (4) on hepatic steatosis and inflammation in Otsuka Long-Evans Tokushima fatty rats and HepG2 cells.

    Science.gov (United States)

    Yoon, Seolah; Kim, Jeongjun; Lee, Hyunghee; Lee, Haerim; Lim, Jonghoon; Yang, Heejeong; Shin, Soon Shik; Yoon, Michung

    2017-01-04

    Hepatic steatosis has risen rapidly in parallel with a dramatic increase in obesity. The aim of this study was to determine whether the herbal composition Gambigyeongsinhwan (4) (GGH(4)), composed of Curcuma longa L. (Zingiberaceae), Alnus japonica (Thunb.) Steud. (Betulaceae), and the fermented traditional Korean medicine Massa Medicata Fermentata, regulates hepatic steatosis and inflammation. The effects of GGH(4) on hepatic steatosis and inflammation in Otsuka Long-Evans Tokushima fatty (OLETF) rats and HepG2 cells were examined using Oil red O, hematoxylin and eosin, and toluidine blue staining, immunohistochemistry, quantitative real-time polymerase chain reaction, and peroxisome proliferator-activated receptor α (PPARα) transactivation assay. Administration of GGH(4) to OLETF rats improved hepatic steatosis and lowered serum levels of alanine transaminase, total cholesterol, triglycerides, and free fatty acids. GGH(4) increased mRNA levels of fatty acid oxidation enzymes (ACOX, HD, CPT-1, and MCAD) and decreased mRNA levels of lipogenesis genes (FAS, ACC1, C/EBPα, and SREBP-1c) in the liver of OLETF rats. In addition, infiltration of inflammatory cells and expression of inflammatory cytokines (CD68, TNFα, and MCP-1) in liver tissue were reduced by GGH(4). Treatment of HepG2 cells with a mixture of oleic acid and palmitoleic acid induced significant lipid accumulation, but GGH(4) inhibited lipid accumulation by regulating the expression of hepatic fatty acid oxidation and lipogenic genes. GGH(4) also increased PPARα reporter gene expression. These effects of GGH(4) were similar to those of the PPARα activator fenofibrate, whereas the PPARα antagonist GW6471 reversed the inhibitory effects of GGH(4) on lipid accumulation in HepG2 cells. These results suggest that GGH(4) inhibits obesity-induced hepatic steatosis and that this process may be mediated by regulation of the expression of PPARα target genes and lipogenic genes. GGH(4) also suppressed obesity

  2. Isolation of major phenolics from Launaea spinosa and their protective effect on HepG2 cells damaged with t-BHP.

    Science.gov (United States)

    Abdallah, Hossam; Farag, Mohamed; Osman, Samir; Kim, Da hye; Kang, Kyungsu; Pan, Cheol-Ho; Abdel-Sattar, Essam

    2016-01-01

    Some Launaea species (Asteraceae) are used traditionally to treat liver oxidative stress. The present study investigates the protective effects of isolated compounds from Launaea spinosa Sch. Bip. (Asteraceae) against oxidative stress on t-BHP-induced HepG2 cells. Major phenolic content from flowering aerial parts of L. spinosa was isolated and identified. The protective effects of isolated compounds (10 and 20 μM) against oxidative stress induced by tert-butyl hydroperoxide (t-BHP) in HepG2 cells were investigated through the measurement of aspartate aminotransferase (AST), alanine transaminase (ALT), and superoxide dismutase (SOD) levels. A new phenolic compound identified as 2,3-diferulyl R,R-(+) methyl tartrate (6), in addition to five known metabolites, esculetin (1), esculetin-7-O-d-glucoside (cichoriin) (2), fertaric acid (3), acacetin-7-O-d-glucoside (4), and acacetin-7-O-d-glucuronic acid (5), were isolated. Oxidant-induced damage by 200 μM t-BHP in HepG2 cells was inhibited by compounds 1, 4, and 5 (10 and 20 μM), or quercetin (10 μM; positive control). The protective effects of compounds 1, 4, and 5 were associated with decreasing in AST, ALT, and SOD levels. Compound 4 (20 μM) decreased the AST level from 128.5 ± 13.9 to 7.9 ±1.8 U/mL. Meanwhile, compound 1 (20 μM) decreased ALT activity from 20.3 ± 7.0 to 7.6 ± 2.4 U/mL, while compound 5 decreased SOD levels from 41.6 ± 9.0 to 28.3 ± 3.4 mU/mg. The major phenolic compounds isolated from L. spinosa displayed a significant cytoprotective effect against oxidative stress, leading to maintenance of the normal redox status of the cell.

  3. Analytical Research to Determine the effects of the Components of ONGABO on the Viability of HepG2 Cancer Cells by Using the Sovereign, Minister, Assistant and Courier Principle ().

    Science.gov (United States)

    Shin, Jeong-Hun; Jun, Seung-Lyul; Hwang, Sung-Yeoun; Ahn, Seong-Hun

    2012-12-01

    This study used the basic principle of Oriental medicine, the sovereign, minister, assistant and courier principle () to investigate the effects of the component of ONGABO, which is composed of Ginseng Radix (Red Ginseng), Angelica Gigantis Radix, Schisandrae Fructus, Cuscuta Semen and Curcumae tuber on the viability of HepG2 cells. Single and mixed extracts of the component of ONGABO were prepared by lypohilizing powder of Red Ginseng (6-year root from Kanghwa), Angelica Gigantis Radix, Schisandrae Fructus, Cuscuta Semen, Curcumae Tuber (from Omniherb Co., Ltd., Korea) at the laboratory of herbal medicine in Woosuk University and were eluted after being macerated with 100% ethanol for three days. The cell viability of HepG2 was determined by using an absorptiometric analysis with PrestoBlue (Invitrogen) reagent after the plate had been incubated for 48 hours. All of the experiments were repeated three times to obtain the average value and standard deviation. The statistical analysis was done and the correlation factor was obtained by using Microsoft Office Excel 2007 and Origin 6.0 software. Although Ginseng Radix (Red Ginseng) and Schisandrae Fructus did not enhance the viability of HepG2 cells, they were shown to provide protection of those cells. On the other hand, Angelica Gigantis Radix decreased the viability of HepG2 cells significantly, Cuscuta Semen and Curcumae Tuber had a small or no effect on the viability of HepG2 cells. In the sovereign, minister, assistant and courier principle (), Ginseng Radix (Red Ginseng) corresponds to the sovereign component because it provides cell protection effects, Angelica Gigantis Radix corresponds to minister medicinal because it kills cells, Schisandrae Fructus corresponds to the assistant medicinal to help red ginseng having cell protect effects. Cuscuta Semen and Curcumae Tuber correspond to the courier medicinal having no effect in cell viability in HepG2. We hope this study provides motivation for advanced research

  4. 20(S)-25-methoxyl-dammarane-3β, 12β, 20-triol negatively regulates activation of STAT3 and ERK pathways and exhibits anti-cancer effects in HepG2 cells.

    Science.gov (United States)

    Ai, Hui-Han; Zhou, Zi-Long; Sun, Lu-Guo; Yang, Mei-Ting; Li, Wei; Yu, Chun-Lei; Song, Zhen-Bo; Huang, Yan-Xin; Wu, Yin; Liu, Lei; Yang, Xiao-Guang; Zhao, Yu-Qing; Bao, Yong-Li; Li, Yu-Xin

    2017-11-01

    The pro-inflammatory cytokine interleukin 6 (IL-6), via activating its downstream JAK/STAT3 and Ras/ERK signaling pathways, is involved in cell growth, proliferation and anti-apoptotic activities in various malignancies. To screen inhibitors of IL-6 signaling, we constructed a STAT3 and ERK dual-pathway responsive luciferase reporter vector (Co.RE). Among several candidates, the natural compound 20(S)-25-methoxyl-dammarane-3β, 12β, 20-triol (25-OCH 3 -PPD, GS25) was identified to clearly inhibit the luciferase activity of Co.RE. GS25 was confirmed to indeed inhibit activation of both STAT3 and ERK pathways and expression of downstream target genes of IL-6, and to predominantly decrease the viability of HepG2 cells via induction of cell cycle arrest and apoptosis. Interestingly, GS25 showed preferential inhibition of HepG2 cell viability relative to normal liver L02 cells. Further investigation showed that GS25 could not induce apoptosis and block activation of STAT3 and ERK pathways in L02 cells as efficiently as in HepG2 cells, which may result in differential effects of GS25 on malignant and normal liver cells. In addition, GS25 was found to potently suppress the expression of endogenous STAT3 at a higher concentration and dramatically induce p38 phosphorylation in HepG2 cells, which could mediate its anti-cancer effects. Finally, we demonstrated that GS25 also inhibited tumor growth in HepG2 xenograft mice. Taken together, these findings indicate that GS25 elicits its anti-cancer effects on HepG2 cells through multiple mechanisms and has the potential to be used as an inhibitor of IL-6 signaling. Thus, GS25 may be developed as a treatment for hepatocarcinoma with low toxicity on normal liver tissues as well as other inflammation-associated diseases.

  5. Cobalt iron oxide nanoparticles induce cytotoxicity and regulate the apoptotic genes through ROS in human liver cells (HepG2).

    Science.gov (United States)

    Ahamed, Maqusood; Akhtar, Mohd Javed; Khan, M A Majeed; Alhadlaq, Hisham A; Alshamsan, Aws

    2016-12-01

    Cobalt iron oxide (CoFe 2 O 4 ) nanoparticles (CIO NPs) have been one of the most widely explored magnetic NPs because of their excellent chemical stability, mechanical hardness and heat generating potential. However, there is limited information concerning the interaction of CIO NPs with biological systems. In this study, we investigated the reactive oxygen species (ROS) mediated cytotoxicity and apoptotic response of CIO NPs in human liver cells (HepG2). Diameter of crystalline CIO NPs was found to be 23nm with a band gap of 1.97eV. CIO NPs induced cell viability reduction and membrane damage, and degree of induction was dose- and time-dependent. CIO NPs were also found to induce oxidative stress revealed by induction of ROS, depletion of glutathione and lower activity of superoxide dismutase enzyme. Real-time PCR data has shown that mRNA level of tumor suppressor gene p53 and apoptotic genes (bax, CASP3 and CASP9) were higher, while the expression level of anti-apoptotic gene bcl-2 was lower in cells following exposure to CIO NPs. Activity of caspase-3 and caspase-9 enzymes was also higher in CIO NPs exposed cells. Furthermore, co-exposure of N-acetyl-cysteine (ROS scavenger) efficiently abrogated the modulation of apoptotic genes along with the prevention of cytotoxicity caused by CIO NPs. Overall, we observed that CIO NPs induced cytotoxicity and apoptosis in HepG2 cells through ROS via p53 pathway. This study suggests that toxicity mechanisms of CIO NPs should be further investigated in animal models. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Targeting the expression of glutathione- and sulfate-dependent detoxification enzymes in HepG2 cells by oxygen in minimal and amino acid enriched medium.

    Science.gov (United States)

    Usarek, Ewa; Graboń, Wojciech; Kaźmierczak, Beata; Barańczyk-Kuźma, Anna

    2016-02-01

    Cancer cells exhibit specific metabolism allowing them to survive and proliferate in various oxygen conditions and nutrients' availability. Hepatocytes are highly active metabolically and thus very sensitive to hypoxia. The purpose of the study was to investigate the effect of oxygen on the expression of phase II detoxification enzymes in hepatocellular carcinoma cells (HepG2) cultured in minimal and rich media (with nonessential amino acids and GSH). The cells were cultured at 1% hypoxia, 10% tissue normoxia, and 21% atmospheric normoxia. The total cell count was determined by trypan blue exclusion dye and the expression on mRNA level by RT-PCR. The result indicated that the expression of glutathione-dependent enzymes (GSTA, M, P, and GPX2) was sensitive to oxygen and medium type. At 1% hypoxia the enzyme expression (with the exception of GSTA) was higher in minimal compared to rich medium, whereas at 10% normoxia it was higher in the rich medium. The expression was oxygen-dependent in both types of medium. Among phenol sulfotransferase SULT1A1 was not sensitive to studied factors, whereas the expression of SULT1A3 was depended on oxygen only in minimal medium. It can be concluded that in HepG2 cells, the detoxification by conjugation with glutathione and, to a lower extent with sulfate, may be affected by hypoxia and/or limited nutrients' availability. Besides, because the data obtained at 10% oxygen significantly differ from those at 21%, the comparative studies on hypoxia should be performed in relation to 10% but not 21% oxygen. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Inhibition of Tumor Growth of Human Hepatocellular Carcinoma HepG2 Cells in a Nude Mouse Xenograft Model by the Total Flavonoids from Arachniodes exilis

    Directory of Open Access Journals (Sweden)

    Huimin Li

    2017-01-01

    Full Text Available A tumor growth model of human hepatocellular carcinoma HepG2 cells in nude mice was employed to investigate the antitumor activity of the total flavonoids extracted from Arachniodes exilis (TFAE in vivo. Several biochemical assays including hematoxylin-eosin (HE staining, immunohistochemistry, and Western blot were performed to elucidate the mechanism of action of total flavonoids extracted from Arachniodes exilis (TFAE. The results showed that TFAE effectively inhibited the tumor growth of hepatocellular carcinoma in nude mice and had no significant effect on body weight, blood system, and functions of liver and kidney. Expression levels of proapoptotic proteins Bax and cleaved caspase-3 remarkably increased while the expressions of Bcl-2, HIF-1α, and VEGF were suppressed by TFAE. These results suggested that the antitumor potential of TFEA was implied by the apoptosis of tumor cells and the inhibition of angiogenesis in tumor tissue.

  8. Moving into advanced nanomaterials. Toxicity of rutile TiO{sub 2} nanoparticles immobilized in nanokaolin nanocomposites on HepG2 cell line

    Energy Technology Data Exchange (ETDEWEB)

    Bessa, Maria João, E-mail: mjbessa8@gmail.com [Department of Environmental Health, Portuguese National Institute of Health, Rua Alexandre Herculano, 321, 4000-055 Porto (Portugal); Costa, Carla, E-mail: cstcosta@gmail.com [Department of Environmental Health, Portuguese National Institute of Health, Rua Alexandre Herculano, 321, 4000-055 Porto (Portugal); EPIUnit - Institute of Public Health, University of Porto, Rua das Taipas 135, 4050-600, Porto (Portugal); Reinosa, Julian, E-mail: jjreinosa@icv.csic.es [Electroceramic Department, Instituto de Cerámica y Vidrio, CSIC, Campus de Cantoblanco, Calle de Kelson, 5, 28049 Madrid (Spain); Pereira, Cristiana, E-mail: cristianacostapereira@gmail.com [Department of Environmental Health, Portuguese National Institute of Health, Rua Alexandre Herculano, 321, 4000-055 Porto (Portugal); EPIUnit - Institute of Public Health, University of Porto, Rua das Taipas 135, 4050-600, Porto (Portugal); Fraga, Sónia, E-mail: teixeirafraga@hotmail.com [Department of Environmental Health, Portuguese National Institute of Health, Rua Alexandre Herculano, 321, 4000-055 Porto (Portugal); EPIUnit - Institute of Public Health, University of Porto, Rua das Taipas 135, 4050-600, Porto (Portugal); Fernández, José, E-mail: jfernandez@icv.csic.es [Electroceramic Department, Instituto de Cerámica y Vidrio, CSIC, Campus de Cantoblanco, Calle de Kelson, 5, 28049 Madrid (Spain); Bañares, Miguel A., E-mail: miguel.banares@csic.es [Catalytic Spectroscopy Laboratory, Instituto de Catálisis y Petroleoquímica, ICP-CSIC, Madrid (Spain); and others

    2017-02-01

    Immobilization of nanoparticles on inorganic supports has been recently developed, resulting in the creation of nanocomposites. Concerning titanium dioxide nanoparticles (TiO{sub 2} NPs), these have already been developed in conjugation with clays, but so far there are no available toxicological studies on these nanocomposites. The present work intended to evaluate the hepatic toxicity of nanocomposites (C-TiO{sub 2}), constituted by rutile TiO{sub 2} NPs immobilized in nanokaolin (NK) clay, and its individual components. These nanomaterials were analysed by means of FE-SEM and DLS analysis for physicochemical characterization. HepG2 cells were exposed to rutile TiO{sub 2} NPs, NK clay and C-TiO{sub 2} nanocomposite, in the presence and absence of serum for different exposure periods. Possible interferences with the methodological procedures were determined for MTT, neutral red uptake, alamar blue (AB), LDH, and comet assays, for all studied nanomaterials. Results showed that MTT, AB and alkaline comet assay were suitable for toxicity analysis of the present materials after slight modifications to the protocol. Significant decreases in cell viability were observed after exposure to all studied nanomaterials. Furthermore, an increase in HepG2 DNA damage was observed after shorter periods of exposure in the absence of serum proteins and longer periods of exposure in their presence. Although the immobilization of nanoparticles in micron-sized supports could, in theory, decrease the toxicity of single nanoparticles, the selection of a suitable support is essential. The present results suggest that NK clay is not the appropriate substrate to decrease TiO{sub 2} NPs toxicity. Therefore, for future studies, it is critical to select a more appropriate substrate for the immobilization of TiO{sub 2} NPs. - Highlights: • Only the MTT and AB assays were found to be suitable for cytotoxicity assessment. • Alkaline comet assay was also appropriate for genotoxicity evaluation

  9. Plasmatic concentration of organochlorine lindane acts as metabolic disruptors in HepG2 liver cell line by inducing mitochondrial disorder

    Energy Technology Data Exchange (ETDEWEB)

    Benarbia, Mohammed el Amine [LUNAM Université, Angers (France); Inserm 1063, Angers (France); Macherel, David [LUNAM Université, Angers (France); UMR 1345 IRHS, Angers (France); Faure, Sébastien; Jacques, Caroline; Andriantsitohaina, Ramaroson [LUNAM Université, Angers (France); Inserm 1063, Angers (France); Malthièry, Yves, E-mail: yves.malthiery@univ-angers.fr [LUNAM Université, Angers (France); Inserm 1063, Angers (France)

    2013-10-15

    Lindane (LD) is a persistent environmental pollutant that has been the subject of several toxicological studies. However, concentrations used in most of the reported studies were relatively higher than those found in the blood of the contaminated area residents and effects of low concentrations remain poorly investigated. Moreover, effects on cell metabolism and mitochondrial function of exposure to LD have received little attention. This study was designed to explore the effects of low concentrations of LD on cellular metabolism and mitochondrial function, using the hepatocarcinoma cell line HepG2. Cells were exposed to LD for 24, 48 and 72 h and different parameters linked with mitochondrial regulation and energy metabolism were analyzed. Despite having any impact on cellular viability, exposure to LD at plasmatic concentrations led to an increase of maximal respiratory capacity, complex I activity, intracellular ATP and NO release but decreased uncoupled respiration to ATP synthesis and medium lactate levels. In addition, LD exposure resulted in the upregulation of mitochondrial biogenesis genes. We suggest that, at plasmatic concentrations, LD acts as a metabolic disruptor through impaired mitochondrial function and regulation with an impact on cellular energetic metabolism. In addition, we propose that a cellular assay based on the analysis of mitochondria function, such as described here for LD, may be applicable for larger studies on the effects of low concentrations of xenobiotics, because of the exquisite sensitivity of this organelle. - Highlights: Our data clearly demonstrated in HepG2 cells that exposure at plasmatic low concentrations of LD were able to: • Impair mitochondrial function • Caused alteration on nucleo-mitochondrial cross-talk • Increase nitric oxide release and protein nitration • Impair cellular energetic metabolism and lipid accumulation.

  10. Live Cell Imaging Confocal Microscopy Analysis of HBV Myr-PreS1 Peptide Binding and Uptake in NTCP-GFP Expressing HepG2 Cells.

    Science.gov (United States)

    König, Alexander; Glebe, Dieter

    2017-01-01

    To obtain basic knowledge about specific molecular mechanisms involved in the entry of pathogens into cells is the basis for establishing pharmacologic substances blocking initial viral binding, infection, and subsequent viral spread. Lack of information about key cellular factors involved in the initial steps of HBV infection has hampered the characterization of HBV binding and entry for decades. However, recently, the liver-specific sodium-dependent taurocholate cotransporting polypeptide (NTCP) has been discovered as a functional receptor for HBV and HDV, thus opening the field for new concepts of basic binding and entry of HBV and HDV. Here, we describe practical issues of a basic in vitro assay system to examine kinetics and mechanisms of receptor-dependent HBV binding, uptake, and intracellular trafficking by live-cell imaging confocal microscopy. The assay system is comprised of HepG2 cells expressing a NTCP-GFP fusion-protein and chemically synthesized, fluorophore-labeled part of HBV surface protein, spanning the first N-terminal 48 amino acids of preS1 of the large hepatitis B virus surface protein.

  11. An Efficient Sonochemical Synthesis of Novel Schiff’s Bases, Thiazolidine, and Pyrazolidine Incorporating 1,8-Naphthyridine Moiety and Their Cytotoxic Activity against HePG2 Cell Lines

    Directory of Open Access Journals (Sweden)

    N. S. Ahmed

    2014-01-01

    Full Text Available Novel Schiff’s bases 4a–e, 5a, 5b, and 6, thiazolidine 7a–d, and pyrazolidine 8 have been synthesized using the versatile synthon 4-hydroxy-2,7-dimethyl-1,8-naphthyridine 1. Reactions carried out under ultrasound irradiation showed higher rates and yields than those done under silent conditions. The newly synthesized compounds were evaluated for HepG2 cell growth inhibition. The results obtained revealed that the tested compounds possess inhibitory effect on the growth of HepG2 liver cancer cells. The results were compared to doxorubicin as a reference drug (IC50: 0.04. Compounds 4a and 7b showed the highest inhibition activity against the HepG2 cell line (IC50: 0.047 and 0.041 µM, resp. among all the tested compounds.

  12. Bactericidal application and cytotoxic activity of biosynthesized silver nanoparticles with an extract of the red seaweed Pterocladiella capillacea on the HepG2 cell line.

    Science.gov (United States)

    El Kassas, Hala Yassin; Attia, Azza Ahmed

    2014-01-01

    Nano-biotechnology is recognized as offering revolutionary changes in various fields of medicine. Biologically synthesized silver nanoparticles have a wide range of applications. Silver nanoparticles (AgNPs) were biosynthesized with an aqueous extract of Pterocladiella (Pterocladia) capillacea, used as a reducing and stabilizing agent, and characterized using UV-VIS spectroscopy, Fourier Transform Infra red (FT-IR) spectroscopy, transmission electron microscopy (TEM) and energy dispersive analysis (EDX). The biosynthesized AgNPs were tested for cytotoxic activity in a human hepatocellular carcinoma (HepG2) cell line cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 1% antibiotic and antimycotic solution and 2 mM glutamine. Bacterial susceptibility to AgNPs was assessed with Staphylococcus aureus, Bacillus subtilis [Gram+ve] and Pseudomonas aeruginosa and Escherichia coli [Gram-ve]. The agar well diffusion technique was adopted to evaluate the bactericidal activity of the biosynthesized AgNPs using Ampicillin and Gentamicin as gram+ve and gram-ve antibacterial standard drugs, respectively. The biosynthesized AgNPs were 11.4±3.52 nm in diameter. FT-IR analysis showed that carbonyl groups from the amino acid residues and proteins could assist in formation and stabilization of AgNPs. The AgNPs showed potent cytotoxic activity against the human hepatocellular carcinoma (HepG2) cell line at higher concentrations. The results also showed that the biosynthesized AgNPs inhibited the entire panel of tested bacteria with a marked specificity towards Bacillus subtillus. Cytotoxic activity of the biosynthesized AgNPs may be due to the presence of alkaloids present in the algal extract. Our AgNPs appear more bactericidal against gram-positive bacteria (B. subtillus).

  13. A Novel Polysaccharide Conjugate from Bullacta exarata Induces G1-Phase Arrest and Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells.

    Science.gov (United States)

    Liao, Ningbo; Sun, Liang; Chen, Jiang; Zhong, Jianjun; Zhang, Yanjun; Zhang, Ronghua

    2017-03-01

    Bullacta exarata has been consumed in Asia, not only as a part of the normal diet, but also as a traditional Chinese medicine with liver- and kidney-benefitting functions. Several scientific investigations involving extraction of biomolecules from this mollusk and pharmacological studies on their biological activities have been carried out. However, little is known regarding the antitumor properties of polysaccharides from B. exarata , hence the polysaccharides from B. exarata have been investigated here. One polysaccharide conjugate BEPS-IA was isolated and purified from B. exarata . It mainly consisted of mannose and glucose in a molar ratio of 1:2, with an average molecular weight of 127 kDa. Thirteen general amino acids were identified to be components of the protein-bound polysaccharide. Methylation and NMR studies revealed that BEPS-IA is a heteropolysaccharide consisting of 1,4-linked-α-d-Glc, 1,6-linked-α-d-Man, 1,3,6-linked-α-d-Man, and 1-linked-α-d-Man residue, in a molar ratio of 6:1:1:1. In order to test the antitumor activity of BEPS-IA, we investigated its effect against the growth of human hepatocellular carcinoma cells HepG2 in vitro. The result showed that BEPS-IA dose-dependently exhibited an effective HepG2 cells growth inhibition with an IC 50 of 112.4 μg/mL. Flow cytometry analysis showed that BEPS-IA increased the populations of both apoptotic sub-G1 and G1 phase. The result obtained from TUNEL assay corroborated apoptosis which was shown in flow cytometry. Western blot analysis suggested that BEPS-IA induced apoptosis and growth inhibition were associated with up-regulation of p53, p21 and Bax, down-regulation of Bcl-2. These findings suggest that BEPS-IA may serve as a potential novel dietary agent for hepatocellular carcinoma.

  14. Repression of the albumin gene in Novikoff hepatoma cells

    International Nuclear Information System (INIS)

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

    1982-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Chi, Hsiang-Cheng; Liao, Chen-Hsin [Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan 333, Taiwan, ROC (China); Huang, Ya-Hui [Medical Research Central, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan, ROC (China); Wu, Sheng-Ming; Tsai, Chung-Ying; Liao, Chia-Jung; Tseng, Yi-Hsin; Lin, Yang-Hsiang; Chen, Cheng-Yi; Chung, I-Hsiao; Wu, Tzu-I [Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan 333, Taiwan, ROC (China); Chen, Wei-Jan [First Cardiovascular Division, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan, ROC (China); Lin, Kwang-Huei, E-mail: khlin@mail.cgu.edu.tw [Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan 333, Taiwan, ROC (China)

    2013-09-13

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

  16. Evaluation of Synergetic Anticancer Activity of Berberine and Curcumin on Different Models of A549, Hep-G2, MCF-7, Jurkat, and K562 Cell Lines

    Directory of Open Access Journals (Sweden)

    Acharya Balakrishna

    2015-01-01

    Full Text Available Ayurvedic system of medicine is using Berberis aristata and Curcuma longa herbs to treat different diseases including cancer. The study was performed to evaluate the synergetic anticancer activity of Berberine and Curcumin by estimating the inhibition of the cell proliferation by cytotoxicity assay using MTT method on specified human cell lines (A549, Hep-G2, MCF-7, Jurkat, and K562. All the cells were harvested from the culture and seeded in the 96-well assay plates at seeding density of 2.0 × 104 cells/well and were incubated for 24 hours. Test items Berberine with Curcumin (1 : 1, Curcumin 95% pure, and Berberine 95% pure were exposed at the concentrations of 1.25, 0.001, and 0.5 mg/mL, respectively, and incubated for a period of 48 hours followed by dispensing MTT solution (5 mg/mL. The cells were incubated at 37 ± 1°C for 4 hours followed by addition of DMSO for dissolving the formazan crystals and absorbance was read at 570 nm. Separate wells were prepared for positive control, controls (only medium with cells, and blank (only medium. The results had proven the synergetic anticancer activity of Berberine with Curcumin inducing cell death greater percentage of >77% when compared to pure curcumin with <54% and pure Berberine with <45% on average on all cell line models.

  17. A comparative assessment of antiproliferative properties of resveratrol and ethanol leaf extract of Anogeissus leiocarpus (DC) Guill and Perr against HepG2 hepatocarcinoma cells.

    Science.gov (United States)

    Olugbami, Jeremiah Olorunjuwon; Damoiseaux, Robert; France, Bryan; Onibiyo, Esther Modupe; Gbadegesin, Michael Adedapo; Sharma, Shivani; Gimzewski, James Kazimierz; Odunola, Oyeronke Adunni

    2017-08-02

    Epidemiological and experimental evidences have shown cancer as a leading cause of death worldwide. Although the folklore use of plants as a reliable source of health-restoring principles is well-documented, the search for more of such plants that are active against diseases, such as cancer, continues. We report here a laboratory-based evidence of the relevance of an ethanol leaf extract of Anogeissus leiocarpus (A2L) in comparison with resveratrol, a natural polyphenol, in cancer therapy. The quantitative assessment of flavonoid and phenolic contents involved quercetin and gallic acid as standards, respectively were determined using spectrophotometry. Cytotoxicity was determined fluorometrically using propidium-iodide-staining method. Antioxidant status, adenosine triphosphate (ATP) levels, caspase activities and mitochondrial integrity were assessed using fluorometry/luminometry. The antioxidant assay demonstrated that A2L possesses a strong antioxidant capacity as compared with the reference compounds, ascorbic acid and butylated hydroxytoluene. This is further buttressed by the significantly high level of phenolics obtained in the quantitative assessment of the extract. A 72-h post-treatment examination indicated that both A2L and resveratrol modulate the proliferation of HepG2 liver carcinoma cells in a time- and concentration-dependent manner. Determination of the total nuclei area, propidium-iodide negative and positive nuclei areas all further buttress the modulation of cell proliferation by A2L and resveratrol with the indication that the observed cell death is due to apoptosis and necrosis at lower and higher concentrations of treatments respectively. At lower concentrations (0.39-3.13 μg/mL), resveratrol possesses higher tendencies to activate caspases 3 and 7. Bioenergetically, both resveratrol and A2L do not adversely affect the cells at lower concentrations (0.39-6.25 μg/mL for resveratrol and 12.5-100.0 μg/mL for A2L) except at higher

  18. (3'R)-hydroxytabernaelegantine C: A bisindole alkaloid with potent apoptosis inducing activity in colon (HCT116, SW620) and liver (HepG2) cancer cells.

    Science.gov (United States)

    Paterna, Angela; Gomes, Sofia E; Borralho, Pedro M; Mulhovo, Silva; Rodrigues, Cecília M P; Ferreira, Maria-José U

    2016-12-24

    Tabernaemontana elegans Stapf. (Apocynaceae) is a medicinal plant traditionally used in African countries to treat cancer. To discover new apoptosis inducing lead compounds from T. elegans and provide scientific validation of the ethnopharmacological use of this plant. Through fractionation, (3'R)-hydroxytaberanelegantine C (1), a vobasinyl-iboga bisindole alkaloid, was isolated from a cytotoxic alkaloid fraction of the methanol extract of T. elegans roots. Its structure was identified by spectroscopic methods, mainly 1D and 2D NMR experiments. Compound 1 was evaluated for its ability to induce apoptosis in HCT116 and SW620 colon and HepG2 liver carcinoma cells. The cell viability of compound 1 was evaluated by the MTS and lactate dehydrogenase (LDH) assays. Induction of apoptosis was analyzed through Guava ViaCount assay, by flow cytometry, caspase-3/7 activity assays and evaluation of nuclear morphology by Hoechst staining. To determine the molecular pathways elicited by 1 exposure, immunoblot analysis was also performed. (3'R)-hydroxytaberanelegantine C (1) displayed strong apoptosis induction activity as compared to 5-fluorouracil (5-FU), the most used anticancer agent in colorectal cancer treatment. In the MTS assay, compound 1 exhibited IC 50 values similar or lower than 5-FU in the three cell lines tested. The IC 50 value of 1 was also calculated in CCD18co normal human colon fibroblasts. The lactate dehydrogenase assay showed increased LDH release by compound 1, and the Guava ViaCount assay revealed that 1 significantly increased the incidence of apoptosis to a further extent than 5-FU. Moreover, the induction of apoptosis was corroborated by evaluation of nuclear morphology by Hoechst staining and caspase-3/7 activity assays of 1 treated cells. As expected, in immunoblot analysis, compound 1 treatment led to poly(ADP-ribose) polymerase cleavage. This was accompanied by decreased anti-apoptotic proteins Bcl-2 and XIAP steady state levels in all three cancer

  19. Caffeine Increases Apolipoprotein A-1 and Paraoxonase-1 but not Paraoxonase-3 Protein Levels in Human-Derived Liver (HepG2) Cells.

    Science.gov (United States)

    Sayılan Özgün, Gülben; Özgün, Eray; Tabakçıoğlu, Kıymet; Süer Gökmen, Selma; Eskiocak, Sevgi; Çakır, Erol

    2017-12-01

    Apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 are antioxidant and anti-atherosclerotic structural high-density lipoprotein proteins that are mainly synthesized by the liver. No study has ever been performed to specifically examine the effects of caffeine on paraoxonase enzymes and on liver apolipoprotein A-1 protein levels. To investigate the dose-dependent effects of caffeine on liver apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 protein levels. In vitro experimental study. HepG2 cells were incubated with 0 (control), 10, 50 and 200 μM of caffeine for 24 hours. Cell viability was evaluated by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 protein levels were measured by western blotting. We observed a significant increase on apolipoprotein A-1 and paraoxonase-1 protein levels in the cells incubated with 50 µM of caffeine and a significant increase on paraoxonase-1 protein level in the cells incubated with 200 µM of caffeine. Our study showed that caffeine does not change paraoxonase-3 protein level, but the higher doses used in our study do cause an increase in both apolipoprotein A-1 and paraoxonase-1 protein levels in liver cells.

  20. Synthesis of PBAD-lipiodol nanoparticles for combination treatment with boric acid in boron neutron capture therapy for hepatoma in-vitro

    International Nuclear Information System (INIS)

    Chou, F.I.; Chung, H.P.; Liu, H.M.; Wen, H.W.; Chi, C.W.; Lin, Shanyang; Lui, W.Y.; Kai, J.J.

    2006-01-01

    This study attempted to increase BNCT efficiency for hepatoma by a combined treatment of phenylboric acid derivative entrapped lipiodol nanoparticles (PBAD-L nanoparticles) with boric acid. The size of PBAD-L nanoparticles were 400-750 nm at the boron concentrations of 0.3-2.7 mg/ml. After 24 hours the boron concentration in PBAD-L nanoparticles treated human hepatoma HepG2 cells was 112 ppm, while that in rat liver Clone 9 cells was 52 ppm. With the use of 25 μg B/ml boric acid, after 6 hours the boron concentration in HepG2 and Clone 9 cells were 75 ppm and 40 ppm, respectively. In a combined treatment, boron concentration in HepG2 cells which were treated with PBAD-L nanoparticles for 18 hours and then combined with boric acid for 6 hours was 158 ppm. After neutron irradiation, the surviving fraction of HepG2 cells treated with PBAD-L nanoparticles was 12.6%, while that in the ones with a combined treatment was 1.3%. In conclusion, the combined treatment provided a higher boron concentration in HepG2 cells than treatments with either PBAD-L nanoparticles or boric acid, resulting in a higher therapeutic efficacy of BNCT in hepatoma cells. (author)

  1. APPLICATION OF CDNA MICROARRAY TECHNOLOGY TO IN VITRO TOXICOLOGY AND THE SELECTION OF GENES FOR A REAL TIME RT-PCR-BASED SCREEN FOR OXIDATIVE STRESS IN HEP-G2 CELLS

    Science.gov (United States)

    Large-scale analysis of gene expression using cDNA microarrays promises therapid detection of the mode of toxicity for drugs and other chemicals. cDNAmicroarrays were used to examine chemically-induced alterations of geneexpression in HepG2 cells exposed to oxidative ...

  2. EFFECTS OF THE ANTIMUTAGENS VANILLIN AND CINNAMALDEHYDE ON SPONTANEOUS MUTATION IN E. COLI LACL STRAINS AND ON GLOBAL GENE EXPRESSION IN SALMONELLA TA104 AND HUMAN HEPG2 CELLS

    Science.gov (United States)

    Effects of the Antimutagens Vanillin and Cinnamaldehyde on Spontaneous Mutation in E. coli lacI Strains and on Global Gene Epression in Salmonella TAlO4 and Human HepG2 Cells In previous work we have shown that vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutag...

  3. (Glyco)sphingolipids are sorted in sub-apical compartments in HepG2 cells : A role for non-Golgi-related intracellular sites in the polarized distribution of (glyco)sphingolipids

    NARCIS (Netherlands)

    van IJzendoorn, SCD; Hoekstra, D

    1998-01-01

    In polarized HepG2 cells, the fluorescent sphingolipid analogues of glucosylceramide (C-6-NBD-GlcCer) and sphingomyelin (C-6-NBD-SM) display a preferential localization at the apical and basolateral domain, respectively, which is expressed during apical to basolateral transcytosis of the lipids (van

  4. The Interactions between ZnO Nanoparticles (NPs and α-Linolenic Acid (LNA Complexed to BSA Did Not Influence the Toxicity of ZnO NPs on HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Yiwei Zhou

    2017-04-01

    Full Text Available Background: Nanoparticles (NPs entering the biological environment could interact with biomolecules, but little is known about the interaction between unsaturated fatty acids (UFA and NPs. Methods: This study used α-linolenic acid (LNA complexed to bovine serum albumin (BSA for UFA and HepG2 cells for hepatocytes. The interactions between BSA or LNA and ZnO NPs were studied. Results: The presence of BSA or LNA affected the hydrodynamic size, zeta potential, UV-Vis, fluorescence, and synchronous fluorescence spectra of ZnO NPs, which indicated an interaction between BSA or LNA and NPs. Exposure to ZnO NPs with the presence of BSA significantly induced the damage to mitochondria and lysosomes in HepG2 cells, associated with an increase of intracellular Zn ions, but not intracellular superoxide. Paradoxically, the release of inflammatory cytokine interleukin-6 (IL-6 was decreased, which indicated the anti-inflammatory effects of ZnO NPs when BSA was present. The presence of LNA did not significantly affect all of these endpoints in HepG2 cells exposed to ZnO NPs and BSA. Conclusions: the results from the present study indicated that BSA-complexed LNA might modestly interact with ZnO NPs, but did not significantly affect ZnO NPs and BSA-induced biological effects in HepG2 cells.

  5. Protein phosphatase 2A mediates JS-K-induced apoptosis by affecting Bcl-2 family proteins in human hepatocellular carcinoma HepG2 cells.

    Science.gov (United States)

    Liu, Ling; Huang, Zile; Chen, Jingjing; Wang, Jiangang; Wang, Shuying

    2018-04-25

    Protein phosphatase 2A (PP2A) is an important enzyme within various signal transduction pathways. The present study was investigated PP2A mediates JS-K-induced apoptosis by affecting Bcl-2 family protein. JS-K showed diverse inhibitory effects in five HCC cell lines, especially HepG2 cells. JS-K caused a dose- and time-dependent reduction in cell viability and increased in levels of LDH release. Meanwhile, JS-K- induced apoptosis was characterized by mitochondrial membrane potential reduction, Hoechst 33342 + /PI + dual staining, release of cytochrome c (Cyt c), and activation of cleaved caspase-9/3. Moreover, JS-K-treatment could lead to the activation of protein phosphatase 2A-C (PP2A-C), decrease of anti-apoptotic Bcl-2 family-protein expression including p-Bcl-2 (Ser70), Bcl-2, Bcl-xL, and Mcl-1 as well as the increase of pro-apoptosis Bcl-2 family-protein including Bim, Bad, Bax, and Bak. Furthermore, JS-K caused a marked increase of intracellular NO levels while pre-treatment with Carboxy-PTIO (a NO scavenger) reduced the cytotoxicity effects and the apoptosis rate. Meanwhile, pre-treatment with Carboxy-PTIO attenuated the JS-K-induced up-regulation of PP2A, Cyt c, and cleaved-caspase-9/3 activation. The silencing PP2A-C by siRNA could abolish the activation of PP2A-C, down-regulation of anti-apoptotic Bcl-2 family-protein (p-Bcl-2, Bcl-2, Bcl-xL, and Mcl-1), increase of pro-apoptosis Bcl-2 family-protein (Bim, Bad, Bax, and Bak) and apoptotic-related protein (Cyt c, cleaved caspase-9/3) that were caused by JS-K in HepG2 cells. In addition, pre-treatment with OA (a PP2A inhibitor) also attenuated the above effects induced by JS-K. In summary, NO release from JS-K induces apoptosis through PP2A activation, which contributed to the regulation of Bcl-2 family proteins. © 2018 Wiley Periodicals, Inc.

  6. Cadmium, lead, and arsenic contamination in paddy soils of a mining area and their exposure effects on human HEPG2 and keratinocyte cell-lines.

    Science.gov (United States)

    Xue, Shengguo; Shi, Lizheng; Wu, Chuan; Wu, Hui; Qin, Yanyan; Pan, Weisong; Hartley, William; Cui, Mengqian

    2017-07-01

    A mining district in south China shows significant metal(loid) contamination in paddy fields. In the soils, average Pb, Cd and As concentrations were 460.1, 11.7 and 35.1mgkg -1 respectively, which were higher than the environmental quality standard for agricultural soils in China (GB15618-1995) and UK Clea Soil Guideline Value. The average contents of Pb, Cd and As in rice were 5.24, 1.1 and 0.7mgkg -1 respectively, which were about 25, 4.5 or 2.5 times greater than the limit values of the maximum safe contaminant concentration standard in food of China (GB 2762-2012), and about 25, 10 or 1 times greater than the limit values of FAO/WHO standard. The elevated contents of Pb, Cd and As detected in soils around the factories, indicated that their spatial distribution was influenced by anthropogenic activity, while greater concentrations of Cd in rice appeared in the northwest region of the factories, indicating that the spatial distribution of heavy metals was also affected by natural factors. As human exposure around mining districts is mainly through oral intake of food and dermal contact, the effects of these metals on the viability and MT protein of HepG2 and KERTr cells were investigated. The cell viability decreased with increasing metal concentrations. Co-exposure to heavy metals (Pb+Cd) increased the metals (Pb or Cd)-mediated MT protein induction in both human HepG2 and KERTr cells. Increased levels of MT protein will lead to greater risk of carcinogenic manifestations, and it is likely that chronic exposure to metals may increase the risk to human health. Nevertheless, when co-exposure to two or more metals occur (such as As+Pb), they may have an antagonistic effect thus reducing the toxic effects of each other. Metal contaminations in paddy soils and rice were influenced by anthropogenic activity; metal co-exposure induced MT protein in human cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Berberine Attenuates Development of the Hepatic Gluconeogenesis and Lipid Metabolism Disorder in Type 2 Diabetic Mice and in Palmitate-Incubated HepG2 Cells through Suppression of the HNF-4α miR122 Pathway.

    Science.gov (United States)

    Wei, Shengnan; Zhang, Ming; Yu, Yang; Lan, Xiaoxin; Yao, Fan; Yan, Xin; Chen, Li; Hatch, Grant M

    2016-01-01

    Berberine (BBR) has been shown to exhibit protective effects against diabetes and dyslipidemia. Previous studies have indicated that BBR modulates lipid metabolism and inhibits hepatic gluconeogensis by decreasing expression of Hepatocyte Nuclear Factor-4α (HNF-4α). However, the mechanism involved in this process was unknown. In the current study, we examined the mechanism of how BBR attenuates hepatic gluconeogenesis and the lipid metabolism alterations observed in type 2 diabetic (T2D) mice and in palmitate (PA)-incubated HepG2 cells. Treatment with BBR for 4 weeks improve all biochemical parameters compared to T2D mice. Treatment of T2D mice for 4 weeks or treatment of PA-incubated HepG2 cells for 24 h with BBR decreased expression of HNF-4α and the microRNA miR122, the key gluconeogenesis enzymes Phosphoenolpyruvate carboxykinase (PEPCK) and Glucose-6-phosphatase (G6Pase) and the key lipid metabolism proteins Sterol response element binding protein-1 (SREBP-1), Fatty acid synthase-1 (FAS-1) and Acetyl-Coenzyme A carboxylase (ACCα) and increased Carnitine palmitoyltransferase-1(CPT-1) compared to T2D mice or PA-incubated HepG2 cells. Expression of HNF-4α in HepG2 cells increased expression of gluconeogenic and lipid metabolism enzymes and BBR treatment or knock down of miR122 attenuated the effect of HNF-4α expression. In contrast, BBR treatment did not alter expression of gluconeogenic and lipid metabolism enzymes in HepG2 cells with knockdown of HNF-4α. In addition, miR122 mimic increased expression of gluconeogenic and lipid metabolism enzymes in HepG2 cells with knockdown of HNF-4α. These data indicate that miR122 is a critical regulator in the downstream pathway of HNF-4α in the regulation of hepatic gluconeogenesis and lipid metabolism in HepG2 cells. The effect of BBR on hepatic gluconeogenesis and lipid metabolism is mediated through HNF-4α and is regulated downstream of miR122. Our data provide new evidence to support HNF-4α and miR122

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

    Directory of Open Access Journals (Sweden)

    Fat-Moon Suk

    2013-01-01

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

  9. Effects of gelling bath on the physical properties of alginate gel beads and the biological characteristics of entrapped HepG2 cells.

    Science.gov (United States)

    Sun, Dongsheng; Liu, Yang; Wu, Hao; Ren, Ying; Ma, Xiaojun; Wu, Huijian; Sun, Guangwei

    2018-03-01

    Optimizing alginate gel beads is necessary to support the survival, proliferation, and function of entrapped hepatocytes. In this study, gelling bath was modified by decreasing calcium ion concentration and increasing sodium ion concentration. Alginate gel beads (using 36% G sodium alginate) prepared in the modified gelling bath had more homogeneous structure and better mass transfer properties compared with the traditional gelling bath that contains only calcium ions. Moreover, alginate gel beads generated in the modified gelling bath could significantly promote the HepG2 cell proliferation and the growth of cell spheroids, and maintain the albumin secretion ability similar to alginate gel beads prepared in the traditional gelling bath with only calcium ions. The mass transfer properties and cell proliferation were similar in ALG beads with different M/G ratio (36% G and 55% G) generated in the modified gelling bath, whereas they were significantly increased compared with alginate gel beads (55% G) in traditional gelling bath. These results indicated that adjusting the gelling bath was a simple and convenient method to enhance the mass transfer properties of alginate gel beads for 3D hepatocyte culture, which might provide more hepatocytes for the bioartificial liver support system. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  10. Schisandra chinensis peptidoglycan-assisted transmembrane transport of lignans uniquely altered the pharmacokinetic and pharmacodynamic mechanisms in human HepG2 cell model.

    Directory of Open Access Journals (Sweden)

    Charng-Cherng Chyau

    Full Text Available Schisandra chinensis (Turz Baill (S. chinensis (SC fruit is a hepatoprotective herb containing many lignans and a large amount of polysaccharides. A novel polysaccharide (called SC-2 was isolated from SC of MW 841 kDa, which exhibited a protein-to-polysaccharide ratio of 0.4089, and showed a characteristic FTIR spectrum of a peptidoglycan. Powder X-ray diffraction revealed microcrystalline structures within SC-2. SC-2 contained 10 monosaccharides and 15 amino acids (essential amino acids of 78.12%w/w. In a HepG2 cell model, SC-2 was shown by MTT and TUNEL assay to be completely non-cytotoxic. A kinetic analysis and fluorescence-labeling technique revealed no intracellular disposition of SC-2. Combined treatment of lignans with SC-2 enhanced the intracellular transport of schisandrin B and deoxyschisandrin but decreased that of gomisin C, resulting in alteration of cell-killing bioactivity. The Second Law of Thermodynamics allows this type of unidirectional transport. Conclusively, SC-2 alters the transport and cell killing capability by a "Catcher-Pitcher Unidirectional Transport Mechanism".

  11. Anthocyanin-Rich Grape Pomace Extract (Vitis vinifera L. from Wine Industry Affects Mitochondrial Bioenergetics and Glucose Metabolism in Human Hepatocarcinoma HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Nathalia F. F. de Sales

    2018-03-01

    Full Text Available Cancer cells demand high ATP provisions to support proliferation, and targeting of energy metabolism is a good strategy to increase their sensitivity to treatments. In Brazil, wine manufacture is expanding, increasing the amount of pomace that is produced. We determined the phenolic composition and antioxidant properties of a dark skin Grape Pomace Extract and its effects on metabolism and redox state in human hepatocarcinoma HepG2 cells. The material and the methods used represented the industrial process since pomace derived from white wine production and the extract concentrated by pilot plant scale reverse osmosis. Grape pomace extract was rich in polyphenols, mainly anthocyanins, and presented high antioxidant capacity. Short-term metabolic effects, irrespective of any cytotoxicity, involved increased mitochondrial respiration and antioxidant capacity and decreased glycolytic metabolism. Long-term incubation was cytotoxic and cells died by necrosis and GPE was not toxic to non-cancer human fibroblasts. To the best of our knowledge, this is the first report to characterize pomace extract from white wine production from Brazilian winemaking regarding its effects on energy metabolism, suggesting its potential use for pharmaceutical and nutraceutical purposes.

  12. Anthocyanin-Rich Grape Pomace Extract (Vitis vinifera L.) from Wine Industry Affects Mitochondrial Bioenergetics and Glucose Metabolism in Human Hepatocarcinoma HepG2 Cells.

    Science.gov (United States)

    de Sales, Nathalia F F; Silva da Costa, Leandro; Carneiro, Talita I A; Minuzzo, Daniela A; Oliveira, Felipe L; Cabral, Lourdes M C; Torres, Alexandre G; El-Bacha, Tatiana

    2018-03-08

    Cancer cells demand high ATP provisions to support proliferation, and targeting of energy metabolism is a good strategy to increase their sensitivity to treatments. In Brazil, wine manufacture is expanding, increasing the amount of pomace that is produced. We determined the phenolic composition and antioxidant properties of a dark skin Grape Pomace Extract and its effects on metabolism and redox state in human hepatocarcinoma HepG2 cells. The material and the methods used represented the industrial process since pomace derived from white wine production and the extract concentrated by pilot plant scale reverse osmosis. Grape pomace extract was rich in polyphenols, mainly anthocyanins, and presented high antioxidant capacity. Short-term metabolic effects, irrespective of any cytotoxicity, involved increased mitochondrial respiration and antioxidant capacity and decreased glycolytic metabolism. Long-term incubation was cytotoxic and cells died by necrosis and GPE was not toxic to non-cancer human fibroblasts. To the best of our knowledge, this is the first report to characterize pomace extract from white wine production from Brazilian winemaking regarding its effects on energy metabolism, suggesting its potential use for pharmaceutical and nutraceutical purposes.

  13. A Novel Polysaccharide Conjugate from Bullacta exarata Induces G1-Phase Arrest and Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Ningbo Liao

    2017-03-01

    Full Text Available Bullacta exarata has been consumed in Asia, not only as a part of the normal diet, but also as a traditional Chinese medicine with liver- and kidney-benefitting functions. Several scientific investigations involving extraction of biomolecules from this mollusk and pharmacological studies on their biological activities have been carried out. However, little is known regarding the antitumor properties of polysaccharides from B. exarata, hence the polysaccharides from B. exarata have been investigated here. One polysaccharide conjugate BEPS-IA was isolated and purified from B. exarata. It mainly consisted of mannose and glucose in a molar ratio of 1:2, with an average molecular weight of 127 kDa. Thirteen general amino acids were identified to be components of the protein-bound polysaccharide. Methylation and NMR studies revealed that BEPS-IA is a heteropolysaccharide consisting of 1,4-linked-α-d-Glc, 1,6-linked-α-d-Man, 1,3,6-linked-α-d-Man, and 1-linked-α-d-Man residue, in a molar ratio of 6:1:1:1. In order to test the antitumor activity of BEPS-IA, we investigated its effect against the growth of human hepatocellular carcinoma cells HepG2 in vitro. The result showed that BEPS-IA dose-dependently exhibited an effective HepG2 cells growth inhibition with an IC50 of 112.4 μg/mL. Flow cytometry analysis showed that BEPS-IA increased the populations of both apoptotic sub-G1 and G1 phase. The result obtained from TUNEL assay corroborated apoptosis which was shown in flow cytometry. Western blot analysis suggested that BEPS-IA induced apoptosis and growth inhibition were associated with up-regulation of p53, p21 and Bax, down-regulation of Bcl-2. These findings suggest that BEPS-IA may serve as a potential novel dietary agent for hepatocellular carcinoma.

  14. Effective delivery of hydrophobic drugs to breast (MCF-7) and Liver (HepG2) cancer cells: A detailed investigation using Cytotoxicity assays, fluorescence imaging and flow cytometry.

    Science.gov (United States)

    Manatunga, Danushika C; de Silva, Rohini M; Nalin de Silva, K M; Neelika Malavige, Gathsaurie; Wijeratne, Dulharie T; Williams, Gareth R; Jayasinghe, Chanika D; Udagama, Preethi V

    2018-04-03

    This study aimed to develop a drug carrier system consisting of a polymer containing hydroxyapatite (HAp) shell and a magnetic core of iron oxide nanoparticles. Doxorubicin and/or curcumin were loaded into the carrier via a simple diffusion deposition approach, with encapsulation efficiencies (EE) for curcumin and doxorubicin of 93.03 ± 0.3% and 97.37 ± 0.12% respectively. The co-loading of curcumin and doxorubicin led to a total EE of 76.02 ± 0.48%. Release studies were carried out at pH 7.4 and 5.3, and revealed higher release was at pH 5.3 expressing the potential application in tumor microenvironments. Cytotoxicity assays, fluorescence imaging and flow cytometry showed the formulations could effectively inhibit the growth of MCF-7 and HEpG2 cancer cells, being more potent than the free drug molecules both in dose and time dependent manner. Additionally, hemolysis tests and cytotoxicity evaluations determined the drug-loaded carriers to be non-toxic towards non-cancerous cells. These formulations thus have great potential in the development of new cancer therapeutics. Copyright © 2018. Published by Elsevier B.V.

  15. Functional Toxicogenomic Assessment of Triclosan in Human HepG2 Cells Using Genome-Wide CRISPR-Cas9 Screening.

    Science.gov (United States)

    Xia, Pu; Zhang, Xiaowei; Xie, Yuwei; Guan, Miao; Villeneuve, Daniel L; Yu, Hongxia

    2016-10-04

    There are thousands of chemicals used by humans and detected in the environment for which limited or no toxicological data are available. Rapid and cost-effective approaches for assessing the toxicological properties of chemicals are needed. We used CRISPR-Cas9 functional genomic screening to identify the potential molecular mechanism of a widely used antimicrobial triclosan (TCS) in HepG2 cells. Resistant genes at IC50 (the concentration causing a 50% reduction in cell viability) were significantly enriched in the adherens junction pathway, MAPK signaling pathway, and PPAR signaling pathway, suggesting a potential role in the molecular mechanism of TCS-induced cytotoxicity. Evaluation of the top-ranked resistant genes, FTO (encoding an mRNA demethylase) and MAP2K3 (a MAP kinase kinase family gene), revealed that their loss conferred resistance to TCS. In contrast, sensitive genes at IC10 and IC20 were specifically enriched in pathways involved with immune responses, which was concordant with transcriptomic profiling of TCS at concentrations of CRISPR-Cas9 fingerprint may reveal the patterns of TCS toxicity at low concentration levels. Moreover, we retrieved the potential connection between CRISPR-Cas9 fingerprint and disease terms, obesity, and breast cancer from an existing chemical-gene-disease database. Overall, CRISPR-Cas9 functional genomic screening offers an alternative approach for chemical toxicity testing.

  16. Synergistic Hepatoprotective and Antioxidant Effect of Artichoke, Fig, Blackberry Herbal Mixture on HepG2 Cells and Their Metabolic Profiling Using NMR Coupled with Chemometrics.

    Science.gov (United States)

    Youssef, Fadia S; Labib, Rola M; Eldahshan, Omayma A; Singab, Abdel Nasser B

    2017-12-01

    The edible plants have long been reported to possess a lot of biological activities. Herein, the hepatoprotective and the antioxidant activities of the aqueous infusion of the edible parts of Cynara cardunculus, Ficus carica, and Morus nigra and their herbal mixture (CFM) was investigated in vitro using CCl 4 induced damage in HepG2 cells. The highest amelioration was observed via the consumption of CFM at 1 mg/ml showing 47.00% and 37.09% decline in aspartate transaminase and alanine transaminase and 77.32% and 101.02% increase in reduced glutathione and superoxide dismutase comparable to CCl 4 treated cells. Metabolic profiling of their aqueous infusions was done using nuclear magnetic resonance spectroscopic experiments coupled with chemometrics particularly hierarchical cluster analysis (HCA) and principal component analysis (PCA). The structural closeness of the various metabolites existing in black berry and the mixture as reflected in the PCA score plot and HCA processed from the 1 H-NMR spectral data could eventually explained the close values in their biological behavior. For fig and artichoke, the existence of different phenolic metabolites that act synergistically could greatly interpret their potent biological behavior. Thus, it can be concluded that a herbal mixture composed of black berry, artichoke, and fig could afford an excellent natural candidate to combat oxidative stress and counteract hepatic toxins owing to its phenolic compounds. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

  17. Effects of individual and combined toxicity of bisphenol A, dibutyl phthalate and cadmium on oxidative stress and genotoxicity in HepG 2 cells.

    Science.gov (United States)

    Li, Xiaohui; Yin, Pinghe; Zhao, Ling

    2017-07-01

    Bisphenol A, dibutyl phthalate and cadmium can be found in environment simultaneously. Several studies suggested that they had genotoxic effect. In this study, mono-exposure and co-exposure treatments, designed by 3 × 3 full factorial, were established to determine the individual toxicity and binary mixtures' combined effects on the oxidative stress and genotoxicity in HepG 2 cells. The highest oxidative damage was observed in the Cd treatments groups. Compared with control groups, the maximum level of reactive oxygen species and malondialdehyde were ∼1.4 fold and ∼2.22 fold respectively. And a minimum level of superoxide dismutase activity was found with the decrease of 43%. The mechanism that excessive oxidative stress led to the DNA damage was inferred. However, cells treated with BPA showed the worst DNA damage rather than Cd, which may because Cd mainly damages DNA repairing mechanism. For the joint effect, different interactions can be found in different biological endpoints for different combinations since different mechanisms have been clarified in mixture toxicity studies. It is sure that the co-exposure groups enhanced cytotoxicity, oxidative stress and genotoxicity compared to the mono-exposures. Synergistic and additive interactions were considered, which means greater threat to organisms when exposed to multiple estrogenic endocrine disruptors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Phytoextract of Indian mustard seeds acts by suppressing the generation of ROS against acetaminophen-induced hepatotoxicity in HepG2 cells.

    Science.gov (United States)

    Parikh, Harita; Pandita, Nancy; Khanna, Aparna

    2015-07-01

    Indian mustard [Brassica juncea (L.) Czern. & Coss. (Brassicaceae)] is reported to possess diverse pharmacological properties. However, limited information is available concerning its hepatoprotective activity and mechanism of action. To study the protective mechanism of mustard seed extract against acetaminophen (APAP) toxicity in a hepatocellular carcinoma (HepG2) cell line. Hepatotoxicity models were established using APAP (2.5-22.5 mM) based on the cytotoxicity profile. An antioxidant-rich fraction from mustard seeds was extracted and evaluated for its hepatoprotective potential. The mechanism of action was elucidated using various in vitro antioxidant assays, the detection of intracellular generation of reactive oxygen species (ROS), and cell cycle analysis. The phytoconstituents isolated via HPLC-DAD were also evaluated for hepatoprotective activity. Hydromethanolic seed extract exhibited hepatoprotective activity in post- and pre-treatment models of 20 mM APAP toxicity and restored the elevated levels of liver indices to normal values (p DAD analysis revealed the presence quercetin, vitamin E, and catechin, which exhibited hepatoprotective activity. A phytoextract of mustard seeds acts by suppressing the generation of ROS in response to APAP toxicity.

  19. [Transmembrane transport behavior of in vitro HepG2 cells of ananas and its effect on lipids and glucose distribution].

    Science.gov (United States)

    Pang, Yu-Nong; Chai, Yu-Shuang; Jiang, Jing-Fei; Wang, Xin-Pei; Yu, Xuan; Lei, Fan; Xing, Dong-Ming; Du, Li-Jun

    2014-08-01

    Pineapple (Ananas comosus) leaves contain mainly phenolic components with antioxidant and hypolipidemic effects. One of the principle components is p-coumaric acid. In this study, the transport behavior of p-coumaric acid, was observed after the administration of pineapple leaf phenols in vitro. Simultaneously, the effect of the phenols on glucose, total cholesterol and triglycerides transportation and metabolism in HepG2 cells was also observed. The results showed that the phenols had good transport characteristics. 5 min after the administration, p-coumaric acid of the phenols could be detected, and the content of p-coumaric acid reached the peak concentration after 60 min of the administration. p-coumaric acid of phenols have time-and dose-dependent manner. While promoting glucose transporter (GLUT4) and low density lipoprotein receptor (LDLR) expression, the phenols decreased intracellular lipid content. This reduction of intracellular lipid content was highly correlated with the promotion of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) expression, while the reduction of intracellular glucose levels was correlated with glycogen synthesis in the cells.

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

    Science.gov (United States)

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

    2016-03-01

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

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

    Science.gov (United States)

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

    2016-10-01

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

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

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    Hai-Shan Peng

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

  3. Egr2 enhances insulin resistance via JAK2/STAT3/SOCS-1 pathway in HepG2 cells treated with palmitate.

    Science.gov (United States)

    Lu, Lin; Ye, Xinhua; Yao, Qing; Lu, Aijiao; Zhao, Zhen; Ding, Yang; Meng, Chuchen; Yu, Wenlong; Du, Yunfeng; Cheng, JinLuo

    2018-05-01

    Insulin resistance is generally responsible for the pathogenesis of type 2 diabetes mellitus (T2DM). Early growth response proteins-2 (Egr2) has been reported to be able to increase the expression of the suppressors of cytokine signaling-1 (SOCS-1), and impair insulin signaling pathway through suppression of insulin receptor substrates (IRS), including IRS-1 and IRS-2. However, whether Egr2 is directly involved in the development of insulin resistance, and how its potential contributions to insulin resistance still remain unknown. Here, our present investigation found that the expression levels of Egr2 were up-regulated when insulin resistance occurs, and knockdown of Egr2 abolished the effect of insulin resistance in HepG2 cells induced with palmitate (PA). Importantly, inhibition of Egr2 decreased the expression of SOCS-1 as well as reduced phosphorylation of JAK2 and STAT3. And, our data indicated that silencing of Egr2 accelerated hepatic glucose uptake and reversed the impaired lipid metabolism upon insulin resistance. In summary, the present study confirms that Egr2 could deteriorate insulin resistance via the pathway of JAK2/STAT3/SOCS-1 and may shed light on resolving insulin resistance and further the pathogenesis of T2DM. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Synthesis, characterization and dose dependent antimicrobial and anti-cancerous activity of phycogenic silver nanoparticles against human hepatic carcinoma (HepG2 cell line

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    N. Supraja

    2016-10-01

    Full Text Available In the present study silver nanoparticles (AgNPs were successfully synthesized using aqueous extract of sea weed, Gracilaria corticata. The aqueous callus extract (5% treated with 1 mM silver nitrate solution resulted in the formation of AgNPs and the surface plasmon resonance (SPR of the formed AgNPs was recorded at 405 nm using UV-Visible spectrophotometer. The molecules involved in the formation of AgNPs were identified by Fourier transform infrared spectroscopy (FT-IR, surface morphology was studied by using scanning electron microscopy (SEM, and X-ray diffraction spectroscopy (XRD was used to determine the crystalline structure. SEM micrograph clearly revealed the size of the AgNPs was in the range of 20–55 nm with spherical, hexagonal in shape and poly-dispersed nature. High positive Zeta potential (22.9 mV of formed AgNPs indicates the stability and XRD pattern revealed the crystal structure of the AgNPs by showing the Bragg’s peaks corresponding to (111, (200, (220 planes of face-centered cubic crystal phase of silver. The synthesized AgNPs exhibited effective anticancerous activity (at doses 6.25 and 12.5 µg/ml of AgNPs against human hepatic carcinoma cell line (HepG2.

  5. Cytoprotective effect exerted by geraniin in HepG2 cells is through microRNA mediated regulation of BACH-1 and HO-1.

    Science.gov (United States)

    Aayadi, Hoda; Mittal, Smriti P K; Deshpande, Anjali; Gore, Makarand; Ghaskadbi, Saroj S

    2017-11-01

    Geraniin, a hydrolysable tannin, used in traditional medicine in Southeast Asia, is known to exhibit various biological activities. As an antioxidant it is known to up-regulate phase II enzyme Heme oxygenase-1 (HO-1). However its mechanism is not clearly understood. Nuclear factor erythroid-derived 2 related factor 2 (Nrf-2) is transcriptionally up-regulated by Extracellular signal-regulated kinase (ERK) 1/2 and retained in nucleus due to inactivated Glycogen synthase kinase 3 beta (GSK-3β). Geraniin additionally down-regulates expression of microRNA 217 and 377 (miR-217 and miR-377) which target HO-1 mRNA. Expression of BTB and CNC homolog 1 (BACH-1), another regulator of HO-1, is also down-regulated by up-regulating microRNA 98 (miR-98), a negative regulator of BACH-1. Thus, geraniin up-regulates HO-1 expression both through activating its positive regulator Nrf-2 and by down-regulating its negative regulator BACH-1. Up-regulation of HO-1 also confers protection to HepG2 cells from tertiary butyl hydroperoxide (TBH) induced cytotoxicity. [BMB Reports 2017; 50(11): 560-565].

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

    Science.gov (United States)

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

    2014-09-01

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

  7. Analytical Research to Determine the effects of the Components of ONGABO on the Viability of HepG2 Cancer Cells by Using the Sovereign, Minister, Assistant and Courier Principle (君臣佐使論

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    Shin Jeong-Hun

    2012-12-01

    Full Text Available Objectives: This study used the basic principle of Oriental medicine, the sovereign, minister, assistant and courier principle (君臣佐使論 to investigate the effects of the component of ONGABO, which is composed of Ginseng Radix (Red Ginseng, Angelica Gigantis Radix, Schisandrae Fructus, Cuscuta Semen and Curcumae tuber on the viability of HepG2 cells. Methods: Single and mixed extracts of the component of ONGABO were prepared by lypohilizing powder of Red Ginseng (6-year root from Kanghwa, Angelica Gigantis Radix, Schisandrae Fructus, Cuscuta Semen, Curcumae Tuber (from Omniherb Co., Ltd., Korea at the laboratory of herbal medicine in Woosuk University and were eluted after being macerated with 100% ethanol for three days. The cell viability of HepG2 was determined by using an absorptiometric analysis with PrestoBlue (Invitrogen reagent after the plate had been incubated for 48 hours. All of the experiments were repeated three times to obtain the average value and standard deviation. The statistical analysis was done and the correlation factor was obtained by using Microsoft Office Excel 2007 and Origin 6.0 software. Results: Although Ginseng Radix (Red Ginseng and Schisandrae Fructus did not enhance the viability of HepG2 cells, they were shown to provide protection of those cells. On the other hand, Angelica Gigantis Radix decreased the viability of HepG2 cells significantly, Cuscuta Semen and Curcumae Tuber had a small or no effect on the viability of HepG2 cells. Conclusions: In the sovereign, minister, assistant and courier principle (君臣佐使論, Ginseng Radix (Red Ginseng corresponds to the sovereign component because it provides cell protection effects, Angelica Gigantis Radix corresponds to minister medicinal because it kills cells, Schisandrae Fructus corresponds to the assistant medicinal to help red ginseng having cell protect effects. Cuscuta Semen and Curcumae Tuber correspond to the courier medicinal having no effect in

  8. Involvement of c-Met- and phosphatidylinositol 3-kinase dependent pathways in arsenite-induced downregulation of catalase in hepatoma cells.

    Science.gov (United States)

    Kim, Soohee; Lee, Seung Heon; Kang, Sukmo; Lee, Lyon; Park, Jung-Duck; Ryu, Doug-Young

    2011-01-01

    Catalase protects cells from reactive oxygen species-induced damage by catalyzing the breakdown of hydrogen peroxide to oxygen and water. Arsenite decreases catalase activity; it activates phosphatidylinositol 3-kinase (PI3K) and its key downstream effector Akt in a variety of cells. The PI3K pathway is known to inhibit catalase expression. c-Met, an upstream regulator of PI3K and Akt, is also involved in the regulation of catalase expression. To examine the involvement of c-Met and PI3K pathways in the arsenite-induced downregulation of catalase, catalase mRNA and protein expression were analyzed in the human hepatoma cell line HepG2 treated with arsenite and either an inhibitor of c-Met (PHA665752 (PHA)) or of PI3K (LY294002 (LY)). Arsenite treatment markedly activated Akt and decreased the levels of both catalase mRNA and protein. Both PHA and LY attenuated arsenite-induced activation of Akt. PHA and LY treatment also prevented the inhibitory effect of arsenite on catalase protein expression but did not affect the level of catalase mRNA. These findings suggest that arsenite-induced inhibition of catalase expression is regulated at the mRNA and post-transcriptional levels in HepG2 cells, and that the post-transcriptional regulation is mediated via c-Met- and PI3K-dependent mechanisms.

  9. Overexpression of thyroid hormone beta1 nuclear receptor is associated with an increased proliferation of human hepatoma cells

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    Lin, K; Lin, Y; McPhie, P [Chang-Gung College of Medicine and Technology, Graduate Institute of Clinical Medicine, Taoyuan (Taiwan, Province of China); Cheng, S [National Cancer Inst., Bethesda, MD (United States)

    1994-12-31

    It is evaluated the expression of thyroid hormone nuclear receptors (TRs) and their possible roles in the carcinogenesis of human hepatocarcinoma. The expression of TR{beta}1 and TR{alpha} genes was evaluated at both the mRNA and protein levels. The expression of TR{beta}1 and TR{alpha}1 mRNAs is similar to those found in normal liver. However, the expression of TR isoform proteins depends on the cell-type. The expression of TRaplha1 protein is low in all cell lines examined. However, TR{Beta}1 protein is overexpressed in Mahlavu, SK-Hep-1, and HA22T, moderately expressed in J5, J7, and J328 and is very low HepG2, Hep3B, and PLC/PRF/5 cells. The proliferation of cells in which TR{beta}1 is overexpressed is stimulated by the thyroid hormone, 3,3`,5- triiodo-L-thyronine. These results suggest that TR{beta}1, not TR{alpha}1, is probably involved in the prolifaration of hepatoma cells.

  10. Overexpression of thyroid hormone beta1 nuclear receptor is associated with an increased proliferation of human hepatoma cells

    International Nuclear Information System (INIS)

    Lin, K.; Lin, Y.; McPhie, P.; Cheng S.

    1994-01-01

    It is evaluated the expression of thyroid hormone nuclear receptors (TRs) and their possible roles in the carcinogenesis of human hepatocarcinoma. The expression of TRβ and TRα genes was evaluated at both the mRNA and protein levels. The expression of TRβ1 and TRα1 mRNAs is similar to those found in normal liver. However, the expression of TR isoform proteins depends on the cell-type. The expression of TRα1 protein is low in all cell lines examined. However, TRβ1 protein is overexpressed in Mahlavu, SK-Hep-1, and HA22T, moderately expressed in J5, J7, and J328 and is very low in HepG2, Hep3B, and PLC/PRF/5 cells. The proliferation of cells in which TRβ1 is overexpressed is stimulated by the thyroid hormone, 3,3',5-triiodo-L-thyronine. These results suggest that TRβ1 not TRα1, is probably involved in the proliferation of hepatoma cells

  11. Protective effects of flavonoids isolated from Korean milk thistle Cirsium japonicum var. maackii (Maxim.) Matsum on tert-butyl hydroperoxide-induced hepatotoxicity in HepG2 cells.

    Science.gov (United States)

    Jung, Hyun Ah; Abdul, Qudeer Ahmed; Byun, Jeong Su; Joung, Eun-Ji; Gwon, Wi-Gyeong; Lee, Min-Sup; Kim, Hyeung-Rak; Choi, Jae Sue

    2017-09-14

    Milk thistle leaves and flowers have been traditionally used as herbal remedy to alleviate liver diseases for decades. Korean milk thistle, Cirsium japonicum var. maackii (Maxim.) Matsum has been employed in traditional folk medicine as diuretic, antiphlogistic, hemostatic, and detoxifying agents. The aim of current investigation was to evaluate hepatoprotective properties of the MeOH extract of the roots, stems, leaves and flowers of Korean milk thistle as well as four isolated flavonoids, luteolin, luteolin 5-O-glucoside, apigenin and apigenin 7-O-glucuronide during t-BHP-induced oxidative stress in HepG2 cells. Hepatoprotective potential of the MeOH extracts and flavonoids derived from Korean milk thistle against t-BHP-induced oxidative stress in HepG2 cells were evaluated following MTT method. Incubating HepG2 cells with t-BHP markedly decreased the cell viability and increased the intracellular ROS generation accompanied by depleted GSH levels. Protein expression of heme oxygenase (HO-1) and nuclear factor-E2-related factor 2 (Nrf-2) was determined by Western blot. Our findings revealed that pretreating HepG2 cells with MeOH extracts and bioactive flavonoids significantly attenuated the t-BHP-induced oxidative damage, followed by increased cell viability in a dose-dependent manner. The results illustrate that excess ROS generation was reduced and GSH levels increased dose-dependently when HepG2 cells were pretreated with four flavonoids. Moreover, Western blotting analysis demonstrated that protein expressions of Nrf-2 and HO-1 were also up-regulated by flavonoids treatment. These results clearly demonstrate that the MeOH extracts and flavonoids from Korean milk thistle protected HepG2 cells against oxidative damage triggered by t-BHP principally by modulating ROS generation and restoring depleted GSH levels in addition to the increased Nrf-2/HO-1 signaling cascade. These flavonoids are potential natural antioxidative biomarkers against oxidative stress

  12. Fibronectin synthesized by a human hepatoma cell line

    International Nuclear Information System (INIS)

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

    1984-01-01

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

  13. Influence of different chemical agents (H2O2, t-BHP and MMS) on the activity of antioxidant enzymes in human HepG2 and hamster V79 cells; relationship to cytotoxicity and genotoxicity.

    Science.gov (United States)

    Slamenova, D; Kozics, K; Melusova, M; Horvathova, E

    2015-01-01

    We investigated activities of antioxidant enzymes (AEs), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in human HepG2 and hamster V79 cells treated with a scale of concentrations of hydrogen peroxide (H2O2), tert-butyl hydroperoxide (t-BHP) and methyl methanesulfonate (MMS). Cytotoxicity and genotoxicity of these substances were evaluated simultaneously. We have found out that H2O2, t-BHP and MMS predictably induce significant concentration-dependent increase of DNA lesions in both cell lines. Cytotoxicity detected in V79 cells with help of PE test was in a good conformity with the level of DNA damage. MTT test has proved unsuitable, except for MMS-treated V79 cells. Compared with human cells HepG2, hamster cells V79 manifested approximately similar levels of SOD and CAT but ten times higher activity of GPx. Across all concentrations tested the most significant increase of activity of the enzyme CAT was found in H2O2- and t-BHP-treated HepG2 cells, of the enzyme SOD in t-BHP- and MMS-treated V79 cells, and of the enzyme GPx in H2O2-treated V79 cells. We suggest that stimulation of enzyme activity by the relevant chemical compounds may result from transcriptional or post-transcriptional regulation of the expression of the genes CAT, SOD and GPx. Several authors suggest that moderate levels of toxic reactants can induce increase of AEs activities, while very high levels of reactants can induce their decrease, as a consequence of damage of the molecular machinery required to induce AEs. Based on a great amount of experiments, which were done and described within this paper, we can say that the above mentioned principle does not apply in general. Only the reactions of t-BHP affected HepG2 cells were consistent with this idea.

  14. Niclosamide suppresses hepatoma cell proliferation via the Wnt pathway

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    Tomizawa M

    2013-11-01

    Full Text Available Minoru Tomizawa,1 Fuminobu Shinozaki,2 Yasufumi Motoyoshi,3 Takao Sugiyama,4 Shigenori Yamamoto,5 Makoto Sueishi,4 Takanobu Yoshida6 1Department of Gastroenterology, 2Department of Radiology, 3Department of Neurology, 4Department of Rheumatology, 5Department of Pediatrics, 6Department of Internal Medicine, National Hospital Organization Shimoshizu Hospital, Yotsukaido City, Chiba, Japan Background: The Wnt pathway plays an important role in hepatocarcinogenesis. We analyzed the association of the Wnt pathway with the proliferation of hepatoma cells using Wnt3a and niclosamide, a drug used to treat tapeworm infection. Methods: We performed an MTS assay to determine whether Wnt3a stimulated proliferation of Huh-6 and Hep3B human hepatoma cell lines after 72 hours of incubation with Wnt3a in serum-free medium. The cells were subjected to hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL after 48 hours of incubation. RNA was isolated 48 hours after addition of Wnt3a or niclosamide, and cyclin D1 expression levels were analyzed by real-time quantitative polymerase chain reaction. The promoter activity of T-cell factor was analyzed by luciferase assay 48 hours after transfection of TOPflash. Western blot analysis was performed with antibodies against β-catenin, dishevelled 2, and cyclin D1. Results: Cell proliferation increased with Wnt3a. Niclosamide suppressed proliferation with or without Wnt3a. Hematoxylin and eosin and TUNEL staining suggested that apoptosis occurred in cells with niclosamide. Cyclin D1 was upregulated in the presence of Wnt3a and downregulated with addition of niclosamide. The promoter activity of T-cell factor increased with Wnt3a, whereas T-cell factor promoter activity decreased with niclosamide. Western blot analysis showed that Wnt3a upregulated β-catenin, dishevelled 2, and cyclin D1, while niclosamide downregulated them. Conclusion: Niclosamide is a potential

  15. HNF-4α regulated miR-122 contributes to development of gluconeogenesis and lipid metabolism disorders in Type 2 diabetic mice and in palmitate-treated HepG2 cells.

    Science.gov (United States)

    Wei, Shengnan; Zhang, Ming; Yu, Yang; Xue, Huan; Lan, Xiaoxin; Liu, Shuping; Hatch, Grant; Chen, Li

    2016-11-15

    Hepatocyte Nuclear Factor-4α (HNF-4α) is a key nuclear receptor protein required for liver development. miR-122 is a predominant microRNA expressed in liver and is involved in the regulation of cholesterol and fatty acid metabolism. HNF-4α is know to regulate expression of miR-122 in liver. We examined how HNF-4α regulated gluconeogenesis and lipid metabolism through miR-122 in vivo and in vitro. Expression of miR-122, HNF-4α, phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), sterol response elementary binding protein-1 (SREBP-1), fatty acid synthase-1 (FAS-1), carnitine palmitoyltransferase-1 (CPT-1) and acetyl Coenzyme A carboxylase alpha (ACCα) were determined in livers of Type 2 diabetic mice and in insulin resistant palmitate-treated HepG2 cells. CPT-1 and phosphorylated ACCα expression were significantly decreased in livers of Type 2 diabetic mice and in palmitate-treated HepG2 cells compared to controls. In contrast, expression of miR-122, HNF-4α, PEPCK, G6Pase, SREBP-1, FAS-1 and ACCα were significantly elevated in liver of Type 2 diabetic mice and in palmitate-treated HepG2 cells compared to controls. Expression of HNF-4α increased whereas siRNA knockdown of HNF-4α decreased miR-122 levels in HepG2 cells compared to controls. In addition, expression of HNF-4α in HepG2 cells increased PEPCK, G6Pase, SREBP-1, FAS-1, ACCα mRNA and protein expression and decreased CPT-1 and p-ACCα mRNA and protein expression compared to controls. Addition of miR-122 inhibitors attenuated the HNF-4α mediated effect on expression of these gluconeogenic and lipid metabolism proteins. The results indicate that HNF-4α regulated miR-122 contributes to development of the gluconeogenic and lipid metabolism alterations observed in Type 2 diabetic mice and in palmitate-treated HepG2 cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Regulatory role of kinases and phosphatases on the internalisation of caveolae in HepG2 cells.

    Science.gov (United States)

    Botos, Erzsébet; Turi, Agnes; Müllner, Nándor; Kovalszky, Ilona; Tátrai, Péter; Kiss, Anna L

    2007-01-01

    The caveolar cycle is thought to be regulated by synchronised function of kinases and phosphatases. Using ocadaic acid--a serine/threonine protein phosphatase inhibitor--and an inhibitor of tyrosine phosphatase (sodium orthovanadate) we have followed the internalisation of caveolae. Since albumin binding to its receptor (gp60) can induce pinching off of caveolae from the plasma membrane, we also used this physiological ligand to induce the internalisation. Our confocal microscopic results show that both ocadaic acid and vanadate treatments have significantly decreased caveolin (caveolin-1 and -2) labelling on the cell surface, while the cytoplasmic labelling became much stronger. Quite often large, strongly labelled "granules" appear at the perinuclear region. Very strong caveolin labelling was detected along the actin-cytoskeleton suggesting that caveolae might move along these filaments. Our electron microscopic results also show an intensive caveolae pinching off from the plasma membrane. After ocadaic acid and vanadate treatments the number of surface connected vesicles (caveolae) decreases. At the same time, large multivesicular bodies (termed caveosomes) appear in the perinuclear area of the cytoplasm. By immunoprecipitation and Western blot analysis we detect an increased tyrosine phosphorylation of a approximately 29kDa protein in ocadaic acid and vanadate treated samples. This protein was identified as caveolin-2. No significant change in the tyrosine phosphorylation of caveolin-1 was found. From these data we can conclude that caveolae internalisation is regulated by phosphorylation of caveolin-2.

  17. Identification of interacting proteins of retinoid-related orphan nuclear receptor gamma in HepG2 cells

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    Ze-Min Huang1,#, Jun Wu2,#, Zheng-Cai Jia1, Yi Tian1, Jun Tang3, Yan Tang1, Ying Wang2, Yu-Zhang Wu1,* & Bing Ni1,*

    2012-06-01

    Full Text Available The retinoid-related orphan nuclear receptor gamma (RORγplays critical roles in regulation of development, immunity andmetabolism. As transcription factor usually forms a proteincomplex to function, thus capturing and dissecting of theRORγ protein complex will be helpful for exploring themechanisms underlying those functions. After construction ofthe recombinant tandem affinity purification (TAP plasmid,pMSCVpuro RORγ-CTAP(SG, the nuclear localization ofRORγ-CTAP(SG fusion protein was verified. Followingisolation of RORγ protein complex by TAP strategy, sevencandidate interacting proteins were identified. Finally, the heatshock protein 90 (HSP90 and receptor-interacting protein 140(RIP140 were confirmed to interplay with RORγ byco-immunoprecipitation. Interference of HSP90 or/and RIP140genes resulted in dramatically decreased expression ofCYP2C8 gene, the RORγ target gene. Data from this studydemonstrate that HSP90 and RIP140 proteins interact withRORγ protein in a complex format and function asco-activators in the RORγ-mediated regulatory processes ofHepG2 cells.

  18. Inhibition of SH2-domain-containing inositol 5-phosphatase (SHIP2) ameliorates palmitate induced-apoptosis through regulating Akt/FOXO1 pathway and ROS production in HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Gorgani-Firuzjaee, Sattar [Department of Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran (Iran, Islamic Republic of); Adeli, Khosrow [Division of Clinical Biochemistry, The Hospital for Sick Children, University of Toronto, Toronto (Canada); Meshkani, Reza, E-mail: rmeshkani@tums.ac.ir [Department of Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran (Iran, Islamic Republic of)

    2015-08-21

    The serine–threonine kinase Akt regulates proliferation and survival by phosphorylating a network of protein substrates; however, the role of a negative regulator of the Akt pathway, the SH2-domain-containing inositol 5-phosphatase (SHIP2) in apoptosis of the hepatocytes, remains unknown. In the present study, we studied the molecular mechanisms linking SHIP2 expression to apoptosis using overexpression or suppression of SHIP2 gene in HepG2 cells exposed to palmitate (0.5 mM). Overexpression of the dominant negative mutant SHIP2 (SHIP2-DN) significantly reduced palmitate-induced apoptosis in HepG2 cells, as these cells had increased cell viability, decreased apoptotic cell death and reduced the activity of caspase-3, cytochrome c and poly (ADP-ribose) polymerase. Overexpression of the wild-type SHIP2 gene led to a massive apoptosis in HepG2 cells. The protection from palmitate-induced apoptosis by SHIP2 inhibition was accompanied by a decrease in the generation of reactive oxygen species (ROS). In addition, SHIP2 inhibition was accompanied by an increased Akt and FOXO-1 phosphorylation, whereas overexpression of the wild-type SHIP2 gene had the opposite effects. Taken together, these findings suggest that SHIP2 expression level is an important determinant of hepatic lipoapotosis and its inhibition can potentially be a target in treatment of hepatic lipoapoptosis in diabetic patients. - Highlights: • Lipoapoptosis is the major contributor to the development of NAFLD. • The PI3-K/Akt pathway regulates apoptosis in different cells. • The role of negative regulator of this pathway, SHIP2 in lipoapoptosis is unknown. • SHIP2 inhibition significantly reduces palmitate-induced apoptosis in HepG2 cells. • SHIP2 inhibition prevents palmitate induced-apoptosis by regulating Akt/FOXO1 pathway.

  19. Inhibition of SH2-domain-containing inositol 5-phosphatase (SHIP2) ameliorates palmitate induced-apoptosis through regulating Akt/FOXO1 pathway and ROS production in HepG2 cells

    International Nuclear Information System (INIS)

    Gorgani-Firuzjaee, Sattar; Adeli, Khosrow; Meshkani, Reza

    2015-01-01

    The serine–threonine kinase Akt regulates proliferation and survival by phosphorylating a network of protein substrates; however, the role of a negative regulator of the Akt pathway, the SH2-domain-containing inositol 5-phosphatase (SHIP2) in apoptosis of the hepatocytes, remains unknown. In the present study, we studied the molecular mechanisms linking SHIP2 expression to apoptosis using overexpression or suppression of SHIP2 gene in HepG2 cells exposed to palmitate (0.5 mM). Overexpression of the dominant negative mutant SHIP2 (SHIP2-DN) significantly reduced palmitate-induced apoptosis in HepG2 cells, as these cells had increased cell viability, decreased apoptotic cell death and reduced the activity of caspase-3, cytochrome c and poly (ADP-ribose) polymerase. Overexpression of the wild-type SHIP2 gene led to a massive apoptosis in HepG2 cells. The protection from palmitate-induced apoptosis by SHIP2 inhibition was accompanied by a decrease in the generation of reactive oxygen species (ROS). In addition, SHIP2 inhibition was accompanied by an increased Akt and FOXO-1 phosphorylation, whereas overexpression of the wild-type SHIP2 gene had the opposite effects. Taken together, these findings suggest that SHIP2 expression level is an important determinant of hepatic lipoapotosis and its inhibition can potentially be a target in treatment of hepatic lipoapoptosis in diabetic patients. - Highlights: • Lipoapoptosis is the major contributor to the development of NAFLD. • The PI3-K/Akt pathway regulates apoptosis in different cells. • The role of negative regulator of this pathway, SHIP2 in lipoapoptosis is unknown. • SHIP2 inhibition significantly reduces palmitate-induced apoptosis in HepG2 cells. • SHIP2 inhibition prevents palmitate induced-apoptosis by regulating Akt/FOXO1 pathway

  20. Bog bilberry (Vaccinium uliginosum L.) extract reduces cultured Hep-G2, Caco-2, and 3T3-L1 cell viability, affects cell cycle progression, and has variable effects on membrane permeability.

    Science.gov (United States)

    Liu, Jia; Zhang, Wei; Jing, Hao; Popovich, David G

    2010-04-01

    Bog bilberry (Vaccinium uliginosum L.) is a blue-pigmented edible berry related to bilberry (Vaccinium myrtillus L.) and the common blueberry (Vaccinium corymbosum). The objective of this study was to investigate the effect of a bog bilberry anthocyanin extract (BBAE) on cell growth, membrane permeability, and cell cycle of 2 malignant cancer cell lines, Caco-2 and Hep-G2, and a nonmalignant murine 3T3-L1 cell line. BBAE contained 3 identified anthocyanins. The most abundant anthocyanin was cyanidin-3-glucoside (140.9 +/- 2.6 microg/mg of dry weight), followed by malvidin-3-glucoside (10.3 +/- 0.3 microg/mg) and malvidin-3-galactoside (8.1 +/- 0.4 microg/mg). Hep-G2 LC50 was calculated to be 0.563 +/- 0.04 mg/mL, Caco-2 LC50 was 0.390 +/- 0.30 mg/mL and 0.214 +/- 0.02 mg/mL for 3T3-L1 cells. LDH release, a marker of membrane permeability, was significantly increased in Hep-G2 cells and Caco-2 cells after 48 and 72 h compared to 24 h. The increase was 21% at 48 h and 57% at 72 h in Caco-2 cells and 66% and 139% in Hep-G2 cells compared to 24 h. However, 3T3-L1 cells showed an unexpected significant lower LDH activity (P < or = 0.05) after 72 h of exposure corresponding to a 21% reduction in LDH release. BBAE treatment increased sub-G1 in all 3 cell lines without influencing cells in the G2/M phase. BBAE treatment reduced the growth and increased the accumulation of sub-G1 cells in 2 malignant and 1 nonmalignant cell line; however, the effect on membrane permeability differs considerably between the malignant and nonmalignant cells and may in part be due to differences in cellular membrane composition.

  1. Relationship between peroxisome proliferator-activated receptor alpha activity and cellular concentration of 14 perfluoroalkyl substances in HepG2 cells.

    Science.gov (United States)

    Rosenmai, Anna Kjerstine; Ahrens, Lutz; le Godec, Théo; Lundqvist, Johan; Oskarsson, Agneta

    2018-02-01

    Peroxisome proliferator-activated receptor alpha (PPARα) is a molecular target for perfluoroalkyl substances (PFASs). Little is known about the cellular uptake of PFASs and how it affects the PPARα activity. We investigated the relationship between PPARα activity and cellular concentration in HepG2 cells of 14 PFASs, including perfluoroalkyl carboxylates (PFCAs), perfluoroalkyl sulfonates and perfluorooctane sulfonamide (FOSA). Cellular concentrations were determined by high-performance liquid chromatography-tandem mass spectrometry and PPARα activity was determined in transiently transfected cells by reporter gene assay. Cellular uptake of the PFASs was low (0.04-4.1%) with absolute cellular concentrations in the range 4-2500 ng mg -1 protein. Cellular concentration of PFCAs increased with perfluorocarbon chain length up to perfluorododecanoate. PPARα activity of PFCAs increased with chain length up to perfluorooctanoate. The maximum induction of PPARα activity was similar for short-chain (perfluorobutanoate and perfluoropentanoate) and long-chain PFCAs (perfluorododecanoate and perfluorotetradecanoate) (approximately twofold). However, PPARα activities were induced at lower cellular concentrations for the short-chain homologs compared to the long-chain homologs. Perfluorohexanoate, perfluoroheptanoate, perfluorooctanoate, perfluorononanoate (PFNA) and perfluorodecanoate induced PPARα activities >2.5-fold compared to controls. The concentration-response relationships were positive for all the tested compounds, except perfluorooctane sulfonate PFOS and FOSA, and were compound-specific, as demonstrated by differences in the estimated slopes. The relationships were steeper for PFCAs with chain lengths up to and including PFNA than for the other studied PFASs. To our knowledge, this is the first report establishing relationships between PPARα activity and cellular concentration of a broad range of PFASs. Copyright © 2017 John Wiley & Sons, Ltd.

  2. Exploiting antidiabetic activity of silver nanoparticles synthesized using Punica granatum leaves and anticancer potential against human liver cancer cells (HepG2).

    Science.gov (United States)

    Saratale, Rijuta G; Shin, Han Seung; Kumar, Gopalakrishnan; Benelli, Giovanni; Kim, Dong-Su; Saratale, Ganesh D

    2018-02-01

    This study first time reports the novel synthesis of silver nanoparticles (AgNPs) using a Punica granatum leaf extract (PGE). The synthesized AgNPs were characterized by various analytical techniques including UV-Vis, Fourier transform infrared (FTIR), X-ray powder diffraction (XRD), X-ray photoelectron spectroscopy (XPS), field emission scanning electron microscopy and energy-dispersive spectra (FESEM-EDS) and high-resolution transmission electron microscopy (HRTEM). FTIR analysis revealed that the involvement of biological macromolecules of P. granatum leaf extract were distributed and involved in the synthesis and stabilization of AgNPs. A surface-sensitive technique of XPS was used to analyse the composition and oxidation state of synthesized AgNPs. The analytical results confirmed that the AgNPs were crystalline in nature with spherical shape. The zeta potential study revealed that the surface charge of synthesized AgNPs was highly negative (-26.6 mV) and particle size distribution was ranging from ∼35 to 60 nm and the average particle size was about 48 nm determined by dynamic light scattering (DLS). The PGE-AgNPs antidiabetic potential exhibited effective inhibition against α-amylase and α-glucosidase (IC 50 ; 65.2 and 53.8 μg/mL, respectively). The PGE-AgNPs showed a dose-dependent response against human liver cancer cells (HepG2) (IC 50 ; 70 μg/mL) indicating its greater efficacy in killing cancer cells. They also possessed in vitro free radical-scavenging activity in terms of ABTS (IC 50 ; 52.2 μg/mL) and DPPH (IC 50 ; 67.1 μg/mL) antioxidant activity. PGE-AgNPs displayed strong antibacterial activity and potent synergy with standard antibiotics against pathogenic bacteria. Thus, synthesized PGE-AgNPs show potential biomedical and industrial applications.

  3. Quantification of homocysteine-related metabolites and the role of betaine-homocysteine S-methyltransferase in HepG2 cells

    Czech Academy of Sciences Publication Activity Database

    Kořínek, M.; Šístek, V.; Mládková, Jana; Mikeš, P.; Jiráček, Jiří; Selicharová, Irena

    2013-01-01

    Roč. 27, č. 1 (2013), s. 111-121 ISSN 0269-3879 R&D Projects: GA ČR(CZ) GAP207/10/1277 Institutional support: RVO:61388963 Keywords : homocysteine * BHMT * LC-MS/MS * HepG2 * metabolites Subject RIV: CE - Biochemistry Impact factor: 1.662, year: 2013

  4. Shifts in dietary carbohydrate-lipid exposure regulate expression of the non-alcoholic fatty liver disease-associated gene PNPLA3/adiponutrin in mouse liver and HepG2 human liver cells.

    Science.gov (United States)

    Hao, Lei; Ito, Kyoko; Huang, Kuan-Hsun; Sae-tan, Sudathip; Lambert, Joshua D; Ross, A Catharine

    2014-10-01

    Patatin-like phospholipase domain containing 3 (PNPLA3, adiponutrin) has been identified as a modifier of lipid metabolism. To better understand the physiological role of PNPLA3/adiponutrin, we have investigated its regulation in intact mice and human hepatocytes under various nutritional/metabolic conditions. PNPLA3 gene expression was determined by real-time PCR in liver of C57BL/6 mice after dietary treatments and in HepG2 cells exposed to various nutritional/metabolic stimuli. Intracellular lipid content was determined in HepG2 cells after siRNA-mediated knockdown of PNPLA3. In vivo, mice fed a high-carbohydrate (HC) liquid diet had elevated hepatic lipid content, and PNPLA3 mRNA and protein expression, compared to chow-fed mice. Elevated expression was completely abrogated by addition of unsaturated lipid emulsion to the HC diet. By contrast, in mice with high-fat diet-induced steatosis, Pnpla3 expression did not differ compared to low-fat fed mice. In HepG2 cells, Pnpla3 expression was reversibly suppressed by glucose depletion and increased by glucose refeeding, but unchanged by addition of insulin and glucagon. Several unsaturated fatty acids each significantly decreased Pnpla3 mRNA, similar to lipid emulsion in vivo. However, Pnpla3 knockdown in HepG2 cells did not alter total lipid content in high glucose- or oleic acid-treated cells. Our results provide evidence that PNPLA3 expression is an early signal/signature of carbohydrate-induced lipogenesis, but its expression is not associated with steatosis per se. Under lipogenic conditions due to high-carbohydrate feeding, certain unsaturated fatty acids can effectively suppress both lipogenesis and PNPLA3 expression, both in vivo and in a hepatocyte cell line. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. The Maillard reaction of a shrimp by-product protein hydrolysate: chemical changes and inhibiting effects of reactive oxygen species in human HepG2 cells.

    Science.gov (United States)

    Zha, Fengchao; Wei, Binbin; Chen, Shengjun; Dong, Shiyuan; Zeng, Mingyong; Liu, Zunying

    2015-06-01

    Recently, much attention has been given to improving the antioxidant activity of protein hydrolysates via the Maillard reaction, but little is known about the cellular antioxidant activity of Maillard reaction products (MRPs) from protein hydrolysates. We first investigated chemical characterization and the cellular antioxidant activity of MRPs in a shrimp (Litopenaeus vannamei) by-product protein hydrolysate (SBH)-glucose system at 110 °C for up to 10 h of heating. Solutions of SBH and glucose were also heated alone as controls. The Maillard reaction greatly resulted in the increase of hydroxymethylfurfural (HMF) and browning intensity, high molecular weight fraction, and reduction of the total amino acid in SBH with the heating time, which correlated well with the free radical scavenging activity of MRPs. MRPs had stronger inhibiting effects on oxidative stress of human HepG2 cells than the original SBH, and its cellular antioxidant activity strongly correlated with free radical scavenging activity, but less affected by the browning intensity and HMF level. The caramelization of glucose partially affected the HMF level and free radical scavenging activity of MRPs, but it was not related to the cellular antioxidant activity. The cellular antioxidant activity of MRPs for 5 h of heating time appeared to reach a maximum level, which was mainly due to carbonyl ammonia condensation reaction. In conclusion, the Maillard reaction is a potential method to increase the cellular antioxidant activity of a shrimp by-product protein hydrolysate, but the higher HMF levels and the lower amino acid content in MRPs should also be considered.

  6. Increase in intracellular free/bound NAD[P]H as a cause of Cd-induced oxidative stress in the HepG2 cells

    International Nuclear Information System (INIS)

    Yang, M.S.; Li, D.; Lin, T.; Zheng, J.J.; Zheng, W.; Qu, J.Y.

    2008-01-01

    The present study shows the use of confocal autofluorescence spectroscopy coupled with the time-resolved fluorescence decay analysis to measure changes in FAD/NAD[P]H and free/bound NAD[P]H in HepG 2 cells at 0.5, 1.5, 3 and 4.5 h after exposure to cadmium chloride (Cd). These changes were compared to changes in GSSG/GSH and production of reactive oxygen radicals (ROS) production. The results demonstrated that both FAD/NAD[P]H and GSSG/GSH increased significantly upon exposure to Cd. The change in GSSG/GSH occurred as early as 1.5 h after treatment while the change in FAD/NAD[P]H did not occur until 3 h after exposure. Production of ROS was also increased at 1.5 h. The ratio of free/bound NAD[P]H was studied. It was demonstrated that free/bound NAD[P]H increased significantly as early as 0.5 h and remained elevated until 4.5 h after treatment with Cd. The present study provides novel data to show that changes in NAD[P]H metabolism precedes the increase in ROS production and cellular oxidative stress (increase GSSG/GSH, FAD/NAD[P]H). It is suggested that Cd causes a release of NAD[P]H, an important cofactor for electron transfer, from its normal protein binding sites. This may result in a disruption of the activity of the enzyme and proteins, and may lead to the subsequent toxic events

  7. Poly (N-isopropylacrylamide)-functionalized dendrimer as a thermosensitive nanoplatform for delivering malloapelta B against HepG2 cancer cell proliferation

    Science.gov (United States)

    Ngan Le, Phung; Chuong Pham, Dinh; Hai Nguyen, Dai; Quyen Tran, Ngoc; Dimitrov, Vladimir; Ivanov, Petko; Nguyen Xuan, Cuong; Nguyen, Hoai Nam; Khoa Nguyen, Cuu

    2017-06-01

    In recent years, nanocarriers have emerged as effective platforms for delivering several kinds of herbal medicine and naturally bioactive compounds. In this study we developed an outstanding thermosensitive dendritic nanocarrier to efficiently deliver malloapelta B (Mall B), which is a water insoluble bioactive compound isolated from leaves of Mallotus apelta—Vietnamese medicinal plant. The thermosensitive poly(N-isopropylacrylamide) (PNIPAM) polymer-conjugated polyamidoamine (PAMAM) dendrimer copolymer was prepared via Michael reaction. The copolymer structures were confirmed by proton nuclear magnectic resonance (1H NMR). Morphology of the nanocarrier was observered around 70-120 nm by transmission electron microscopy (TEM). Size distributions were measured by dynamic light scattering (DLS) of the nanocarrier and its Mall B-loaded performed at 146.8 nm and 194.5 nm, respectively. The PNIPAM-g-PAMAM-based nanocarrier exhibited higher Mall B loading efficiency (DL  =  59.93  ±  0.19%) and entrapment efficiency (EE  =  89.98  ±  2.06%) as compared to PNIPAM (DL  =  52.54  ±  0.45% and EE  =  66.45  ±  2.78%). In vitro release indicated that approximately 30% amount of the loaded Mall B released at pH 5.5 after 54 h tracking. At the same time, 12.5% amount of the molecules released at pH 7.4.Cytotoxicity assay results showed that the Mall B-loaded nanocarrier significantly inhibited HepG2 cancer cell proliferation. These obtained results indicated that the nanocarrier could solve hydrophobic property of Mall B for further medicine applications.

  8. HCV core protein-induced down-regulation of microRNA-152 promoted aberrant proliferation by regulating Wnt1 in HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Shifeng Huang

    Full Text Available Hepatitis C virus (HCV has been reported to regulate cellular microRNAs (miRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma (HCV-HCC, but HCV core-regulated miRNAs are largely unknown. Our preliminary experiments revealed significant down-regulation of microRNA-152 (miR-152 by HCV core protein in HepG2 cells. Through target gene prediction softwares, Wnt1 was predicted to be a potential target of miR-152. The present study was initiated to investigate whether miR-152 is aberrantly regulated by the HCV core protein, and involved in the regulation of the aberrant proliferation of HCV-HCC cells.MiR-152 levels were examined by stem-loop real-time RT-PCR (SLqRT-PCR. Cell proliferation was analyzed by MTT and colony formation assay. Cell cycle analysis was performed by flow cytometry. Luciferase reporter assay was conducted to confirm miRNA-target association. Wnt1 expression was determined by real-time qPCR and Western blotting.HCV core protein significantly suppressed miR-152 expression, and led to significant Wnt1 up-regulation with a concomitant aberrantly promoted proliferation. Moreover, we validated that miR-152 inhibition promoted, while miR-152 mimics inhibited cell proliferation. Using, qRT-PCR and western blot, Wnt1 was demonstrated to be regulated by miR-152. Luciferase activity assay showed that while miR-152 mimics significantly reduced the luciferase activity by 83.76% (P<0.0001, miR-152 inhibitor showed no effect on luciferase reporter. Most notably, salvage expression of miR-152 after Ad-HCV core infection for 24 h almost totally reversed the proliferation-promoting effect of the HCV core protein, and meanwhile, reduced the expression of both Wnt1 mRNA and protein to basal levels.These findings provide important evidence that the reduced miR-152 expression by HCV core protein can indirectly lose an inhibitory effect on Wnt1, which might, at least partially lead to cell

  9. Data in support of fumosorinone, a novel PTP1B inhibitor, activates insulin signaling in insulin-resistance HepG2 cells and shows anti-diabetic effect in diabetic KKAy mice

    Directory of Open Access Journals (Sweden)

    Du-Qiang Luo

    2015-09-01

    Full Text Available This data article contains data related to the research article entitled “Fumosorinone, a novel PTP1B inhibitor, activates insulin signaling in insulin-resistance HepG2 cells and shows anti-diabetic effect in diabetic KKAy mice” in the Toxicology and Applied Pharmacology [1]. Fumosorinone (FU is a new inhibitor of protein phosphatase 1B inhibitor, which was isolated from insect pathogenic fungi Isaria fumosorosea. FU was found to inhibit PTP1B activity in our previous study [2]. PTP1B is the physiological antagonist of the insulin signalling pathway. Inhibition of PTP 1B may increase insulin sensitivity [3]. PTP1B has been considered promising as an insulin-sensitive drug target for the prevention and the treatment of insulin-based diseases [4]. We determined the effect of FU on the glucose consumption of IR HepG2 cells. FU caused significant enhancement in glucose consumption by insulin-resistant HepG2 cells compared with control cells.

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Science.gov (United States)

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

    2008-02-15

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

  12. Effects of defined mixtures of persistent organic pollutants (POPs) on multiple cellular responses in the human hepatocarcinoma cell line, HepG2, using high content analysis screening

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Jodie [Institute for Global Food Security, School of Biological Sciences, Queen' s University Belfast, Northern Ireland (United Kingdom); Berntsen, Hanne Friis; Zimmer, Karin Elisabeth [Norwegian University of Life Sciences, Oslo (Norway); Frizzell, Caroline [Institute for Global Food Security, School of Biological Sciences, Queen' s University Belfast, Northern Ireland (United Kingdom); Verhaegen, Steven; Ropstad, Erik [Norwegian University of Life Sciences, Oslo (Norway); Connolly, Lisa, E-mail: l.connolly@qub.ac.uk [Institute for Global Food Security, School of Biological Sciences, Queen' s University Belfast, Northern Ireland (United Kingdom)

    2016-03-01

    Persistent organic pollutants (POPs) are toxic substances, highly resistant to environmental degradation, which can bio-accumulate and have long-range atmospheric transport potential. Most studies focus on single compound effects, however as humans are exposed to several POPs simultaneously, investigating exposure effects of real life POP mixtures on human health is necessary. A defined mixture of POPs was used, where the compound concentration reflected its contribution to the levels seen in Scandinavian human serum (total mix). Several sub mixtures representing different classes of POPs were also constructed. The perfluorinated (PFC) mixture contained six perfluorinated compounds, brominated (Br) mixture contained seven brominated compounds, chlorinated (Cl) mixture contained polychlorinated biphenyls and also p,p’-dichlorodiphenyldichloroethylene, hexachlorobenzene, three chlordanes, three hexachlorocyclohexanes and dieldrin. Human hepatocarcinoma (HepG2) cells were used for 2 h and 48 h exposures to the seven mixtures and analysis on a CellInsight™ NXT High Content Screening platform. Multiple cytotoxic endpoints were investigated: cell number, nuclear intensity and area, mitochondrial mass and membrane potential (MMP) and reactive oxygen species (ROS). Both the Br and Cl mixtures induced ROS production but did not lead to apoptosis. The PFC mixture induced ROS production and likely induced cell apoptosis accompanied by the dissipation of MMP. Synergistic effects were evident for ROS induction when cells were exposed to the PFC + Br mixture in comparison to the effects of the individual mixtures. No significant effects were detected in the Br + Cl, PFC + Cl or total mixtures, which contain the same concentrations of chlorinated compounds as the Cl mixture plus additional compounds; highlighting the need for further exploration of POP mixtures in risk assessment. - Highlights: • High content analysis (HCA) is a novel approach for determining toxicity of

  13. Effects of defined mixtures of persistent organic pollutants (POPs) on multiple cellular responses in the human hepatocarcinoma cell line, HepG2, using high content analysis screening

    International Nuclear Information System (INIS)

    Wilson, Jodie; Berntsen, Hanne Friis; Zimmer, Karin Elisabeth; Frizzell, Caroline; Verhaegen, Steven; Ropstad, Erik; Connolly, Lisa

    2016-01-01

    Persistent organic pollutants (POPs) are toxic substances, highly resistant to environmental degradation, which can bio-accumulate and have long-range atmospheric transport potential. Most studies focus on single compound effects, however as humans are exposed to several POPs simultaneously, investigating exposure effects of real life POP mixtures on human health is necessary. A defined mixture of POPs was used, where the compound concentration reflected its contribution to the levels seen in Scandinavian human serum (total mix). Several sub mixtures representing different classes of POPs were also constructed. The perfluorinated (PFC) mixture contained six perfluorinated compounds, brominated (Br) mixture contained seven brominated compounds, chlorinated (Cl) mixture contained polychlorinated biphenyls and also p,p’-dichlorodiphenyldichloroethylene, hexachlorobenzene, three chlordanes, three hexachlorocyclohexanes and dieldrin. Human hepatocarcinoma (HepG2) cells were used for 2 h and 48 h exposures to the seven mixtures and analysis on a CellInsight™ NXT High Content Screening platform. Multiple cytotoxic endpoints were investigated: cell number, nuclear intensity and area, mitochondrial mass and membrane potential (MMP) and reactive oxygen species (ROS). Both the Br and Cl mixtures induced ROS production but did not lead to apoptosis. The PFC mixture induced ROS production and likely induced cell apoptosis accompanied by the dissipation of MMP. Synergistic effects were evident for ROS induction when cells were exposed to the PFC + Br mixture in comparison to the effects of the individual mixtures. No significant effects were detected in the Br + Cl, PFC + Cl or total mixtures, which contain the same concentrations of chlorinated compounds as the Cl mixture plus additional compounds; highlighting the need for further exploration of POP mixtures in risk assessment. - Highlights: • High content analysis (HCA) is a novel approach for determining toxicity of

  14. Mitochondrial oxidative stress in human hepatoma cells exposed to stavudine

    International Nuclear Information System (INIS)

    Velsor, Leonard W.; Kovacevic, Miro; Goldstein, Mark; Leitner, Heather M.; Lewis, William; Day, Brian J.

    2004-01-01

    The toxicity of nucleoside reverse transcriptase inhibitors (NRTIs) is linked to altered mitochondrial DNA (mtDNA) replication and subsequent disruption of cellular energetics. This manifests clinically as elevated concentrations of lactate in plasma. The mechanism(s) underlying how the changes in mtDNA replication lead to lactic acidosis remains unclear. It is hypothesized that mitochondrial oxidative stress links the changes in mtDNA replication to mitochondrial dysfunction and ensuing NRTIs toxicity. To test this hypothesis, changes in mitochondrial function, mtDNA amplification efficiency, and oxidative stress were assessed in HepG2-cultured human hepatoblasts treated with the NRTI stavudine (2',3'-didehydro-2',3'-deoxythymidine or d4T) for 48 h. d4T produced significant mitochondrial dysfunction with a 1.5-fold increase in cellular lactate to pyruvate ratios. In addition, d4T caused a dose-dependent decrease in mtDNA amplification and a correlative increase in abundance of markers of mitochondrial oxidative stress. Manganese (III) meso-tetrakis (4-benzoic acid) porphyrin, MnTBAP, a catalytic antioxidant, ameliorated or reversed d4T-induced changes in cell injury, energetics, mtDNA amplification, and mitochondrial oxidative stress. In conclusion, d4T treatment elevates mitochondrial reactive oxygen species (ROS), enhances mitochondrial oxidative stress, and contributes mechanistically to NRTI-induced toxicity. These deleterious events may be potentiated in acquired immunodeficiency syndrome (AIDS) by human immunodeficiency virus (HIV) infection itself, coinfection (e.g., viral hepatitis), aging, substance, and alcohol use

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    The authors have studied the internalization of the insulin receptor (IR) in rat hepatoma cells (Fao). The cells were surface-iodinated at 4 0 C, stimulated with insulin at 37 0 C, and then cooled rapidly, trypsinized at 4 0 C and solubilized. The IR was immunoprecipitated with a specific antibody, and internalization of the IR was assessed by the appearance of trypsin-resistant bands on SDS-PAGE. Insulin induced the internalization of surface receptors with a t 1/2 of 9-10 mins; cells not exposed to insulin internalized less than 20% of the IR during 1 h at 37 0 C. Further experiments demonstrated that the accumulation of trypsin-resistant IR paralleled a loss of receptor from the cell surface. Insulin-stimulated cells were chilled and iodinated at 4 0 C, followed by solubilization, immunoprecipitation and SDS-PAGE; alternatively, insulin-stimulated cells were chilled, surface-bound ligand removed by washing the cells at pH 4.2, and specific [ 125 I]insulin binding measured at 4 0 C. Both techniques confirmed the disappearance of IR from the cell surface at rates comparable to the insulin-stimulated internalization described above. The total amount of phosphotyrosine-containing IR, as assessed by immunoprecipitation with an anti-phosphotyrosine antibody, remained constant during this time interval, suggesting that active kinase is translocated into the cell. In summary, the authors data indicate that insulin binding increases the rate of IR internalization of Fao cells. This relocation may facilitate the interaction of the activated tyrosine kinase in the IR with intracellular substrates, thus transmitting the insulin signal to metabolic pathways

  16. Cytotoxicity and Expression of c-fos, HSP70, and GADD45/153 Proteins in Human Liver Carcinoma (HepG2 Cells Exposed to Dinitrotoluenes

    Directory of Open Access Journals (Sweden)

    Paul B. Tchounwou

    2005-08-01

    Full Text Available Dinitrotoluenes (DNTs are byproducts of the explosive trinitrotoluene (TNT, and exist as a mixture of 2 to 6 isomers, with 2,4-DNT and 2,6-DNT being the most significant. The main route of human exposure at ammunition facilities is inhalation. The primary targets of DNTs toxicity are the hematopoietic system, cardiovascular system, nervous system and reproductive system. In factory workers, exposure to DNTs has been linked to many adverse health effects, including: cyanosis, vertigo, headache, metallic taste, dyspnea, weakness and lassitude, loss of appetite, nausea, and vomiting. Other symptoms including pain or parasthesia in extremities, abdominal discomfort, tremors, paralysis, chest pain, and unconsciousness have been documented. An association between DNTs exposure and increased risk of hepatocellular carcinomas and subcutaneous tumors in rats, as well as renal tumors in mice, has been established. This research was therefore designed targeting the liver to assess the cellular and molecular responses of human liver carcinoma cells following exposure to 2,4-DNT and 2,6-DNT. Cytotoxicity was evaluated using the MTT assay. Upon 48 hrs of exposure, LC50 values of 245 + 14.72μg/mL, and 300 + 5.92μg/mL were recorded for 2,6-DNT and 2,4-DNT respectively, indicating that both DNTs are moderately toxic, and 2,6-DNT is slightly more toxic to HepG2 cells than 2,4-DNT. A dose response relationship was recorded with respect to the cytotoxicity of both DNTs. Western blot analysis resulted in a significant expression (p<0.05 of the 70-kDa heat shock protein in 2,6-DNT-treated cells compared to the control cells and at the 200 μg/mL dose for 2,4-DNT. A statistically significant expression in c-fos was also observed at the 200 and 250 μg/mL treatment level for 2,4- and 2,6-DNT, respectively. However, no statistically significant expression of this protooncogene-related protein was observed at the doses of 0, 100, or 300

  17. Inhibition of HBV replication by delivering the dual-gene expression vector pHsa-miR16-siRNA in HepG2.2.15 cells.

    Science.gov (United States)

    Wei, Wei; Wang, Su-Fei; Yu, Bing; Ni, Ming

    2017-12-01

    This study aimed to construct the dual-gene expression vector pHsa-miR16-siRNA which can express human miR-16 and HBV X siRNA, and examine its regulatory effect on HBV gene expression in the HepG2.2.15 cell line. The expression vectors siR-1583 and pHsa-miR16-siRNA were designed and constructed. HepG2.2.15 cells were transfected with the empty vector, siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively. ELISA was performed to measure the expression of HBsAg and HBeAg in the culture supernatant 48 and72 h post transfection. Fluorescence quantitative PCR was used to measure the HBV mRNA degradation efficiency and HBV DNA copy number. The results showed that the expression of HBV genes was significantly inhibited in HepG2.2.15 cells transfected with siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively, when compared with that in cells transfected with the empty vectors, with the inhibitory effect of pHsa-miR16-siRNA being the most significant. ELISA showed that the inhibitory rates of HBsAg and HBeAg in pHsa-miR16-siRNA transfected cells were correspondingly 87.3% and 85.0% at 48 h, and 88.6% and 86.5% at 72 h post transfection (PHBV mRNA decreased by 80.2% (t=-99.22, PHBV DNA by 92.8% (t=-73.06, PHBV DNA copy number by 89.8% (t=-47.13, PHBV more efficiently than a single-gene expression vector.

  18. SM22α-induced activation of p16INK4a/retinoblastoma pathway promotes cellular senescence caused by a subclinical dose of γ-radiation and doxorubicin in HepG2 cells

    International Nuclear Information System (INIS)

    Kim, Tae Rim; Lee, Hee Min; Lee, So Yong; Kim, Eun Jin; Kim, Kug Chan; Paik, Sang Gi; Cho, Eun Wie; Kim, In Gyu

    2010-01-01

    Research highlights: → SM22α overexpression in HepG2 cells leads cells to a growth arrest state, and the treatment of a subclinical dose of γ-radiation or doxorubicin promotes cellular senescence. → SM22α overexpression elevates p16 INK4a followed by pRB activation, but there are no effects on p53/p21 WAF1/Cip1 pathway. → SM22α-induced MT-1G activates p16 INK4a /pRB pathway, which promotes cellular senescence by damaging agents. -- Abstract: Smooth muscle protein 22-alpha (SM22α) is known as a transformation- and shape change-sensitive actin cross-linking protein found in smooth muscle tissue and fibroblasts; however, its functional role remains uncertain. We reported previously that SM22α overexpression confers resistance against anti-cancer drugs or radiation via induction of metallothionein (MT) isozymes in HepG2 cells. In this study, we demonstrate that SM22α overexpression leads cells to a growth arrest state and promotes cellular senescence caused by treatment with a subclinical dose of γ-radiation (0.05 and 0.1 Gy) or doxorubicin (0.01 and 0.05 μg/ml), compared to control cells. Senescence growth arrest is known to be controlled by p53 phosphorylation/p21 WAF1/Cip1 induction or p16 INK4a /retinoblastoma protein (pRB) activation. SM22α overexpression in HepG2 cells elevated p16 INK4a followed by pRB activation, but did not activate the p53/p21 WAF1/Cip1 pathway. Moreover, MT-1G, which is induced by SM22α overexpression, was involved in the activation of the p16 INK4a /pRB pathway, which led to a growth arrest state and promoted cellular senescence caused by damaging agents. Our findings provide the first demonstration that SM22α modulates cellular senescence caused by damaging agents via regulation of the p16 INK4a /pRB pathway in HepG2 cells and that these effects of SM22α are partially mediated by MT-1G.

  19. SM22{alpha}-induced activation of p16{sup INK4a}/retinoblastoma pathway promotes cellular senescence caused by a subclinical dose of {gamma}-radiation and doxorubicin in HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Tae Rim; Lee, Hee Min; Lee, So Yong; Kim, Eun Jin; Kim, Kug Chan [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Paik, Sang Gi [Department of Biology, School of Biosciences and Biotechnology, Chungnam National University, Daejeon (Korea, Republic of); Cho, Eun Wie, E-mail: ewcho@kribb.re.kr [Daejeon-KRIBB-FHCRC Cooperation Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Kim, In Gyu, E-mail: igkim@kaeri.re.kr [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2010-09-10

    Research highlights: {yields} SM22{alpha} overexpression in HepG2 cells leads cells to a growth arrest state, and the treatment of a subclinical dose of {gamma}-radiation or doxorubicin promotes cellular senescence. {yields} SM22{alpha} overexpression elevates p16{sup INK4a} followed by pRB activation, but there are no effects on p53/p21{sup WAF1/Cip1} pathway. {yields} SM22{alpha}-induced MT-1G activates p16{sup INK4a}/pRB pathway, which promotes cellular senescence by damaging agents. -- Abstract: Smooth muscle protein 22-alpha (SM22{alpha}) is known as a transformation- and shape change-sensitive actin cross-linking protein found in smooth muscle tissue and fibroblasts; however, its functional role remains uncertain. We reported previously that SM22{alpha} overexpression confers resistance against anti-cancer drugs or radiation via induction of metallothionein (MT) isozymes in HepG2 cells. In this study, we demonstrate that SM22{alpha} overexpression leads cells to a growth arrest state and promotes cellular senescence caused by treatment with a subclinical dose of {gamma}-radiation (0.05 and 0.1 Gy) or doxorubicin (0.01 and 0.05 {mu}g/ml), compared to control cells. Senescence growth arrest is known to be controlled by p53 phosphorylation/p21{sup WAF1/Cip1} induction or p16{sup INK4a}/retinoblastoma protein (pRB) activation. SM22{alpha} overexpression in HepG2 cells elevated p16{sup INK4a} followed by pRB activation, but did not activate the p53/p21{sup WAF1/Cip1} pathway. Moreover, MT-1G, which is induced by SM22{alpha} overexpression, was involved in the activation of the p16{sup INK4a}/pRB pathway, which led to a growth arrest state and promoted cellular senescence caused by damaging agents. Our findings provide the first demonstration that SM22{alpha} modulates cellular senescence caused by damaging agents via regulation of the p16{sup INK4a}/pRB pathway in HepG2 cells and that these effects of SM22{alpha} are partially mediated by MT-1G.

  20. Fumosorinone, a novel PTP1B inhibitor, activates insulin signaling in insulin-resistance HepG2 cells and shows anti-diabetic effect in diabetic KKAy mice

    International Nuclear Information System (INIS)

    Liu, Zhi-Qin; Liu, Ting; Chen, Chuan; Li, Ming-Yan; Wang, Zi-Yu; Chen, Ruo-song; Wei, Gui-xiang; Wang, Xiao-yi; Luo, Du-Qiang

    2015-01-01

    Insulin resistance is a characteristic feature of type 2 diabetes mellitus (T2DM) and is characterized by defects in insulin signaling. Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of the insulin signaling pathways, and its increased activity and expression are implicated in the pathogenesis of insulin resistance. Therefore, the inhibition of PTP1B is anticipated to become a potential therapeutic strategy to treat T2DM. Fumosorinone (FU), a new natural product isolated from insect fungi Isaria fumosorosea, was found to inhibit PTP1B activity in our previous study. Herein, the effects of FU on insulin resistance and mechanism in vitro and in vivo were investigated. FU increased the insulin-provoked glucose uptake in insulin-resistant HepG2 cells, and also reduced blood glucose and lipid levels of type 2 diabetic KKAy mice. FU decreased the expression of PTP1B both in insulin-resistant HepG2 cells and in liver tissues of diabetic KKAy mice. Furthermore, FU increased the phosphorylation of IRβ, IRS-2, Akt, GSK3β and Erk1/2 in insulin-resistant HepG2 cells, as well as the phosphorylation of IRβ, IRS-2, Akt in liver tissues of diabetic KKAy mice. These results showed that FU increased glucose uptake and improved insulin resistance by down-regulating the expression of PTP1B and activating the insulin signaling pathway, suggesting that it may possess antidiabetic properties. - Highlights: • Fumosorinone is a new PTP1B inhibitor isolated from insect pathogenic fungi. • Fumosorinone attenuated the insulin resistance both in vitro and in vivo. • Fumosorinone decreased the expression of PTP1B both in vitro and in vivo. • Fumosorinone activated the insulin signaling pathway both in vitro and in vivo

  1. Fumosorinone, a novel PTP1B inhibitor, activates insulin signaling in insulin-resistance HepG2 cells and shows anti-diabetic effect in diabetic KKAy mice

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhi-Qin [College of Life Sciences, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei University, Baoding 071002 (China); College of Pharmaceutical Sciences, key laboratory of pharmaceutical quality control of Hebei province, Hebei University, Baoding 071002 (China); Liu, Ting; Chen, Chuan [College of Life Sciences, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei University, Baoding 071002 (China); Li, Ming-Yan; Wang, Zi-Yu; Chen, Ruo-song; Wei, Gui-xiang; Wang, Xiao-yi [College of Pharmaceutical Sciences, key laboratory of pharmaceutical quality control of Hebei province, Hebei University, Baoding 071002 (China); Luo, Du-Qiang, E-mail: duqiangluo999@126.com [College of Life Sciences, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei University, Baoding 071002 (China)

    2015-05-15

    Insulin resistance is a characteristic feature of type 2 diabetes mellitus (T2DM) and is characterized by defects in insulin signaling. Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of the insulin signaling pathways, and its increased activity and expression are implicated in the pathogenesis of insulin resistance. Therefore, the inhibition of PTP1B is anticipated to become a potential therapeutic strategy to treat T2DM. Fumosorinone (FU), a new natural product isolated from insect fungi Isaria fumosorosea, was found to inhibit PTP1B activity in our previous study. Herein, the effects of FU on insulin resistance and mechanism in vitro and in vivo were investigated. FU increased the insulin-provoked glucose uptake in insulin-resistant HepG2 cells, and also reduced blood glucose and lipid levels of type 2 diabetic KKAy mice. FU decreased the expression of PTP1B both in insulin-resistant HepG2 cells and in liver tissues of diabetic KKAy mice. Furthermore, FU increased the phosphorylation of IRβ, IRS-2, Akt, GSK3β and Erk1/2 in insulin-resistant HepG2 cells, as well as the phosphorylation of IRβ, IRS-2, Akt in liver tissues of diabetic KKAy mice. These results showed that FU increased glucose uptake and improved insulin resistance by down-regulating the expression of PTP1B and activating the insulin signaling pathway, suggesting that it may possess antidiabetic properties. - Highlights: • Fumosorinone is a new PTP1B inhibitor isolated from insect pathogenic fungi. • Fumosorinone attenuated the insulin resistance both in vitro and in vivo. • Fumosorinone decreased the expression of PTP1B both in vitro and in vivo. • Fumosorinone activated the insulin signaling pathway both in vitro and in vivo.

  2. Bitter melon extract ameliorates palmitate-induced apoptosis via inhibition of endoplasmic reticulum stress in HepG2 cells and high-fat/high-fructose-diet-induced fatty liver

    Directory of Open Access Journals (Sweden)

    Hwa Joung Lee

    2018-03-01

    Full Text Available Background: Bitter melon (BM improves glucose level, lipid homeostasis, and insulin resistance in vivo. However, the preventive mechanism of BM in nonalcoholic fatty liver disease (NAFLD has not been elucidated yet. Aim & Design: To determine the protective mechanism of bitter melon extract (BME, we performed experiments in vitro and in vivo. BME were treated palmitate (PA-administrated HepG2 cells. C57BL/6J mice were divided into two groups: high-fat/high-fructose (HF/HFr without or with BME supplementation (100 mg/kg body weight. Endoplasmic reticulum (ER stress, apoptosis, and biochemical markers were then examined by western blot and real-time PCR analyses. Results: BME significantly decreased expression levels of ER-stress markers (including phospho-eIF2α, CHOP, and phospho-JNK [Jun N-terminal kinases] in PA-treated HepG2 cells. BME also significantly decreased the activity of cleaved caspase-3 (a well known apoptotic-induced molecule and DNA fragmentation. The effect of BME on ER stress–mediated apoptosis in vitro was similarly observed in HF/HFr-fed mice in vivo. BME significantly reduced HF/HFr-induced hepatic triglyceride (TG and serum alanine aminotransferase (ALT as markers of hepatic damage in mice. In addition, BME ameliorated HF/HFr-induced serum TG and serum-free fatty acids. Conclusion: These data indicate that BME has protective effects against ER stress mediated apoptosis in HepG2 cells as well as in HF/HFr-induced fatty liver of mouse. Therefore, BME might be useful for preventing and treating NAFLD.

  3. A novel anti-alpha-fetoprotein single-chain variable fragment displays anti-tumor effects in HepG2 cells as a single agent or in combination with paclitaxel.

    Science.gov (United States)

    Ji, Xiaonan; Shen, Yanli; Sun, Hao; Gao, Xiangdong

    2016-08-01

    Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

  4. In vitro transfection of the hepatitis B virus PreS2 gene into the human hepatocarcinoma cell line HepG2 induces upregulation of human telomerase reverse transcriptase

    International Nuclear Information System (INIS)

    Liu Hua; Luan Fang; Ju Ying; Shen Hongyu; Gao Lifen; Wang Xiaoyan; Liu Suxia; Zhang Lining; Sun Wensheng; Ma Chunhong

    2007-01-01

    The preS2 domain is the minimal functional unit of transcription activators that is encoded by the Hepatitis B virus (HBV) surface (S) gene. It is present in more than one-third of the HBV-integrates in HBV induced hepatocarcinoma (HCC). To further understand the functional role of PreS2 in hepatocytes, a PreS2 expression plasmid, pcS2, was constructed and stably transfected into HepG2 cells. We conducted growth curve and colony-forming assays to study the impact of PreS2 expression on cell proliferation. Cells transfected with PreS2 proliferated more rapidly and formed colonies in soft agar. PreS2 expressing cells also induced upregulation of human telomerase reverse transcriptase (hTERT) and telomerase activation by RT-PCR and the modified TRAP assay. Blocking expression of hTERT with antisense oligonuleotide reversed the growth rate in cells stably transfected with PreS2. Our data suggest that PreS2 may increase the malignant transformation of human HCC cell line HepG2 by upregulating hTERT and inducing telomerase activation

  5. In vitro transfection of the hepatitis B virus PreS2 gene into the human hepatocarcinoma cell line HepG2 induces upregulation of human telomerase reverse transcriptase

    Energy Technology Data Exchange (ETDEWEB)

    Hua, Liu [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Fang, Luan [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Ying, Ju [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Hongyu, Shen [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Lifen, Gao [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Xiaoyan, Wang [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Suxia, Liu [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Lining, Zhang [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Wensheng, Sun [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Chunhong, Ma [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Key Laboratory for Experimental Teratology, Ministry of Education (China)]. E-mail: machunhong@sdu.edu.cn

    2007-04-06

    The preS2 domain is the minimal functional unit of transcription activators that is encoded by the Hepatitis B virus (HBV) surface (S) gene. It is present in more than one-third of the HBV-integrates in HBV induced hepatocarcinoma (HCC). To further understand the functional role of PreS2 in hepatocytes, a PreS2 expression plasmid, pcS2, was constructed and stably transfected into HepG2 cells. We conducted growth curve and colony-forming assays to study the impact of PreS2 expression on cell proliferation. Cells transfected with PreS2 proliferated more rapidly and formed colonies in soft agar. PreS2 expressing cells also induced upregulation of human telomerase reverse transcriptase (hTERT) and telomerase activation by RT-PCR and the modified TRAP assay. Blocking expression of hTERT with antisense oligonuleotide reversed the growth rate in cells stably transfected with PreS2. Our data suggest that PreS2 may increase the malignant transformation of human HCC cell line HepG2 by upregulating hTERT and inducing telomerase activation.

  6. Preparation of a radioactive boron compound (B-I-131-lipiodol) for neutron capture therapy of hepatoma

    International Nuclear Information System (INIS)

    Chou, F.I.; Chung, H.P.; Chung, R.J.; Wen, H.W.; Wei, Y.Y.; Kai, J.J.; Lui, W.Y.; Chi, C.W.

    2000-01-01

    In our research, a radioactive boron compound, B-I-131-lipiodol, that can be selectively retained in hepatoma cells was prepared. Combining the effect of α particles produced by boron neutron capture reaction with the β particles released by radionuclides in the radioactive boron compounds will produce a synergistic killing effect on cancer cells. Human hepatoma HepG2 cell cultures were used to examine the stability and the intracellular distribution of the radioactive boron drug. Microscopes were used to examine the interaction and retention of B-I-131-lipiodol globules in the individual hepatoma cell. Moreover, ICP-AES and NaI scintillation counter were performed to determine boron concentrations and I-131 radioactivity, respectively. Results showed that B-I-131-lipiodol with a boron concentration and a specific radioactivity ranged from 500-2000 ppm and 0.05-10 mCi/mL respectively was stably retained in serum. The radiochemical purity of B-I-131-lipiodol was 98%. After supplement with a medium containing B-I-131-lipiodol, the HepG2 cells had intracellular B-I-131-lipiodol globules in the cytoplasm as seen by inverted light microscope, the I-131 and boron can be stably retained in HepG2 cells. (author)

  7. Simultaneous determination of reactive oxygen and nitrogen species in mitochondrial compartments of apoptotic HepG2 cells and PC12 cells based on microchip electrophoresis-laser-induced fluorescence.

    Science.gov (United States)

    Chen, Zhenzhen; Li, Qingling; Sun, Qianqian; Chen, Hao; Wang, Xu; Li, Na; Yin, Miao; Xie, Yanxia; Li, Hongmin; Tang, Bo

    2012-06-05

    Determination of intracellular bioactive species will afford beneficial information related to cell metabolism, signal transduction, cell function, and disease treatment. In this study, the first application of a microchip electrophoresis-laser-induced fluorescence (MCE-LIF) method for concurrent determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS), i.e., superoxide (O(2)(-•)) and nitric oxide (NO) in mitochondria, was developed using fluorescent probes 2-chloro-1,3-dibenzothiazolinecyclohexene (DBZTC) and 3-amino,4-aminomethyl-2',7'-difluorescein (DAF-FM), respectively. Potential interference of intracellular dehydroascorbic acid (DHA) and ascorbic acid (AA) for NO detection with DAF-FM was eliminated through oxidation of AA with the addition of ascorbate oxidase, followed by subsequent MCE separation. Fluorescent products of O(2)(-•) and NO, DBZTC oxide (DBO), and DAF-FM triazole (DAF-FMT) showed excellent baseline separation within 1 min with a running buffer of 40 mM Tris solution (pH 7.4) and a separating electric field of 500 V/cm. The levels of DBO and DAF-FMT in mitochondria isolated from normal HepG2 cells and PC12 cells were evaluated using this method. Furthermore, the changes of DBO and DAF-FMT levels in mitochondria isolated from apoptotic HepG2 cells and PC12 cells could also be detected. The current approach was proved to be simple, fast, reproducible, and efficient. Measurement of the two species with the method will be beneficial to understand ROS/RNS distinctive functions. In addition, it will provide new insights into the role that both species play in biological systems.

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

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Guoxing [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Shi, Hui [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin (China); Li, Jiong; Yang, Zhe [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Fang, Runping; Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin (China); Zhang, Weiying, E-mail: zhwybao@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China)

    2016-04-08

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

  9. Apoptosis Induction by Polygonum minus is related to antioxidant capacity, alterations in expression of apoptotic-related genes, and S-phase cell cycle arrest in HepG2 cell line.

    Science.gov (United States)

    Mohd Ghazali, Mohd Alfazari; Al-Naqeb, Ghanya; Krishnan Selvarajan, Kesavanarayanan; Hazizul Hasan, Mizaton; Adam, Aishah

    2014-01-01

    Polygonum minus (Polygonaceae) is a medicinal herb distributed throughout eastern Asia. The present study investigated antiproliferative effect of P. minus and its possible mechanisms. Four extracts (petroleum ether, methanol, ethyl acetate, and water) were prepared by cold maceration. Extracts were subjected to phytochemical screening, antioxidant, and antiproliferative assays; the most bioactive was fractionated using vacuum liquid chromatography into seven fractions (F1-F7). Antioxidant activity was measured via total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) assays. Antiproliferative activity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Most active fraction was tested for apoptosis induction and cell cycle arrest in HepG2 cells using flow cytometry and confocal microscopy. Apoptotic-related gene expression was studied by RT-PCR. Ethyl acetate extract was bioactive in initial assays. Its fraction, F7, exhibited highest antioxidant capacity (TPC; 113.16 ± 6.2 mg GAE/g extract, DPPH; EC50: 30.5 ± 3.2 μg/mL, FRAP; 1169 ± 20.3 μmol Fe (II)/mg extract) and selective antiproliferative effect (IC50: 25.75 ± 1.5 μg/mL). F7 induced apoptosis in concentration- and time-dependent manner and caused cell cycle arrest at S-phase. Upregulation of proapoptotic genes (Bax, p53, and caspase-3) and downregulation of antiapoptotic gene, Bcl-2, were observed. In conclusion, F7 was antiproliferative to HepG2 cells by inducing apoptosis, cell cycle arrest, and via antioxidative effects.

  10. Gymnaster koraiensis and its major components, 3,5-di-O-caffeoylquinic acid and gymnasterkoreayne B, reduce oxidative damage induced by tert-butyl hydroperoxide or acetaminophen in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Eun Hye Jho

    2013-10-01

    Full Text Available We investigated the protective effects of Gymnaster koraiensisagainst oxidative stress-induced hepatic cell damage. We usedtwo different cytotoxicity models, i.e., the administration oftert-butyl hydroperoxide (t-BHP and acetaminophen, in HepG2cells to evaluate the protective effects of G. koraiensis. The ethylacetate (EA fraction of G. koraiensis and its major compound,3,5-di-O-caffeoylquinic acid (DCQA, exerted protective effectsin the t-BHP-induced liver cytotoxicity model. The EA fractionand DCQA ameliorated t-BHP-induced reductions in GSHlevels and exhibited free radical scavenging activity. The EAfraction and DCQA also significantly reduced t-BHP-inducedDNA damage in HepG2 cells. Furthermore, the hexane fractionof G. koraiensis and its major compound, gymnasterkoreayne B(GKB, exerted strong hepatoprotection in the acetaminopheninducedcytotoxicity model. CYP 3A4 enzyme activity wasstrongly inhibited by the extract, hexane fraction, and GKB. Thehexane fraction and GKB ameliorated acetaminophen-inducedreductions in GSH levels and protected against cell death. [BMBReports 2013; 46(10: 513-518

  11. Cell death induced by Morarah and Khaltita in hepatoma cancer cells (Huh-7).

    Science.gov (United States)

    Baig, Saeeda; Alamgir, Mohiuddin

    2009-10-01

    To compare the combined and isolated growth inhibitory effects of Morarah and Khaltita (herbs) on hepatoma cell lines (Huh-7), through induction of apoptosis or necrosis. Comparative controlled in-vitro study. The Molecular Biology Laboratory, The Aga Khan University, Karachi, from June to December 2006. The growth of hepatoma cell lines (Huh-7) was checked by adding Khaltita and Morarah to the cells before culture in a 24 well plate. Six wells were selected and labeled for each of the four variables (controls, Khaltita, Morarah and mixture). After 2 days, cells were studied under an inverted phase contrast microscope and fields were recorded. Approximately four fields per slide of higher intensity were selected randomly to determine the dead cell density, and the procedure was repeated 10 or more times. Frequency and percentages were calculated for dead or alive cells in controls, Morarah, Khaltita and their mixture. Chi-square was used to compare the qualitative variables. P-values < 0.05 were considered significant. Morarah and Khaltita were found to induce statistically significant (p < 0.001) cell death in hepatoma cell lines (Huh-7). At a magnification of 40x, the controls showed 1% dead cells compared to 91% in Morarah, 83% in Khaltita and 73% in combined mixture of Khaltita and Morarah. At magnification of 20x, the controls showed 4% dead cells compared to 44% in Morarah, 47% in Khaltita and 49% in the combined mixture of Khaltita and Morarah. Morarah and Khaltita induced cell death in cultured hepatoma cells (Huh-7).

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

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    Yuki Haga

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

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

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

    2017-03-01

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

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

  15. In Vitro Characterization and Evaluation of the Cytotoxicity Effects of Nisin and Nisin-Loaded PLA-PEG-PLA Nanoparticles on Gastrointestinal (AGS and KYSE-30), Hepatic (HepG2) and Blood (K562) Cancer Cell Lines.

    Science.gov (United States)

    Goudarzi, Fariba; Asadi, Asadollah; Afsharpour, Maryam; Jamadi, Robab Hassanvand

    2018-05-01

    The aim of this study was an in vitro evaluation and comparison of the cytotoxic effects of free nisin and nisin-loaded PLA-PEG-PLA nanoparticles on gastrointestinal (AGS and KYSE-30), hepatic (HepG2), and blood (K562) cancer cell lines. To create this novel anti-cancer drug delivery system, the nanoparticles were synthesized and then loaded with nisin. Subsequently, their biocompatibility, ability to enter cells, and physicochemical properties, including formation, size, and shape, were studied using hemolysis, fluorescein isothiocyanate (FITC), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and scanning electron microscopy (SEM), respectively. Then, its loading efficiency and release kinetics were examined to assess the potential impact of this formulation for the nanoparticle carrier candidacy. The cytotoxicities of nisin and nisin-loaded nanoparticles were evaluated by using the MTT and Neutral Red (NR) uptake assays. Detections of the apoptotic cells were done via Ethidium Bromide (EB)/Acridine Orange (AO) staining. The FTIR spectra, SEM images, and DLS graph confirmed the formations of the nanoparticles and nisin-loaded nanoparticles with spherical, distinct, and smooth surfaces and average sizes of 100 and 200 nm, respectively. The loading efficiency of the latter nanoparticles was about 85-90%. The hemolysis test represented their non-cytotoxicities and the FITC images indicated their entrance inside the cells. An increase in the percentage of apoptotic cells was observed through EB/AO staining. These results demonstrated that nisin had a cytotoxic effect on AGS, KYSE-30, HepG2, and K562 cancer cell lines, while the cytotoxicity of nisin-loaded nanoparticles was more than that of the free nisin.

  16. Effects of Uptake of Hydroxyapatite Nanoparticles into Hepatoma Cells on Cell Adhesion and Proliferation

    Directory of Open Access Journals (Sweden)

    Meizhen Yin

    2014-01-01

    Full Text Available Hydroxyapatite nanoparticles (nano-HAPs were prepared by homogeneous precipitation, and size distribution and morphology of these nanoparticles were determined by laser particle analysis and transmission electron microscopy, respectively. Nano-HAPs were uniformly distributed, with rod-like shapes sizes ranging from 44.6 to 86.8 nm. Attached overnight, suspended, and proliferating Bel-7402 cells were repeatedly incubated with nano-HAPs. Inverted microscopy, transmission electron microscopy, and fluorescence microscopy were used to observe the cell adhesion and growth, the culture medium containing nano-HAPs, the cell ultrastructure, and intracellular Ca2+ labeled with a fluo-3 calcium fluorescent probe. The results showed that nano-HAPs inhibited proliferation of Bel-7402 cells and, caused an obvious increase in the concentration of intracellular Ca2+, along with significant changes in the cell ultrastructure. Moreover, nano-HAPs led suspended cells and proliferating cells after trypsinized that did not attach to the bottom of the culture bottle died. Nano-HAPs continuously entered these cells. Attached, suspended, and proliferating cells endocytosed nano-HAPs, and nanoparticle-filled vesicles were in the cytoplasm. Therefore, hepatoma cellular uptake of nano-HAPs through endocytosis was very active and occurred continuously. Nano-HAPs affected proliferation and adhesion of hepatoma cells probably because uptake of nano-HAPs blocked integrin-mediated cell adhesion, which may have potential significance in inhibiting metastatic cancer cells to their target organ.

  17. SH2 domain-containing inositol 5-phosphatase (SHIP2) regulates de-novo lipogenesis and secretion of apoB100 containing lipoproteins in HepG2 cells.

    Science.gov (United States)

    Gorgani-Firuzjaee, Sattar; Khatami, Shohreh; Adeli, Khosrow; Meshkani, Reza

    2015-09-04

    Hepatic de-novo lipogenesis and production of triglyceride rich VLDL are regulated via the phosphoinositide 3-kinase cascade, however, the role of a negative regulator of this pathway, the SH2 domain-containing inositol 5-phosphatase (SHIP2) in this process, remains unknown. In the present study, we investigated the molecular link between SHIP2 expression and metabolic dyslipidemia using overexpression or suppression of SHIP2 gene in HepG2 cells. The results showed that overexpression of the wild type SHIP2 gene (SHIP2-WT) led to a higher total lipid content (28%) compared to control, whereas overexpression of the dominant negative SHIP2 gene (SHIP2-DN) reduced total lipid content in oleate treated cells by 40%. Overexpression of SHIP2-WT also led to a significant increase in both secretion of apoB100 containing lipoproteins and de-novo lipogenesis, as demonstrated by an enhancement in secreted apoB100 and MTP expression, increased intra and extracellular triglyceride levels and enhanced expression of lipogenic genes such as SREBP1c, FAS and ACC. On the other hand, overexpression of the SHIP2-DN gene prevented oleate-induced de-novo lipogenesis and secretion of apoB100 containing lipoproteins in HepG2 cells. Collectively, these findings suggest that SHIP2 expression level is a key determinant of hepatic lipogenesis and lipoprotein secretion, and its inhibition could be considered as a potential target for treatment of dyslipidemia. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Mechanisms involved in growth inhibition induced by clofibrate in hepatoma cells

    International Nuclear Information System (INIS)

    Muzio, Giuliana; Maggiora, Marina; Trombetta, Antonella; Martinasso, Germana; Reffo, Patrizia; Colombatto, Sebastiano; Canuto, Rosa Angela

    2003-01-01

    Low concentrations of some peroxisome proliferators have been found to decrease apoptosis in rat liver cells, whereas higher but pharmacological concentrations have been found to inhibit cell proliferation or to induce apoptosis in human and rat hepatoma cells. The highly deviated JM2 rat hepatoma cell line was used to examine the mechanisms underlying the inhibitory effect on cell proliferation. Clofibrate chiefly inhibited cell proliferation in these cells. Parallel to the decrease in cell proliferation there was an increase of peroxisome proliferator activated receptor (PPAR) gamma and of protein phosphatase 2A, whose importance was confirmed, respectively, by using antisense oliginucleotides (AS-ODN) or okadaic acid. The increase of protein phosphatase 2A induced by PPARgamma caused a decrease of MAPK, an intracellular signaling transduction pathway, as shown by evaluation of Erk1,2 and c-myc. In light of these results, clofibrate, like conventional synthetic ligands of PPARgamma, may be regarded as a possible prototype anti-tumour drug

  19. α-Methyl artoflavanocoumarin from Juniperus chinensis exerts anti-diabetic effects by inhibiting PTP1B and activating the PI3K/Akt signaling pathway in insulin-resistant HepG2 cells.

    Science.gov (United States)

    Jung, Hee Jin; Seong, Su Hui; Ali, Md Yousof; Min, Byung-Sun; Jung, Hyun Ah; Choi, Jae Sue

    2017-12-01

    Diabetes mellitus is one of the greatest global health issues and much research effort continues to be directed toward identifying novel therapeutic agents. Insulin resistance is a challenging integrally related topic and molecules capable of overcoming it are of considerable therapeutic interest in the context of type 2 diabetes mellitus (T2DM). Protein tyrosine phosphatase 1B (PTP1B) negatively regulates insulin signaling transduction and is regarded a novel therapeutic target in T2DM. Here, we investigated the inhibitory effect of α-methyl artoflavanocoumarin (MAFC), a natural flavanocoumarin isolated from Juniperus chinensis, on PTP1B in insulin-resistant HepG2 cells. MAFC was found to potently inhibit PTP1B with an IC 50 of 25.27 ± 0.14 µM, and a kinetics study revealed MAFC is a mixed type PTP1B inhibitor with a K i value of 13.84 µM. Molecular docking simulations demonstrated MAFC can bind to catalytic and allosteric sites of PTP1B. Furthermore, MAFC significantly increased glucose uptake and decreased the expression of PTP1B in insulin-resistant HepG2 cells, down-regulated the phosphorylation of insulin receptor substrate (IRS)-1 (Ser307), and dose-dependently enhanced the protein levels of IRS-1, phosphorylated phosphoinositide 3-kinase (PI3K), Akt, and ERK1. These results suggest that MAFC from J. chinensis has therapeutic potential in T2DM by inhibiting PTP1B and activating insulin signaling pathways.

  20. Quercitrin from Toona sinensis (Juss. M.Roem. Attenuates Acetaminophen-Induced Acute Liver Toxicity in HepG2 Cells and Mice through Induction of Antioxidant Machinery and Inhibition of Inflammation

    Directory of Open Access Journals (Sweden)

    Van-Long Truong

    2016-07-01

    Full Text Available Quercitrin is found in many kinds of vegetables and fruits, and possesses various bioactive properties. The aim of the present study was to elucidate hepatoprotective mechanisms of quercitrin isolated from Toona sinensis (Juss. M.Roem. (syn. Cedrela sinensis Juss., using acetaminophen (APAP-treated HepG2 cell and animal models. In an in vitro study, quercitrin suppressed the production of reactive oxygen species and enhanced expression of nuclear factor E2-related factor 2 (Nrf2, activity of antioxidant response element (ARE-reporter gene, and protein levels of NADPH: quinone oxidoreductase 1 (NQO1, catalase (CAT, glutathione peroxidase (GPx, and superoxide dismutase 2 (SOD-2 in APAP-treated HepG2 cells. In an in vivo study, Balb/c mice were orally administered with 10 or 50 mg/kg of quercitrin for 7 days and followed by the injection with single dose of 300 mg/kg APAP. Quercitrin decreased APAP-caused elevation of alanine aminotransferase and aspartate aminotransferase levels, liver necrosis, the expression of pro-inflammatory factors including inducible nitric oxide synthase, cyclooxygenase 2 and inerleukin-1β, and phosphorylation of kinases including c-Jun N-terminal kinase and p38. Quercitrin restored protein levels of Nrf2, NQO1 and activities and expressions of CAT, GPx, SOD-2. The results suggested that quercitrin attenuates APAP-induced liver damage by the activation of defensive genes and the inhibition of pro-inflammatory genes via the suppressions of JNK and p38 signaling.

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

    Science.gov (United States)

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

    2017-07-01

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

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

    Science.gov (United States)

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

    2002-11-01

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

  3. Effects of a fish oil-based emulsion on rat hepatoma cell invasion in culture.

    Science.gov (United States)

    Hagi, Akifumi; Nakayama, Mitsuo; Miura, Yutaka; Yagasaki, Kazumi

    2007-01-01

    Total parenteral nutrition containing a lipid emulsion is often employed after surgical tumor resection. This study investigated the effects of a fish oil-based infusion on rat hepatoma cell invasion. Rat ascites hepatoma cell line AH109A was precultured with a fish oil-based or safflower oil-based emulsion for 48 h. Changes in membranous fatty acid composition were evaluated by gas chromatography. The invasiveness of hepatoma cells was assessed by coculturing with mesentery-derived mesothelial cells. To examine ex vivo effects of the fish oil-based infusion on hepatoma invasion, sera were prepared from rats infused with fish oil- or safflower oil-based emulsion and the effects of these sera were assessed. To clarify the mechanism of inhibition of invasion by the fish oil-based emulsion, the effects of prostaglandin (PG) E(2) and PGE(3) on invasion were examined. Pretreatment with the fish oil-based emulsion reduced invasiveness without affecting growth compared with the safflower oil-based emulsion. Pretreatment with the sera from rats infused with the fish oil-based emulsion also reduced invasiveness compared with the sera from rats infused with the safflower oil-based emulsion. The addition of PGE(2) eliminated the inhibitory effect of the fish oil-based emulsion, and the addition of PGE(3) reduced the invasiveness of hepatoma cells pretreated with the safflower oil-based emulsion. These results suggest that the fish oil-based emulsion may have anti-invasive effects. Changes in the membranous fatty acid composition and consequent changes in the prostaglandins produced may be involved in this inhibitory effect.

  4. Cellular Interactions and Biological Responses to Titanium Dioxide Nanoparticles in HepG2 and BEAS-2B Cells: Role of Cell Culture Media

    Science.gov (United States)

    ABSTRACT We have shown previously that the composition of the biological medium used in vitro can affect the cellular interaction and biological response of titanium dioxide nanoparticles (nano-TiO2) in human lung epithelial cells. However, it is unclear if these effects are co...