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Sample records for huh6 hep3b hepg2

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

    Science.gov (United States)

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

    2010-02-19

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

  2. Cytotoxic, apoptotic, and sensitization properties of ent-kaurane-type diterpenoids from Croton tonkinensis Gagnep on human liver cancer HepG2 and Hep3b cell lines.

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    Pham, Minh Quan; Iscache, Anne Laure; Pham, Quoc Long; Gairin, Jean Edouard

    2016-04-01

    Human hepatocellular carcinoma (HCC) is the most common type of liver cancer, the second most common cause of death from cancer worldwide. A very poor prognosis and a lack of effective treatments make liver cancer a major public health problem, notably in less developed regions, particularly in eastern Asia. This fully justifies the search of new molecules and therapeutic strategies against HCC. Ent-kaurane diterpenoids are natural compounds displaying a broad spectrum of potential therapeutic effects including anticancer activity. In this study, we analyzed the pharmacological properties of a family of ent-kaurane diterpenoids from Croton tonkinensis Gagnep in human HepG2 and Hep3b cell lines, used as cellular reference models for in vitro evaluation of new molecules active on HCC. A structure-related cytotoxicity was observed against both HCC cell lines, enlighting the role of the 16-en-15-one skeleton of ent-kaurane diterpenoids. Cytotoxicity was closely correlated to apoptosis, evidenced by concentration-dependent subG1 cell accumulation, and increased annexin V expression. In addition, subtoxic concentration of ent-kaurane diterpenoid dramatically enhanced the sensitivity of HCC cells to doxorubicin. All together, our data bring strong support to the potential interest of ent-kaurane diterpenoids, alone or in combination with a cytotoxic agent, in cancer and more precisely against HCC.

  3. HepG2、Hep3B细胞中肿瘤干细胞相关标志分子的表达%Expression of cancer stem cell-associated markers in liver cancer cell lines HepG2 and Hep3B

    Institute of Scientific and Technical Information of China (English)

    贾茜; 高建; 张小丽; 向颖; 邓涛

    2012-01-01

    Objective To explore the expression of cancer stem cell ( CSC) -associated markers CD90, CD133, octamer4 (Oct4) and ATP-binding cassette transporter G2 (ABCG2) in liver cancer cell lines HepG2 and Hep3B, and to preliminarily analyze the significance. Methods Liver CSCs were separated from the HepG2 and Hep3B cell lines by flow cytometry, and were cultured in serum-free medium to form spheres. The liver cancer cells were assigned as a control group. Cell proliferation capacity was examined by single-cell clone formation assay. Cell viability was detected by MTT assay after treated with doxorubicin. The mRNA and protein expression levels of CD90, CD133, Oct4 and ABCG2 were detected by real-time PCR and Western blotting , respectively. Results The single cell proliferation capacity of the liver CSCs was stronger than that of the liver cancer cells. The single-cell clone formation assay showed that the colony formation rate of the Hep3B cells was lower than that of the Hep3B CSCs after cultured for 14 d [8/27 (30% ) vs 12/23 (52% ) , P <0. 05 ] , and the colony formation rate of the HepG2 cells was also lower than that of the HepG2 CSCs [7/38 (18% ) vs 9/26 (35%), P <0. 05]. MTT assay showed that the cell viability significantly increased in the liver CSCs compared with that in the liver cancer cells after treated with doxorubicin for 48 h [ HepG2 cells (38. 17 ± 6. 92)% vs HepG2 CSCs (69. 88 ±5. 43)% , P <0. 05; Hep3B cells (50. 16 ±4. 89)% vs Hep3B CSCs (78.53 ± 7. 86 ) % , P < 0. 05 ]. The Real-time PCR results showed that the mRNA expression levels of CD90, CD133, Oct4 and ABCG2 were significantly increased in the liver CSCs compared with those in the parental cells (P < 0. 05 ) . Western blotting results showed that the protein expression levels of Oct4 and ABCG2 significantly increased in the liver CSCs compared with those in the parental cells ( P < 0. 05 ) . Conclusion The CSC-associated markers CD90, CD133, Oct4 and ABCG2 are highly expressed in liver CSCs

  4. Peroxiredoxin 3 is resistant to oxidation-induced apoptosis of Hep-3b cells.

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    Wang, Y-G; Li, L; Liu, C-H; Hong, S; Zhang, M-J

    2014-06-01

    Although peroxiredoxin 3 (PRX3) was reported to be overexpressed in liver cancer, the precise function of PRX3 in the development and/or progression of liver cancer remained to be obscure. The present study was conducted to investigate the response of PRX3 to oxidative stress in hepatocellular carcinoma (HCC) cells. After successful knockdown of PRX3 expression by small interfering RNA, we treated HCC cell lines Hep-3b and Hep-G2 with gradient concentrations of H2O2 and detected cell proliferation, apoptosis, and the level of reactive oxygen species (ROS) in the cells. After low-dose (5-20 μmol/l) H2O2 treatment, the ROS level was significantly higher in PRX3-knockdown Hep-3b cells than in controls. In addition, PRX3 down-regulation resulted in decreased proliferation, increased apoptosis, and increased caspase 3 activity of Hep-3b cells. We did not notice significant difference between PrxIII knockdown and control Hep-G2 cells in ROS level, cell viability or apoptosis. Our results suggest that PRX3 is an indispensable ROS scavenger that protects tumor cells against oxidative damage and subsequent apoptosis, which provides a clue that PRX3 may be involved in the chemotherapeutic resistance of liver cancer. The underlying mechanism for PRX3 function needs further investigation.

  5. Selenium regulation of glutathione peroxidase in human hepatoma cell line Hep3B.

    Science.gov (United States)

    Baker, R D; Baker, S S; LaRosa, K; Whitney, C; Newburger, P E

    1993-07-01

    Glutathione peroxidase is an important enzyme in cellular antioxidant defense systems, detoxifying peroxides and hydroperoxides. As a component of the glutathione cycle, it protects the liver from reactive oxygen metabolites. Selenocysteine is present at the catalytic site of glutathione peroxidase, and selenium availability regulates glutathione peroxidase enzyme activity. Hep3B cells, a well-differentiated human hepatoma-derived cell line, exhibited time-dependent decrease in glutathione peroxidase activity (nmol NADPH oxidized/min/mg protein, mean +/- SE) when incubated in selenium-free medium for 10 days (Day 0, 21.8 +/- 7.3; Day 2, 10.9 +/- 1.2; Day 4, 7.9 +/- 0.8; Day 6, 4.0 +/- 0.7; Day 8, 4.5 +/- 0.6; Day 10, 1.6 +/- 0.4). With the reintroduction of selenium, glutathione peroxidase activity returned. A second human hepatoma cell line, HepG2, demonstrated a similar pattern when depleted of and then repleted with selenium. To assess protein synthesis, glutathione peroxidase activity was measured in deficient and replete Hep3B cells incubated with and without selenium and with and without cycloheximide. Deficient cells (mean +/- SE) (4.9 +/- 0.2) showed an increase in glutathione peroxidase activity after 24 h in selenium-containing medium (11.6 +/- 0.2), but not when cycloheximide was included in the medium (6.9 +/- 0.5) or when cycloheximide and no selenium was included (5.3 +/- 0.8). Replete Hep3B cells (40.1 +/- 1.1) demonstrated decreased glutathione peroxidase after 24 h in medium without selenium (34.0 +/- 1.4), medium with both cycloheximide and selenium (34.0 +/- 2.6), and medium without selenium and containing cycloheximide (37.6 +/- 1.3). These data suggest that protein synthesis is needed for selenium repletion to exert control on glutathione peroxidase activity. Using a cDNA for human glutathione peroxidase (GPx1), selenium-deficient and replete Hep3B cell RNA was analyzed by Northern blot. mRNA for GPx was quantified by densitometry. The steady

  6. Down-regulation of Sonic hedgehog signaling pathway activity is involved in 5-fluorouracil-induced apoptosis and motility inhibition in Hep3B cells

    Institute of Scientific and Technical Information of China (English)

    Qiyu Wang; Shuhong Huang; Ling Yang; Ling Zhao; Yuxia Yin; Zhongzhen Liu; Zheyu Chen; Hongwei Zhang

    2008-01-01

    The Sonic hedgehog (SHh) pathway plays a critical role in normal embryogenesis and carcinogenesis, but its function in cancer cells treated with 5-fluorouracil (5-FU) remains unknown. We examined the expression of a subset of SHh signaling pathway genes, including SHh, SMO, PTC1, Su(Fu) and HIP in human hepatocellular carcinoma (HCC) cell lines,Hep3B and HepG2, treated with 5-FU by reverse transcriptionpolymerase chain reaction. Using trypan blue analysis,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, we also detected the apoptosis of Hep3B cells resulting from the transfection of pCS2-Gli1 expression vector combined with 5-FU treatment.The motility of the cells was detected by scratch wound closure assay. The expression and subcellular location of PTC1 protein in Hep3B cells treated by 5-FU were also investigated by Western blot analysis and immunofluorescent microscopy. The results indicated that the expression of SHh pathway target molecules at both messenger RNA and protein levels are evidently down-regulated in Hep3B cells treated with 5-FU. The overexpression of Gli1 restores cell viability and, to some extent, the migration abilities inhibited by 5-FU.Furthermore, 5-FU treatment affects the subcellular localization of PTC1 protein, a key member in SHh signaling pathway. Our data showed that the down-regulation of SHh signaling pathway activity was involved in 5-FU-induced apoptosis and the inhibition of motility in hedgehog-activated HCC cell lines. This implies that the combination of SHh signaling pathway inhibitor and 5-FU-based chemotherapy might represent a more promising strategy against HCC.

  7. Effects of Lu-Do-Huang Extract (LDHE) on Apoptosis Induction in Human Hep3B Cells.

    Science.gov (United States)

    Huang, Hui-Yu; Chen, Li-Han; Liu, Chen-Wei; Chien, Ting-Yi; Yu, Yu-Ping; Kao, Yu-Yu; Yang, Jo-Hsuan; Tsai, Ying-Chieh

    2015-06-30

    Lu-Do-Huang (Pracparatum mungo) is a fermented mung bean [corrected] (Vigna radiata) and has long been used as a traditional and functional food in Traditional Chinese Medicine, especially for treating a variety of liver disorders. The present study aimed to evaluate the apoptotic effects of Lu-Do-Huang ethanol extract (LDHE) on Hep3B cells, a human hepatoma cell line. A variety of cellular assays, flow cytometry and immunoblotting were used. Our results showed that LDHE significantly inhibited Hep3B cells growth. Additionally, the cell cycle assay showed that LDHE prevented Hep3B cell entry into S phase and led to an arrest of Hep3B cells in the G₀/G₁ phase. LDHE induced Hep3B cells to undergo apoptosis as determined through Hep3B cell morphology changes, increase of apoptotic bodies, apoptotic cells, DNA fragmentations and caspase activity. We further examined the protein expression of TRADD, FADD, and Bax to verify the possible apoptotic pathways. The results indicated that LDHE-induced apoptosis in Hep3B cells might be mediated [corrected] by an extrinsic signaling pathway leading to an induction of apoptosis in Hep3B cells. In conclusion, LDHE induced apoptosis and cell cycle arrest in Hep3B cells. Our data provide the evidences regarding the anti-hepatoma potential of LDHE in Hep3B cells.

  8. Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B

    Institute of Scientific and Technical Information of China (English)

    Xin-Bo Xue; Kun Chen; Cong-Jun Wang; Jian-Wei Zheng; Yuan Yu; Zhi-Hai Peng; Zai-De Wu

    2008-01-01

    BACKGROUND: Melanoma differentiation associated gene-7 (mda-7) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of various intracellular signaling pathways. In this study, we investigated the potential effect of mda-7 on human hepatocellular carcinoma (HCC) in vitro. METHODS: Cells from the human HCC cell line Hep3B and the human liver cell line L-02 were assigned to three groups. One was cultured in Dulbecco's modiifed Eagle's medium without serum (control). The others were transfected with adenovirus expressing the mda-7 gene (Ad.mda-7) or adenovirus vector serving as negative control (Ad.vec). The expression of MDA-7 and Bcl-2 proteins in Hep3B and L-02 cells was conifrmed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The methyl thiazolyl tetrazolium colorimetric assay and lfow cytometry were used to assess tumor cell proliferation and the cell cycle. Hoechst and Annexin-V/propidium iodide staining were used to study mda-7 gene expression in Hep3B and L-02 cells. The expression of MDA-7, Bcl-2 and Bax proteins were detected by Western blotting. RESULTS: The mda-7 gene was expressed in Hep3B and L-02 cells. The protein concentrations of MDA-7 in supernatants were 790 and 810 pg/ml, respectively. mda-7 induced Hep3B growth suppression and apoptosis, compared with Ad.mda-7 and control (P CONCLUSIONS: mda-7 selectively induces growth inhibi-tion and apoptosis in the HCC cell line Hep3B but not in the normal liver cell line L-02 via downregulating the anti-apoptosis protein Bcl-2. It could be an ideal gene for gene therapy in HCC.

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

    Science.gov (United States)

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

    2009-01-01

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

  10. Comparative proteomic analysis of drug sodium iron chlorophyllin addition to Hep 3B cell line.

    Science.gov (United States)

    Zhang, Jun; Wang, Wenhai; Yang, Fengying; Zhou, Xinwen; Jin, Hong; Yang, Peng-yuan

    2012-09-21

    The human hepatoma 3B cell line was chosen as an experimental model for in vitro test of drug screening. The drugs included chlorophyllin and its derivatives such as fluo-chlorophyllin, sodium copper chlorophyllin, and sodium iron chlorophyllin. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method was used in this study to obtain the primary screening results. The results showed that sodium iron chlorophyllin had the best LC(50) value. Proteomic analysis was then performed for further investigation of the effect of sodium iron chlorophyllin addition to the Hep 3B cell line. The proteins identified from a total protein extract of Hep 3B before and after the drug addition were compared by two-dimensional-gel-electrophoresis. Then 32 three-fold differentially expressed proteins were successfully identified by MALDI-TOF-TOF-MS. There are 29 unique proteins among those identified proteins. These proteins include proliferating cell nuclear antigen (PCNA), T-complex protein, heterogeneous nuclear protein, nucleophosmin, heat shock protein A5 (HspA5) and peroxiredoxin. HspA5 is one of the proteins which are involved in protecting cancer cells against stress-induced apoptosis in cultured cells, protecting them against apoptosis through various mechanisms. Peroxiredoxin has anti-oxidant function and is related to cell proliferation, and signal transduction. It can protect the oxidation of other proteins. Peroxiredoxin has a close relationship with cancer and can eventually become a disease biomarker. This might help to develop a novel treatment method for carcinoma cancer.

  11. Mitochondrial dysfunction induces EMT through the TGF-β/Smad/Snail signaling pathway in Hep3B hepatocellular carcinoma cells.

    Science.gov (United States)

    Yi, Eui-Yeun; Park, Shi-Young; Jung, Seung-Youn; Jang, Won-Jun; Kim, Yung-Jin

    2015-11-01

    Mitochondrial dysfunction has been found to be associated with various pathological conditions, particularly cancer. However, the mechanisms underlying tumor malignancy induced by mitochondrial dysfunction are not fully understood. In the present study, the effects of mitochondrial dysfunction on epithelial-mesenchymal transition (EMT), were investigated using mitochondrial-depleted ρ(0) cells derived from the Hep3B hepatocarcinoma cell line. The Hep3B/ρ(0) cells displayed the EMT phenotype with more aggressive migration and higher invasiveness compared to their parental cells. The Hep3B/ρ(0) cells also showed typical expression pattern of EMT markers such as vimentin and E-cadherin. These phenotypes in Hep3B/ρ(0) cells were mediated by increased transforming growth factor-β (TGF-β) through the canonical Smad-dependent signaling pathway. Additionally, TGF-β signaling was activated via induction of c-Jun/AP-1 expression and activity. Therefore, mitochondrial dysfunction induces EMT through TGF-β/Smad/Snail signaling via c-Jun/AP-1 activation. These results indicate that mitochondrial dysfunction plays an important role in the EMT process and could be a novel therapeutic target for malignant cancer therapy.

  12. A methyl jasmonate derivative, J-7, induces apoptosis in human hepatocarcinoma Hep3B cells in vitro.

    Science.gov (United States)

    Park, Cheol; Jin, Cheng-Yun; Kim, Gi-Young; Cheong, JaeHun; Jung, Jee H; Yoo, Young Hyun; Choi, Yung Hyun

    2010-10-01

    The pro-apoptotic activity of J-7, a synthetic methyl jasmonate derivative, on the Hep3B human hepatocarcinoma cell line was investigated. Treatment of Hep3B cells with J-7 resulted in growth inhibition and the induction of apoptosis as measured by trypan blue-excluding cells, MTT assay, nuclear staining, DNA fragmentation, and flow cytometry analysis. The increased apoptotic events in Hep3B cells caused by J-7 were associated with the alteration in the ratio of Bax/Bcl-2 protein expression. J-7 treatment induced the expression of death receptor-related proteins such as death receptor 5, which triggered the activation of caspase-8 and the down-regulation of the whole Bid expression. In addition, the apoptosis induction by J-7 was correlated with the activation of caspase-9 and caspase-3, down-regulation IAP family proteins such as XIAP and cIAP-1, and concomitant degradation of poly (ADP-ribose) polymerase. However, the cytotoxic and apoptotic effects induced by J-7 were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role that caspase-3 plays in the process. Furthermore, blocking the extracellular signal-regulated protein kinase and c-Jun N-terminal kinase pathways showed increased apoptosis and the activation of caspases in J-7-induced apoptosis. The results indicated that J-7 induces the apoptosis of Hep3B cells through a signaling cascade of death-receptor-mediated extrinsic as well as mitochondria-mediated intrinsic caspase pathways, which are associated with the activation of the mitogen-activated protein kinases signal pathway.

  13. Lin28/let-7/Bcl-xL pathway: the underlying mechanism of drug resistance in Hep3B cells.

    Science.gov (United States)

    Tian, Nan; Han, Ziwu; Li, Zhaohui; Zhou, Mingjie; Fan, Chunlei

    2014-09-01

    Hepatocellular carcinoma (HCC) is highly resistant to chemotherapeutic drugs, which markedly reduces the effect of chemotherapy. Lin28 has been shown to contribute to tumor relapse after chemotherapy; however, the relationship between Lin28 and chemotherapy drug resistance is unknown. In the present study, we established a drug-resistant Hep3B cell line to investigate the association between Lin28 and drug resistance in HCC, and we identified the underlying mechanisms. We found that the expression of Lin28 was closely associated with resistance to paclitaxel. The drug‑resistant Hep3B cell line, which expresses high levels of Lin28, is more resistant to paclitaxel and other anticancer drugs than the parental cell line. Moreover, further studies showed that dysregulation of Lin28 inhibited let-7 family microRNA levels and upregulated the anti-apoptotic protein Bcl-xL, which is a target of let-7. Our results indicate that the Lin28/let-7/Bcl-xL pathway underlies the drug resistance of Hep3B cells.

  14. The targeted inhibition of mitochondrial Hsp90 overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis

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    Yan, Chunlan [Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan, 602-714 (Korea, Republic of); Department of Physiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058 (China); Oh, Joon Seok; Yoo, Seung Hee; Lee, Jee Suk [Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan, 602-714 (Korea, Republic of); Yoon, Young Geol [Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan, 602-714 (Korea, Republic of); Department of Biomedical Science, Institute for Biomedical and Health Sciences, Jungwon University, Chungbuk, 367-805 (Korea, Republic of); Oh, Yoo Jin; Jang, Min Seok [Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan, 602-714 (Korea, Republic of); Lee, Sang Yeob [Department of Rheumatology, Dong-A University College of Medicine, Busan, 602-714 (Korea, Republic of); Yang, Jun [Department of Toxicology, Hangzhou Normal University School of Public Health, Hangzhou, Zhejiang, 310036 China (China); Lee, Sang Hwa [Department of Microbiology and, Dong-A University College of Medicine, Busan, 602-714 (Korea, Republic of); Kim, Hye Young [Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan, 602-714 (Korea, Republic of); Yoo, Young Hyun, E-mail: yhyoo@dau.ac.kr [Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan, 602-714 (Korea, Republic of)

    2013-01-01

    Previous studies have reported that a Gamitrinib variant containing triphenylphosphonium (G-TPP) binds to mitochondrial Hsp90 and rapidly inhibits its activity, thus inducing the apoptotic pathway in the cells. Accordingly, G-TPP shows a potential as a promising drug for the treatment of cancer. A cell can die from different types of cell death such as apoptosis, necrosis, necroptosis, and autophagic cell death. In this study, we further investigated the mechanisms and modes of cell death in the G-TPP-treated Hep3B and U937 cell lines. We discovered that G-TPP kills the U937 cells through the apoptotic pathway and the overexpression of Bcl-2 significantly inhibits U937 cell death to G-TPP. We further discovered that G-TPP kills the Hep3B cells by activating necroptosis in combination with the partial activation of caspase-dependent apoptosis. Importantly, G-TPP overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. We also observed that G-TPP induces compensatory autophagy in the Hep3B cell line. We further found that whereas there is a Bcl-2-Beclin 1 interaction in response to G-TPP, silencing the beclin 1 gene failed to block LC3-II accumulation in the Hep3B cells, indicating that G-TPP triggers Beclin 1-independent protective autophagy in Hep3B cells. Taken together, these data reveal that G-TPP induces cell death through a combination of death pathways, including necroptosis and apoptosis, and overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. These findings are important for the therapeutic exploitation of necroptosis as an alternative cell death program to bypass the resistance to apoptosis. Highlights: ► G-TPP binds to mitochondrial Hsp90. ► G-TPP induces apoptosis in U937 human leukemia cancer cells. ► G-TPP induces combination of death pathways in Hep3B cell. ► G-TPP overcomes the resistance conferred by Bcl-2 in Hep3B cells via necroptosis. ► G-TPP triggers Beclin 1-independent

  15. Autophagy inhibition contributes to the synergistic interaction between EGCG and doxorubicin to kill the hepatoma Hep3B cells.

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    Li Chen

    Full Text Available (--Epigallocatechin-3-O-gallate(EGCG, the highest catechins from green tea, has promisingly been found to sensitize the efficacy of several chemotherapy agents like doxorubicin (DOX in hepatocellular carcinoma (HCC treatment. However, the detailed mechanisms by which EGCG augments the chemotherapeutic efficacy remain unclear. Herein, this study was designed to determine the synergistic impacts of EGCG and DOX on hepatoma cells and particularly to reveal whether the autophagic flux is involved in this combination strategy for the HCC. Electron microscopy and fluorescent microscopy confirmed that DOX significantly increased autophagic vesicles in hepatoma Hep3B cells. Western blot and trypan blue assay showed that the increasing autophagy flux by DOX impaired about 45% of DOX-induced cell death in these cells. Conversely, both qRT-PCR and western blotting showed that EGCG played dose-dependently inhibitory role in autophagy signaling, and that markedly promoted cellular growth inhibition. Amazingly, the combined treatment caused a synergistic effect with 40 to 60% increment on cell death and about 45% augmentation on apoptosis versus monotherapy pattern. The DOX-induced autophagy was abolished by this combination therapy. Rapamycin, an autophagic agonist, substantially impaired the anticancer effect of either DOX or combination with EGCG treatment. On the other hand, using small interference RNA targeting chloroquine autophagy-related gene Atg5 and beclin1 to inhibit autophagy signal, hepatoma cell death was dramatically enhanced. Furthermore, in the established subcutaneous Hep3B cells xenograft tumor model, about 25% reduction in tumor growth as well as 50% increment of apoptotic cells were found in combination therapy compared with DOX alone. In addition, immunohistochemistry analysis indicated that the suppressed tendency of autophagic hallmark microtubule-associated protein light chain 3 (LC3 expressions was consistent with thus combined

  16. Studies on the apoptosis inducing and cell cycle regulatory effect of Cuscuta reflexa Roxb chloroform extract on human hepatocellular carcinoma cell line, Hep 3B

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    Ramesh J. Praseeja

    2015-03-01

    Full Text Available Summary. Preliminary studies in our lab showed the anti-hepatocellular carcinoma (HCC activity of Cuscuta reflexa in Hep 3B cell line. This study aims to detail the mechanism behind the anti-HCC effect of C. reflexa on HCC cell line. The chloroform extract of C. reflexa (CRCE reduced the proliferation of Hep 3B cells in a dose and time dependent manner without showing much cytotoxic effect on non-hepatocyte cell lines, HEK 293 and RAW 264.7. C. reflexa induced apoptosis in Hep 3B cells as evidenced by DNA fragmentation, Annexin V-FITC apoptotic assay, PARP cleavage, Caspase activation etc. Treatment with this extract significantly increased the percentage of apoptotic cells. It also caused G1 arrest, associated with apoptotic induction. The active fractions present in CRCE were also isolated by TLC.Industrial relevance. Hepatocellular carcinoma (HCC has gained major clinical interest because of its worldwide increasing incidence and carries a poor survival rate. A complete cure for this disease is not available today. C. reflexa is ethnomedically used for the treatment of various diseases especially jaundice. In this study we have evaluated the apoptosis inducing and associated cell cycle regulatory effect of CRCE in HCC cell line (Hep 3B. Results showed that CRCE is able to induce apoptosis in Hep 3B cells in a dose and time dependent manner through the intrinsic mitochondrial apoptotic pathway. CRCE is a potential candidate for further development as an anti-HCC drug. The HPTLC fingerprint profile of CRCE was analyzed and the active fraction was isolated. Further studies are needed to identify and characterize the isolated active fraction for the development of active anti-HCC drug.Keywords. Hepatocellular carcinoma; Hep 3B; Cuscuta reflexa; Apoptosis; Cell cycle arrest

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

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

    2017-04-01

    JS-K is a novel anticancer nitric oxide (NO) prodrug effective against a variety of cancer cells, including the inhibition of AM-1 hepatoma cell growth in rats. To further evaluate anticancer effects of JS-K, human hepatoma Hep3B cells were treated with JS-K and the compound control JS-43-126 at various concentrations (0-100μM) for 24h, and cytotoxicity was determined by the MTS assay. The compound control JS-43-126 was not cytotoxic to Hep3B cells at concentrations up to 100μM, while the LC50 for JS-K was about 10μM. To examine the molecular mechanisms of antitumor effects of JS-K, Hep3B cells were treated with 1-10μM of JS-K for 24h, and then subjected to gene expression analysis via real time RT-PCR and protein immunostain via confocal images. JS-K is a GST-α targeting NO prodrug, and decreased immunostaining for GST-α was associated with JS-K treatment. JS-K activated apoptosis pathways in Hep3B cells, including induction of caspase-3, caspase-9, Bax, TNF-α, and IL-1β, and immunostaining for caspase-3 was intensified. The expressions of thrombospondin-1 (TSP-1) and the tissue inhibitors of metalloproteinase-1 (TIMP-1) were increased by JS-K at both transcript and protein levels. JS-K treatment also increased the expression of differentiation-related genes CD14 and CD11b, and depressed the expression of c-myc in Hep3B cells. Thus, multiple molecular events appear to be associated with anticancer effects of JS-K in human hepatoma Hep3B cells, including activation of genes related to apoptosis and induction of genes involved in antiangiogenesis and tumor cell migration. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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

    Science.gov (United States)

    Wills, P J; Asha, V V

    2009-03-18

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

  19. Anti-proliferative and apoptosis-triggering potential of disulfiram and disulfiram-loaded polysorbate 80-stabilized PLGA nanoparticles on hepatocellular carcinoma Hep3B cell line.

    Science.gov (United States)

    Hoda, Muddasarul; Pajaniradje, Sankar; Shakya, Garima; Mohankumar, Kumaravel; Rajagopalan, Rukkumani

    2016-08-01

    There is an emerging trend to restudy known drugs for their anti-cancer potential. One such anti-alcoholic drug, disulfiram, with significant anti-cancer potential was studied for its efficacy against Hep3B cell lines, an in vitro model of hepatocellular carcinoma. Simultaneously, we intended to study the effect of polysorbate 80-stabilized PLGA nanoparticles and its DSF-loaded counterpart. Cell and nuclear staining, comet assay, flow cytometry and Western blots were performed. Results suggest that cell proliferation was inhibited by DSF and its PLGA nanoparticles through cell cycle arrest, triggering activation of apoptotic pathways that culminates with cell death. DSF loaded nanoparticles when compared with free DSF, showed significantly lesser effect due to its sustained drug-releasing property, while empty nanoparticles showed negligible influence on Hep3B cells. Our results suggest that DSF alone contributes to cell death, while polysorbate 80-stabilized PLGA nanoparticles show sustained drug release patterns that would potentially lower dosage regimens.

  20. Quinoline-azetidinone hybrids: Synthesis and in vitro antiproliferation activity against Hep G2 and Hep 3B human cell lines.

    Science.gov (United States)

    Alegaon, S G; Parchure, P; Araujo, L D; Salve, P S; Alagawadi, K R; Jalalpure, S S; Kumbar, V M

    2017-04-01

    In search of new heterocyclic anticancer agents, a new quinoline-azetidinone hybrid template have been designed, synthesized and screened for their cytotoxic activity against human cancer cell lines such as Hep G2, and Hep 3B by the MTT assay and results were compared with paclitaxel, 5-fluorouracil and doxorubicin. Interestingly, some of the compounds were found significantly active against both cell lines. The compound 6f (IC50=0.04±0.01µM) exhibited potent antiproliferation activity against Hep G2 cell line, and 6j compound (IC50=0.66±0.01µM) demonstrated potent antiproliferation activity against Hep 3B cell line and provide to be more potent as cytotoxic agents than standard drugs. Morphological changes suggest the induction of apoptosis and describe the mechanism of action of these hybrid antitumor agents. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Effect of various factors and substrates on the growth of a human hepatoblastoma cell line, HuH-6 in a serum-free medium.

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    Tokiwa,Takayoshi

    1989-12-01

    Full Text Available The effect of various factors and substrates on the growth of a human hepatoblastoma cell line, HuH-6, which was inoculated at low density in a serum-free medium was examined. Several supplements were required to enhance cell growth of HuH-6. These included cholera toxin (CT, glucagon (Glu and selenium (Se. Type IV collagen (C-IV provided the most conductive environment tested for cell growth. These results suggest that CT, Glu, Se, and C-IV are important stimulators for the continuous growth of HuH-6 in a serum-free medium at low density.

  2. Effects of the Geiji-Bokryung-Hwan on carrageenan-induced inflammation in mice and cyclooxygenase-2 in hepatoma cells of HepG2 and Hep3B.

    Science.gov (United States)

    Park, Won-Hwan; Joo, Shin-Tak; Park, Kwan-Kyu; Chang, Young-Chae; Kim, Cheorl-Ho

    2004-02-01

    We investigated the effects of a Korean traditional prescription, Geiji-Bokryung-Hwan (GBH) consisting of five herbs of Cinnamomi Ramulus (Korean name Geiji), Poria cocos (Bokryung), Moutan Cortex Radicis (Modanpi). Paeoniae Radix (Jakyak) and Persicae Semen (Doin) on tumor growth-inhibitory activity and cancer chemopreventive activity in assays representing three major stages of carcinogenesis. Effects of the GBH extracts on carrageenan-induced edema inflammation using female (C57BL/6XC3H) F1 (B6C3F1) mice and tumorigenesis were examined. Finally, cyclooxygenase metabolites were determined after extracts treatment. These data suggest that GBH extracts merits investigation as a potential cancer chemopreventive agent in humans.

  3. The protective effects of epigallocatechin gallate on lipopolysa ccharide-induced hepatotoxicity: an in vitro study on Hep3B cells

    Directory of Open Access Journals (Sweden)

    Murat Karamese

    2016-05-01

    Full Text Available Objective(s: In the present study, our aim was to investigate the possible protective effects of epigallocatechin gallate (EGCG on lipopolysaccharide (LPS-induced hepatotoxicity by using Hep3B human hepatoma cells. Specifically, the study examines the role of some proinflammatory markers and oxidative damage as possible mechanisms of LPS-associated cytotoxicity. Consequently, the hepatocellular carcinoma cell line Hep3B was chosen as a model for investigation of LPS toxicity and the effect of EGCG on LPS-exposed cells. Materials and Methods:The Hep3B human hepatoma cells were used for this study. The cytotoxic effects of chemicals (EGCG and LPS, AST and ALT levels, SOD and CAT activities, GSH-Px level and TNF-alpha and IL-6 levels were detected by using different biochemical and molecular methods. LPS and EGCG were applied to cells at various times and doses. Results:The highest treatment dose of EGCG (400 µM led to a dramatic decrease in SOD level and increase in CAT and GSH levels. Additionally, the highest dose of EGCG also led to a dramatic increase in TNF-alpha and IL-6 levels. On the other hand, effective doses of EGCG (200 and 100 µM normalized all related parameters levels. Conclusion:LPS caused hepatotoxicity, but interestingly, a high dose of EGCG was found to be a cytotoxic agent in this study. However, other two doses of EGCG led to a decrease in both inflammatory cytokine levels and antioxidant enzyme levels. Further studies should examine the effect of EGCG on secondary cellular signaling pathways.

  4. A novel cantharidin analog N-Benzylcantharidinamide reduces the expression of MMP-9 and invasive potentials of Hep3B via inhibiting cytosolic translocation of HuR

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji-Yeon; Chung, Tae-Wook; Choi, Hee-Jung [Division of Applied Medicine, School of Korean Medicine, Pusan National University, Yangsan, Gyeongnam (Korea, Republic of); Lee, Chang Hyun [Department of Anatomy, College of Korean Medicine, Woosuk University, Wanju-gun, Jeonbuk (Korea, Republic of); Eun, Jae Soon; Han, Young Taek [College of Pharmacy, Woosuk University, Wanju-gun, Jeonbuk (Korea, Republic of); Choi, Jun-Yong [Department of Internal Medicine, Korean Medicine Hospital, School of Korean Medicine, Pusan National University, Yangsan 626-870 (Korea, Republic of); Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan, Gyeongnam (Korea, Republic of); Kim, So-Yeon; Han, Chang-Woo [Department of Internal Medicine, Korean Medicine Hospital, School of Korean Medicine, Pusan National University, Yangsan 626-870 (Korea, Republic of); Jeong, Han-Sol, E-mail: jhsol33@pusan.ac.kr [Division of Applied Medicine, School of Korean Medicine, Pusan National University, Yangsan, Gyeongnam (Korea, Republic of); Ha, Ki-Tae, E-mail: hagis@pusan.ac.kr [Division of Applied Medicine, School of Korean Medicine, Pusan National University, Yangsan, Gyeongnam (Korea, Republic of); Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan, Gyeongnam (Korea, Republic of)

    2014-05-02

    Highlights: • We examined the inhibition of N-Benzylcantharidinamide on MMP-9-mediated invasion. • Unlike cantharidin, N-Benzylcantharidinamide has very low toxicity on Hep3B cells. • The reduced MMP-9 expression was due to HuR-mediated decrease of mRNA stability. • We suggest N-Benzylcantharidinamide as a novel inhibitor of MMP-9-related invasion. - Abstract: Invasion and metastasis are major causes of malignant tumor-associated mortality. The present study aimed to investigate the molecular events underlying inhibitory effect of N-Benzylcantharidinamide, a novel synthetic analog of cantharidin, on matrix metalloproteinase-9 (MMP-9)-mediated invasion in highly metastatic hepatocellular carcinoma Hep3B cells. In this investigation, among six analogs of cantharidin, only N-Benzylcantharidinamide has the inhibitory action on MMP-9 expression at non-toxic dose. The MMP-9 expression and invasion of Hep3B cells were significantly suppressed by treatment of N-Benzylcantharidinamide in a dose-dependent manner. On the other hand, the transcriptional activity of MMP-9 promoter and nuclear levels of NF-κB and AP-1 as the main transcriptional factors inducing MMP-9 expression were not affected by it although the level of MMP-9 mRNA was reduced by treatment of N-Benzylcantharidinamide. Interestingly, the stability of MMP-9 mRNA was significantly reduced by N-Benzylcantharidinamide-treatment. In addition, the cytosolic translocation of human antigen R (HuR), which results in the increase of MMP-9 mRNA stability through interaction of HuR with 3′-untranslated region of MMP-9 mRNA, was suppressed by treatment of N-Benzylcantharidinamide, in a dose-dependent manner. Taken together, it was demonstrated, for the first time, that N-Benzylcantharidinamide suppresses MMP-9 expression by reducing HuR-mediated MMP-9 mRNA stability for the inhibition of invasive potential in highly metastatic Hep3B cells.

  5. Paclitaxel-sensitization enhanced by curcumin involves down-regulation of nuclear factor-κB and Lin28 in Hep3B cells.

    Science.gov (United States)

    Zhou, Mingjie; Li, Zhaohui; Han, Ziwu; Tian, Nan

    2015-01-01

    Although paclitaxel is an effective chemotherapeutic drug used in the treatment of many tumors, hepatoma cells, in particular, are known to be highly resistant to it. Previously, we discovered that Lin28 was closely associated with resistance to paclitaxel in Hep3B cells. The nuclear factor-kappa B (NF-κB) transcription factor, which plays an important role in tumor survival, directly activates Lin28 expression through a binding site on the first intron. Curcumin, a non-toxic anti-inflammatory agent, inhibits NF-κB activity in vitro. In this study, we reported that a combination of curcumin and paclitaxel exhibited synergistic anti-proliferative and pro-apoptosis effects on Hep3B cells, and curcumin down-regulated paclitaxel-induced enhanced expression of Lin28 and NF-κB activation. Furthermore, our results revealed that curcumin reduced Lin28 levels via mechanisms directly mediated by inhibition of NF-κB activity. These mechanism-based observations evidence that curcumin enhances the sensitivity of hepatoma cells to paclitaxe, and strongly support the notion that paclitaxel in combination with curcumin may provide a superior therapeutic index for HCC chemotherapy.

  6. Piper betle leaf extracts induced human hepatocellular carcinoma Hep3B cell death via MAPKs regulating the p73 pathway in vitro and in vivo.

    Science.gov (United States)

    Wu, Pei-Fang; Tseng, Hsien-Chun; Chyau, Charng-Cherng; Chen, Jing-Hsien; Chou, Fen-Pi

    2014-12-01

    Extracts of Piper betle leaf (PBLs) are rich in bioactive compounds with potential chemopreventive ability. In this study, Hep3B cells which are p53 null were used to investigate the anti-tumor effect of PBLs in the cell and in the xenograft model. The results revealed that PBLs (0.1 to 1 mg mL(-1)) induced a dose- and time-dependent increase of cell toxicity. The underlying mechanisms as evidenced by flow cytometry and western blot analysis showed that PBLs triggered ATM, cAbl, and p73 expressions and activated JNK and p38 pathways that subsequently led to cell cycle arrest and mitochondria-dependent apoptosis. PBLs also inhibited tumor growth in Hep3B-bearing mice via inducing the MAPK-p73 pathway. Our results demonstrated the in vitro and in vivo anti-tumor potential of PBLs, supporting their application as a novel chemopreventive agent for the treatment of human hepatocellular carcinoma (HCC) in the future via targeting the p73 pathway.

  7. Hexane extracts of garlic cloves induce apoptosis through the generation of reactive oxygen species in Hep3B human hepatocarcinoma cells.

    Science.gov (United States)

    Kim, Hye Jeong; Han, Min Ho; Kim, Gi Young; Choi, Young-Whan; Choi, Yung Hyun

    2012-11-01

    Garlic (Allium sativum) compounds have recently received increasing attention due to their cancer chemopreventive properties, and their anticancer activities are extensively reported in many cancer cell lines. However, the anticancer activity and the signaling pathway associated with the induction of apoptosis by extracts of garlic cloves have not been elucidated. In this study, we examined the effects of hexane extracts of garlic cloves (HEGCs) on reactive oxygen species (ROS) production and the association of these effects with apoptotic cell death, using a Hep3B human hepatocarcinoma cell line in vitro. The results demonstrated that HEGCs mediate ROS production, and that this mediation is followed by a collapse of mitochondrial membrane potential (MMP, ΔΨm), the downregulation of anti-apoptotic Bcl-2 and Bcl-xL and the activation of caspase-9 and -3. HEGCs also promoted the activation of caspase-8 and the downregulation of Bid, a BH3-only pro-apoptotic member of the Bcl-2. However, the apoptotic phenomena displayed by HEGCs were significantly diminished by the presence of z-VAD-fmk (non-selective caspase inhibitor). Moreover, N-acetyl-L-cysteine (NAC), a widely used ROS scavenger, effectively blocked the HEGC-induced apoptotic effects via the inhibition of ROS production and MMP collapse. These observations clearly indicate that HEGC-induced ROS are key mediators of MMP collapse, which leads to the induction of apoptosis, followed by caspase activation.

  8. Neferine isolated from Nelumbo nucifera enhances anti-cancer activities in Hep3B cells: molecular mechanisms of cell cycle arrest, ER stress induced apoptosis and anti-angiogenic response.

    Science.gov (United States)

    Yoon, Jin-Soo; Kim, Hwa-Mi; Yadunandam, Anandam Kasin; Kim, Nan-Hee; Jung, Hyun-Ah; Choi, Jae-Sue; Kim, Chi-Yeon; Kim, Gun-Do

    2013-08-15

    Hepatocellular carcinoma (HCC) is one of the most aggressive malignant diseases and is highly resistant to conventional chemotherapy. Neferine, a major bisbenzylisoquinoline alkaloid derived from the embryos of Nelumbo nucifera, has been reported a few physiological activities. However, the mechanisms of anticancer effects are not well understood and its detailed activities on Hep3B cells have not been determined. Our results suggest that neferine exhibited cytotoxicity against HCC Hep3B cells, but not against HCC Sk-Hep1 and THLE-3, a normal human liver cell line. In addition, consistent with the induction of G1/S phase cell population in flow cytometry, downregulation of c-Myc, cyclin D1, D3, CDK4, E2F-1, as well as dephosphorlyation of cdc2 by western blot analysis, as evidenced by the appearance of cell cycle arrest, were observed in Hep3B cells treated with neferine. Our results demonstrated neferine induced ER stress and apoptosis, acting through multiple signaling cascades by the activation of Bim, Bid, Bax, Bak, Puma, caspases-3, -6, -7, -8 and PARP, and the protein expression levels of Bip, calnexin, PDI, calpain-2 and caspase-12 were also upregulated dramatically by neferine treatment. Overexpression of GFP-LC3B by neferine resulted in a diffuse cytosolic GFP fluorescence and the strong fluorescent spots, representing autophagosomes. The significant reduction of the migration in Hep3B cells and the capillary tube-like formation of HUVECs by neferine were also determined. These observations reveal that the therapeutic potential of neferine in treating HCC Hep3B cells, containing copies of hepatitis B virus (HBV) genomes.

  9. Hepatoma cell line HepG2.2.15 demonstrates distinct biological features compared with parental HepG2

    Institute of Scientific and Technical Information of China (English)

    Ran Zhao; Tian-Zhen Wang; Dan Kong; Lei Zhang; Hong-Xue Meng; Yang Jiang; Yi-Qi Wu; Zu-Xi Yu; Xiao-Ming Jin

    2011-01-01

    AIM: To investigate the biological features of hepatitis B virus (HBV)-transfected HepG2.2.15 cells. METHODS: The cell ultrastructure, cell cycle and apop-tosis, and the abilities of proliferation and invasion of HBV-transfected HepG2.2.15 and the parent HepG2 cells were examined by electron microscopy, flow cytometry, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trans-well assay. Oncogenicity of the two cell lines was compared via subcutaneous injection and orthotopic injection or implantation in nude mice, and the pathological analysis of tumor formation was performed. Two cytoskeletal proteins were detected by Western blotting.RESULTS: Compared with HepG2 cells, HepG2.2.15 cells showed organelle degeneration and filopodia disappear-ance under electron microscope. HepG2.2.15 cells pro-liferated and migrated slowly in vitro, and hardly formed tumor and lung metastasis in nude mice. Flow cytom-etry showed that the majority of HepG2.2.15 cells were arrested in G1 phase, and apoptosis was minor in both cell lines. Furthermore, the levels of cytoskeletal pro-teins F-actin and Ezrin were decreased in HepG2.2.15 cells.CONCLUSION: HepG2.2.15 cells demonstrated a low-er proliferation and invasion ability than the HepG2 cells due to HBV transfection.

  10. Peroxiredoxin Ⅱ基因在Hep3B细胞中的生物学功能和作用机制%The biological function of peroxiredoxin Ⅱ on Hep3B cells and its underlying mechanism

    Institute of Scientific and Technical Information of China (English)

    岳海英; 岳惠芬; 李瑗; 刘银坤; 代智; 郭坤; 孙瑞霞; 曹骥; 崔杰峰; 苏建家; 杨春; 欧超

    2008-01-01

    目的 探讨peroxiredoxin Ⅱ (Prx Ⅱ)基因在肝癌发生发展中的作用及其机制.方法 化学合成两对针对Prx Ⅱ基因的小干扰RNA,即Prx Ⅱ-1和Prx Ⅱ-2,分别用脂质体LipofectamineTM 2000介导转入人肝癌细胞系Hep3B.通过实时荧光定量PCR和Western blot检测确定Prx Ⅱ在转染细胞中mRNA和蛋白表达水平受到明显抑制后,对Prx Ⅱ基因沉默的Hep3B细胞分别进行细胞生长、细胞凋亡、细胞集落形成等检测,观察Prx Ⅱ对Hep3B细胞生物学功能的影响;通过二氯荧光素双乙酸和硫代巴比妥酸试验,检测细胞内过氧化代谢产物活性氧类和丙二醛的水平,探讨Prx Ⅱ的作用机制.结果 与阴性对照组和空白对照组相比,Prx Ⅱ基因沉默的Hep3B细胞生长受到抑制,生长抑制率在转染96h后达40%以上;细胞凋亡增加;细胞形成集落的能力降低,Prx Ⅱ-1组和PrxⅡ-2组的细胞集落形成数分别为42.0±2.8和40.5±0.7,明显低于阴性对照组和空白对照组的121.5±2.1和130.0±1.4 (P<0.05).Prx Ⅱ基因沉默的Hep3B细胞内活性氧和丙二醛水平明显增加(P<0.05).结论 Prx Ⅱ具有促进Hep3B细胞生长的生物学功能,其机制可能与通过抗氧化作用形成有利于肿瘤细胞生长的微环境有关.%Objective To study the biological function and its possible underlying mechanism of peroxiredoxin Ⅱ (PrxⅡ) in liver cancer cell line Hep3B. Methods Two pairs of double-stranded small interfering RNA (siRNA) targeted on PrxⅡ gene were transfected into Hep3B cells using LipofectamineTM 2000. After confirming the inhibited effects of these siRNAs through Quant SYBR Green polymerase chain reaction and Western blot, the biological characters of Hep3B cell were analyzed by flow cytometry analysis, MTT and colony formation assays. Furthermore, dichlorodihydrofluorescein diacetate (DCFH-DA) and thiobarbituric acid (TBA) assays, for measuring the products of oxidative reaction, such as the reactive

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

    Science.gov (United States)

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

    2015-01-01

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

  12. Synthesis of platinum(II) and palladium(II) complexes with 9,9-dihexyl-4,5-diazafluorene and their in vivo antitumour activity against Hep3B xenografted mice.

    Science.gov (United States)

    Wang, Q-W; Lam, P-L; Wong, R S-M; Cheng, G Y-M; Lam, K-H; Bian, Z-X; Ho, C-L; Feng, Y-H; Gambari, R; Lo, Y-H; Wong, W-Y; Chui, C-H

    2016-11-29

    Two complexes dichloro(9,9-dihexyl-4,5-diazafluorene)platinum(II) (Pt-DHF) and dichloro(9,9-dihexyl-4,5-diazafluorene)palladium(II) (Pd-DHF) were synthesized and their in vivo antitumour activity was investigated using an athymic nude mice model xenografted with human Hep3B carcinoma cells. Pt-DHF- and Pd-DHF-treated groups showed significant tumour growth inhibition (with about 9-fold and 3-fold tumour growth retardation) when compared with the vehicle control group. The liver toxicology effects on the animals of the two compounds were investigated. Pt-DHF and Pd-DHF-treated groups had a lower alanine transaminase and aspartate transaminase values than those of the vehicle treated group as the animals from the vehicle control group had very heavy hepatoma burden. We assume that both complexes could be further investigated as effective antitumour agents and it is worthwhile to study their underlying working mechanism.

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2017-08-01

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

  16. Mycotoxins’ Activity at Toxic and Sub-Toxic Concentrations: Differential Cytotoxic and Genotoxic Effects of Single and Combined Administration of Sterigmatocystin, Ochratoxin A and Citrinin on the Hepatocellular Cancer Cell Line Hep3B

    Directory of Open Access Journals (Sweden)

    Nikolia Αnninou

    2014-02-01

    Full Text Available Food safety organizations indicate the likelihood of constant human and animal exposure to mycotoxin mixtures as a possible negative public health impact. Risk assessment demonstrates that certain mycotoxins of Aspergillus and Penicillium spp. are toxic and hold a significant genotoxic efficacy at nanomolar concentrations. The aim of the current study was to investigate the potential cytogenetic effects of sterigmatocystin (STER, ochratoxin A (OTA and citrinin (CTN alone or in combination, at pM to μΜ concentrations, on the human hepatocellular cancer cell line Hep3B. MTT reduction, mitotic divisions, cell cycle delays and sister chromatid exchange rates (SCE were determined as endpoints of metabolic activity, cytotoxicity, cytostaticity, and genotoxicity, respectively. All mycotoxin treatments induce SCE rates from 10−12 M, while their cytotoxic and cytostatic potential varies. In PRI and MI assays, but not at MTT, STER alone or in combination with OTA + CTN appeared cytostatic and cytotoxic, even at 10−12 M, while CTN alone and all other combinations displayed substantial cellular survival inhibition in doses ≥ 10−8 M. Co-administration of STER + OTA or STER + CTN in concentrations ≤ 10−1 M, increased the MI and MTT activity, while it did not affect the PRI. Mycotoxin co-treatments revealed in general similar-to-additive or antagonistic genotoxic and cytotoxic effects. Our results for the first time describe that STER alone or in combination with OTA and/or CTN share a cytotoxic and cytogenetic potential even at picoMolar concentrations on human hepatoma cells in vitro.

  17. Apoptosis and its pathway in X gene-transfected HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Na Lin; Hong-Ying Chen; Dan Li; Sheng-Jun Zhang; Zhi-Xin Cheng; Xiao-Zhong Wang

    2005-01-01

    AIM: To investigate the effect of hepatitis B virus (HBV) X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells.METHODS: The HBV X gene eukaryon expression vector pcDNVA3-Xwas transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by PT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells in.three groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL,and c-myc in each group.RESULTS: HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferation capacity was obviously lower in HepG2/pcDNA3-X cells (0.08910±0.003164) than in HepG2 (0.14410±0.004927)and HepG2/pcDNA3 cells (0.12150±0.007159) (P<0.05and P<0.01). Analyzed by TUNEL, cell apoptosis was much more in HepG2/pcDNA3-X cells (980/2 000) than HepG2 (420/2 000), HepG2/pcDNA3 cells (520/2 000) (P<0.05 and P<0.01). Evaluated by semi-quantified RT-PCR, the expression level of Fas/FasL was significantly higher in HepG2 cells transfected with HBx than in HepG2 and HepG2/pcDNA3 cells (P<0.05 and P<0.01). Bax/Bcl-xL expression level was also elevated in HepG2/pcDNA3-X cells (P<0.05and P<0.01). Expression of c-myc was markedly higher in HepG2/pcDNA3-X cells than in HepG2 and HepG2/pcDNA3 cells (P<0.05 and P<0.01).CONCLUSION: HBV X gene can impair cell proliferation capacity, improve cell apoptosis, and upregulate expression of apoptosis factors. The intervention of HBV X gene on the expression of apoptosis factors may be a possible mechanism responsible for the change in cell apoptosis and proliferation.

  18. Study on preliminary mechanism of apoptosis in HepG-2 by CSA

    Institute of Scientific and Technical Information of China (English)

    YU Lei; MU Ke; WANG Wei; CUI Rong-tian; JI Yu-bin; ZOU Xiang

    2008-01-01

    t Objective To study on the mechanism of killing and apoptosis inducing effect of total alkaloid in the CSA(Capparis spinosa L. alkaloid, CSA)on human hepatoearcinoma cell Line HepG-2. Methods The killing effect of the CSA on human hepatoeareinoma cell Line HepG-2 was measured by MTT method. Morphological observation of the HepG-2 cells was completed by fluorescence microscope. The apoptosis indueing effect and changing of mitoehondria membrane potential of the CSA on the HepG-2 cells were measured by flow cytometry. In addition, effect of intraeellular Ca2+ level of the CSA on the HepG-2 cells was studied by laser confocal microscope. Results The CSA has obvious cytotoxicity on the HepG-2 and seems to be dose-dependent, and its IC50 value is 162.4 μg·mL-1. The HepG-2 cells have characteristic morphologic changes of apoptosis by the function of CSA, and the apoptosis percentage is higher than the natural one. The progress of cells cycle from S phase to G2 phase has been blocked, and the mitochondria membrane potential is markedly decreased, and the intraecllular Ca2+ level is increased by the function of CSA. Conclusions The CSA has obviously killing and apoptosis inducing effect on human hepatoearcinoma cell Line HepG-2 by the mechanism of decreasing the mitoehondria membrane potential and increasing the intracellular Ca2+ level.

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

    Science.gov (United States)

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

    2016-06-02

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

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

    Science.gov (United States)

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

    2016-01-01

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

  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

    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...... of the cell periphery. On the other hand the cells on bare glass substrate display spheroid morphology. Further analysis using ToF-SIMS imaging shows that the HepG2 cells on glycopolymer surfaces is enriched with protein fragment along the cell periphery which is absent in the case of cells on bare glass...... substrate. It is suggested that the interaction of the galactose units of the polymer brush with the asialoglycoprotein receptor (ASGPR) of HepG2 cells has resulted in the protein enrichment along the cell periphery....

  2. Establishment of hepatocellular carcinoma multidrug resistant monoclone cell line HepG2/mdr1

    Institute of Scientific and Technical Information of China (English)

    CHEN Yong-bing; XIE Jian-guo; YANG Jia-yin; YAN Lü-nan; YAN Mao-lin; GONG Jian-ping; XIA Ren-pin; LIU Li-xin; LI Ning; LU Shi-chun; ZHANG Jing-guang; ZENG Dao-bing

    2007-01-01

    Background The multidrug resistance (MDR) associated with the expression of the mdr1 gene and its product P-glycoprotein is a major factor in the prognosis of hepatocellular carcinoma cell (HCC) patients treated with chemotherapy. Our study was to establish a stable HCC MDR cell line where a de novo acquisition of multidrug resistance specifically related to overexpression of a transgenic mdr1.Methods The 4.5-kb mdr1 cDNA obtained from the plasmid pHaMDR1-1 was cloned into the PCI-neo mammalian expression vector, later was transferred by liposome to human hepatocarcinoma cell line HepG2. Then the transfected HepG2 cells resisting G418 were clustered and cultured and the specific fragment of mdr1 cDNA, mRNA and the P-glycoprotein (Pgp) in these HepG2 cells were detected by PCR, RT-PCR and flow cytometry, respectively. The accumulation of the daunorubicin was determinated by flow cytometry simultaneously. The nude mice model of grafting tumour was established by injecting subcutaneously HepG2/mdr1 cells in the right axilla. When the tumour diameter reached 5 mm, adriamycin was injected into peritoneal cavity. The size and growth inhibition of tumour were evaluated.Results The mdr1 expression vector was constructed successfully and the MDR HCC line HepG2/mdr1 developed.The PCR analysis showed that the specific fragment of mdr1 cDNA in HepG2/mdr1 cells, but not in the control group HepG2 cells. Furthermore, the content of the specific fragment of mdr1 mRNA and Pgp expression in HepG2/mdr1 cells were (59.7±7.9)% and (12.28±2.09)%, respectively, compared with (16.9±3.2)% and (3.07±1.06)% in HepG2 cells.In the nude mice HCC model, the tumour genes of both groups were identified. After ADM therapy, the mean size of HepG2 cell tumours was significantly smaller than HepG2/mdr1 cell tumours.Conclusion The approach using the transfer of mdr1 cDNA may be applicable to the development of MDR hepatocarcinoma cell line, whose MDR mechanism is known. This would provide the experimental basis of MDR research.

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

    OpenAIRE

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

    2004-01-01

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

  4. 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.

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Science.gov (United States)

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

    2017-04-01

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

  7. Enhancement of the Growth Inhibiting Effect to Liver Tumor Cell Hep3B by Combining Parthenolide with ZD55-TRAIL%PTL联合ZD55-TRAIL对肝癌细胞Hep3B生长抑制的研究

    Institute of Scientific and Technical Information of China (English)

    李晓艳; 马晖; 潘强; 杨新燕; 吴丽琴; 陈侃

    2013-01-01

    联合运用NF-KB小分子抑制剂小白菊内酯(parthenolide,PTL)和携带TRAIL的溶瘤腺病-毒ZD55-TRAIL在体外杀伤人肝癌细胞株Hep3B,以评估两者联用的实验疗效.首先用Adeasy系统包装ZD55-TRAIL病毒;分别用MTT、结晶紫和Hoechst33342荧光染色实验检测ZD55-TRAIL、ZD55-TRAIL和PTL联合运用对肝癌细胞株Hep3B和正常肝细胞QSG-7701的增殖抑制作用.结果表明:10 MOI ZD55-TRAIL联合PTL处理72 h后,Hep3B细胞的存活率仅为44%,但对QSG-7701细胞无明显损伤;而10 MOI的ZD55-TRAIL和PTL分别单独处理后细胞存活率为70%和66%;结晶紫实验及Hoechst33342染色结果表明联合处理组的Hep3B细胞凋亡特征更加明显.溶瘤腺病毒ZD55-TRAIL联合PTL在体外对肝癌细胞Hep3B具有显著的杀伤性,而对QSG-7701细胞无明显毒副作用.%This study aimed at studying the anti-tumor effect in hepatocarcinoma cell Hep3B cells by combining parthenolide (PTL) with ZD55-TRAIL.The experiment was constructed oncolytic adenovirus ZD55-TRAIL by Adeasy systerm.Then MTT,crystal violet and Hoechst33342 staining assay were used to assess the growth inhibition effects of single or combination therapy on liver tumor cell Hep3B and human normal cell QSG-7701.The results showed that 10 MOI ZD55-TRAIL combining with PTL treated for 72 h,Hep3B cells' survival rate is only 44%,and no significant damage to the QSG-7701 cells,while in ZD55-TRAIL and PTL 10 MOI separately treated cells,the survival rate is 70% and 66%,respectively.The crystal violet and Hoechst33342 staining results showed that the combined treatment group Hep3B apoptotic features more obviously.Oncolytic adenovirus ZD55-TRAIL joint PTL had significant destruction in vitro on hepatoma cells Hep3B,but no significant side effects to QSG-7701 cells.

  8. Initial study on apoptosis in HepG-2 Human heptocarcinoma cell line by CSS

    Institute of Scientific and Technical Information of China (English)

    YU Lei; CUI Rong-tian; MO Ke; WANG Wei; JI Yu-bin; ZOU Xiang

    2008-01-01

    Objective To discuss on mechanism of the killing and apoptosis inducing effect induced by total alkaloid in the CSS(Capparis spinosa L. saponin, CSS)on human hepatocarcinoma cell Line HepG-2. Methods The killing effect of the CSS on human hepatocarcinoma cell Line HepG-2 was observed by MTT method. Morphological observation of the HepG-2 cells was completed by fluorescence microscope. This test was signed to observe the changes of the cell cycle of HepG-2 cells affected by the CSS by PI single-staining, and to observe if there were typical apoptosis peaks. The apoptosis inducing effect and changing of mitochondria membrane potential of the CSS on the HepG-2 cells were studied by flow cytometry. The effect of intraceUular Ca2+ level of CSS on the HepG-2 cells was measured by laser confocal microscope. Results CSS has growth inhibiting on the HepG-2 and seems to be enhanced with the increasing concentration of CSS, and its IC50 value was 46.16 μg·mL-1. The HepG-2 cells are characteristic apoptosis morphologic changed, and the apoptosis percentage is increased to 66.652 % in the 50 μg·mL-1 dosage group. The cells cycle has been changed obviously that the progresses of cells cycle of G1 period and G2 period in high dosage group have been blocked, and the cellular proportion in G2 period is decreased by the function of CSS for 24 h. The mitochondria membrane potential of HepG-2 cells induced by CSS is decreased in various degrees. In addition, the intracellular Ca2+ level is increased by the function of CSS in the middle and high dose groups. Conclusions The CSS has obviously killing and apoptosis inducing effect on human hepatoearcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.

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

    Science.gov (United States)

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

    2017-05-01

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

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

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

    2017-06-01

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

  12. Ammonia metabolism capacity of HepG2 cells with high expression of human glutamine synthetase

    Institute of Scientific and Technical Information of China (English)

    Nan-Hong Tang; Xiao-Qian Wang; Xiu-Jin Li; Yan-Ling Chen

    2008-01-01

    BACKGROUND:Currently, one of the tough problems for the application of bioartiifcial liver (BAL) is the shortage of suitable hepatocytes. There are reports on different types of BAL assistance developed with porcine hepatocytes and HepG2 C3A cells, but their defects are obvious. In recent years, some studies focus more on liver cells with features of human origin and improved detoxiifcation. In this study, a hepatocyte line with high expression of human glutamine synthetase (hGS) was raised and its capacity for ammonia metabolism was investigated. METHODS:hGS cDNA and alpha-fetoprotein transcription regulatory element (AFP-TRE) were cloned with the designed primers. The eukaryotic expression vectors, pLNChGS and pLNAFhGS, were constructed and transfected into PA317 cells. Recombinant retroviruses (Retro-hGS and Retro-AFhGS) were produced and then infected into HepG2 cells. G418-resistant cell clones, HepG2/pLNChGS and HepG2/pLNAFhGS, were selected and ampliifed. Then hGS mRNA was measured by semi-quantitative RT-PCR;hGS enzymatic activity and ammonia metabolism analysis in different concentration of NH4+were detected with a quantitative biochemistry kit. The cell proliferation was also detected by MTT chromatometry. RESULTS:The expression of hGS mRNA in HepG2/pLNChGS cells (8.306±0.336) and HepG2/pLNAFhGS cells (21.358±1.716) was much stronger than in control cells (P CONCLUSION:The constructed hepatocytes (HepG2 cells) with speciifc high-expression of hGS have a powerful ability to degrade ammonia in vitro, and provide necessary experimental data for the selection of biomaterials in BAL.

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

    Science.gov (United States)

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

    2012-01-01

    AIM: To investigate the effects of tectorigenin on human hepatocellular carcinoma (HCC) HepG2 cells. METHODS: Tectorigenin, one of the main components of rhizome of Iris tectorum, was prepared by simple methods, such as extraction, filtration, concentration, precipitation and recrystallization. HepG2 cells were incubated with tectorigenin at different concentrations, and their viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was detected by morphological observation of nuclear change, agarose gel electrophoresis of DNA ladder, and flow cytometry with Hoechst 33342, Annexin V-EGFP and propidium iodide staining. Generation of reactive oxygen species was quantified using DCFH-DA. Intracellular Ca2+ was monitored by Fura 2-AM. Mitochondrial membrane potential was monitored using Rhodamine 123. Release of cytochrome c from mitochondria to cytosol was detected by Western blotting. Activities of caspase-3, -8 and -9 were investigated by Caspase Activity Assay Kit. RESULTS: The viability of HepG2 cells treated by tectorigenin decreased in a concentration- and time-dependent manner. The concentration that reduced the number of viable HepG2 cells by 50% (IC50) after 12, 24 and 48 h of incubation was 35.72 mg/L, 21.19 mg/L and 11.06 mg/L, respectively. However, treatment with tectorigenin at 20 mg/L resulted in a very slight cytotoxicity to L02 cells after incubation for 12, 24 or 48 h. Tectorigenin at a concentration of 20 mg/L greatly inhibited the viability of HepG2 cells and induced the condensation of chromatin and fragmentation of nuclei. Tectorigenin induced apoptosis of HepG2 cells in a time- and dose-dependent manner. Compared with the viability rate, induction of apoptosis was the main mechanism of the anti-proliferation effect of tectorigenin in HepG2 cells. Furthermore, tectorigenin-induced apoptosis of HepG2 cells was associated with the generation of reactive oxygen species, increased intracellular [Ca2+]i, loss of mitochondrial membrane potential, translocation of cytochrome c, and activation of caspase-9 and -3. CONCLUSION: Tectorigenin induces apoptosis of HepG2 cells mainly via mitochondrial-mediated pathway, and produces a slight cytotoxicity to L02 cells. PMID:22553399

  14. Midkine accumulated in nucleolus of HepG2 cells involved in rRNA transcription

    Institute of Scientific and Technical Information of China (English)

    Li-Cheng Dai; Jian-Zhong Shao; Li-Shan Min; Yong-Tao Xiao; Li-Xin Xiang; Zhi-Hong Ma

    2008-01-01

    AIM: To invesgate the ultrastructural location of midkine (MK) in nucleolus and function corresponding to its location. METHODS: To investigate the ultrastructural location of MK in nucleolus with immunoelectronic microscopy. To study the role that MK plays in ribosomal biogenesis by real-time PCR. The effect of MK on anti-apoptotic activity of HepG2 cells was studied with FITC-conjugated annexin V and propidium iodide PI double staining through FACS assay. RESULTS: MK mainly localized in the granular component (GC), dense fibrillar component (DFC) and the border between the DF-C and fibrillar center (FC). The production of 45S precursor rRNA level was decreased significantly in the presence of IK antisense oligonucleotide in the HepG2 cells. Furthermore, it was found that exogenous MK could protect HepG2 from apoptosis significantly. CONCLUSION: NK was constitutively translocated to the nucleolus of HepG2 cells, where it accumulated and mostly distributed at DFC, GC components and at the region between FC and DFC, MK played an important role in rRNA transcription, ribosome biogenesis, and cell proliferation in HepG2 cells. MK might serve as a molecular target for therapeutic intervention of human carcinomas.

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2017-06-01

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

  17. 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)

  18. Anticancer and apoptosis-inducing effects of Moringa concanensis using hepG2 cell lines

    Directory of Open Access Journals (Sweden)

    V. Balamurugan

    2014-12-01

    Full Text Available The objective of the present investigation is focused on the anticancer activity of the ethanolic crude extract of Moringa concanensis leaf and bark against HepG2 cell line. The study was facilitated by collecting the plant sample and subjected to ethanol crude extraction. The anticancer activity of the crude extracted sample against HepG2 cell line was examined by MTT assay. The study confirms that the leaf crude extract of M. concanensis has pronounced anticancer potential against HepG2 cell lines while compared to that of the bark extract. The plant investigated possesses remarkable anticancer activity and hence isolation of the compound contributing to the activity may lead to develop at a novel and natural phytomedicine for the disease.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2016-11-25

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

  1. Selective Cytotoxicity of Goniothalamin against Hepatoblastoma HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Noorjahan B. Alitheen

    2011-04-01

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

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

    Science.gov (United States)

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

    2004-12-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  4. Synthesis of apoptotic chalcone analogues in HepG2 human hepatocellular carcinoma cells.

    Science.gov (United States)

    Park, Cheon-Soo; Ahn, Yongchel; Lee, Dahae; Moon, Sung Won; Kim, Ki Hyun; Yamabe, Noriko; Hwang, Gwi Seo; Jang, Hyuk Jai; Lee, Heesu; Kang, Ki Sung; Lee, Jae Wook

    2015-12-15

    Eight chalcone analogues were prepared and evaluated for their cytotoxic effects in human hepatoma HepG2 cells. Compound 5 had a potent cytotoxic effect. The percentage of apoptotic cells was significantly higher in compound 5-treated cells than in control cells. Exposure to compound 5 for 24h induced cleavage of caspase-8 and -3, and poly (ADP-ribose) polymerase (PARP). Our findings suggest that compound 5 is the active chalcone analogue that contributes to cell death in HepG2 cells via the extrinsic apoptotic pathway.

  5. PPARγ pathway activation results in apoptosis and COX-2 inhibition in HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Ming-Yi Li; Hua Deng; Jia-Ming Zhao; Dong Dai; Xiao-Yu Tan

    2003-01-01

    AIM: To investigate whether troglitazone (TGZ), theperoxisome proliferator-activated receptor (PPAR) gammaligand, can induce apoptosis and inhibit cell proliferation inhuman liver cancer cell line HepG2 and to explore themolecular mechanisms. METHODS: [3-(4,5)-dimethyithiazol-2-yl]-2,5-diphenyltetrazolium bromide (NTT), [3H] Thymidine incorporation,Hochest33258 staining, DNA ladder, enzyme-linkedimmunosorbent assay (ELISA), RT-PCR, Northern and Western blotting analyses were employed to investigate the effect of TGZ on HepG2 cells and related molecular mechanisms.RESULTS: TGZ was found to inhibit the growth of HepG2cells and to induce apoptosis. During the process, the expression of COX-2 mRNA and protein and Bcl-2 protein was down-regulated, while that of Bax and Bak proteins was up-regulated, and the activity of caspase-3 was elevated.Furthermore, the level of PGE2 was decreased transiently after 12 h of treatment with 30 gM troglitazone. CONCLUSION: TGZ inhibits cell proliferation and induces apoptosis in HepG2 cells, which may be associated with the activation of caspase-3-like proteases, down-regulation of the expression of COX-2 mRNA and protein, Bcl-2 protein,the elevation of PGE2 levels, and up-regulation of the expressions of Bax and Bak proteins.

  6. Octreotide induces caspase activation and apoptosis inhuman hepatoma HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Nikos J Tsagarakis; Ioannis Drygiannakis; Antonis G Batistakis; George Kolios; Elias A Kouroumalis

    2011-01-01

    AIM: To investigate the role of octreotide on cellular proliferation and apoptosis of human hepatoma (HepG2) cells.METHODS: We studied cellular proliferation, apoptosis and the possible internal caspase-mediated apoptosis pathway involved, after treatment of HepG2 carcinomacells with octreotide in comparison with the apoptosis caused by tumor necrosis factor-α (TNF-α). Activities of caspase-3, caspase-9, caspase-8 and caspase-2 were studied, while apoptosis was investigated through detection of DNA fragmentation and through identification of apoptotic cells with the annexin-V/propidium iodide flow cytometric method.RESULTS: After an initial increase in HepG2 cellular proliferation, a significant inhibition was observed with 10-8 mol/L octreotide, while TNF-α dose-dependentlydecreased proliferation. Early and late apoptosis was significantly increased with both substances. Octreotide significantly increased caspase-3, caspase-8 andcaspase-2 activity. TNF-α significantly increased only caspase-2. Cellular proliferation was decreased after treatment with octreotide or TNF-α alone but, in contrast to TNF-α, octreotide decreased proliferation onlyat concentrations of 10-8 mol/L, while lower concentrations increased proliferation.CONCLUSION: Our findings are suggestive of caspasemediated signaling pathways of octreotide antitumor activity in HepG2 cells, and indicate that measurementsof serum octreotide levels may be important, at least in clinical trials, to verify optimal therapeutic drug concentrations.

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

    DEFF Research Database (Denmark)

    Hansen, Toke Peter Krogager

    lipidmetabolismen når HepG2-SF celler blev inkuberet med elaidinsyre sammenlignet med oleinsyre eller stearinsyre. Den mest fremtrædende ændring var en opregulering af enzymer som syntetiserer kolesterol og fedtsyrer, hvilken indikerede aktivering af sterol regulatory element-binding proteins (SREBPs). Dog blev...

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

    Science.gov (United States)

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

    2014-04-01

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

  9. 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.

  10. 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.

  11. 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...

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

    Science.gov (United States)

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

    2011-01-01

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

  13. 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

    of the cell periphery. On the other hand the cells on bare glass substrate display spheroid morphology. Further analysis using ToF-SIMS imaging shows that the HepG2 cells on glycopolymer surfaces is enriched with protein fragment along the cell periphery which is absent in the case of cells on bare glass...

  14. Polarization Restricts Hepatitis C Virus Entry into HepG2 Hepatoma Cells

    NARCIS (Netherlands)

    Mee, Christopher J.; Harris, Helen J.; Farquhar, Michelle J.; Wilson, Garrick; Reynolds, Gary; Davis, Christopher; van IJzendoorn, Sven C. D.; Balfe, Peter; McKeating, Jane A.

    2009-01-01

    The primary reservoir for hepatitis C virus (HCV) replication is believed to be hepatocytes, which are highly polarized with tight junctions (TJ) separating their basolateral and apical domains. HepG2 cells develop polarity over time, resulting in the formation and remodeling of bile canalicular

  15. 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...

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

    Science.gov (United States)

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

    2016-01-13

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

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

    Science.gov (United States)

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

    2009-02-01

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

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

    Science.gov (United States)

    Buranrat, Benjaporn; Mairuae, Nootchanat; Kanchanarach, Watchara

    2017-04-01

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

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

    Science.gov (United States)

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

    2017-08-15

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

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

    Science.gov (United States)

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

    2015-03-01

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

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

    Science.gov (United States)

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

    2014-11-01

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

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

    Science.gov (United States)

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

    2014-07-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Minqian Shen

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

  5. Gene Transfection Mediated by Ultrasound and Pluronic P85 in HepG2 Cells

    Institute of Scientific and Technical Information of China (English)

    WANG Fen; LI Kaiyan; CHEN Yunchao; DENG Yuan; HONG Kai

    2007-01-01

    In order to assess whether gene transfection could be mediated by ultrasound in associa- tion with P85 and find the appropriate parameters of ultrasound irradiation, the effects of ultrasound with or without P85 on gene transfection of HepG2 cells were examined. The HepG2 cells were irra- diated by ultrasound at 1 MHz, 0.4-2.0 W/cm2 and 50% duty cycle with plasmid encoding enhanced green fluorescent protein (EGFP) as a report gene. Forty-eight h later, the expression of EGFP was detected under the fluorescence microscopy. Transfection efficacy was quantitatively assessed by flow cytometry, and cell viability was evaluated by trypan blue exclusion. The results showed that the transfection efficacy was increased with the increases in ultrasound output power and the ideal trans- fection efficacy was achieved in HepG2 cells irradiated by ultrasound at 0.8 W/cm2 for 30 s. The transfection efficacy in ulstrasound+P85 group was three times higher than in single ultrasound group [(17.63±1.07)% vs (5.57±0.56)%, P<0.051. The cell viability was about 81% and 62% in ultrasound group and ultrasound+P85 group respectively. It was concluded that ultrasound in combination with P85 could mediate the gene transfection of HepG2 cells, ideal transfection efficacy was achieved by ultrasound irradiation at 0.8 W/cm2 for 30 s, and P85 could somewhat increase the damage to cells caused by ultrasound.

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

    Science.gov (United States)

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

    2011-02-23

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

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

    Science.gov (United States)

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

    2013-01-01

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

  8. Knockdown of nucleophosmin induces S-phase arrest in HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Qing-Qing Wang; Zhi-Yi Zhang; Jian-Yong Xiao; Chun Yi; Lin-Zi Li; Yan Huang; Jing-Ping Yun

    2011-01-01

    Nucleophosmin/B23 (NPM) is a universally expressed nucleolar phosphoprotein that participates in proliferation,apoptosis,ribosome assembly,and centrosome duplication; however,the role of NPM in cell cycle regulation is not well characterized.We investigated the mechanism by which NPM is involved in cell cycle regulation.NPM was knocked down using siRNA in HepG2 hepatoblastoma cells.NPM translocation following actinomycin D (ActD) treatment was investigated using immunofluorescent staining.Expression of NPM and other factors involved in cell cycle regulation was examined by Westem blotting.Cell cycle distribution was measured using flow cytometry to detect 5-ethynyl-2'-deoxyuddine (EdU) incorporation.Cell proliferation was quantified by the MTT assay.Knockdown of NPM increased the percentage of HepG2 calls in S phase and led to decreased expression of P53 and P21Cp1/WAF1.S-phase arrest in HepG2 cells was significantly enhanced by ActD treatment.Furthermore,knockdown of NPM abrogated ActD-induced G2/M phase call cycle arrest.Taken together,these data demonstrate that inhibition of NPM has a significant effect on the cell cycle.

  9. SUMO-1 Enhancing the p53-induced HepG2 Cell Apoptosis

    Institute of Scientific and Technical Information of China (English)

    LU Xingrong; YI Jilin

    2005-01-01

    Summary: In order to investigate the effect of small ubiquitin-like modifier-1 (SUMO-1) on the p53-induced HepG2 cell apoptosis, HepG2 cells were transfected by recombinant plasmids as pwtp53, pMDM2 and pSUMO-1 respectively. Western blot was employed to detect the protein expression of the transfected recombinant plasmids and the rate of apoptosis was measured by flow cytometry. The results showed that in cells transfected with pwtp53 and pwtp53+pSUMO-1, the apoptosis rate was (16.79±1.62) % and (18.15±1.36) % respectively, while transfected with pwtp53+pMDM2, the rate was decreased to (5.17±1.23) %. The apoptosis rate was (14.06±1.84) % in the cells transfected with pwtp53+pMDM2+pSUMO-1, significantly higher than that in the cells Transfected with pwtp53+pMDM2 (P<0.01). The apoptosis rates in the cells were all less than 2 % and had no significant difference among the groups. It was suggested that in the HepG2 cells, SUMO-1 can increase the apoptosis induced by wild-type p53 through binding to p53 protein, post-translational modification and inhibiting the p53 degradation by MDM2.

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

    Science.gov (United States)

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

    2010-03-01

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

  11. Tea pigments induce cell-cycle arrest and apoptosis in HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Xu-Dong Jia; Chi Han; Jun-Shi Chen

    2005-01-01

    AIM: To investigate the molecular mechanisms by which tea pigments exert preventive effects on liver carcinogenesis.METHODS: HepG2 cells were seeded at a density of 5×105/well in six-well culture dishes and incubated overnight. The cells then were treated with various concentrations of tea pigments over 3 d, harvested by trypsinization, and counted using a hemocytometer. Flow cytometric analysis was performed by a flow cytometer after propidium iodide labeling. Bcl-2 and p21WAF1 proteins were determined by Western blotting. In addition, DNA laddering assay was performed on treated and untreated cultured HepG2 cells.RESULTS: Tea pigments inhibited the growth of HepG2 cells in a dose-dependent manner. Flow-cytometric analysis showed that tea pigments arrested cell cycle progression at G1 phase. DNA laddering was used to investigate apoptotic cell death, and the result showed that 100 mg/L of tea pigments caused typical DNA laddering. Our study also showed that tea pigments induced upregulation of p21WAF1 protein and downregulation of Bcl-2 protein.CONCLUSION: Tea pigments induce cell-cycle arrest and apoptosis. Tea pigments may be used as an ideal chemopreventive agent.

  12. Potentiation of resveratrol-induced apoptosis by matrine in human hepatoma HepG2 cells.

    Science.gov (United States)

    Ou, Xiuyuan; Chen, Yan; Cheng, Xinxin; Zhang, Xumeng; He, Qiyang

    2014-12-01

    Resveratrol, a natural polyphenolic phytochemical, has received considerable attention due to its potential chemopreventive and chemotherapeutic properties. In the present study, we first evaluated the growth-inhibitory effect of resveratrol on HepG2 cells and explored the underlying molecular mechanisms. Resveratrol inhibited proliferation and induced apoptosis in HepG2 cells via activation of caspase-9 and caspase-3, upregulation of the Bax/Bcl-2 ratio and induction of p53 expression. Cell cycle analysis demonstrated that resveratrol arrested cell cycle progression in the G1 and S phase. We further focused on the combination of matrine, a natural component extracted from the traditional Chinese medical herb Sophora flavescens Ait., as a mechanism to potentiate the growth-inhibitory effect of resveratrol on HepG2 cells. Both MTT and colony formation assay results indicated that the combined treatment of resveratrol and matrine exhibited a synergistic antiproliferative effect. In addition, resveratrol-induced apoptosis was significantly enhanced by matrine, which could be attributed to activation of caspase-3 and caspase-9, downregulation of survivin, induction of reactive oxygen species (ROS) generation and disruption of mitochondria membrane potential (Δψm). Our findings suggest that the combination treatment of resveratrol and matrine is a promising novel anticancer strategy for liver cancer; it also provides new insights into the mechanisms of combined therapy.

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

    Science.gov (United States)

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

    2008-09-10

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

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

    Science.gov (United States)

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

    2009-02-01

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

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

    Science.gov (United States)

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

    2015-05-29

    Recent clinical studies have linked polymorphisms in the xeroderma pigmentosum group D (XPD) gene, a key repair gene involved in nucleotide excision repair, to increased risk of hepatocellular carcinoma (HCC). However, the cellular effects of XPD expression in cultured HCC cells remain largely uncharacterized. Therefore, the aim of this study was to characterize the in vitro cellular effects of XPD expression on the HCC cell line HepG2. HepG2 cells were transfected as follows to create four experimental groups: pEGFP-N2/XPD plasmid (XPD) group, EGFP-N2 plasmid (N2) control group, lipofectamine™ 2000 (lipid) control group, and non-transfected (CON) control group. An MTT cell proliferation assay, Annexin V-APC apoptosis assay, colony formation assay, scratch wound migration assay, Transwell migration assay, and Western blotting of the autophagic proteins LC3 and p62 were conducted. XPD expression significantly inhibited HepG2 cell proliferation (pHepG2 cell apoptosis (pHepG2 colony formation (pHepG2 cells' migratory ability (pHepG2 cells' invasive capacity (pHepG2 cells in vitro. Further in vivo pre-clinical studies and clinical trials are needed to validate XPD's potential as a tumor-suppressive gene therapy.

  16. The impact of beta-elemene on beta-tubulin of human hepatoma hepg2 cells

    Institute of Scientific and Technical Information of China (English)

    Yuqiu Mao; Liying Ban; Jielin Zhang; Li Hou; Xiaonan Cui

    2014-01-01

    Objective:The aim of this study was to investigate the impact of beta-elemene injection on the growth and beta-tubulin of human hepatocarcinoma HepG2 cells. Methods:cellproliferation was assessed by MTT assay. cellcycle distribution was detected by flow cytometry (FCM). The mRNA expression of beta-tubulin was measured by RT-PCR. West-ern blot analysis was used to determine protein expression of beta-tubulin and the polymerization of beta-tubulin. Results:Beta-elemene injection inhibited HepG2 cells proliferation in a dose-and time-dependent manner;FCM analysis indicated beta-elemene injection induced cellcycle arrested at S phase. RT-PCR and western-blot analysis showed that beta-elemene injection down-regulated beta-tubulin expression at both mRNA and protein levels, presenting a dose-dependent manner. Moreover, beta-elemene injection reduced the polymerization of microtubules in a dose-dependent manner. Conclusion:Beta-elemene injection can inhibit the proliferation of hepatoma HepG2 cells, the mechanism might be partly related to the down-regulation of beta-tubulin and inhibition of microtubular polymerization.

  17. Construction and characterization of a yeast two-hybrid cDNA library from a FAT10-overexpressing human hepatic carcinoma cell line Hep3B%类泛素FAT10高表达肝癌细胞株Hep3B酵母双杂交cDNA文库的构建

    Institute of Scientific and Technical Information of China (English)

    余新; 刘天; 德洪波; 李国惠; 邵江华

    2011-01-01

    AIM: To construct a yeast two-hybrid cDNA library from a FAT10-overexpressing human hepatic carcinoma cell line Hep3B.METHODS: Total RNA was prepared from Hep3B cells and used to purify poly (A) mRNA.Double-stranded cDNA was synthesized from the purified mRNA, ligated to EcoR Ⅰ adaptor,digested with EcoR Ⅰ/Xho Ⅰ enzymes, and then cloned into the pGADT7 vector.The recombinant vector was transformed into E.coli DH10B to obtain a primary cDNA library.The primary library was amplified and used to determine the size of cDNA inserts through enzyme digestion.RESULTS: The primary cDNA library contained 1.03 × 106independent clones.The titer of the cDNA library was estimated to be 2.50 × 106 cfu/mL, and that of the amplified library was 3.60 × 109 cfu/mL.The size of the inserts varied from 0.5 to 3.5 kb, with an average value of about 2.0 kb.CONCLUSION: A yeast two-hybrid cDNA library has been successfully generated from FAT10-over-expressing Hep3B ceils and can be used for future screening of proteins interacting with FAT10.%目的:构建类泛素FAT10高表达肝癌细胞株Hep3B的酵母双杂交用cDNA文库.方法:从肝癌细胞Hep3B中提取总RNA,分离mRNA.利用反转录酶M-MLV与Oligo(dT)AnchorPrimer合成1 Strand cDNA,用E.coli DNAPolymerase与E.coli DNA Ligase将RNA链置换成DNA链,合成2 Strand cDNA.将双链cDNA与EcoR Ⅰ Adaptor连接,然后用EcoR Ⅰ /Xho Ⅰ进行酶切.使用Spin Column除去短链cDNA与pGADT7载体连接,转化入E.coli DH10B,建成原始文库.然后对其进行扩增并随机挑取单菌落,酶切鉴定重组子插入片段大小.结果:提取的总RNA降解少且分子完整;RNA纯度高,相对分子质量为400-5000 bp;成功合成双链cDNA,均符合建库要求:库容量达到1.03×10克隆,原始文库滴度为2.50×10 cfu/L,扩增后的文库滴度为3.60×10 cfu/L.插入片段大小分布为0.5-3.5 kb,平均长度约为2.0 kb.结论:所构建文库的各项指标均达到要求,为筛选FAT10作用蛋白奠定了重要基础.

  18. Nuclear matrix associated protein PML: an arsenic trioxide apoptosis therapeutic target protein in HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    于鼎; 王子慧; 朱立元; 邱殷庆

    2003-01-01

    Objective To investigate arsenic trioxide (As2O3)-induced apoptosis and the effects on cell nuclear matrix related protein promyelocytic leukaemia (PML). Methods HepG2 cells were cultured in MEM medium and treated with 0.5, 2, 5 and 10 μmol/L As2O3 for either 24 h or 96 h at each concentration. In situ terminal deoxynucleotidyl transferase (TdT) labeling (TUNEL) and DNA ladders were used to detect apoptosis. Confocal microscopy and Western blotting were used to observe the expression of PML. Results The growth rates of HepG2 cells were slower in the As2O3 treated than the untreated control group. DNA ladder and TUNEL positive apoptotic cells could be detected in As2O3 treated groups. The expression of PML decreased in HepG2 cells with 2 μmol/L As2O3 treatment. Confocal images demonstrated that the expression of PML protein in HepG2 cell nuclei decreased after treatment with 2 μmol/L As2O3, and micropunctates characteristic of PML protein in HepG2 cell nuclei disappeared after treatment with 5 μmol/L As2O3.Conclusions Our results show that arsenic trioxide can significantly inhibit the growth of HepG2 cells in vitro. As2O3 induces apoptosis in HepG2 tumor cells in a time and concentration dependent manner. As2O3 may degrade the PML protein in HepG2 cell nuclei. The decreased expression of PML in As2O3 treated tumor cells is most likely to be caused by apoptosis. Nuclear matrix associated protein PML could be the target of As2O3 therapy.

  19. Early Transcriptional Responses of HepG2-A 16 Liver Cells to Infection by Plasmodium falciparum Sporozoites

    Science.gov (United States)

    2011-07-29

    286, ’JC 30, pp Early Transcriptional Responses of HepG2-A 16 Liver Cells to Infection by Plasmodium falciparum Sporozoites*[i] Received for...7500 and󈧏Sun BioMedical Technologies Inc., Ridgecrest, California 93555 Invasion of hepatocytes by Plasmodium sporozoites depos- ited by Anopheles...expression profiling of human HepG2-A16liver cells infected with Plasmodium falciparum sporozoites to understand the host early cellular events and

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2016-05-10

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

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

    Science.gov (United States)

    Li, Xiaoyu; Ma, Junguo; Wang, Jianji

    2015-10-01

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

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

    Science.gov (United States)

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

    2016-07-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-05-07

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

  6. 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 (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.

  7. 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.

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

    Science.gov (United States)

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

    2017-01-01

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

  9. 冬凌草甲素通过激活ATR通路诱导HEPG2细胞凋亡实验研究%Oridonin Induced the HEPG2 Cells Apoptosis by the Activation of the ATR Pathway

    Institute of Scientific and Technical Information of China (English)

    周泽雄; 曲珍仪; 刘颖

    2016-01-01

    Objective:To research oridonin induce the HEPG2 cells apoptosis and its the related mechanism.Methods:Using MTT and crystal violet staining to study oridonin inhibited HEPG2 cells proliferative,Western Blotting to detect the change of ATR,H2AX,γ-H2AX,P53 and so on protein after oridonin treated HEPG2 cells.Results:MTT method and crystal violet staining show that oridonin can significantly inhibited HEPG2 cells,and the inhibition effect in certain concentrations with a dosedependent manner.Western blotting showed that the ATR,P-P53,γ-H2AX protein activity and express has significant enhanced during oridonin induced the HEPG2 cells apoptosis.Conclusion:Oridonin induced the HEPG2 cells apoptosis may be cause by the phosphorylation of H2AX protein further activate the ATR signaling pathways lead to cell death.%目的:探讨冬凌草甲素抑制HEGP2肝癌细胞增殖及其机制研究方法:MTT法检测抑制率、结晶紫检测冬凌草甲素诱导HEPG2肝癌细胞抑制增殖作用、Western blotting检测不同浓度冬凌草甲素作用HEPG2肝癌细胞后ATR、H2AX、γ-H2AX、P53等蛋白的变化.结果:MTT法、结晶紫法显示冬凌草甲素对HEPG2肝癌细胞能够明显抑制,且抑制作用在一定浓度范围内呈计量依赖性;Western blotting结果显示冬凌草甲素在诱导细胞凋亡过程中ATR、P-P53、γ-H2AX表达水平及活性显著增强.结论:冬凌草甲素诱导HEPG2肝癌细胞发生凋亡可能是通过引起H2AX蛋白磷酸化进一步激活ATR信号通路引起细胞凋亡.

  10. Apoptosis induced by nucleosides in the human hepatoma HepG2

    Institute of Scientific and Technical Information of China (English)

    Suh-Ching Yang; Che-Lin Chiu; Chi-Chang Huang; Jiun-Rong Chen

    2005-01-01

    AIM: To investigate the apoptotic effects of nucleosides on the human hepatoma HepG2.METHODS: The nucleosides included inosine (I), cytidine(C), uridine (U), thymidine (T), adenosine (A), and guanosine (G). Cells were incubated by the mediums with or without nucleosides at 37 ℃ in a 50 mL/L CO2 humidified atmosphere.RESULTS: It was found that the cell viabilities were significantly decreased, when cells were treated with 30 mmol/L I, 30 mmol/L C, 30 mmol/L U, 30 mmol/L T,0.5 mmol/L A, and 0.5 mmol/L G after 12 h incubation (P<0.05). About the apoptotic phenomenon, the cell percentages of sub-G1 cells were significantly increased in the mediums containing nucleosides such as C, U, T,A, and G (P<0.05). Furthermore, the caspase-3 activity was increased, when the cells were incubated with T(P<0.05). The protein expressions of p53 and p21 showed no difference in each group. To investigate the mechanism of apoptosis induced by nucleosides, it was found that the contents of soluble Fas ligand contents were increased in HepG2 cells following I, U, T, and A treatment (P<0.05).But, TNF-α and cytochrome c were undetectable.CONCLUSION: Thymidine may induce the apoptosis in HepG2, but the effective dosages and reactive time must be investigated in the future study. However, the apoptosis-inducing abilities of other nucleosides were still unclear in this study.

  11. Cellular Trafficking of Thymosin Beta-4 in HEPG2 Cells Following Serum Starvation

    Science.gov (United States)

    Pichiri, Giuseppina; Coni, Pierpaolo; Nemolato, Sonia; Cabras, Tiziana; Fanari, Mattia Umberto; Sanna, Alice; Di Felice, Eliana; Messana, Irene; Castagnola, Massimo; Faa, Gavino

    2013-01-01

    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 regulate the pore permeability. PMID:23967050

  12. 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.

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

    Science.gov (United States)

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

    2008-01-17

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

  14. Lipid synthesis and secretion in HepG2 cells is not affected by ACTH

    Directory of Open Access Journals (Sweden)

    Nilsson-Ehle Peter

    2010-05-01

    Full Text Available Abstract Apolipoprotein B (apoB containing lipoproteins, i.e. VLDL, LDL and Lp(a, are consequently lowered by ACTH treatment in humans. This is also seen as reduced plasma apoB by 20-30% and total cholesterol by 30-40%, mostly accounted for by a decrease in LDL-cholesterol. Studies in hepatic cell line (HepG2 cells showed that apoB mRNA expression is reduced in response to ACTH incubation and is followed by a reduced apoB secretion, which may hypothesize that ACTH lowering apoB containing lipoproteins in humans may be mediated by the inhibition of hepatic apoB synthesis. This was recently confirmed in vivo in a human postprandial study, where ACTH reduced transient apoB48 elevation from the small intestine, however, the exogenic lipid turnover seemed unimpaired. In the present study we investigated if lipid synthesis and/or secretion in HepG2 cells were also affected by pharmacological levels of ACTH to accompany the reduced apoB output. HepG2 cells were incubated with radiolabelled precursors ([14C]acetate and [3H]glycerol either before or during ACTH stimuli. Cellular and secreted lipids were extracted with chloroform:methanol and separated by the thin layer chromatography (TLC, and [14C]labelled cholesterol and cholesteryl ester and [3H]labelled triglycerides and phospholipids were quantitated by the liquid scintillation counting. It demonstrated that ACTH administration did not result in any significant change in neither synthesis nor secretion of the studied lipids, this regardless of presence or absence of oleic acid, which is known to stabilize apoB and enhance apoB production. The present study suggests that ACTH lowers plasma lipids in humans mainly mediated by the inhibition of apoB synthesis and did not via the reduced lipid synthesis.

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

    Science.gov (United States)

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

    2015-10-27

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

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

    Science.gov (United States)

    Lee, Ah Ron; Han, Sung Nim

    2016-07-01

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

  17. 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 regulate the pore permeability.

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

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    Zakaria Yusmazura

    2009-06-01

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

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

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    Xuan Liu

    2015-05-01

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

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

    Science.gov (United States)

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

    2015-05-26

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2017-02-21

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

  3. Transfection of p27 kip1 enhances radiosensitivity induced by 60Coγ-irradiation in hepatocellular carcinoma HepG2 cell line

    Institute of Scientific and Technical Information of China (English)

    Xiao-Xiang Guan; Long-Bang Chen; Gui-Xia Ding; Wei De; Ai-Hua Zhang

    2004-01-01

    AIM: To study the cell cycle alterations of human hepatoma cell line HepG2 in vitro after 60Co γ-irradiation and further to examine the mechanisms underlying the enhancement of radiosensitivity to γ-irradiation in HepG2 transiently transfected with wild type p27kip1.METHODS: The proliferation of HepG2 cells was evaluated with MTT assay, and the cell cycle profile and apoptosis were assessed by cell morphology, DNA fragmentation analysis and flow cytometry. HepG2 cells were transfected with p27kip1 wild type by using Lipofectamine (LF2000), and the expression and subcellular localization of p27kip1 in HepG2were detected by immunocytochemistry.RESULTS: 60Co γ-irradiation inhibited the growth of HepG2cells in a dose-dependent manner. Apoptosis of HepG2 cells was induced 48 h after γ ray exposure. Furthermore research was carried out to induce exogenous expression of p27kip1in HepG2. The expression of p27kip1 induced G0/G1 phase arrest in HepG2 cells. The overexpression of p27kip1 enhanced 60Co γ-irradiation-induced radiosensitivity in HepG2 cells.CONCLUSION: Overexpression of p27kip1 is a rational approach to improve conventional radiotherapy outcomes, which may be a possible strategy for human hepatoma therapy.

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

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

    2014-08-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

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    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.

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

    Science.gov (United States)

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

    2016-01-04

    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.

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

    Science.gov (United States)

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

    2010-05-01

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

  9. Novel dammarane saponins from Gynostemma pentaphyllum and their cytotoxic activities against HepG2 cells.

    Science.gov (United States)

    Piao, Xiang-Lan; Xing, Shao-Fang; Lou, Cai-Xia; Chen, Dao-Jin

    2014-10-15

    Two new dammarane saponins, 2α,3β,12β-trihydroxydammar-20(22),24-diene-3-O-[β-D-glucopyranoxyl(1→2)-β-D-6″-O-acetylglucopyranoside (1, namely damulin C) and 2α,3β,12β-trihydroxydammar-20(21),24-diene-3-O-[β-D-glucopyranoxyl(1→2)-β-D-6″-O-acetylglucopyranoside (2, namely damulin D), were isolated from the ethanol extract of Gynostemma pentaphyllum, which had been heat processed by steaming at 125 °C. The NMR spectroscopic data of the novel saponins were completely assigned by using a combination of 2D NMR experiments including (1)H-(1)H COSY, HSQC, and HMBC. Their cytotoxic activities of human liver adenocarcinoma HepG2 cells were evaluated in vitro. They showed cytotoxicities against HepG2 cell line with IC50 of 40±0.7 and 38±0.5 μg/ml, respectively. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

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    María Roel

    2015-07-01

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

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

    Science.gov (United States)

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

    2009-08-26

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

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

    Science.gov (United States)

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

    2010-04-01

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

  13. Proanthocyanidins modulate microRNA expression in human HepG2 cells.

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    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2017-04-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Science.gov (United States)

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

    2017-02-01

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

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

    Science.gov (United States)

    Zhou, Lei; Zhang, Heng; Wu, Jie

    2016-05-01

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

  20. Synthesis and Evaluation of New Pyrazoline Derivatives as Potential Anticancer Agents in HepG-2 Cell Line

    Directory of Open Access Journals (Sweden)

    Weijie Xu

    2017-03-01

    Full Text Available Cancer is a major public health concern worldwide. Adverse effects of cancer treatments still compromise patients’ quality of life. To identify new potential anticancer agents, a series of novel pyrazoline derivatives were synthesized and evaluated for cytotoxic effects on HepG-2 (human liver hepatocellular carcinoma cell line and primary hepatocytes. Compound structures were confirmed by 1H-NMR, mass spectrometry, and infrared imaging. An in vitro assay demonstrated that several compounds exerted cytotoxicity in the micromolar range. Benzo[b]thiophen-2-yl-[5-(4-hydroxy-3,5-dimethoxy-phenyl-3-(2-hydroxy-phenyl-4,5-dihydo-pyrazol-1-yl]-methanone (b17 was the most effective anticancer agent against HepG-2 cells owing to its notable inhibitory effect on HepG-2 with an IC50 value of 3.57 µM when compared with cisplatin (IC50 = 8.45 µM and low cytotoxicity against primary hepatocytes. Cell cycle analysis and apoptosis/necrosis evaluation using this compound revealed that b17 notably arrested HepG-2 cells in the G2/M phase and induced HepG-2 cells apoptosis. Our findings indicate that compound b17 may be a promising anticancer drug candidate.

  1. Effect of shikonin on multidrug resistance in HepG2: The role of SIRT1.

    Science.gov (United States)

    Jin, Yong-Dong; Ren, Yi; Wu, Ming-Wei; Chen, Ping; Lu, Jin

    2015-07-01

    Overexpression of SIRT1 is considered to enhance the resistance of HepG2 cells to irradiation. Shikonin, a naturally occurring naphthoquinone compound, displays anticancer effects and circumvents cancer drug resistance. This study investigated the MDR reversal effect of shikonin induced by the overexpression of SIRT1. The overexpression of SIRT1 in HepG2 cells was established by lentivirus infection. Five days after transduction, real-time quantitative polymerase chain reaction and western blotting were used to detect the expression of SIRT1 and MDR1/P-gp. Drug resistance was also evaluated by flow cytometry after rhodamine-123 staining. On day 5, the multidrug resistance cells were treated by shikonin (10(-7), 10(-6), and 10(-5) µmol/L) one time. The cell viability was detected by the MTT assay, and apoptosis was evaluated by Hoechst 33342 staining and caspase-3 activity 24 h after shikonin treatment. Overexpression of SIRT1 decreased rhodamine-123 staining and successfully produced the R-HepG2 cell line. Compared with HepG2, the expression of MDR1/P-gp mRNA (3.45 ± 0.35) and protein (1.40 ± 0.05) were both upregulated in R-HepG2. Shikonin inhibited cell viability (from 93.9 ± 2.1 to 66.7 ± 1.5%), induced apoptosis of R-HepG2 (apoptotic ratio from 3.5 ± 0.8 to 47.5 ± 2.7%, caspase-3 activity from 103.5 ± 1.9 to 329.2 ± 14.9%, respectively), downregulated the mRNA and protein expression of SIRT1 and MDR1/P-gp, and decreased rhodamin 123 efflux. In the present study, we demonstrated that shikonin is able to overcome drug resistance in hepatocellular carcinoma cells, and the mechanism is related to the SIRT1-MDR1/P-gp signaling pathway.

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Piret, Jean-Pascal; Vankoningsloo, Sebastien; Noel, Florence; Saout, Christelle; Toussaint, Olivier [Research Unit in Cellular Biology (URBC), Narilis, University of Namur, 5000 Namur (Belgium); Mendoza, Jorge Mejia; Lucas, Stephane, E-mail: olivier.toussaint@fundp.ac.be [Research Center for the Physics of Matter and Radiation (PMR), Narilis, University of Namur, 5000 Namur (Belgium)

    2011-07-06

    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.

  4. 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.

  5. Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin

    Institute of Scientific and Technical Information of China (English)

    Xiang-Wen Tan; Hong Xia; Jin-Hua Xu; Jian-Guo Cao

    2009-01-01

    AIM: To investigate the effect of 5-allyl-7-gen-difluoromethylenechrysin (ADFMChR) on apoptosis of human liver carcinoma HepG2 cell line and the molecular mechanisms involved. METHODS: HepG2 cells and L-02 cells were cultured in vitro and the inhibitory effect of ADFMChR on their proliferation was measured by MTT assay. The apoptosis of HepG2 cells was determined by flow cytometry (FCM) using propidium iodide (PI) fluorescence staining.DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of ADFMChR on the proxisome proliferator-activated receptor γ (PPARγ), NF-κB, Bcl-2 and Bax protein expression of HepG2 cells were analyzed by Western blotting. RESULTS: MTT assay showed that ADFMChR significantly inhibited proliferation of HepG2 cells in a dosedependent manner, with little effect on growth of L-02 cells, and when IC50 was measured as 8.45 μmol/L and 191.55 μmol/L respectively, the potency of ADFMChR to HepG2 cells, was found to be similar to 5-fluorouracil (5-FU, IC50 was 9.27 μmol/L). The selective index of ADFMChR cytotoxicity to HepG2 cells was 22.67 (191.55/8.45), higher than 5-FU (SI was 7.05 (65.37/9.27). FCM with PI staining demonstrated that the apoptosis rates of HepG2 cells treated with 3.0, 10.0 and 30.0 μmol/L ADFMChR for 48 h were 5.79%, 9.29% and 37.8%, respectively, and were significantly higher when treated with 30.0 μmol/L ADFMChR than when treated with 30.0 μmol/L ChR (16.0%) ( P < 0.05) and were similar to those obtained with 30.0 μmol/L 5-FU (41.0%). DNA agarose gel electrophoresis showed that treatment of HepG2 cells with 10.0 μmol/L ADFMChR for 48 h and 72 h resulted in typical DNA ladders which could be reversed by 10.00 μmol/L GW9662, a blocker of PPARγ. Western blotting analysis revealed that after 24 h of treatment with 3.0, 10.0, 30.0 μmol/L ADFMChR, PPARγ and Bax protein expression in HepG2 cells increased but Bcl-2 and NF-κB expression decreased; however, pre-incubation with 10.0 μmol/L GW9662 could efficiently antagonize and weaken the regulatory effect of 3.0, 30.0 μmol/L ADFMChR on PPARγ and NF-κB protein expression in HepG2 cells. CONCLUSION: ADFMChR induces apoptosis of HepG2 cell lines by activating PPARγ, inhibiting protein expression of Bcl-2 and NF-κB, and increasing Bax expression.

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

    Science.gov (United States)

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

    2014-12-15

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

  7. 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-loaded chitosan nanoparticles (TP-CNPs) were prepared. • The antitumor effects of TP-CNPs were evaluated in HepG2 cells. • TP-CNPs could inhibit the proliferation of HepG2. • Cell apoptosis and cell cycle analysis reveal the antitumor mechanism.

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

    Science.gov (United States)

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

    2016-03-01

    The aim of the present study was to investigate the effect of solanine on promoting human hepatocellular carcinoma HepG2 cells to produce reactive oxygen species (ROS), and the molecular mechanisms leading to tumor cell apoptosis. Solanine was administered to HepG2 cells in vitro. A selection of probes targeting various cellular localizations of ROS were used to detect ROS expression using flow cytometry. The expression levels of apoptosis-associated proteins, including apoptosis signal-regulating kinase 1 (ASK1) and thioredoxin binding protein 2 (TBP-2), and proliferation-associated proteins, including histone deacetylase 1 (HDAC1), were detected using western blotting. The percentage of cells undergoing apoptosis was measured using an Annexin V-fluorescein isothiocyanate/propidium iodide assay, and cell morphology was examined using Wright's stain followed by inverted microscopy analysis. ROS detection probes 2',7'-dichlorofluorescin diacetate and dihydrorhodamine 123 identified that abundant ROS, including hydroxyl radical (OH(-)) and hydrogen peroxide (H2O2), were produced in the cytoplasm and mitochondria of the solanine-treated HepG2 cells compared with the control cells (Psolanine treatment compared with the control cells (P>0.05). Western blotting results revealed that solanine upregulated the expression levels of ASK1 and TBP-2 and enhanced their kinase activities, whereas solanine decreased the expression level of the proliferation-associated protein, HDAC1. The cell apoptotic rate was significantly increased (Psolanine-treated HepG2 cells compared with the control cells. (Psolanine induces HepG2 cells to produce ROS, mainly OH(-) and H2O2, in a mitochondria-dependent and -independent manner. In addition, solanine stimulates the expression of ASK1 and TBP-2, and their kinase activities, but inhibits the expression of proliferation-associated proteins, such as HDAC1, thus contributing to HepG2 cell apoptosis.

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Science.gov (United States)

    Melušová, Martina; Jantová, Soňa; Horváthová, Eva

    2014-12-01

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

  11. [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.

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

    Science.gov (United States)

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

    2017-07-01

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

  13. Alcohol Dehydrogenase 5 Is a Source of Formate for De Novo Purine Biosynthesis in HepG2 Cells.

    Science.gov (United States)

    Bae, Sajin; Chon, James; Field, Martha S; Stover, Patrick J

    2017-04-01

    Background: Formate provides one-carbon units for de novo purine and thymidylate (dTMP) synthesis and is produced via both folate-dependent and folate-independent pathways. Folate-independent pathways are mediated by cytosolic alcohol dehydrogenase 5 (ADH5) and mitochondrial aldehyde dehydrogenase 2 (ALDH2), which generate formate by oxidizing formaldehyde. Formate is a potential biomarker of B-vitamin-dependent one-carbon metabolism.Objective: This study investigated the contributions of ADH5 and ALDH2 to formate production and folate-dependent de novo purine and dTMP synthesis in HepG2 cells.Methods:ADH5 knockout and ALDH2 knockdown HepG2 cells were cultured in folate-deficient [0 nM (6S) 5-formyltetrahydrofolate] or folate-sufficient [25 nM (6S) 5-formyltetrahydrofolate] medium. Purine biosynthesis was quantified as the ratio of [(14)C]-formate to [(3)H]-hypoxanthine incorporated into genomic DNA, which indicates the contribution of the de novo purine synthesis pathway relative to salvage synthesis. dTMP synthesis was quantified as the ratio of [(14)C]-deoxyuridine to [(3)H]-thymidine incorporation into genomic DNA, which indicates the capacity of de novo dTMP synthesis relative to salvage synthesis.Results: The [(14)C]-formate-to-[(3)H]-hypoxanthine ratio was greater in ADH5 knockout than in wild-type HepG2 cells, under conditions of both folate deficiency (+30%; P HepG2 cells, indicating decreased use of exogenous formate, or increased endogenous formate synthesis, for de novo purine biosynthesis.Conclusions: In HepG2 cells, ADH5 is a source of formate for de novo purine biosynthesis, especially during folate deficiency when folate-dependent formate production is limited. Formate is also shown to be limiting in the growth of HepG2 cells. © 2017 American Society for Nutrition.

  14. Relationship of HepG2 cell sensitivity to continuous low dose-rate irradiation with ATM phosphorylation

    Institute of Scientific and Technical Information of China (English)

    Quelin Mei; Jianyong Yang; Duanming Du; Zaizhong Cheng; Pengcheng liu

    2008-01-01

    Objective: To investigate the change of ATM phosphorylation in HepG2 cells and its effect on HepG2 cell survival under a continuous low dose-rate irradiation.Methods: HepG2 cells were exposed to equivalent doses of irradiation delivered at either a continuous low dose-rate (7.76 cGy/h) or a high dose-rate (4500 cGy/h).The ATM phosphorylated proteins and surviving fraction of HepG2 cell after low dose-rate irradiation were compared with that after equivalent doses of high dose-rate irradiation.Results: The phosphorylation of ATM protein was maximal at 0.5 Gy irradiation delivered at either a high dose-rate or a continuous low dose-rate.As the radiation dose increased, the phosphorylation of ATM protein decreased under continuous low dose-rate irradiation.However, the phosphorylation of ATM protein was remained stable under high dose-rate irradiation.When the phosphorylation of ATM protein under continuous low dose-rate irradiation was equal to that under high dose-rate irradiation, there was no significant difference in the surviving fraction of HepG2 cells between two ir-radiation methods (P>0.05).When the phosphorylation of ATM protein significantly decreased after continuous low dose-rate irradiation compared with that after high dose-rate irradiation, increased amounts of cell killing was found in low dose-rate irradiation (P<0.01).Conclusion: Continuous low dose-rate irradiation increases HepG2 cells radiosensitivity compared with high dose-rate irradiation.The increased amounts of cell killing following continuous low dose-rate exposures are associated with reduced ATM phosphorylated protein.

  15. Heterologous expression of human cytochrome P450 2E1 in HepG2 cell line

    Institute of Scientific and Technical Information of China (English)

    Jian Zhuge; Ye Luo; Ying-Nian Yu

    2003-01-01

    AIM: Human cytochrome P-450 2E1 (CYP2E1) takes part in the biotransformation of ethanol, acetone, many smallmolecule substrates and volatile anesthetics. CYP2E1 is involved in chemical activation of many carcinogens,procarcinogens, and toxicants. To assess the metabolic and toxicological characteristics of CYP2E1, we cloned CYP2E1 cDNA and established a HepG2 cell line stably expressing recombinant CYP 2E1.METHODS: Human CYP2E1 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR)from total RNAs extracted from human liver and cloned into pGEM-T vector. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP2E1 to HepG2 cells. The expression of CYP2E1 mRNA was validated by RT-PCR. The enzyme activity of CYP2E1 catalyzing oxidation of 4-nitrophenol in postmitochondrial supernate (S9) fraction of the cells was determined by spectrophotometry. The metabolic activation of HepG2-CYP2E1 cells was assayed by N-nitrosodiethylamine (NDEA)cytotoxicity and micronucleus test.RESULTS: The cloned CYP2E1 cDNA segment was identical to that reported by Umeno et al(GenBank access No.J02843). HepG2-CYP2E1 cells expressed CYP2E1 mRNA and had 4-nitrophenol hydroxylase activity (0.162±0.025nmol.min-1.mg-1 S9 protein), which were undetectable in parent HepG2 cells. HepG2-CYP2E1 cells increased the cytotoxicity and micronucleus rate of NDEA in comparison with those of HepG2 cells.CONCLUSION: The cDNA of human CYP2E1 can be successfully cloned, and a cell line, HepG2-CYP2E1, which can efficiently express mRNA and has CYP2E1 activity, is established. The cell line is useful for testing the cytotoxicity,mutagenicity and metabolism of xenobiotics, which may possibly be activated or metabolized by CYP2E1.

  16. 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

    , an increase in nuclear size and granularity was observed in the G1 and S phases of HL-60 treated and HepG2-treated cells. Apoptosis was also stimulated by resveratrol in a concentration-dependent manner in HL-60 and HepG2 cells. In conclusion, resveratrol inhibits cell proliferation in a concentration......- and time-dependent manner by interfering with different stages of the cell cycle. Furthermore, resveratrol treatment causes stimulation of apoptosis as well as an increase in nuclear size and granularity....

  17. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  19. 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.

  20. Characterization of dengue virus entry into HepG2 cells

    Directory of Open Access Journals (Sweden)

    Suksanpaisan Lukkana

    2009-02-01

    Full Text Available Abstract Background Despite infections by the dengue virus being a significant problem in tropical and sub-tropical countries, the mechanism by which the dengue virus enters into mammalian cells remains poorly described. Methods A combination of biochemical inhibition, dominant negative transfection of Eps15 and siRNA mediated gene silencing was used to explore the entry mechanism of dengue into HepG2 cells. Results Results were consistent with entry via multiple pathways, specifically via clathrin coated pit mediated endocytosis and macropinocytosis, with clathrin mediated endocytosis being the predominant pathway. Conclusion We propose that entry of the dengue virus to mammalian cells can occur by multiple pathways, and this opens the possibility of the virus being directed to multiple cellular compartments. This would have significant implications in understanding the interaction of the dengue virus with the host cell machinery.

  1. Retroendocytosis of high density lipoproteins by the human hepatoma cell line, HepG2

    Energy Technology Data Exchange (ETDEWEB)

    Kambouris, A.M.; Roach, P.D.; Calvert, G.D.; Nestel, P.J. (CSIRO, Division of Human Nutrition, Adelaide (Australia))

    1990-07-01

    When human HepG2 hepatoma cells were pulsed with 125I-labeled high density lipoproteins (HDL) and chased in fresh medium, up to 65% of the radioactivity released was precipitable with trichloroacetic acid. Cell-internalized 125I-HDL contributed to the release of acid-precipitable material; when cells were treated with trypsin before the chase to remove 125I-HDL bound to the outer cell membrane, 50% of the released material was still acid-precipitable. Characterization of the radioactive material resecreted by trypsinized cells revealed the presence of particles that were similar in size and density to mature HDL and contained intact apolipoproteins (apo) A-I and A-II. The release of internalized label occurred at 37 degrees C but not at 4 degrees C. Monensin, which inhibits endosomal recycling of receptors, decreased the binding of 125I-HDL to cells by 75%, inhibited the release of internalized radioactivity as acid-precipitable material by 80%, and increased the release of acid-soluble material by 90%. In contrast, the lysosomal inhibitor chloroquine increased the association of 125I-HDL to cells by 25%, inhibited the release of precipitable material by 10%, and inhibited the release of acid-soluble radioactivity by 80%. Pre-incubation with cholesterol caused a 50% increase in the specific binding, internalization, and resecretion of HDL label. Cholesterol affected the release of acid-precipitable label much more (+90%) than that of acid-soluble material (+20%). Taken together, these findings suggest that HepG2 cells can bind, internalize, and resecrete HDL by a retroendocytotic process. Furthermore, the results with cholesterol and monensin indicate that a regulated, recycling, receptor-like molecule is involved in the binding and intracellular routing of HDL.

  2. 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.

  3. Pregnane X receptor protects HepG2 cells from BaP-induced DNA damage.

    Science.gov (United States)

    Naspinski, Christine; Gu, Xinsheng; Zhou, Guo-Dong; Mertens-Talcott, Susanne U; Donnelly, Kirby C; Tian, Yanan

    2008-07-01

    Pregnane X receptor (PXR) is a nuclear receptor that coordinately regulates transcriptional expression of both phase I and phase II metabolizing enzymes. PXR plays an important role in the pharmacokinetics of a broad spectrum of endogenous and xenobiotic compounds and appears to have evolved in part to protect organisms from toxic xenobiotics. Metabolism of benzo[a]pyrene (BaP), a well-established carcinogen and ubiquitous environmental contaminant, can result in either detoxification or bioactivation to its genotoxic forms. Therefore, PXR could modulate the genotoxicity of BaP by changing the balance of the metabolic pathways in favor of BaP detoxification. To examine the role of PXR in BaP genotoxicity, BaP-DNA adduct formation was measured by 32P-postlabeling in BaP-treated parental HepG2 cells and human PXR-transfected HepG2 cells. The presence of transfected PXR significantly reduced the level of adducts relative to parental cells by 50-65% (p BaP. To analyze potential PXR-regulated detoxification pathways in liver cells, a panel of genes involved in phase I and phase II metabolism and excretion was surveyed with real-time quantitative reverse transcription PCR. The messenger RNA levels of CYP1A2, GSTA1, GSTA2, GSTM1, UGT1A6, and BCRP (ABCG2) were significantly higher in cells overexpressing PXR, independent of exposure to BaP. In addition, the total GST enzymatic activity, which favors the metabolic detoxification of BaP, was significantly increased by the presence of PXR (p BaP exposure. Taken together, these results suggest that PXR plays an important role in protection against DNA damage by polycyclic aromatic hydrocarbons (PAHs) such as BaP, and that these protective effects may be through a coordinated regulation of genes involved in xenobiotic metabolism.

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

    Science.gov (United States)

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

    2011-02-01

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

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

    Science.gov (United States)

    Luo, Yi; Rana, Payal; Will, Yvonne

    2012-06-01

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

  6. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Barillet, Sabrina; Jugan, Mary-Line; Simon-Deckers, Angelique; Carriere, Marie [Laboratoire Pierre Suee, CEA-CNRS UMR9956, IRAMIS, CEA Saclay, 91191 Gif sur Yvette (France)], E-mail: marie.carriere@cea.fr; Leconte, Yann; Herlin-Boime, Nathalie; Mayne-l' Hermite, Martine; Reynaud, Cecile [Laboratoire Francis Perrin, CEA-CNRS URA2453, IRAMIS, CEA Saclay, 91191 Gif sur Yvette (France)

    2009-05-01

    While emerging nanotechnologies have seen significant development in recent years, knowledge on exposure levels as well as data on toxicity of nanoparticles are still quite limited. Indeed, there is a general agreement that development of nanotechnologies may lead to considerable dissemination of nanoparticles in the environment. Nevertheless, questions relative to toxicity versus innocuousness of such materials still remain. Our present study has thus been carried out with the purpose of assessing some aspects of toxicological capacities of three kinds of nano-sized particles: TiO{sub 2} and SiC nanoparticles, as well as multi-walled carbon nanotubes (CNT). In order to address the question of their potential toxicity toward living cells, we chose several cellular models. Assuming inhalation as the most probable exposure scenario, we used A549 alveolar epithelial cells as a model for mammalian primary target organ (lung). Furthermore, we considered that nanoparticles that would deposit into the pulmonary system may be translocated to the circulatory system. Thus, we decided to study the effect of nanoparticles on potentially secondary target organs: liver (WIF-B9, Can-10, HepG2) and kidneys (NRK-52E, LLC-PK1). Herein, we will focus our attention on results obtained on the HepG2 cell line exposed to SiC nanoparticles. Scarce literature exists on SiC nanotoxicology. According to the authors that have already carried out studies on this particular nanoparticle, it would seem that SiC nanoparticles do not induce cytotoxicity. That is one of the reasons of the potential use of these nanoparticles as biological labels [1]. We thus were interested in acquiring more data on biological effects induced by SiC nanoparticles. Furthermore, one of the particular aspects of the present study lies in the fact that we tried to specify the influence of physico-chemical characteristics of nanoparticles on toxicological endpoints (cytotoxicity and genotoxicity)

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

    Science.gov (United States)

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

    2015-04-10

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

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

    Science.gov (United States)

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

    2015-01-01

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

  10. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  12. Selection of scFvs specific for the HepG2 cell line using ribosome display

    Indian Academy of Sciences (India)

    Lei Zhou; Wei-Ping Mao; Juan Fen; Hong-Yun Liu; Chuan-Jing Wei; Wen-Xiu Li; Feng-Yun Zhou

    2009-06-01

    The aim of this study was to construct a ribosome display library of single chain variable fragments (scFvs) associated with hepatocarcinoma and screen such a library for hepatocarcinoma-binding scFvs. mRNA was isolated from the spleens of mice immunized with hepatocellular carcinoma cell line HepG2. Heavy and k chain genes (VH and k) were amplified separately by RT-PCR, and an anti-HepG2 VH/k chain ribosome display library was constructed by assembling VH and k into the VH/k chain with a specially constructed linker by SOE-PCR. The VH/k chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. In order to isolate specific scFvs, recognizing HepG2 negative selection on a normal hepatocyte line WRL-68 was carried out before three rounds of positive selection on HepG2. After three rounds of panning, cell enzyme-linked immunosorbent assay (ELISA) showed that one of the scFvs had high affinity for the HepG2 cell and lower affinity for the WRL-68 cell. In this study, we successfully constructed a native ribosome display library. Such a library would prove useful for direct intact cell panning using ribosome display technology. The selected scFv had a potential value for hepatocarcinoma treatment.

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

    Science.gov (United States)

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

    2016-08-25

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

  14. 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) ...

  15. Citreoviridin Induces Autophagy-Dependent Apoptosis through Lysosomal-Mitochondrial Axis in Human Liver HepG2 Cells.

    Science.gov (United States)

    Wang, Yuexia; Liu, Yanan; Liu, Xiaofang; Jiang, Liping; Yang, Guang; Sun, Xiance; Geng, Chengyan; Li, Qiujuan; Yao, Xiaofeng; Chen, Min

    2015-08-06

    Citreoviridin (CIT) is a mycotoxin derived from fungal species in moldy cereals. In our previous study, we reported that CIT stimulated autophagosome formation in human liver HepG2 cells. Here, we aimed to explore the relationship of autophagy with lysosomal membrane permeabilization and apoptosis in CIT-treated cells. Our data showed that CIT increased the expression of LC3-II, an autophagosome biomarker, from the early stage of treatment (6 h). After treatment with CIT for 12 h, lysosomal membrane permeabilization occurred, followed by the release of cathepsin D in HepG2 cells. Inhibition of autophagosome formation with siRNA against Atg5 attenuated CIT-induced lysosomal membrane permeabilization. In addition, CIT induced collapse of mitochondrial transmembrane potential as assessed by JC-1 staining. Furthermore, caspase-3 activity assay showed that CIT induced apoptosis in HepG2 cells. Inhibition of autophagosome formation attenuated CIT-induced apoptosis, indicating that CIT-induced apoptosis was autophagy-dependent. Cathepsin D inhibitor, pepstatin A, relieved CIT-induced apoptosis as well, suggesting the involvement of the lysosomal-mitochondrial axis in CIT-induced apoptosis. Taken together, our data demonstrated that CIT induced autophagy-dependent apoptosis through the lysosomal-mitochondrial axis in HepG2 cells. The study thus provides essential mechanistic insight, and suggests clues for the effective management and treatment of CIT-related diseases.

  16. 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...

  17. Estrogen receptor alpha augments changes in hemostatic gene expression in HepG2 cells treated with estradiol and phytoestrogens.

    Science.gov (United States)

    Kelly, Lynne A; Seidlova-Wuttke, Dana; Wuttke, Wolfgang; O'Leary, John J; Norris, Lucy A

    2014-01-15

    Phytoestrogens are popular alternatives to estrogen therapy however their effects on hemostasis in post-menopausal women are unknown. The aim of this study was to determine the effect of the phytoestrogens, genistein, daidzein and equol on the expression of key genes from the hemostatic system in human hepatocyte cell models and to determine the role of estrogen receptors in mediating any response seen. HepG2 cells and Hep89 cells (expressing estrogen receptor alpha (ERα)) were incubated for 24 h with 50 nM 17β-estradiol, genistein, daidzein or equol. Tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), Factor VII, fibrinogen γ, protein C and protein S mRNA expression were determined using TaqMan PCR. Genistein and equol increased tPA and PAI-1 expression in Hep89 cells with fold changes greater than those observed for estradiol. In HepG2 cells (which do not express ERα), PAI-1 and tPA expression were unchanged. Increased expression of Factor VII was observed in phytoestrogen treated Hep89 cells but not in similarly treated HepG2s. Prothrombin gene expression was increased in equol and daidzein treated HepG2 cells in the absence of the classical estrogen receptors. These data suggest that phytoestrogens can regulate the expression of coagulation and fibrinolytic genes in a human hepatocyte cell line; an effect which is augmented by ERα.

  18. Anticancer Effect of Nemopilema nomurai Jellyfish Venom on HepG2 Cells and a Tumor Xenograft Animal Model

    Directory of Open Access Journals (Sweden)

    Hyunkyoung Lee

    2017-01-01

    Full Text Available Various kinds of animal venoms and their components have been widely studied for potential therapeutic applications. This study evaluated whether Nemopilema nomurai jellyfish venom (NnV has anticancer activity. NnV strongly induced cytotoxicity of HepG2 cells through apoptotic cell death, as demonstrated by alterations of chromatic morphology, activation of procaspase-3, and an increase in the Bax/Bcl-2 ratio. Furthermore, NnV inhibited the phosphorylation of PI3K, PDK1, Akt, mTOR, p70S6K, and 4EBP1, whereas it enhanced the expression of p-PTEN. Interestingly, NnV also inactivated the negative feedback loops associated with Akt activation, as demonstrated by downregulation of Akt at Ser473 and mTOR at Ser2481. The anticancer effect of NnV was significant in a HepG2 xenograft mouse model, with no obvious toxicity. HepG2 cell death by NnV was inhibited by tetracycline, metalloprotease inhibitor, suggesting that metalloprotease component in NnV is closely related to the anticancer effects. This study demonstrates, for the first time, that NnV exerts highly selective cytotoxicity in HepG2 cells via dual inhibition of the Akt and mTOR signaling pathways, but not in normal cells.

  19. Effect of baicalin-copper on the induction of apoptosis in human hepatoblastoma cancer HepG2 cells.

    Science.gov (United States)

    Li, Xiaoli; Zou, Kaili; Gou, Jing; Du, Qin; Li, Dejuan; He, Xiaoyan; Li, Zhubo

    2015-03-01

    The medical properties of baicalin have been well known for many years. However, the discovery that baicalin in the presence of metal ions is more effective than baicalin alone changed the course of drug research. The present study was designed to investigate the effect and possible mechanism of apoptosis induced by baicalin-copper in a human hepatoblastoma cancer cell line (HepG2) and in vivo. This study demonstrated that baicalin-copper suppresses the proliferation of HepG2 cells in a dose-dependent manner. Intraperitoneal injection of baicalin-copper resulted in a significant decrease in tumor growth in xenografts in nude mice. Acridine orange staining and flow cytometry analysis demonstrated that baicalin-copper induced apoptosis in HepG2 cells and caused cells to arrest in G2-M phase of the cell cycle. Furthermore, baicalin-copper treatment significantly increased the Bax/Bcl-2 ratio and p38 levels, as well as decreased the expression of caspase-3, p-PI3K, p-Akt and p-mTOR (P copper induces apoptosis in HepG2 cells by down-regulating the PI3K/Akt/mTOR signaling pathway.

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

    Science.gov (United States)

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

    2006-04-15

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

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

    Science.gov (United States)

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

  2. Xanthine oxidase inhibition by allopurinol increases in vitro pyrazinamide-induced hepatotoxicity in HepG2 cells.

    NARCIS (Netherlands)

    Tostmann, A.; Aarnoutse, R.E.; Peters, W.H.M.; Richard, P.N.; Boeree, M.J.

    2010-01-01

    Despite the important role of pyrazinamide in tuberculosis treatment, little is known about the mechanism of pyrazinamide-induced hepatotoxicity. We inhibited xanthine oxidase in HepG2 cells by using a nontoxic concentration of allopurinol, a well-known xanthine-oxidase inhibitor. This increased in

  3. 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...

  4. Oncostatin M regulates membrane traffic and stimulates bile canalicular membrane biogenesis in HepG2 cells

    NARCIS (Netherlands)

    Van der Wouden, Johanna M.; Van IJzendoorn, Sven C.D.; Hoekstra, Dick

    2002-01-01

    Hepatocytes are the major epithelial cells of the liver and they display membrane polarity: the sinusoidal membrane representing the basolateral surface, while the bile canalicular membrane is typical of the apical membrane. In polarized HepG2 cells an endosomal organelle, SAC, fulfills a prominent

  5. The binding of human lipoprotein lipase treated VLDL by the human hepatoma cell line HepG2

    NARCIS (Netherlands)

    Mulder, M.; Wit, E.de; Havekes, L.M.

    1991-01-01

    It has been suggested that besides the LDL-receptor, hepatocytes possess an apo E or remnant receptor. To evaluate which hepatic lipoprotein receptor is involved in VLDL remnant catabolism, we studied the binding of VLDL remnants to HepG2 cells. Native VLDL was obtained from type IIb hyperlipidemic

  6. 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) ...

  7. Zebularine upregulates expression of CYP genes through inhibition of DNMT1 and PKR in HepG2 cells

    Science.gov (United States)

    Nakamura, Kazuaki; Aizawa, Kazuko; Aung, Kyaw Htet; Yamauchi, Junji; Tanoue, Akito

    2017-01-01

    Drug-induced hepatotoxicity is one of the major reasons cited for drug withdrawal. Therefore, it is of extreme importance to detect human hepatotoxic candidates as early as possible during the drug development process. In this study, we aimed to enhance hepatocyte functions such as CYP gene expression in HepG2 cells, one of the most extensively used cell lines in evaluating hepatotoxicity of chemicals and drugs. We found that zebularine, a potent inhibitor of DNA methylation, remarkably upregulates the expression of CYP genes in HepG2 cells. In addition, we revealed that the upregulation of CYP gene expression by zebularine was mediated through the inhibition of both DNA methyltransferase 1 (DNMT1) and double-stranded RNA-dependent protein kinase (PKR). Furthermore, HepG2 cells treated with zebularine were more sensitive than control cells to drug toxicity. Taken together, our results show that zebularine may make HepG2 cells high-functioning and thus could be useful for evaluating the hepatotoxicity of chemicals and drugs speedily and accurately in in-vitro systems. The finding that zebularine upregulates CYP gene expression through DNMT1 and PKR modulation sheds light on the mechanisms controlling hepatocyte function and thus may aid in the development of new in-vitro systems using high-functioning hepatocytes. PMID:28112215

  8. 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.

  9. An autophagic process is activated in HepG2 cells to mediate BDE-100-induced toxicity.

    Science.gov (United States)

    Pereira, Lilian Cristina; Duarte, Filipe Valente; Varela, Ana Teresa Inácio Ferreira; Rolo, Anabela Pinto; Palmeira, Carlos Manuel Marques; Dorta, Daniel Junqueira

    2017-02-01

    To reduce flammability and meet regulatory requirements, Brominated Flame Retardants (BFRs) are added to a wide variety of consumer products including furniture, textiles, electronics, and construction materials. Exposure to polybrominated phenyl ethers (PBDEs) adversely affects the human health. Bearing in mind that (i) PBDEs are potentially toxic, (ii) the mechanism of PBDE toxicity is unclear, and (iii) the importance of the autophagy to the field of toxicology is overlooked, this study investigates whether an autophagic process is activated in HepG2 cells (human hepatoblastoma cell line) to mediate BDE-100-induced toxicity. HepG2 cells were exposed with BDE-100 at three concentrations (0.1, 5, and 25μM), selected from preliminary toxicity tests, for 24 and 48h. To assess autophagy, immunocytochemistry was performed after exposure of HepG2 cells to BDE-100. Labeling of HepG2 cells with 100nM LysoTracker Red DND-99 aided examination of lysosome distribution. Proteins that are key to the autophagic process (p62 and LC3) were evaluated by western blotting. DNA was isolated and quantified to assess mitochondrial DNA copy number by qPCR on the basis of the number of DNA copies of a mitochondrial encoded gene normalized against a nuclear encoded gene. Conversion of LC3-I to LC3-II increased in HepG2 cells. Pre-addition of 100nM wortmannin decreased the amount of LC3 in the punctuate form and increased nuclear fragmentation (apoptotic feature). HepG2 cells exposed to BDE-100 presented increased staining with the lysosomal dye and had larger LC3 and p62 content after pre-treatment with ammonium chloride. The mitochondrial DNA copy number decreased, which probably constituted an attempt of the cell to manage mitochondrial damage by selective mitochondrial degradation (mitophagy). In conclusion, an autophagic process is activated in HepG2 cells to mediate BDE-100-induced toxicity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. Using the stable HSPA1A promoter-driven luciferase reporter HepG2 cells to assess the overall toxicity of coke oven emissions

    Institute of Scientific and Technical Information of China (English)

    信丽丽

    2013-01-01

    Objective Using the stable HSPA1A(HSP70-1) promoter-driven luciferase reporter HepG2 cells(HepG2/HSPA1A cells) to assess the overall toxicity of coke oven emissions. Methods The stable HepG2/HSPA1A cells were treated with different concentrations of coke oven

  11. Development and validation of a high-content screening in vitro micronucleus assay in CHO-k1 and HepG2 cells

    NARCIS (Netherlands)

    Westerink, W.M.; Schirris, T.J.J.; Horbach, G.J.; Schoonen, W.G.

    2011-01-01

    In the present study an automated image analysis assisted in vitro micronucleus assay was developed with the rodent cell line CHO-k1 and the human hepatoma cell line HepG2, which are both commonly used in regulatory genotoxicity assays. The HepG2 cell line was chosen because of the presence in these

  12. Inhibition of MEK/ERK activation attenuates autophagy and potentiates pemetrexed-induced activity against HepG2 hepatocellular carcinoma cells.

    Science.gov (United States)

    Tong, Yongxi; Huang, Haijun; Pan, Hongying

    2015-01-02

    Identification of efficient chemo-therapeutic/chemo-preventive agents for treatment of hepatocellular carcinoma (HCC) is important. In this study, we examined the activity of pemetrexed, an anti-folate chemotherapy drug, against HepG2 human HCC cells. Pemetrexed treatment in vitro exerted weak but significant cytotoxic activity against HepG2 cells. When analyzing the possible pemetrexed-resistance factors, we indentified that pemetrexed treatment in HepG2 cells induced cyto-protective autophagy activation, evidenced by GFP-light chain 3B (LC3B) puncta formation, p62 downregulation and Beclin-1/LC3B-II upregulation. Correspondingly, autophagy inhibitors, including bafliomycin A1, 3-methyladenine and chloroquine, enhanced pemetrexed-induced cytotoxicity against HepG2 cells. Further, RNAi-mediated knockdown of Beclin-1 in HepG2 cells also increased pemetrexed sensitivity. Pemetrexed activated MEK (mitogen-activated protein kinase/ERK kinase)/ERK (extracellular-signal-regulated kinase) signaling in HepG2 cells, which was required for autophagy induction. Pharmacological inhibition of MEK/ERK activation attenuated pemetrexed-induced autophagy, enhanced HepG2 cell death and apoptosis. In summary, pemetrexed activates MEK/ERK-dependent cyto-protective autophagy, and inhibition of this pathway potentiates pemetrexed's activity in HepG2 cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Development and validation of a high-content screening in vitro micronucleus assay in CHO-k1 and HepG2 cells

    NARCIS (Netherlands)

    Westerink, W.M.; Schirris, T.J.J.; Horbach, G.J.; Schoonen, W.G.

    2011-01-01

    In the present study an automated image analysis assisted in vitro micronucleus assay was developed with the rodent cell line CHO-k1 and the human hepatoma cell line HepG2, which are both commonly used in regulatory genotoxicity assays. The HepG2 cell line was chosen because of the presence in these

  14. 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.

  15. Investigation of the anti-cancer effect of quercetin on HepG2 cells in vivo

    Science.gov (United States)

    Li, Lin; Yao, Wenxiu; Xiong, Zhujuan; Zhou, Xiang

    2017-01-01

    Quercetin, a natural polyphenolic flavonoid compound, can inhibit the growth of several malignant cancers. However, the mechanism still remains unclear. Our previous findings have suggested that quercetin can significantly inhibit HepG2 cell proliferation and induce cell apoptosis in vitro. It can also affect cell cycle distribution and significantly decrease cyclin D1 expression. In this study, we investigated the anti-cancer effect of quercetin on HepG2 tumor-bearing nude mice and its effect on cyclin D1 expression in the tumor tissue. First, the nude murine tumor model was established by subcutaneous inoculation of HepG2 cells, then quercetin was administered intraperitoneally, and the mice injected with saline solution were used as controls. The daily behavior of the tumor-bearing mice was observed and differences in tumor growth and survival rate were monitored. The expression of cyclin D1 in isolated tumor sections was evaluated by immunohistochemistry. We found that HepG2 tumor became palpable in the mice one-week post-inoculation. Tumors in the control group grew rapidly and the daily behavior of the mice changed significantly, including listlessness, poor feeding and ataxia. The mice in quercetin-treated group showed delayed tumor growth, no significant changes in daily behavior, and the survival rate was significantly improved. Finally, we observed increased tumor necrosis and a lighter cyclin D1 staining with reduced staining areas. Our findings thus suggest that quercetin can significantly inhibit HepG2 cell proliferation, and this effect may be achieved through the regulation of cyclin D1 expression. PMID:28264020

  16. GRK2 negatively regulates IGF-1R signaling pathway and cyclins' expression in HepG2 cells.

    Science.gov (United States)

    Wei, Zhengyu; Hurtt, Reginald; Gu, Tina; Bodzin, Adam S; Koch, Walter J; Doria, Cataldo

    2013-09-01

    G protein coupled receptor kinase 2 (GRK2) plays a central role in the regulation of a variety of important signaling pathways. Alternation of GRK2 protein level and activity casts profound effects on cell physiological functions and causes diseases such as heart failure, rheumatoid arthritis, and obesity. We have previously reported that overexpression of GRK2 has an inhibitory role in cancer cell growth. To further examine the role of GRK2 in cancer, in this study, we investigated the effects of reduced protein level of GRK2 on insulin-like growth factor 1 receptor (IGF-1R) signaling pathway in human hepatocellular carcinoma (HCC) HepG2 cells. We created a GRK2 knockdown cell line using a lentiviral vector mediated expression of GRK2 specific short hairpin RNA (shRNA). Under IGF-1 stimulation, HepG2 cells with reduced level of GRK2 showed elevated total IGF-1R protein expression as well as tyrosine phosphorylation of receptor. In addition, HepG2 cells with reduced level of GRK2 also demonstrated increased tyrosine phosphorylation of IRS1 at the residue 612 and increased phosphorylation of Akt, indicating a stronger activation of IGF-1R signaling pathway. However, HepG2 cells with reduced level of GRK2 did not display any growth advantage in culture as compared with the scramble control cells. We further detected that reduced level of GRK2 induced a small cell cycle arrest at G2/M phase by enhancing the expression of cyclin A, B1, and E. Our results indicate that GRK2 has contrasting roles on HepG2 cell growth by negatively regulating the IGF-1R signaling pathway and cyclins' expression.

  17. 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.

  18. 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.

  19. 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. • The galactosylated functionalized PCL scaffold is a potential candidate for liver tissue engineering.

  20. Cytotoxic effect of Agaricus bisporus and Lactarius rufus β-D-glucans on HepG2 cells.

    Science.gov (United States)

    Pires, Amanda do Rocio Andrade; Ruthes, Andrea Caroline; Cadena, Silvia Maria Suter Correia; Acco, Alexandra; Gorin, Philip Albert James; Iacomini, Marcello

    2013-07-01

    The cytotoxic activity of β-D-glucans isolated from Agaricus bisporus and Lactarius rufus fruiting bodies was evaluated on human hepatocellular carcinoma cells (HepG2). NMR and methylation analysis suggest that these β-d-glucans were composed of a linear (1→6)-linked and a branched (1→3), (1→6)-linked backbone, respectively. They both decreased cell viability at concentrations of up to 100 μg mL(-1), as shown by MTT assay. The amount of LDH released and the analysis of cell morphology corroborated these values and also showed that the β-D-glucan of L. rufus was more cytotoxic to HepG2 cells than that of A. bisporus. The treatment of HepG2 cells with L. rufus and A. bisporus β-D-glucans at a dose of 200 μg mL(-1) for 24h promoted an increase of cytochrome c release and a decrease of ATP content, suggesting that these polysaccharides could promote cell death by apoptosis. Both β-D-glucans were tested against murine primary hepatocytes at a dose of 200 μg mL(-1). The results suggest that the L. rufus β-d-glucan was as cytotoxic for hepatocytes as for HepG2 cells, whereas the A. bisporus β-D-glucan, under the same conditions, was cytotoxic only for HepG2 cells, suggesting cell selectivity. These results open new possibilities for use of mushroom β-D-glucans in cancer therapy.

  1. ssDNA Aptamer Specifically Targets and Selectively Delivers Cytotoxic Drug Doxorubicin to HepG2 Cells.

    Directory of Open Access Journals (Sweden)

    Ge Yu

    Full Text Available Hepatocellular carcinoma (HCC is the third leading cause of death due to cancer worldwide with over 500,000 people affected annually. Although chemotherapy has been widely used to treat patients with HCC, alternate modalities to specifically deliver therapeutic cargos to cancer cells have been sought in recent years due to the severe side effects of chemotherapy. In this respect, aptamer-based tumor targeted drug delivery has emerged as a promising approach to increase the efficacy of chemotherapy and reduce or eliminate drug toxicity. In this study, we developed a new HepG2-specific aptamer (HCA#3 by a procedure known as systematic evolution of ligands by exponential enrichment (SELEX and exploited its role as a targeting ligand to deliver doxorubicin (Dox to HepG2 cells in vitro. The selected 76-base nucleotide aptamer preferentially bound to HepG2 hepatocellular carcinoma cells but not to control cells. The aptamer HCA#3 was modified with paired CG repeats at the 5'-end to carry and deliver a high payload of intercalated Dox molecules at the CG sites. Four Dox molecules (mol/mol were fully intercalated in each conjugate aptamer-Dox (ApDC molecule. Biostability analysis showed that the ApDC molecules are stable in serum. Functional analysis showed that ApDC specifically targeted and released Dox within HepG2 cells but not in control cells, and treatment with HCA#3 ApDC induced HepG2 cell apoptosis but had minimal effect on control cells. Our study demonstrated that HCA#3 ApDC is a promising aptamer-targeted therapeutic that can specifically deliver and release a high doxorubicin payload in HCC cells.

  2. Recombinant production of native human α-1-antitrypsin protein in the liver HepG2 cells.

    Science.gov (United States)

    Jaberie, Hajar; Naghibalhossaini, Fakhraddin

    2016-10-01

    Alpha-1 antitrypsin (A1AT) deficiency is associated with emphysema and liver disease. Only plasma-derived A1AT protein is available for augmentation therapy. Recombinant A1AT (recA1AT) protein expressed in various types of available hosts are either non-glycosylated or aberrantly glycosylated resulting into reduced stability and biological activity. To overcome these limitations, we have used the human liver HepG2 cell line to produce recA1AT protein. HepG2 cells were transfected by A1AT cDNA and cell populations were generated that stably overexpressed A1AT protein. Real-time RT-PCR and rocket immunoelectrophoresis of cell culture supernatants indicated that the transfection resulted more than two-fold increase in A1AT production compared to that of control parental cells. Immunoblot analysis showed that both plasma and HepG2-produced A1AT proteins have identical molecular weight in either glycosylated or deglycosylated form. Partial digestion with PNGase F indicated that the three N-glycosylation sites of recA1AT, like the native A1AT protein in plasma, are occupied. Recombinant A1AT also like the native A1AT was thermostable and could efficiently inhibit trypsin proteolytic activity against BSA and BAPNA chromogenic substrate. The recombinant HepG2 cells cultured in media containing B27 serum free supplement released recA1AT at the same level as in the serum containing media. RecA1AT production in HepG2 cells grown under serum free condition at a large scale could provide a reliable source of the native protein suitable for therapeutic use in human.

  3. Cytotoxicity of various chemicals and mycotoxins in fresh primary duck embryonic fibroblasts: a comparison to HepG2 cells.

    Science.gov (United States)

    Chen, Xi; Murdoch, Rhonda; Shafer, Daniel J; Ajuwon, Kolapo M; Applegate, Todd J

    2016-11-01

    To screen cost-effectively the overall toxicity of a sample, particularly in the case of food and feed ingredient quality control, a sensitive bioassay is necessary. With the wide variety of cytotoxicity assays, performance comparison between assays using different cells has become of interest. Fresh primary duck embryonic fibroblasts (DEF) were hypothesized to be a sensitive tool for in vitro cytotoxicity screening; cell viability of DEF in response to various cytotoxins was determined and compared with response of HepG2 cells. The IC50 values by the alamar blue assay in the DEF cells had a high correlation (R(2)  = 0.96) with those obtained in HepG2 cells. Within the same toxin, primary DEF yielded significantly lower IC50 values than that obtained from HepG2 cells using the MTT and alamar blue assay. Additionally, primary DEF responded to all mycotoxins tested using the alamar blue assay, while HepG2 was less sensitive, particularly at short exposure times. The estimated IC50 for aflatoxin B1 , fumonisins B1 and deoxynivalenol in DEF after 72 h incubation were 3.69, 4.19 and 1.26 μg ml(-1) , respectively. Results from the current study suggest that primary DEF are more sensitive to cytotoxins and mycotoxins compared to HepG2, and thus may have great potential as an effective tool for cytotoxicity assessment. The question remains whether in vitro IC50 values can accurately predict in vivo toxicity; however, the current study accentuates the need for further attention to identify sensitive cell models for in vitro cytotoxicity screening and subsequent exploration of species-specific prediction models for in vivo toxicity. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  4. 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 palmitate/ CsA induced toxicity. ► Palmitate sensitizes cells to the toxicity induced by CsA at therapeutic exposure. ► Elevated free fatty acids may predispose the patients to CsA-induced toxicity.

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

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    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 secretion of ApoM by protein kinase C but independently of the classical androgen receptor.

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

    Science.gov (United States)

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

    2016-04-13

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

  7. Insulin resistance contributes to multidrug resistance in HepG2 cells via activation of the PERK signaling pathway and upregulation of Bcl-2 and P-gp.

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    Liu, Xinyue; Li, Linjing; Li, Jing; Cheng, Yan; Chen, Jing; Shen, Minghui; Zhang, Shangdi; Wei, Hulai

    2016-05-01

    Liver tumorigenesis frequently causes insulin resistance which may be used as an independent risk factor for evaluation of survival and post-surgery relapse of liver cancer patients. In the present study, HepG2/IR, an insulin resistant HepG2 cell line, was established by exposing HepG2 cells to 0.5 µmol/l of insulin for 72 h, and comparison of HepG2/IR with the parental HepG2 cells indicated that the HepG2/IR cells showed significantly enhanced resistance to the most frequently used chemotherapeutics for solid tumors, such as cisplatin, 5-fluorouracil, vincristine and mitomycin. Flow cytometric analysis of cisplatin-treated HepG2/IR cells showed a significantly decreased hypodiploid peak and a significantly downregulated expression level of pro-apoptotic protein caspase-3 compared with the parental HepG2 cells. Our data further showed swollen endoplasmic reticulum (ER) in the cisplatin-treated HepG2/IR cells with significantly increased levels of glucose-regulated protein 78 (GRP78), phosphorylated protein kinase R-like ER kinase (p-PERK) and P-glycoprotein (P-gp). There was also an upregulated expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) whereas no significant change was observed for CCAAT-enhancer-binding protein homologous protein (CHOP), which is known to be induced by ER stress and to mediate apoptosis. Our results demonstrated that insulin resistance in HepG2 cells promoted a protective unfolded protein response and upregulated the expression of ER chaperone protein GRP78, which resulted in the phosphorylation of PERK kinase to activate the PERK-mediated ER stress signal transduction pathway and the upregulation of Bcl-2 and P-gp, leading to the inhibition of the caspase-3-dependent apoptosis pathway and to the survival of liver tumor cells.

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

    Science.gov (United States)

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

    2016-01-01

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

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

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    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.

  10. 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.

  11. Antioxidant potential of herbs extracts and impact on HepG2 cells viability

    Directory of Open Access Journals (Sweden)

    Anna Gramza-Michałowska

    2008-12-01

    Full Text Available Mercury poisoning is responsible for inducing serious adverse effects in living organisms. One of protection factors could be substances proven to possess high antioxidant and metal chelating activity – plant polyphenols. There are many sources of polyphenols in plant kingdom but the most interesting for food industry could be widely consumed herbs. Aim of the research was to evaluate antioxidative potential of selected plant extracts and its influence on HepG2 cells in different conditions. Ethanolic herbs extracts were characterised by total polyphenol content. Antioxidant activity was estimated with use of DPPH• and ABTS+• radicals scavenging methods and FRAP. Research included cells viability estimation by the MTT assay and cells exposition to HgCl2, chemical agent inducing cell death. Analysis of herbs extracts antioxidative activity showed best potential represented thyme and marjoram, highest FRAP was evaluated in samples with mint and marjoram extracts. On the basis of received results it was found that examined plant extracts showed weak protection against Hg presence in examined cells environment.

  12. Cytotoxicity evaluation of symmetrically branched glycerol trimer in human hepatocellular carcinoma HepG2 cells.

    Science.gov (United States)

    Miyamoto, Licht; Watanabe, Masashi; Kono, Mai; Matsushita, Tsuyoshi; Hattori, Hatsuhiko; Ishizawa, Keisuke; Nemoto, Hisao; Tsuchiya, Koichiro

    2012-01-01

    An appropriate balance between lipophilicity and hydrophilicity is necessary for pharmaceuticals to achieve fine Absorption, Distribution, Metabolism and Excretion (ADME) properties including absorption and distribution, in particular. We have designed and proposed symmetrically branched oligoglycerols (BGL) as an alternative approach to improve the lipophilic-hydrophilic balance. We have previously shown that stability in circulation and water-solubility of such molecules as proteins, liposomes and hydrophobic compounds are much improved by conjugation to BGL. Albeit these successful applications of BGL, little was known whether BGL could be used in safety. Thus we conducted evaluation of the cytotoxicity of a representative BGL, symmetrically branched glycerol trimer (BGL003) in the cultured cells to clarify its biological safeness. Here we demonstrate that water-solubility of an extremely hydrophobic agent, fenofibrate was more than 2,000-fold improved just by conjugated with BGL003. BGL003 did not exhibit any significant cytotoxicity in human hepatocarcinoma HepG2 cells. Thus BGL003 should be safe and suitable strategy to endow hydrophobic molecules with much hydrophilicity.

  13. Bile acids reduce endocytosis of high-density lipoprotein (HDL) in HepG2 cells.

    Science.gov (United States)

    Röhrl, Clemens; Eigner, Karin; Fruhwürth, Stefanie; Stangl, Herbert

    2014-01-01

    High-density lipoprotein (HDL) transports lipids to hepatic cells and the majority of HDL-associated cholesterol is destined for biliary excretion. Cholesterol is excreted into the bile directly or after conversion to bile acids, which are also present in the plasma as they are effectively reabsorbed through the enterohepatic cycle. Here, we provide evidence that bile acids affect HDL endocytosis. Using fluorescent and radiolabeled HDL, we show that HDL endocytosis was reduced in the presence of high concentrations of taurocholate, a natural non-cell-permeable bile acid, in human hepatic HepG2 and HuH7 cells. In contrast, selective cholesteryl-ester (CE) uptake was increased. Taurocholate exerted these effects extracellularly and independently of HDL modification, cell membrane perturbation or blocking of endocytic trafficking. Instead, this reduction of endocytosis and increase in selective uptake was dependent on SR-BI. In addition, cell-permeable bile acids reduced HDL endocytosis by farnesoid X receptor (FXR) activation: chenodeoxycholate and the non-steroidal FXR agonist GW4064 reduced HDL endocytosis, whereas selective CE uptake was unaltered. Reduced HDL endocytosis by FXR activation was independent of SR-BI and was likely mediated by impaired expression of the scavenger receptor cluster of differentiation 36 (CD36). Taken together we have shown that bile acids reduce HDL endocytosis by transcriptional and non-transcriptional mechanisms. Further, we suggest that HDL endocytosis and selective lipid uptake are not necessarily tightly linked to each other.

  14. Does Resveratrol Improve Insulin Signalling in HepG2 Cells?

    Science.gov (United States)

    Norouzzadeh, Marjan; Amiri, Fatemehsadat; Saboor-Yaraghi, Ali Akbar; Shemirani, Farnoosh; Kalikias, Yas; Sharifi, Loghman; Seyyedsalehi, Monireh Sadat; Mahmoudi, Maryam

    2017-04-01

    Diabetes mellitus is a common metabolic disorder with high global prevalence. It is characterized by a decrease in insulin secretion or a decrease in insulin sensitivity or both. The aim of the present study was to investigate the effects of resveratrol treatment on the expression of the genes involved in insulin signalling cascade, such as Forkhead box protein O1 (FoxO1), 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and mammalian target of rapamycin (mTOR). HepG2 cells were cultured in serum-free medium with high concentrations of glucose and insulin and then were treated with resveratrol (5, 10 and 20 µM) for 24 and 48 hours. Complementary deoxyribonucleic acids (cDNAs) were synthesized followed by RNA extraction. Real-time quantitative reverse transcription polymerase chain reaction was used to analyze the expression of FoxO1, PDPK1 and mTOR. Resveratrol increased the expression of PDPK1, mTOR and FoxO1. No significant difference was seen among differing dosages of resveratrol, but treatments for 48 hours exerted the greatest effectiveness. Our results were consistent with other studies showing the beneficial effects of resveratrol on diabetes. However, considering the effects of resveratrol in increasing FoxO1 and gluconeogenic gene expression, long-term usage of resveratrol should be investigated in greater depth in future studies. Copyright © 2016 Canadian Diabetes Association. Published by Elsevier Inc. All rights reserved.

  15. 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.

  16. Ovothiol Isolated from Sea Urchin Oocytes Induces Autophagy in the Hep-G2 Cell Line

    Directory of Open Access Journals (Sweden)

    Gian Luigi Russo

    2014-07-01

    Full Text Available Ovothiols are histidine-derived thiols isolated from sea urchin eggs, where they play a key role in the protection of cells toward the oxidative burst associated with fertilization by controlling the cellular redox balance and recycling oxidized glutathione. In this study, we show that treatment of a human liver carcinoma cell line, Hep-G2, with ovothiol A, isolated from Paracentrotus lividus oocytes, results in a decrease of cell proliferation in a dose-dependent manner. The activation of an autophagic process is revealed by phase contrast and fluorescence microscopy, together with the expression of the specific autophagic molecular markers, LC3 II and Beclin-1. The effect of ovothiol is not due to its antioxidant capacity or to hydrogen peroxide generation. The concentration of ovothiol A in the culture media, as monitored by HPLC analysis, decreased by about 24% within 30 min from treatment. The proliferation of normal human embryonic lung cells is not affected by ovothiol A. These results hint at ovothiol as a promising bioactive molecule from marine organisms able to inhibit cell proliferation in cancer cells.

  17. The toxicity of extracts of plant parts of Moringa stenopetala in HEPG2 cells in vitro.

    Science.gov (United States)

    Mekonnen, Negussu; Houghton, Peter; Timbrell, John

    2005-10-01

    The cytotoxicity of extracts from a widely used species of plant, Moringa stenopetala, was assessed in HEPG2 cells, by measuring the leakage of lactate dehydrogenase (LDH) and cell viability. The functional integrity of extract-exposed cells was determined by measuring intracellular levels of ATP and glutathione (GSH). The ethanol extracts of leaves and seeds increased significantly (p leaf and seed extracts. At a concentration of 500 microg/mL, the water extract of leaves increased (p leaf extract decreased GSH levels at a concentration of 500 microg/mL (p Moringa stenopetala show that they contain toxic substances that are extractable with organic solvents or are formed during the process of extraction with these solvents. The significant depletion of ATP and GSH only occurred at concentrations of extract that caused leakage of LDH. Further investigation with this plant in order to identify the constituents extracted and their individual toxic effects both in vivo and in vitro is warranted. This study also illustrates the utility of cell culture for screening plant extracts for potential toxicity.

  18. The color and size of chili peppers (Capsicum annuum) influence Hep-G2 cell growth.

    Science.gov (United States)

    Popovich, David G; Sia, Sharon Y; Zhang, Wei; Lim, Mon L

    2014-11-01

    Four types of chili (Capsicum annuum) extracts, categorized according to color; green and red, and size; small and large were studied in Hep-G2 cells. Red small (RS) chili had an LC50 value of 0.378 ± 0.029 compared to green big (GB) 1.034 ± 0.061 and green small (GS) 1.070 ± 0.21 mg/mL. Red big (RB) was not cytotoxic. Capsaicin content was highest in RS and produced a greater percentage sub-G1 cells (6.47 ± 1.8%) after 24 h compared to GS (2.96 ± 1.3%) and control (1.29 ± 0.8%) cells. G2/M phase was reduced by GS compared to RS and control cells. RS at the LC50 concentration contained 1.6 times the amount of pure capsaicin LC50 to achieve the same effect of capsaicin alone. GS and GB capsaicin content at the LC50 value was lower (0.2 and 0.66, respectively) compared to the amount of capsaicin to achieve a similar reduction in cell growth.

  19. Anticancer effect of 20(S)-ginsenoside Rh2 on HepG2 liver carcinoma cells: Activating GSK-3β and degrading β-catenin.

    Science.gov (United States)

    Shi, Qingqiang; Shi, Xueping; Zuo, Gei; Xiong, Wei; Li, Haixing; Guo, Pei; Wang, Fen; Chen, Yi; Li, Jing; Chen, Di-Long

    2016-10-01

    20(S)-ginsenoside Rh2 [(S)Rh2] possesses potential to prevent cancer in vitro as well as in vivo, but the underlying mechanism is still unknown. First, we infected HepG2 cells with lentivirus which carries β‑catenin. We detected the pharmacological effects of (S)Rh2 on HepG2 and HepG2‑β‑catenin cells and found that the IC50 of (S)Rh2 exposure on HepG2-β-catenin cells was higher than HepG2 cells. Flow cytometry (FCM) indicated that (S)Rh2 could be arrested in G0/G1 phase and induce early apoptosis in HepG2 and HepG2‑β‑catenin cells. Second, ELISA kit was used to check the activity of glycogen synthase kinase‑3β (GSK‑3β), which was upregulated by (S)Rh2. GSK‑3β inhibitor BIO, was used to verify that (S)Rh2 activated GSK‑3β. PCR and western blotting results indicated that (S)Rh2 could degrade the expression of β‑catenin, which combined with TCF in the nucleus and activate transcription of Wnt target genes, such as Bax, Bcl‑2, cyclin D1, MMP3, which were checked by chromatin immunoprecipitation (ChIP), PCR and western blotting. The results showed that the expression of Bax mRNA and proteins increased, while the cyclin D1, Bcl‑2, MMP3 mRNA and proteins were downregulated in HepG2 and HepG2‑β‑catenin cells which was induced by (S)Rh2. By contrast, with the HepG2-β-catenin + (S)Rh2 group, the expression of other mRNA and proteins in HepG2 + (S)Rh2 group changed significantly. In vivo, experiments were performed using a nude mouse xenograft model to investigate the (S)Rh2 effect. So these results suggested that (S)Rh2 could suppress proliferation, promote apoptosis and inhibit metastasis of HepG2, decrease weight of tumor by downregulating β‑catenin through activating GSK‑3β and the pharmacological effect of (S)Rh2 on HepG2 cells might be weakened by overexpression of β‑catenin.

  20. Differential expression of genes in HepG2 cells caused by UC001kfo RNAi as shown by RNA-seq.

    Science.gov (United States)

    Pan, Yan-Feng; Su, Tong; Chen, Li-Dan; Qin, Tao

    2017-08-01

    The differential expression of genes in HepG2 cells caused by UC001kfo RNAi was investigated using RNA-seq. HepG2 cells were infected by Lenti-shUC001kfo lentivirus particles. The expression of UC001kfo mRNA in the HepG2-shUC001kfo cell line was detected by real-time PCR. RNA-seq technology was used to identify the difference in the expression of genes regulated by lncRNA UC001kfo in the HepG2 cell line. Gene ontology and signaling pathway analysis were performed to reveal the biological functions of the genes encoding of significantly different mRNAs. The results showed that mRNAs were differentially expressed between the HepG2-shUC001kfo cell line and the HepG2 cell line. The UC001kfo mRNA was significantly down-regulated in the stable cell line HepG2-shUC001kfo (PHepG2 cell line after the down-regulation of lncRNA-UC001kfo. Some took part in the extracellular matrix, cell adhesion, motility, growth, and localization. The genes encoding of differentially expressed mRNAs may participate in cell invasion and metastasis.

  1. [PCR analysis of the absolute number of copies of human chromosome 18 transcripts in liver and HepG2 cells].

    Science.gov (United States)

    Kiseleva, Y Y; Ptitsyn, K G; Tikhonova, O V; Radko, S P; Kurbatov, L K; Vakhrushev, I V; Zgoda, V G; Ponomarenko, E A; Lisitsa, A V; Archakov, A I

    2017-03-01

    Using reverse transcription in conjunction with the quantitative real-time PCR or digital droplet PCR, the transcriptome profiling of human chromosome 18 has been carried out in liver hepatocytes and hepatoblastoma cells (HepG2 cell line) in terms of the absolute number of each transcript per cell. The transcript abundance varies within the range of 0.006 to 9635 and 0.011 to 4819 copies per cell for HepG2 cell line and hepatocytes, respectively. The expression profiles for genes of chromosome 18 in hepatocytes and HepG2 cells were found to significantly correlate: the Spearman's correlation coefficient was equal to 0.81. The distribution of frequency of transcripts over their abundance was bimodal for HepG2 cells and unimodal for liver hepatocytes. Bioinformatic analysis of the differential gene expression has revealed that genes of chromosome 18, overexpressed in HepG2 cells compared to hepatocytes, are associated with cell division and cell adhesion processes. It is assumed that the enhanced expression of those genes in HepG2 cells is related to the proliferation activity of cultured cells. The differences in transcriptome profiles have to be taken into account when modelling liver hepatocytes with cultured HepG2 cells.

  2. [CXCR3 monoclonal antibody inhibits the proliferation and migration of MCF-7 cells and HepG2 cells in vitro].

    Science.gov (United States)

    Zhu, Yuqiang; Wang, Zhiyao; Zhu, Ying; Wang, Jing; Cai, Lei; Shen, Hui; Kong, Yong; Qiu, Yuhua

    2015-11-01

    To study the effects of chemokine (C-X-C motif) receptor 3 (CXCR3) monoclonal antibody (mAb) on the proliferation and migration of MCF-7 and HepG2 cells in vitro. Ascites of CXCR3 mAb was prepared at first. MCF-7 and HepG2 cells with high expressions of CXCR3 were screened by flow cytometry. MTT assay was used to detect the effects of CXCR3 mAb on the proliferation of MCF-7 and HepG2 cells in vitro in the absence/presence of interferon-inducible T-cell alpha chemoattractant (I-TAC). Transwell™ assay was performed to investigate the effects of CXCR3 mAb on the migration of MCF-7 and HepG2 cells in vitro in the absence/presence of I-TAC. The expression rate of CXCR3 on MCF-7 and HepG2 cells were 83.5% and 96.2%, respectively. 50 mg/mL CXCR3 mAb significantly inhibited the proliferation and migration of MCF-7 and HepG2 cells, and also inhibited the promoting effect of I-TAC on the proliferation and migration of MCF-7 and HepG2 cells in vitro. CXCR3 mAb can significantly inhibit the proliferation and migration of the tumor cells highly expressing CXCR3 in vitro.

  3. 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.

  4. Chemical characterization of Pleurotus eryngii polysaccharide and its tumor-inhibitory effects against human hepatoblastoma HepG-2 cells.

    Science.gov (United States)

    Ren, Daoyuan; Wang, Ning; Guo, Jianjun; Yuan, Li; Yang, Xingbin

    2016-03-15

    This study was designed to investigate the chemical characterization and antitumor effects of Pleurotus eryngii polysaccharides (PEP). The crude PEP was fractionated into two fractions, namely PEP-1 and PEP-2. HPLC analysis showed that PEP-1 and PEP-2 were heteropolysaccharides mainly composed of glucose with the average molecular weights of 2.54×10(4)Da (PEP-1) and 4.63×10(5)Da (PEP-2), respectively. High molecular mass PEP-2 was shown to exhibit stronger growth inhibition against human hepatoblastoma HepG-2 cells in comparison with PEP-1. Flow cytometric analysis showed that PEP-2 exerted a stimulatory effect on apoptosis of HepG-2 cells, and induced the cell-cycle arrest at the S-phase, with the observation of intracellular ROS production. These findings suggest that the polysaccharides, especially PEP-2, are very important nutritional ingredients responsible for the anticancer health benefits of P. eryngii.

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Science.gov (United States)

    Tang, Jie; Wang, Chuan; Jiang, Zheng

    2015-06-01

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

  7. The role of oxidative stress in citreoviridin-induced DNA damage in human liver-derived HepG2 cells.

    Science.gov (United States)

    Bai, Yuntao; Jiang, Li-Ping; Liu, Xiao-Fang; Wang, Dong; Yang, Guang; Geng, Cheng-Yan; Li, Qiujuan; Zhong, Lai-Fu; Sun, Qinghua; Chen, Min

    2015-05-01

    We hypothesize that citreoviridin (CIT) induces DNA damage in human liver-derived HepG2 cells through an oxidative stress mechanism and that N-acetyl-l-cysteine (NAC) protects against CIT-induced DNA damage in HepG2 cells. CIT-induced DNA damage in HepG2 cells was evaluated by alkaline single-cell gel electrophoresis assay. To elucidate the genotoxicity mechanisms, the level of oxidative DNA damage was tested by immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG); the intracellular generation of reactive oxygen species (ROS) and reduced glutathione (GSH) were examined; mitochondrial membrane potential and lysosomal membranes' permeability were detected; furthermore, protective effects of NAC on CIT-induced ROS formation and CIT-induced DNA damage were evaluated in HepG2 cells. A significant dose-dependent increment in DNA migration was observed at tested concentrations (2.50-10.00 µM) of CIT. The levels of ROS, 8-OHdG formation were increased by CIT, and significant depletion of GSH in HepG2 cells was induced by CIT. Destabilization of lysosome and mitochondria was also observed in cells treated with CIT. In addition, NAC significantly decreased CIT-induced ROS formation and CIT-induced DNA damage in HepG2 cells. The data indicate that CIT induces DNA damage in HepG2 cells, most likely through oxidative stress mechanisms; that NAC protects against DNA damage induced by CIT in HepG2 cells; and that depolarization of mitochondria and lysosomal protease leakage may play a role in CIT-induced DNA damage in HepG2 cells. © 2014 The Authors. Published by Wiley Periodicals Inc.

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

    Science.gov (United States)

    Wang, Lili; Ai, Wenchao; Zhai, Yanwu; Li, Haishan; Zhou, Kebin; Chen, Huiming

    2015-09-02

    Cerium oxide nanoparticles (nano-CeO₂) have been reported to cause damage and apoptosis in human primary hepatocytes. Here, we compared the toxicity of three types of nano-CeO₂ with different nanocrystal morphologies (cube-, octahedron-, and rod-like crystals) in human hepatocellular carcinoma cells (HepG2). The cells were treated with the nano-CeO₂ at various concentrations (6.25, 12.5, 25, 50, 100 μg/mL). The crystal structure, size and morphology of nano-CeO₂ were investigated by X-ray diffractometry and transmission electron microscopy. The specific surface area was detected using the Brunauer, Emmet and Teller method. The cellular morphological and internal structure were observed by microscopy; apoptotic alterations were measured using flow cytometry; nuclear DNA, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and glutathione (GSH) in HepG2 cells were measured using high content screening technology. The scavenging ability of hydroxyl free radicals and the redox properties of the nano-CeO₂ were measured by square-wave voltammetry and temperature-programmed-reduction methods. All three types of nano-CeO₂ entered the HepG2 cells, localized in the lysosome and cytoplasm, altered cellular shape, and caused cytotoxicity. The nano-CeO₂ with smaller specific surface areas induced more apoptosis, caused an increase in MMP, ROS and GSH, and lowered the cell's ability to scavenge hydroxyl free radicals and antioxidants. In this work, our data demonstrated that compared with cube-like and octahedron-like nano-CeO₂, the rod-like nano-CeO₂ has lowest toxicity to HepG2 cells owing to its larger specific surface areas.

  9. HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

    Science.gov (United States)

    El-Awady, Mostafa K; Tabll, Ashraf A; El-Abd, Yasmine S; Bahgat, Mahmoud M; Shoeb, Hussein A; Youssef, Samar S; Din, Noha G Bader El; Redwan, El-Rashdy M; El-Demellawy, Maha; Omran, Moataza H; El-Garf, Wael T; Goueli, Said A

    2006-01-01

    AIM: To establish a cell culture system with long-term replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro. METHODS: HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect naïve cells. The presence of minus (antisense) RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques (flow cytometry and Western blot) respectively. RESULTS: The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies (anti-core, and anti-E1). Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION: HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells. PMID:16937465

  10. Comparative cytotoxicity of dolomite nanoparticles in human larynx HEp2 and liver HepG2 cells.

    Science.gov (United States)

    Ahamed, Maqusood; Alhadlaq, Hisham A; Ahmad, Javed; Siddiqui, Maqsood A; Khan, Shams T; Musarrat, Javed; Al-Khedhairy, Abdulaziz A

    2015-06-01

    Dolomite is a natural mineral of great industrial and commercial importance. With the advent of nanotechnology, natural minerals including dolomite in the form of nanoparticles (NPs) are being utilized in various applications to improve the quality of products. However, safety or toxicity information of dolomite NPs is largely lacking. This study evaluated the cytotoxicity of dolomite NPs in two widely used in vitro cell culture models: human airway epithelial (HEp2) and human liver (HepG2) cells. Concentration-dependent decreased cell viability and damaged cell membrane integrity revealed the cytotoxicity of dolomite NPs. We further observed that dolomite NPs induce oxidative stress in a concentration-dependent manner, as indicated by depletion of glutathione and induction of reactive oxygen species (ROS) and lipid peroxidation. Quantitative real-time PCR data demonstrated that the mRNA level of tumor suppressor gene p53 and apoptotic genes (bax, CASP3 and CASP9) were up-regulated whereas the anti-apoptotic gene bcl-2 was down-regulated in HEp2 and HepG2 cells exposed to dolomite NPs. Moreover, the activity of apoptotic enzymes (caspase-3 and caspase-9) was also higher in both kinds of cells treated with dolomite NPs. It is also worth mentioning that HEp2 cells seem to be marginally more susceptible to dolomite NPs exposure than HepG2 cells. Cytotoxicity induced by dolomite NPs was efficiently prevented by N-acetyl cysteine treatment, which suggests that oxidative stress is primarily responsible for the cytotoxicity of dolomite NPs in both HEp2 and HepG2 cells. Toxicity mechanisms of dolomite NPs warrant further investigations at the in vivo level.

  11. HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

    Institute of Scientific and Technical Information of China (English)

    Mostafa K El-Awady; Moataza H Omran; Wael T El-Garf; Said A Goueli; Ashraf A Tabll; Yasmine S El-Abd; Mahmoud M Bahgat; Hussein A Shoeb; Samar S Youssef; Noha G Bader El Din; El-Rashdy M Redwan; Maha El-Demellawy

    2006-01-01

    AIM: To establish a cell culture system with longterm replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro. METHODS: HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect naive cells. The presence of minus (antisense) RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques (flow cytometry and Western blot) respectively. RESULTS: The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies (anti-core, and anti-E1). Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION: HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.

  12. Prediction of Drug-Induced Liver Injury in HepG2 Cells Cultured with Human Liver Microsomes.

    Science.gov (United States)

    Choi, Jong Min; Oh, Soo Jin; Lee, Ji-Yoon; Jeon, Jang Su; Ryu, Chang Seon; Kim, Young-Mi; Lee, Kiho; Kim, Sang Kyum

    2015-05-18

    Drug-induced liver injury (DILI) via metabolic activation by drug-metabolizing enzymes, especially cytochrome P450 (CYP), is a major cause of drug failure and drug withdrawal. In this study, an in vitro model using HepG2 cells in combination with human liver microsomes was developed for the prediction of DILI. The cytotoxicity of cyclophosphamide, a model drug for bioactivation, was augmented in HepG2 cells cultured with microsomes in a manner dependent on exposure time, microsomal protein concentration, and NADPH. Experiments using pan- or isoform-selective CYP inhibitors showed that CYP2B6 and CYP3A4 are responsible for the bioactivation of cyclophosphamide. In a metabolite identification study employing LC-ESI-QTrap and LC-ESI-QTOF, cyclophosphamide metabolites including phosphoramide mustard, a toxic metabolite, were detected in HepG2 cells cultured with microsomes, but not without microsomes. The cytotoxic effects of acetaminophen and diclofenac were also potentiated by microsomes. The potentiation of acetaminophen cytotoxicity was dependent on CYP-dependent metabolism, and the augmentation of diclofenac cytotoxicity was not mediated by either CYP- or UDP-glucuronosyltransferase-dependent metabolism. The cytotoxic effects of leflunomide, nefazodone, and bakuchiol were attenuated by microsomes. The detoxication of leflunomide by microsomes was attributed to mainly CYP3A4-dependent metabolism. The protective effect of microsomes against nefazodone cytotoxicity was dependent on both CYP-mediated metabolism and nonspecific protein binding. Nonspecific protein binding but not CYP-dependent metabolism played a critical role in the attenuation of bakuchiol cytotoxicity. The present study suggests that HepG2 cells cultured with human liver microsomes can be a reliable model in which to predict DILI via bioactivation by drug metabolizing enzymes.

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

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    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.

  14. Apoptosis induction by silica nanoparticles mediated through reactive oxygen species in human liver cell line HepG2

    Energy Technology Data Exchange (ETDEWEB)

    Ahmad, Javed [Department of Zoology, College of Science, King Saud University, Riyadh 11451 (Saudi Arabia); Ahamed, Maqusood, E-mail: maqusood@gmail.com [King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451 (Saudi Arabia); Akhtar, Mohd Javed [Fibre Toxicology, CSIR-Indian Institute of Toxicology Research, Lucknow-226001 (India); Alrokayan, Salman A. [King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451 (Saudi Arabia); Siddiqui, Maqsood A.; Musarrat, Javed; Al-Khedhairy, Abdulaziz A. [Department of Zoology, College of Science, King Saud University, Riyadh 11451 (Saudi Arabia)

    2012-03-01

    Silica nanoparticles are increasingly utilized in various applications including agriculture and medicine. In vivo studies have shown that liver is one of the primary target organ of silica nanoparticles. However, possible mechanisms of hepatotoxicity caused by silica nanoparticles still remain unclear. In this study, we explored the reactive oxygen species (ROS) mediated apoptosis induced by well-characterized 14 nm silica nanoparticles in human liver cell line HepG2. Silica nanoparticles (25–200 μg/ml) induced a dose-dependent cytotoxicity in HepG2 cells. Silica nanoparticles were also found to induce oxidative stress in dose-dependent manner indicated by induction of ROS and lipid peroxidation and depletion of glutathione (GSH). Quantitative real-time PCR and immunoblotting results showed that both the mRNA and protein expressions of cell cycle checkpoint gene p53 and apoptotic genes (bax and caspase-3) were up-regulated while the anti-apoptotic gene bcl-2 was down-regulated in silica nanoparticles treated cells. Moreover, co-treatment of ROS scavenger vitamin C significantly attenuated the modulation of apoptotic markers along with the preservation of cell viability caused by silica nanoparticles. Our data demonstrated that silica nanoparticles induced apoptosis in human liver cells, which is ROS mediated and regulated through p53, bax/bcl-2 and caspase pathways. This study suggests that toxicity mechanisms of silica nanoparticles should be further investigated at in vivo level. -- Highlights: ► We explored the mechanisms of toxicity caused by silica NPs in human liver HepG2 cells. ► Silica NPs induced a dose-dependent cytotoxicity in HepG2 cells. ► Silica NPs induced ROS generation and oxidative stress in a dose-dependent manner. ► Silica NPs were also modulated apoptosis markers both at mRNA and protein levels. ► ROS mediated apoptosis induced by silica NPs was preserved by vitamin C.

  15. Molecular mechanisms of apoptosis induced by Scorpio water extract in human hepatoma HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Kang-Beom Kwon; Eun-Kyung Kim; Jung-Gook Lim; Eun-Sil Jeong; Byung-Cheul Shin; Young-Se Jeon; Kang-San Kim; Eun-A Seo; Do-Gon Ryu

    2005-01-01

    AIM: To clarify the mechanism underlying the anti-mutagenic and anti-cancer activities of Scorpio water extract (SWE).METHODS: Human hepatoma HepG2 cells were incubated with various concentrations of SWE. After 24-h incubation,cytotoxicity and apoptosis evaluations were determined by MTT and DNA fragmentation assay, respectively. After treatment with SWE, mitochondrial membrane potential(MMP) was determined by measuring the retention of the dye 3,3'-dihexyloxacarbocyanine (DiOC6(3)) and the protein expression including cytochrome C and poly-(ADPribose) polymerase (PARP)were measured by Western blotting. Caspase-3 and -9 enzyme activities were measured using specific fluorescence dyes such as Ac-DEVD-AFC and Ac-LEHD-AFC.RESULTS: We found that treatment with SWE induced apoptosis as confirmed by discontinuous DNA fragmentation in cultured human hepatoma HepG2 cells. Our investigation also showed that SWE-induced apoptosis of HepG2 cells were associated with intracellular events including disruption of MMP, increased translocation of cytochrome C from mitochondria to cytosol, activation of caspase-3,and PARP. Pre-treatment of N-acetyl-Asp-Glu-Val-Asp-CHO(Ac-DEVD-CHO), a caspase-3 specific inhibitor, or cydosporin A (CsA), an inhibitor of MMP disruption, completely abolished SWE-induced DNA fragmentation.CONCLUSION: These results suggest that SWE possibly causes mitochondrial damage, leading to cytochrome C release into cytosol and activation of caspases resulting in PARP cleavage and execution of apoptotic cell death in HepG2 cells. These results further suggest that Scorpio may be a valuable agent of therapeutic intervention of human hepatomas.

  16. Heat Killed Attenuated Leishmania Induces Apoptosis of HepG2 Cells Through ROS Mediated p53 Dependent Mitochondrial Pathway

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    Dipayan Bose

    2016-03-01

    Full Text Available Background/Aims: Cytotoxic effect of attenuated Leishmania on liver cancer cells by inducing ROS generation. Methods: Spectrophotometric study to analyze cell death and levels of different active caspases. Flow cytometric study was done to analyze apoptosis induction and ROS generation and levels of different protein. Western blot analysis was performed to study the levels of protein. Confocal microscopy was done to ascertain the expression of different apoptotic markers. Results: We have now observed that attenuated Leishmania donovani UR6 also has potentiality towards growth inhibition of HepG2 cells and investigated the mechanism of action. The effect is associated with increased DNA fragmentation, rise in number of annexinV positive cells, and cell cycle arrest at G1 phase. The detection of unregulated levels of active PARP, cleaved caspases 3 and 9, cytosolic cytochrome C, Bax, and Bad, along with the observed downregulation of Bcl-2 and loss of mitochondrial membrane potential suggested the involvement of mitochondrial pathway. Enhanced ROS and p53 levels regulate the apoptosis of HepG2 cells. NAC was found to inhibit p53 production but PFT-α has no effect on ROS generation. In conclusion, Leishmania donovani UR6 efficiently induces apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. Conclusion: It has been reported earlier that some parasites show prominent cytotoxic effect and prevent tumor growth. From our study we found that Leishmania donovani UR6 efficiently induced apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. This study has rejuvenated the age old idea of bio-therapy.

  17. Cytotoxic and apoptotic effects of six herbal plants against the human hepatocarcinoma (HepG2 cell line

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    Nonpunya Apiyada

    2011-10-01

    Full Text Available Abstract Background Six plants from Thailand were evaluated for their cytotoxicity and apoptosis induction in human hepatocarcinoma (HepG2 as compared to normal African green monkey kidney epithelial cell lines. Methods Ethanol-water crude extracts of the six plants were tested with neutral red assay for their cytotoxicity after 24 hours of exposure to the cells. Apoptotic induction was tested in the HepG2 cells with diamidino-2-phenylindole staining. DNA fragmentation, indicative of apoptosis, was analyzed with agarose gel electrophoresis. Alkylation, indicative of DNA damage, was also evaluated in vitro by 4-(4'-nitrobenzyl pyridine assay. Results The extract of Pinus kesiya showed the highest selectivity (selectivity index = 9.6 and potent cytotoxicity in the HepG2 cell line, with an IC50 value of 52.0 ± 5.8 μg/ml (mean ± standard deviation. Extract of Catimbium speciosum exerted cytotoxicity with an IC50 value of 55.7 ± 8.1 μg/ml. Crude extracts from Glochidion daltonii, Cladogynos orientalis, Acorus tatarinowii and Amomum villosum exhibited cytotoxicity with IC50 values ranging 100-500 μg/ml. All crude extracts showed different alkylating abilities in vitro. Extracts of P. kesiya, C. speciosum and C. orientalis caused nuclei morphological changes and DNA laddering. Conclusion The extracts of C. speciosum, C. orientalis and P. kesiya induced apoptosis. Among the three plants, P. kesiya possessed the most robust anticancer activity, with specific selectivity against HepG2 cells.

  18. HNF-4alpha Negatively Regulates Hepcidin Expression Through BMPR1A in HepG2 Cells.

    Science.gov (United States)

    Shi, Wencai; Wang, Heyang; Zheng, Xuan; Jiang, Xin; Xu, Zheng; Shen, Hui; Li, Min

    2016-09-23

    Hepcidin synthesis is reported to be inadequate according to the body iron store in patients with non-alcoholic fatty liver disease (NAFLD) undergoing hepatic iron overload (HIO). However, the underlying mechanisms remain unclear. We hypothesize that hepatocyte nuclear factor-4α (HNF-4α) may negatively regulate hepcidin expression and contribute to hepcidin deficiency in NAFLD patients. The effect of HNF-4α on hepcidin expression was observed by transfecting specific HNF-4α small interfering RNA (siRNA) or plasmids into HepG2 cells. Both direct and indirect mechanisms involved in the regulation of HNF-4α on hepcidin were detected by real-time PCR, Western blotting, chromatin immunoprecipitation (chIP), and reporter genes. It was found that HNF-4α suppressed hepcidin messenger RNA (mRNA) and protein expressions in HepG2 cells, and this suppressive effect was independent of the potential HNF-4α response elements. Phosphorylation of SMAD1 but not STAT3 was inactivated by HNF-4α, and the SMAD4 response element was found essential to HNF-4α-induced hepcidin reduction. Neither inhibitory SMADs, SMAD6, and SMAD7 nor BMPR ligands, BMP2, BMP4, BMP6, and BMP7 were regulated by HNF-4α in HepG2 cells. BMPR1A, but not BMPR1B, BMPR2, ActR2A, ActR2B, or HJV, was decreased by HNF-4α, and HNF4α-knockdown-induced stimulation of hepcidin could be entirely blocked when BMPR1A was interfered with at the same time. In conclusion, the present study suggests that HNF-4α has a suppressive effect on hepcidin expression by inactivating the BMP pathway, specifically via BMPR1A, in HepG2 cells.

  19. Micropatterned culture of HepG2 spheroids using microwell chip with honeycomb-patterned polymer film.

    Science.gov (United States)

    Yamazaki, Hidekazu; Gotou, Shun; Ito, Koju; Kohashi, Souichi; Goto, Yuki; Yoshiura, Yukiko; Sakai, Yusuke; Yabu, Hiroshi; Shimomura, Masatsugu; Nakazawa, Kohji

    2014-10-01

    Microwell chip culture is a promising technique for the generation of homogenous spheroids. We investigated the relationship between the structure of the bottom surface of microwell chip and the properties of HepG2 spheroid. We developed a microwell chip, the bottom surface of which consisted of a honeycomb-patterned polymer film (honeycomb film) that had a regular porous structure (HF chip). The chip comprised 270 circular microwells; each microwell was 600 μm in diameter and 600 μm in depth. At the center of the honeycomb film, an area, 200 μm in diameter, was modified with collagen to facilitate cell adhesion. With the exception of the collagen-coated area, the entire microwell was modified with polyethylene glycol to eliminate cell adhesion. HepG2 cells formed uniform spheroids when cultured in the microwells of HF chip. Furthermore, the cells passed through the porous structure of honeycomb film and formed spheroids at its opposite side. The spheroid growth of HepG2 cells cultured in HF chip was greater than that when the cells were culture in a microwell chip, the bottom surface of which was made of poly-methylmethacrylate (PMMA chip). The albumin secretion activity of HepG2 spheroids in HF chip was equal to that in PMMA chip. These results indicate that the microwell bottom with a porous structure enhances the cell growth and maintains well the spheroid function. Thus, HF chip is a promising platform for spheroid cell culture.

  20. Implications of altered glutathione metabolism in aspirin-induced oxidative stress and mitochondrial dysfunction in HepG2 cells.

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    Haider Raza

    Full Text Available We have previously reported that acetylsalicylic acid (aspirin, ASA induces cell cycle arrest, oxidative stress and mitochondrial dysfunction in HepG2 cells. In the present study, we have further elucidated that altered glutathione (GSH-redox metabolism in HepG2 cells play a critical role in ASA-induced cytotoxicity. Using selected doses and time point for ASA toxicity, we have demonstrated that when GSH synthesis is inhibited in HepG2 cells by buthionine sulfoximine (BSO, prior to ASA treatment, cytotoxicity of the drug is augmented. On the other hand, when GSH-depleted cells were treated with N-acetyl cysteine (NAC, cytotoxicity/apoptosis caused by ASA was attenuated with a significant recovery in oxidative stress, GSH homeostasis, DNA fragmentation and some of the mitochondrial functions. NAC treatment, however, had no significant effects on the drug-induced inhibition of mitochondrial aconitase activity and ATP synthesis in GSH-depleted cells. Our results have confirmed that aspirin increases apoptosis by increased reactive oxygen species production, loss of mitochondrial membrane potential and inhibition of mitochondrial respiratory functions. These effects were further amplified when GSH-depleted cells were treated with ASA. We have also shown that some of the effects of aspirin might be associated with reduced GSH homeostasis, as treatment of cells with NAC attenuated the effects of BSO and aspirin. Our results strongly suggest that GSH dependent redox homeostasis in HepG2 cells is critical in preserving mitochondrial functions and preventing oxidative stress associated complications caused by aspirin treatment.

  1. Quercetin modulates Nrf2 and glutathione-related defenses in HepG2 cells: Involvement of p38.

    Science.gov (United States)

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

    2012-01-25

    Dietary flavonoid quercetin has been suggested as a cancer chemopreventive agent, but the mechanisms of action remain unclear. This study investigated the influence of quercetin on p38-MAPK and the potential regulation of the nuclear transcription factor erythroid-2p45-related factor (Nrf2) and the cellular antioxidant/detoxifying defense system related to glutathione (GSH) by p38 in HepG2 cells. Incubation of HepG2 cells with quercetin at a range of concentrations (5-50μM) for 4 or 18h induced a differential effect on the modulation of p38 and Nrf2 in HepG2 cells, 50μM quercetin showed the highest activation of p38 at 4h of treatment and values of p38 similar to those of control cells after 18 h of incubation, together with the inhibition of Nrf2 at both incubation times. Quercetin (50μM) induced a time-dependent activation of p38, which was in concert with a transient stimulation of Nrf2 to provoke its inhibition afterward. Quercetin also increased GSH content, mRNA levels of glutamylcysteine-synthetase (GCS) and expression and/or activity of glutathione-peroxidase, glutathione-reductase and GCS after 4h of incubation, and glutathione-S-transferase after 18h of exposure. Further studies with the p38 specific inhibitor SB203580 showed that the p38 blockage restored the inhibited Nrf2 transcription factor and the enzymatic expression and activity of antioxidant/detoxificant enzymes after 4h exposure. In conclusion, p38-MAPK is involved in the mechanisms of the cell response to quercetin through the modulation of Nrf2 and glutathione-related enzymes in HepG2 cells.

  2. 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.

  3. A novel AMPK activator, WS070117, improves lipid metabolism discords in hamsters and HepG2 cells

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    Hao Linghua

    2011-04-01

    Full Text Available Abstract Background WS070117 is a novel small molecule compound that significantly improves lipid metabolism disorders in high-fat-diet (HFD induced hyperlipidemia in hamsters. Methods and Results We evaluated liver/body weight ratio, liver histology, serum and hepatic lipid content in HFD-fed hamsters treated with WS070117 for 8 weeks. Comparing with HFD fed hamsters, WS070117 (2 mg/kg per day and above reduced serum triglyceride (TAG, total cholesterol (TC, low density lipoprotein cholesterol (LDL-C and hepatic cholesterol and triglyceride contents. Oil Red O staining of liver tissue also showed that WS070117 improved lipid accumulation. We then carried out an experiment in the oleic acid (OLA-induced steatosis model in HepG2 cell to investigate the lipid-lowering effect of WS070117. Oleic acid (0.25 mM markedly induced lipid accumulation in HepG2 cells, but WS070117 (10 μM inhibited cellular lipid accumulation. In OLA-treated HepG2 cells, WS070117 (above 1 μM treatment reduced lipid contents which synthesized from [1-14C] labeled acetic acid. Because WS070117 is an analog of adenosine, we evaluated the effect of WS070117 on AMP-activated protein kinase (AMPK signaling. The results showed that the activation of AMPK in OLA-induced steatosis in HepG2 cells was up-regulated by treatment with 0.1, 1 and 10 μM WS070117. The hepatic cellular AMPK phosphorylation is also up regulated by WS070117 (6 and 18 mg/kg treatment in HFD fed hamsters. Conclusion These new findings identify WS070117 as a novel molecule that regulates lipid metabolism in the hyperlipidemia hamster model. In vitro and in vivo studies suggested that WS070117 may regulate lipid metabolism through stimulating the activation of AMPK and its downstream pathways.

  4. Antisense oligonucleotide inhibition of hepatitis C virus genotype 4 replication in HepG2 cells

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    Omran Moataza H

    2006-06-01

    Full Text Available Abstract Background Hepatitis C (HCV viral infection is a serious medical problem in Egypt and it has a devastating impact on the Egyptian economy. It is estimated that over 15% of Egyptians are infected by the virus and thus finding a cure for this disease is of utmost importance. Current therapies for hepatitis C virus (HCV genotype 4 with interferon/ribavirin have not been successful and thus the development of alternative therapy for this genotype is disparately needed. Results Although previous studies utilizing viral subgenomic or full cDNA fragments linked to reporter genes transfected into adhered cells or in a cell free system showed promise, demonstration of efficient viral replication was lacking. Thus, we utilized HepG2 cells infected with native HCV RNA genomes in a replication competent system and used antisense phosphorothioate Oligonucleotides (S-ODN against stem loop IIId and the AUG translation start site of the viral polyprotein precursor to monitor viral replication. We were able to show complete arrest of intracellular replication of HCV-4 at 1 uM S-ODN, thus providing a proof of concept for the potential antiviral activity of S-ODN on native genomic replication of HCV genotype 4. Conclusion We have successfully demonstrated that by using two S-ODNs [(S-ODN1 (nt 326–348 and S-ODN-2 (nt 264–282], we were able to completely inhibit viral replication in culture, thus confirming earlier reports on subgenomic constructs and suggesting a potential therapeutic value in HCV type 4.

  5. Mangiferin, a Dietary Xanthone Protects Against Mercury-Induced Toxicity in HepG2 Cells

    Science.gov (United States)

    Agarwala, Sobhika; Rao, B. Nageshwar; Mudholkar, Kaivalya; Bhuwania, Ridhirama; Rao, B. S. Satish

    2012-01-01

    Mercury is one of the noxious heavy metal environmental toxicants and is a cause of concern for human exposure. Mangiferin (MGN), a glucosylxanthone found in Mangifera indica, reported to have a wide range of pharmacological properties. The objective of this study was to evaluate the cytoprotective potential of MGN, against mercury chloride (HgCl2) induced toxicity in HepG2 cell line. The cytoprotective effect of MGN on HgCl2 induced toxicity was assessed by colony formation assay, while antiapoptotic effect by fluorescence microscopy, flow cytometric DNA analysis, and DNA fragmentation pattern assays. Further, the cytoprotective effect of MGN against HgCl2 toxicity was assessed by using biochemical parameters like reduced glutathione (GSH), glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) by spectrophotometrically, mitochondrial membrane potential by flowcytometry and the changes in reactive oxygen species levels by DCFH-DA spectrofluoremetric analysis. A significant increase in the surviving fraction was observed with 50 µM of MGN administered two hours prior to various concentrations of HgCl2. Further, pretreatment of MGN significantly decreased the percentage of HgCl2 induced apoptotic cells. Similarly, the levels of ROS generated by the HgCl2 treatment were inhibited significantly (P < 0.01) by MGN. MGN also significantly (P < 0.01) inhibited the HgCl2 induced decrease in GSH, GST, SOD, and CAT levels at all the post incubation intervals. Our study demonstrated the cytoprotective potential of MGN, which may be attributed to quenching of the ROS generated in the cells due to oxidative stress induced by HgCl2, restoration of mitochondrial membrane potential and normalization of cellular antioxidant levels. PMID:20629087

  6. HepG2 cells recovered from apoptosis show altered drug responses and invasiveness

    Institute of Scientific and Technical Information of China (English)

    Shan-Shan Wang; Xin Xie; Chung Sing Timothy Wong

    2014-01-01

    BACKGROUND: Cancer  relapse,  associated  with  increased drug resistance and rate of metastasis, often follows completion of chemotherapy but the cancer escape mechanisms are still incompletely understood. Percutaneous ethanol injection (PEI) has been used for treating hepatocellular carcinoma (HCC) for decades, while the recurrence after PEI treatment remains a major limitation. Recent evidence mounted that cancer cells could survive from chemical induced apoptosis, suggesting a potential route through which cancer relapse may occur. This study focuses on the consequence of HepG2 recovery from ethanol-induced apoptotic event. METHODS: The  model  of  HepG2  recovery  from  ethanol-induced apoptotic event was established by live cell imaging, BrdU assay and Western blotting. MTT assay, wound healing assay and invasion assay were used to investigate the behavior of HepG2 after recovery. RESULTS: HepG2 cells could recover from ethanol-induced apoptosis. These cells changed their behaviors such as drug resistance, mobility and invasiveness. On average, the recovered HepG2 cell clones were found to be 46% more resistant to ethanol and 84% higher in mobility. The recovered clones became 58.2% more sensitive to 5-lfuorouracil. CONCLUSIONS: HepG2  cells  can  recover  from  ethanol-induced apoptotic event. These cells became more resistant to ethanol and more invasive. Although the recovered cell clones were more resistant to ethanol, they became more sensitive to 5-lfuorouracil treatment.

  7. Quercetin Induces Antiproliferative Activity Against Human Hepatocellular Carcinoma (HepG2) Cells by Suppressing Specificity Protein 1 (Sp1).

    Science.gov (United States)

    Lee, Ra Ham; Cho, Jin Hyoung; Jeon, Young-Joo; Bang, Woong; Cho, Jung-Jae; Choi, Nag-Jin; Seo, Kang Seok; Shim, Jung-Hyun; Chae, Jung-Il

    2015-02-01

    Preclinical Research Quercetin, found in red onions and red apple skin can induce apoptosis insome malignant cells. However, the apoptotic effect of quercetin in hepatocellular carcinoma HepG2 cells via regulation of specificity protein 1 (Sp1) has not been studied. Here, we demonstrated that quercetin decreased cell growth and induce apoptosis in HepG2 cells via suppression of Sp1 using 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, 4',6-diamidino-2-phenylindole (DAPI) staining, Annexin V, and Western blot analysis, an effect that was dose- and time-dependent manner. Treatment of HepG2 cells with quercetin reduced cell growth and induced apoptosis, followed by regulation of Sp1 and Sp1 regulatory protein. Taken together, the results suggest that quercetin can induce apoptotic cell death by regulating cell cycle and suppressing antiapoptotic proteins. Therefore, quercetin may be useful for cancer prevention. Drug Dev Res 76 : 9-16, 2015. © 2015 Wiley Periodicals, Inc.

  8. Hyperthermia inhibits hypoxia-induced epithelial-mesenchymal transition in HepG2 hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Guang-Jin Yuan; Qian-wen Li; Shun-Lin Shan; Wu-Ming Wang; Sen Jiang; Xi-Ming Xu

    2012-01-01

    AIM:TO investigate the effect of hyperthermia on hypoxia-induced epithelial-mesenchymal transition (EMT)in HepG2 hepatocellular carcinoma (HCC) cells,and its mechanism.METHODS:Cells were treated with hyperthermia at 43 ℃ for 0.5 h,followed by incubation under hypoxic or normoxic conditions for 72 h.Cell morphology was observed.Expressions of E-cadherin and vimentin were determined by immunofluorescence assay or Western blot.The protein and mRNA expressions of Snail were also determined by Western blot and reverse transcription-polymerase chain reaction.Cell migratory capacity was evaluated.RESULTS:Hypoxia induced EMT in HepG2 cells,which was evidenced by morphological,molecular and functional changes,including the formation of a spindle shape and the loss of cell contact.The expression of E-cadherin was decreased but the expression of vimentin was increased; also,the migratory capability was increased by 2.2 ± 0.20-fold as compared with normoxia.However,those effects were inhibited by hyperthermia pretreatment.Furthermore,protein synthesis and mRNA expression of Snail in the cells were enhanced by hypoxia as compared with normoxia,and also significantly inhibited by hyperthermia pretreatment.CONCLUSION:Hyperthermia may inhibit hypoxiainduced EMT in HepG2 HCC cells,and the mechanism may involve inhibition of induced expression of Snail.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Phellinus linteus mushroom protects against tacrine-induced mitochondrial impairment and oxidative stress in HepG2 cells.

    Science.gov (United States)

    Gao, Chunpeng; Zhong, Laifu; Jiang, Liping; Geng, Chengyan; Yao, Xiaofeng; Cao, Jun

    2013-06-15

    Tacrine (THA) was the first drug licensed for the treatment of Alzheimer's disease. Unfortunately, reversible hepatotoxicity is evident in about 30% of patients and limits its clinical use. The intracellular mechanisms have not yet been elucidated. Phellinus linteus (PL) is a mushroom that has long been used as a folk medicine. In our previous study, we found that PL could decrease the reactive oxygen species (ROS) formation in HepG2 cells. Presently, protective effects of PL on tacrine-induced ROS formation and mitochondria dysfunction were evaluated. The results showed that PL significantly reduced tacrine-induced ROS production, disruption of ΔΨm, 8-OHdG formation in mitochondrial DNA, and cytotoxicity in HepG2 cells. These data suggest that PL could attenuate the cytotoxicity and mitochondria dysfunction induced by tacrine in HepG2 cells. The protection is probably mediated by an antioxidant protective mechanism. Consumption of PL may be a plausible way to prevent tacrine-induced hepatotoxicity.

  11. Fucoidan induces apoptosis of HepG2 cells by down-regulating p-Stat3.

    Science.gov (United States)

    Roshan, Sadia; Liu, Yun-yi; Banafa, Amal; Chen, Hui-jie; Li, Ke-xiu; Yang, Guang-xiao; He, Guang-yuan; Chen, Ming-jie

    2014-06-01

    Fucoidan is one of the main bioactive components of polysaccharides. The current study was focused on the anti-tumor effects of fucoidan on human heptoma cell line HepG2 and the possible mechanisms. Fucoidan treatment resulted in cell cycle arrest and apoptosis of HepG2 cells in a dose-dependent manner detected by MTT assay, flow cytometry and fluorescent microscopy. The results of flow cytometric analysis revealed that fucoidan induced G2/M arrest in the cell cycle progression. Hoechst 33258 and Annexin V/PI staining results showed that the apoptotic cell number was increased, which was associated with a dose-dependent up-regulation of Bax and down-regulation of Bcl-2 and p-Stat3. In parallel, the up-regulation of p53 and the increase in reactive oxygen species were also observed, which may play important roles in the inhibition of HepG2 growth by fucoidan. In the meantime, Cyclin B1 and CDK1 were down-regulated by fucoidan treatment. Down-regulation of p-Stat3 by fucoidan resulted in apoptosis and an increase in ROS in response to fucoidan exposure. We therefore concluded that fucoidan induces apoptosis through the down-regulation of p-Stat3. These results suggest that fucoidan may be used as a novel anti-cancer agent for hepatocarcinoma.

  12. Effect of TSLC1 Gene on Proliferation, Invasion and Apoptosis of Human Hepatocellular Carcinoma Cell Line HepG2

    Institute of Scientific and Technical Information of China (English)

    QIN Li; ZHU Wentao; XU Tao; HAO Youhua; ZHANG Zhengmao; TIAN Yongjun; YANG Dongliang

    2007-01-01

    The recombinant plasmid pCI-TSLC1 carrying TSLC1 gene was stably transfected into human hepatocellular carcinoma cell line HepG2. Cell proliferation was analyzed by MTT assay. The ability of migration was determined by transwell and FACSort flow cytometry was used to detect the cell cycle distribution and apoptosis. Western blotting revealed that H4 expressed higher amounts of TSLC1 protein than H15 and H0 did. The growth of TSLC1-transfected cells was significantly sup- pressed in vitro, and the ability of migration was reduced as well. The re-expression of TSLC1 could induce cell apoptosis. It was concluded that TSLC1 strongly inhibited the growth and ability of mi- gration of HepG2 cell line in vitro and also induced apoptosis, suggesting that TSLC1 could reduce the tumorigenicity of human hepatocellular carcinoma cell line HepG2 in vitro, which provided a ba-sis for further exploring the roles of TSLC1 in hepatocellular cellular carcinoma.

  13. Protective role of metabolism by intestinal microflora in butyl paraben-induced toxicity in HepG2 cell cultures.

    Science.gov (United States)

    Khanal, Tilak; Kim, Hyung Gyun; Jin, Sun Woo; Shim, Eol; Han, Hwa Jeong; Noh, Keumhan; Park, Sunkyoung; Lee, Dae Hun; Kang, Wonku; Yeo, Hee Kyung; Kim, Dong Hyun; Jeong, Tae Cheon; Jeong, Hye Gwang

    2012-09-03

    Parabens are alkyl esters of p-hydroxybenzoic acid (BA), including methyl paraben (MP), ethyl paraben, propyl paraben (PP), and butyl paraben (BP). In the present study, possible role of metabolism by fecalase in BP-induced cytotoxicity was investigated in HepG2 cell cultures. As an intestinal bacterial metabolic system, a human fecalase prepared from human fecal specimen was employed. Among the parabens tested, cytotoxicity of BP was most severe. BA, the de-esterified metabolite, did not induce cytotoxicity when compared to other parabens. When BP was incubated with fecalase, it rapidly disappeared, in association with reduced cytotoxicity in HepG2 cells. In addition, BP incubated with fecalase significantly caused an increase in Bcl-2 expression together with a decrease in Bax expression and cleaved caspase-3. Moreover, anti-apoptotic effect by the incubation of BP with fecalase was also confirmed by the TUNEL assay. Furthermore, BP induced a sustained activation of the phosphorylation of JNK only when it was treated alone. Meanwhile, BP-induced cell death was reversed by the pre-incubation of BP with either fecalase or SP600125. Taken together, the findings suggested that metabolism of BP by human fecalase might have protective effects against BP-induced toxicity in HepG2 cells.

  14. 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.

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

    Institute of Scientific and Technical Information of China (English)

    Sasipawan Machana; Natthida Weerapreeyakul; Sahapat Barusrux

    2012-01-01

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

  16. DNA binding and apoptotic induction ability of harmalol in HepG2: Biophysical and biochemical approaches.

    Science.gov (United States)

    Sarkar, Sarita; Bhattacharjee, Paromita; Bhadra, Kakali

    2016-10-25

    Harmalol administration caused remarkable reduction in proliferation of HepG2 cells with GI50 of 14.2 μM, without showing much cytotoxicity in embryonic liver cell line, WRL-68. Data from circular dichroism (CD) and differential scanning calorimetric (DSC) analysis of harmalol-CT DNA complex shows conformational changes with prominent CD perturbation and stabilization of CT DNA by 8 °C. Binding constant and stoichiometry was calculated using the above biophysical techniques. The Scatchard plot constructed from CD data showed cooperative binding, from which the cooperative binding affinity (K'ω) of 4.65 ± 0.7 × 10(5) M(-1), and n value of 4.16 were deduced. The binding parameter obtained from DSC melting data was in good agreement with the above CD data. Furthermore, dose dependent apoptotic induction ability of harmalol was studied in HepG2 cells using different biochemical assays. Generation of ROS, DNA damage, changes in cellular external and ultramorphology, alteration of membrane, formation of comet tail, decreased mitochondrial membrane potential and a significant increase in Sub Go/G1 population made the cancer cell, HepG2, prone to apoptosis. Up regulation of p53 and caspase 3 further indicated the apoptotic role of harmalol.

  17. Caffeine dose-dependently induces thermogenesis but restores ATP in HepG2 cells in culture.

    Science.gov (United States)

    Riedel, Annett; Pignitter, Marc; Hochkogler, Christina M; Rohm, Barbara; Walker, Jessica; Bytof, Gerhard; Lantz, Ingo; Somoza, Veronika

    2012-09-01

    Caffeine has been hypothesised as a thermogenic agent that might help to maintain a healthy body weight. Since very little is known about its actions on cellular energy metabolism, we investigated the effect of caffeine on mitochondrial oxidative phosphorylation, cellular energy supply and thermogenesis in HepG2 cells, and studied its action on fatty acid uptake and lipid accumulation in 3T3-L1 adipocytes at concentrations ranging from 30-1500 μM. In HepG2 cells, caffeine induced a depolarisation of the inner mitochondrial membrane, a feature of mitochondrial thermogenesis, both directly and after 24 h incubation. Increased concentrations of uncoupling protein-2 (UCP-2) also indicated a thermogenic activity of caffeine. Energy generating pathways, such as mitochondrial respiration, fatty acid oxidation and anaerobic lactate production, were attenuated by caffeine treatment. Nevertheless, HepG2 cells demonstrated a higher energy charge potential after exposure to caffeine that might result from energy restoration through attenuation of energy consuming pathways, as typically found in hibernating animals. In 3T3-L1 cells, in contrast, caffeine increased fatty acid uptake, but did not affect lipid accumulation. We provide evidence that caffeine stimulates thermogenesis but concomitantly causes energy restoration that may compensate enhanced energy expenditure.

  18. Role of mitochondrial permeability transition in human hepatocellular carcinoma Hep-G2 cell death induced by rhein.

    Science.gov (United States)

    Du, Qiong; Bian, Xiao-Lan; Xu, Xiao-Le; Zhu, Bin; Yu, Bo; Zhai, Qing

    2013-12-01

    Rhein, a compound found as a glucoside in the root of rhubarb, is currently a subject of interest for its antitumor properties. The apoptosis of tumor cell lines induced by rhein was observed, and the involvement of mitochondria was established; however, the role of mitochondrial permeability transition (MPT) remains unknown. Here we report that MPT plays an important role in the apoptosis of human hepatocellular carcinoma Hep-G2 cells induced by rhein. After adding rhein to the isolated hepatic mitochondria, swelling effects and the leakage of Ca(2+) were observed. These alterations were suppressed by cyclosporin A (CsA), an MPT inhibitor. Furthermore, in Hep-G2 cells, the decrease of ATP production, the loss of mitochondrial transmembrane potential (MTP), the release of cytochrome c (Cyto c), and the activation of caspase 3 were also observed. These toxic effects of rhein can also be attenuated by CsA as well. Moreover, TUNEL assay confirmed that in the presence of CsA, rhein-induced apoptosis was largely inhibited. These results suggest that MPT plays a critical role in the pathogenesis of Hep-G2 cell injury induced by rhein, and imply that MPT may contribute to the anti-cancer activity of rhein. © 2013.

  19. Curcumin up-regulates LDL receptor expression via the sterol regulatory element pathway in HepG2 cells.

    Science.gov (United States)

    Dou, Xiaobing; Fan, Chunlei; Wo, Like; Yan, Jin; Qian, Ying; Wo, Xingde

    2008-09-01

    Plasma low-density lipoprotein-cholesterol (LDL-C) is mainly taken up and cleared by the hepatocellular LDL receptor (LDL-R). LDL-R gene expression is regulated by the sterol regulatory element binding proteins (SREBPs). Previous studies have shown that curcumin reduces plasma LDL-C and has hypolipidemic and anti-atherosclerotic effects. Herein, we investigated the effect of curcumin on LDL-R expression and its molecular mechanism in HepG2 cells. Curcumin increased LDL-R expression (mRNA and protein) and the resultant uptake of DiI-LDL in a dose- and time-dependent manner. Using a GFP reporter system in a transfected HepG2/SRE-GFP cell line, we found that curcumin activated the sterol regulatory element of the LDL-R promoter. In HepG2/Insig2 cells, curcumin reversed the inhibition of LDL-R expression induced by Insig2 overexpression. These data demonstrate that curcumin increases LDL-R protein expression and uptake activity via the SREBPs pathway. These findings contribute to our further understanding of the cholesterol-lowering and anti-atherosclerotic effects of curcumin.

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

    Directory of Open Access Journals (Sweden)

    Kyoung Jin Nho

    2012-01-01

    Full Text Available 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.

  1. 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.

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

    Science.gov (United States)

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

    2015-07-01

    The aim of the present study was to observe the effect and molecular mechanism of taurine (Tau) on the cell proliferation and apoptosis of human hepatocellular carcinoma (HHCC) HepG2 cells. HHCC HepG2 cells were used as target cells, and the cell survival rate was assessed using a multi-time-step method. The p53 upregulated modulator of apoptosis (PUMA) gene was transiently transfected by lipofection and subsequently silenced with specific small interfering (si)RNA. The cell apoptosis rate was detected by flow cytometry, and protein expression levels were analyzed with western blotting. Addition of 20-160 mM Tau was shown to have a significant inhibitory effect on cell proliferation, while promoting the induction of HHCC HepG2 cell apoptosis (PHepG2 cells. In addition, transfection of the PUMA gene increased the protein expression of B-cell lymphoma-2-associated X and reduced the expression of B-cell lymphoma-2 (PHepG2 cells (PHepG2 cells.

  3. Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report

    Science.gov (United States)

    Khaoustov, V. I.; Risin, D.; Pellis, N. R.; Yoffe, B.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

  4. Enhancement of amygdalin activated with β-D-glucosidase on HepG2 cells proliferation and apoptosis.

    Science.gov (United States)

    Zhou, Cunshan; Qian, Lichun; Ma, Haile; Yu, Xiaojie; Zhang, Youzuo; Qu, Wenjuan; Zhang, Xiaoxu; Xia, Wei

    2012-09-01

    The growth inhibition and induction of apoptosis brought by amygdalin and activated with β-D-glucosidase were tested for cytoactivity in HepG2 cells. The MTT viability assay showed that all samples had effects on HepG2 proliferation in dose and time response manners. IC50 of stand-alone amygdalin and activation with β-D-glucosidase on the proliferation of HepG2 cells for 48 h were 458.10 mg/mL and 3.2 mg/mL, respectively. Moreover, apoptotic cells were determined by AO/EB (acridine orange/ethidium bromide) fluorescent staining method and Annexin V-FITC/PI staining flow cytometry cell cycle analysis. With increasing of amygdalin concentration and the incubation time, the apoptotic rate was heightened. Compared with the control, there was significant difference (pamygdalin had no strong anti-HepG2 activity; however the ingredients of amygdalin activated with β-D-glucosidase had a higher and efficient anti-HepG2 activity. It was therefore suggested that this combination strategy may be applicable for treating tumors with a higher activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. [Hepatitis B virus X promotes HepG2 cell cycle progression and growth via downregulation expression of p16 protein].

    Science.gov (United States)

    Mai, Li; Yang, Lin; Kuang, Jian-yu; Zhu, Jian-yun; Kang, Yan-hong; Zhang, Fu-cheng; Xie, Qi-feng; Gao, Zhi-liang

    2013-08-01

    To investigate the effects and related mechanisms of hepatitis B virus X (HBx) protein on cell cycle and growth in hepatocellular carcinoma. A human hepatocyte HepG2 cell line stably expressing a green fluorescent protein (GFP)-tagged HBx (HepG2/GFP-HBx cells) was used for the experiment, and HepG2 parental and HepG2/GFP cells was used as the controls. Effect of HBx on cell growth was evaluated by the MTT cell proliferation assay and on cell cycle progression by flow cytometry analysis of cells with or without treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR; 5 pmol/L). Effect of HBx expression on promoter methylation status of the p16INK4A tumor-suppressor gene was detected by methylation-specific polymerase chain reaction and on p16 protein level was analyzed with western blotting. The HepG2/GFP-HBx cells showed significantly higher cell proliferation at 72 hrs of culture (3.225+/-0.038 A490) than either control (HepG2: 2.012+/-0.022 A490, t = -46.86, P less than 0.001; HepG2/GFP: 2.038+/-0.029 A490, t = 42.51, P less than 0.001). The HepG2/GFP-HBx cells also showed significantly lower proportion of cells in the G0/G1 phase (16.45%+/-0.45%) than either control (HepG2: 44.81%+/-1.36%, t = -34.202, P less than 0.001; HepG2/GFP: 42.76%+/-1.58%, t = -28.88, P less than 0.001). However, 5-Aza-CdR treatment did lead to a significant amount of HepG2/GFP-HBx cells being arrested in the G0/G1 phase (33.25%+/-0.79%, t = 31.85, P less than 0.001). The p16INK4A promoter was methylated in the HepG2/GFP-HBx cells, and became demethylation after treatment with 5-Aza-CdR. However, no methylation of p16INK4A promoter was observed in both HepG2 and HepG2/GFP cells. The p16 protein level was significantly lower in the HepG2/GFP-HBx (vs. HepG2 and HepG2/GFP cells) and this level increased after treatment with 5-Aza-CdR. HBx protein promotes hepatocellular carcinoma cell cycle progression and growth by shortening the G0/G1 phase, and the underlying mechanism may involve inducing p16INK4A promoter methylation and downregulating p16 protein expression.

  6. Transcriptional down regulation of hTERT and senescence induction in HepG2 cells by chelidonine

    Science.gov (United States)

    Kazemi Noureini, Sakineh; Wink, Michael

    2009-01-01

    AIM: To investigate the potential effects of chelidonine, the main alkaloid of Chelidonium majus, on telomerase activity and its regulation in HepG2 cells. METHODS: Cytotoxicity of chelidonine for HepG2 cells was determined by neutral red assay. A modified polymerase chain reaction (PCR)-based telomerase repeat amplification protocol was used to estimate relative telomerase activity in chelidonine-treated cells in comparison with the untreated control cells. Relative expression level of the catalytic subunit of telomerase (hTERT) gene and P-glycoprotein (pgp) were estimated using semi-quantitative real-time reverse transcription-PCR (RT-PCR). Cell senescence in treated cells was demonstrated using a β-galactosidase test. RESULTS: Cytotoxicity of chelidonine in HepG2 cells was not dose-dependent and tended to reach plateau immediately after the living cells were reduced in number to slightly higher than 50%. However, 12 μmol/L concentration of chelidonine was considered as LD50, where the maximal attainable effects were realized. Real-time RT-PCR data showed that the expression of pgp increased three-fold in chelidonine treated HepG2 cells in comparison with the untreated controls. Morphologically, treated HepG2 cells showed apoptotic features after 24 h and a small fraction of cells appeared with single blister cell death. The relative expression level of Bcl-2 dropped to less than 50% of control cells at a sub-apoptotic concentration of chelidonine and subsequently increased to higher than 120% at LD50. Telomerase activity was reduced considerably after administration of very low doses of chelidonine, whereas higher concentrations of chelidonine did not remarkably enhance the effect. Real-time RT-PCR experiments indicated a drastic decrease in expression level of hTERT subunit of telomerase under treatment with chelidonine. Repeated treatment of cells with very low doses of chelidonine caused a decline in growth rate by 4 wk and many of the cells appeared to be aged with large volume and dark staining in the β-galactosidase assay. CONCLUSION: Chelidonine reduces telomerase activity through down-regulation of hTERT expression. Senescence induction might not be directly caused by reducing telomerase activity as it occurs after a few population doublings. PMID:19653337

  7. Stable expression of human cytochrome P450 2D6*10 in HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Jian Zhuge; Ying-Nian Yu; Xiao-Dan Wu

    2004-01-01

    AIM: Over 90% of drugs are metabolized by the cytochrome P-450 (CYP) family of liver isoenzymes. The most important enzymes are CYP1A2, 3A4, 2C9/19, 2D6 and 2E1. Although CYP2D6 accounts for <2% of the total CYP liver enzyme content, it mediates metabolism in almost 25% of drugs. In order to study its enzymatic activity for drug metabolism, its cDNA was cloned and a HepG2 cell line stably expressing CYP2D6 was established.METHODS: Human CYP2D6 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR)from total RNA extracted from human liver tissue and cloned into pGEM-T vector, cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A cell line was established by transfecting the recombinant plasmid of pREP9-CYP2D6 to hepatoma HepG2 cells. Expression of mRNA was validated by RT-PCR.Enzyme activity of catalyzing dextromethorphan O-demethylation in postmitochondrial supernant (S9) fraction of the cells was determined by high performance liquid chromatography (HPLC).RESULTS: The cloned cDNA had 4 base differences, e.g.100 C→T, 336 T→C, 408 C→G and 1 457 G→C, which resulted in P34S, and S486T amino acid substitutions, and two samesense mutations were 112 F and 136 V compared with that reported by Kimura et al(GenBank accession number: M33388). P34S and S486T amino acid substitutions were the characteristics of CYP2D6*10 allele. The relative activity of S9 fraction of HepG2-CYP2D6*10 metabolized detromethorphan O-demethylation was found to be 2.31±0.19 nmol.min-1.mg-1 S9 protein (n=3), but was undetectable in parental HepG2 cells.CONCLUSION: cDNA of human CYP2D6*10can be successfully doned. A cell line, HepG2-CYP2D6*10, expressing CYP2D6*10 mRNA and having metabolic activity, has been established.

  8. Transcriptional down regulation of hTERT and senescence induction in HepG2 cells by chelidonine

    Institute of Scientific and Technical Information of China (English)

    Sakineh Kazemi Noureini; Michael Wink

    2009-01-01

    AIM: To investigate the potential effects of chelidonine, the main alkaloid of Chelidonium majus, on telomerase activity and its regulation in HepG2 cells. METHODS: Cytotoxicity of chelidonine for HepG2 cells was determined by neutral red assay. A modified polymerase chain reaction (PCR)-based telomerase repeat amplification protocol was used to estimate relative telomerase activity in chelidonine-treated cells in comparison with the untreated control cells. Relative expression level of the catalytic subunit of telomerase (hTERT) gene and P-glycoprotein (pgp) were estimated using semi-quantitative real-time reverse transcription-PCR (RT-PCR). Cell senescence in treated cells was demonstrated using a β-galactosidase test. RESULTS: Cytotoxicity of chelidonine in HepG2 cells was not dose-dependent and tended to reach plateau immediately after the living cells were reduced in number to slightly higher than 50%. However, 12 μmol/L concentration of chelidonine was considered as LD50, where the maximal attainable effects were realized. Real-time RT-PCR data showed that the expression of pgp increased three-fold in chelidonine treated HepG2cells in comparison with the untreated controls. Morphologically, treated HepG2 cells showed apoptotic features after 24 h and a small fraction of cells appeared with single blister cell death. The relative expression level of Bcl-2 dropped to less than 50% of control cells at a sub-apoptotic concentration of chelidonine and subsequently increased to higher than 120% at LD50. Telomerase activity was reduced considerably after administration of very low doses of chelidonine, whereas higher concentrations of chelidonine did not remarkably enhance the effect. Real-time RT-PCR experiments indicated a drastic decrease in expression level of hTERT subunit of telomerase under treatment with chelidonine. Repeated treatment of cells with very low doses of chelidonine caused a decline in growth rate by 4 wk and many of the cells appeared to be aged with large volume and dark staining in the β-galactosidase assay. CONCLUSION: Chelidonine reduces telomerase activity through down-regulation of hTERT expression. Senescence induction might not be directly caused by reducing telomerase activity as it occurs after a few population doublings.

  9. The expression of efflux and uptake transporters are regulated by statins in Caco-2 and hepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Alice Cristina RODRIGUES; Rui CURI; Fabiana Dalla Vecchia GENVIGIR; Mario Hiroyuki HIRATA; Rosario Dominguez Crespo HIRATA

    2009-01-01

    Aim:Statin disposition and response are greatly determined by the activities of drug metabolizing enzymes and effiux/uptake transporters.There is little information on the regulation of these proteins in human ceils after statin therapy.In this study,the effects of atorvastatin and simvastatin on mRNA expression of efflux (ABCB1,ABCG2 and ABCC2) and uptake (SLCO1B1,SLCO2B1 and SLC22A1) drug transporters in Caco-2 and HepG2 cells were investigated.Methods:Quantitative real-time PCR was used to measure mRNA levels after exposure of HepG2 and Caco-2 cells to statins Results:Differences in mRNA basal levels of the transporters were as follows:ABCC2>ABCG2>ABCB1>SLCOIB1>>>SLC22A1>SLC 02B1 for HepG2 cells,and SLCO2B1>>ABCC2>ABCB1>ABCG2>>>SLC22A1 for Caco-2 cells.While for HepG2 cells,ABCC2,ABCG2 and SLCO2B1 mRNA levels were significantly up-regulated at 1,10 and 20 μmol/L after 12 or 24 h treatment,in Caco-2 cells,only the efflux transporter ABCB1 was significantly down-regulated by two-fold following a 12 h treatment with atorvastatin.Interestingly,whereas treatment with simvastatin had no effect on mRNA levels of the transporters in HepG2 cells,in Caco-2 cells the statin signifi cantly down-regulated ABCB1,ABCC2,SLC22A1,and SLCO2B1 mRNA levels after 12 or 24 h treatment.Conclusion:These findings reveal that statins exhibits differential effects on mRNA expression of drug transporters,and this effect depends on the cell type.Furthermore,alterations in the expression levels of drug transporters in the liver and/or intestine may contribute to the variability in oral disposition of statins.

  10. Knockdown of Decoy Receptor 3 Impairs Growth and Invasiveness of Hepatocellular Carcinoma Cell Line of HepG2

    Institute of Scientific and Technical Information of China (English)

    Xiao-Na Zhou; Guang-Ming Li; Ying-Chen Xu; Tuan-Jie Zhao; Ji-Xiang Wu

    2016-01-01

    Background:Decoy receptor 3 (DcR3) binds to Fas ligand (FasL) and inhibits FasL-induced apoptosis.The receptor is overexpressed in hepatocellular carcinoma (HCC),and it is associated with the growth and metastatic spread of tumors.DcR3 holds promises as a new target for the treatment of HCC,but little is known regarding the molecular mechanisms underlying the oncogenic properties of DcR3.The present work,therefore,examined the role of DcR3 in regulating the growth and invasive property of liver cancer cell HepG2.Methods:HepG2 cells were stably transfected with lentivirus-based short hairpin RNA vector targeting DcR3.After the knockdown of DcR3 was confirmed,cell proliferation,clone formation,ability of migrating across transwell membrane,and wound healing were assessed in vitro.Matrix metalloproteinase-9 (MMP 9) and vascular epithelial growth factor (VEGF)-C and D expressions of the DcR3 knockdown were also studied.Comparisons between multiple groups were done using one-way analysis of variance (ANOVA),while pairwise comparisons were performed using Student's t test.P < 0.05 was regarded statistically significant.Results:DcR3 was overexpressed in HepG2 compared to other HCC cell lines and normal hepatocyte Lo-2.Stable knockdown of DcR3 slowed down the growth of HepG2 (P < 0.05) and reduced the number of clones formed by 50% compared to those without DcR3 knockdown (P < 0.05).The knockdown also reduced the migration of HepG2 across transwell matrix membrane by five folds compared to the control (P < 0.05) and suppressed the closure of scratch wound (P < 0.05).In addition,the messenger RNA levels of MMP 9,VEGF-C,and VEGF-D were significantly suppressed by DcR3 knockdown by 90% when compared with the mock control (P < 0.05).Conclusions:Loss of DcR3 impaired the growth and invasive property of HCC cell line of HepG2.Targeting DcR3 may be a potential therapeutic approach for the treatment of HCC.

  11. Anti-tumor effects of bemiparin in HepG2 and MIA PaCa-2 cells.

    Science.gov (United States)

    Alur, İhsan; Dodurga, Yavuz; Seçme, Mücahit; Elmas, Levent; Bağcı, Gülseren; Gökşin, İbrahim; Avcı, Çığır Biray

    2016-07-10

    Recent researches have demonstrated improved survival in oncologic patients treated with low molecular weight heparins (LMWHs) which are anticoagulant drugs. We evaluated "second generation" LMWH bemiparin and its in vitro anti-tumor effects on HepG2 hepatocellular carcinoma and MIA PaCa-2 cancer cells. The aim of the study is to investigate anti-cancer mechanism of bemiparin in HepG2 and Mia-Paca-2 cancer cells. Cytotoxic effects of bemiparin were determined by XTT assay. IC50 dose of bemiparin was found to be 200 IU/mL in the 48th hour in the MiaPaCa-2 cell line and 50 IU/mL in the 48th hour in the HepG2 cell line. CCND1 (cyclin D1), CDK4, CDK6, p21, p16, p53, caspase-3, caspase-9, caspase-8, Bcl-2, BID, DR4, DR5, FADD, TRADD, Bax, gene mRNA expressions were evaluated by Real-time PCR. Real-time PCR analysis showed that CCND1 expression was reduced in HepG2 dose the group cells when compared with the control group cells and p53, caspase-3, caspase p21, caspase-8 and expressions were increased in the dose group cells when compared with the control group cells. CCND1, CDK4 and CDK6 expressions were reduced in MIA PaCa-2 dose group cells when compared with the control group cells and p53 expression was increased in the dose group cells when compared with the control group cells. Other expressions of genes were found statistically insignificant both of cell lines. It was found that bemiparin in HepG2 and MIA PaCa-2 cells suppressed invasion, migration, and colony formation by using matrigel invasion chamber, and colony formation assay, respectively. In conclusion, it is thought that bemiparin indicates anti-tumor activity by affecting cell cycle arrest, apoptosis, invasion, migration, and colony formation on cancer cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Protective effects of marein on high glucose-induced glucose metabolic disorder in HepG2 cells.

    Science.gov (United States)

    Jiang, Baoping; Le, Liang; Zhai, Wei; Wan, Wenting; Hu, Keping; Yong, Peng; He, Chunnian; Xu, Lijia; Xiao, Peigen

    2016-08-15

    Our previous study has shown that Coreopsis tinctoria increases insulin sensitivity and regulates hepatic metabolism in high-fat diet (HFD)-induced insulin resistance rats. However, it is unclear whether or not marein, a major compound of C. tinctoria, could improve insulin resistance. Here we investigate the effect and mechanism of action of marein on improving insulin resistance in HepG2 cells. We investigated the protective effects of marein in high glucose-induced human liver carcinoma cell HepG2. In kinase inhibitor studies, genistein, LY294002, STO-609 and compound C were added to HepG2 cells 1h before the addition of marein. Transfection with siRNA was used to knock down LKB1, and 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG), an effective tracer, was used to detect glucose uptake. The results showed for the first time that marein significantly stimulates the phosphorylation of AMP-activated protein kinase (AMPK) and the Akt substrate of 160kDa (AS160) and enhanced the translocation of glucose transporter 1 (GLUT1) to the plasma membrane. Further study indicated that genistein (an insulin receptor tyrosine kinase inhibitor) altered the effect of marein on glucose uptake, and both LY294002 (a phosphatidylinositol 3-kinase inhibitor) and compound C (an AMP-activated protein kinase inhibitor) significantly decreased marein-stimulated 2-NBDG uptake. Additionally, marein-stimulated glucose uptake was blocked in the presence of STO-609, a CaMKK inhibitor; however, marein-stimulated AMPK phosphorylation was not blocked by LKB1 siRNA in HepG2 cells. Marein also inhibited the phosphorylation of insulin receptor substrate (IRS-1) at Ser 612, but inhibited GSK-3β phosphorylation and increased glycogen synthesis. Moreover, marein significantly decreased the expression levels of FoxO1, G6Pase and PEPCK. Consequently, marein improved insulin resistance induced by high glucose in HepG2 cells through CaMKK/AMPK/GLUT1 to promote glucose uptake, through IRS/Akt/GSK-3β to increase glycogen synthesis, and through Akt/FoxO1 to decrease gluconeogenesis. Marein could be a promising leading compound for the development of hypoglycemic agent or developed as an adjuvant drug for diabetes mellitus. Copyright © 2016. Published by Elsevier GmbH.

  13. Effect of continuous irradiation with 125I radioactive seeds in inhibiting HepG2 cell proliferation and related mechanisms

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    CIDAN Wangjiu

    2016-10-01

    Full Text Available Objective To investigate the effect of continuous irradiation with 125I radioactive seeds in inhibiting HepG2 cell proliferation and possible mechanisms in inducing apoptosis. Methods Human hepatoma cell line HepG2 was selected as the research object and exposed to continuous irradiation with 125I radioactive seeds. The initial dose rate was 5.32 cGy/h, and HepG2 cells were exposed to a dose of 0, 2, or 4 Gy. A light microscope and Hoechst33258 staining were used to observe the morphological change of HepG2 cells, the colony-forming assay was used to calculate plating efficiency, the scratch test was used to evaluate the change in migration ability, flow cytometry was used to measure cell apoptosis, and Western Blot was used to measure the expression of apoptosis-related proteins. An analysis of variance was used for comparison between multiple groups, and the least significant difference method was used for further comparison between any two groups. Results After HepG2 cells were exposed to 125I radioactive seeds at a dose of 0 Gy, 2 Gy, or 4 Gy, the 4 Gy group showed a reduction in cell density and an increase in the number of spherical free dead cells, as was shown by the light microscope; Hoechst33258 staining showed that the 4 Gy group had typical features of apoptotic cells, such as karyopyknosis, fragmentation, and margination. The colony-forming assay showed that there were significant differences in plating efficiency between the control group, 2 Gy group, and 4 Gy group (120.00%±3.61%, 112.00%±2.00%, and 45.00%±3.61%, F=508.90, P<0.001. The scratch test showed that there were significant differences in cell migration rate between the control group, 2 Gy group, and 4 Gy group (21.24%±4.36%, 19.93%±3.37%, and 11.42%±0.65%, F=8.29, P<0.001. The results of flow cytometry for cell apoptosis showed that there were significant differences in cell apoptosis rate between the control group, 2 Gy group, and 4 Gy group (4.33%±0.67%, 6.90%±1.31%, and 17.03%±1.43%, F=110.01, P<0.001. Western blot showed that 125I irradiation significantly inhibited the expression of tumor proliferation-related protein PCNA, apoptosis-related protein Survivin, and Bcl-2 and promoted the expression of pro-apoptotic proteins Bak and Bax. Conclusion 125I radioactive seeds inhibit the growth of HepG2 cells through promoting HepG2 cell apoptosis and inhibiting proliferation and migration. The possible mechanism may be related to the inhibition of the expression of apoptosis-related proteins.

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

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    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 evidence that coexisting carnitine deficiency masks some indicators of biotin deficiency and support the potential importance of the ratios of acylcarnitines arising from the acyl-CoA substrates and products for biotin-dependent carboxylases in detecting the biotin deficiency that is masked by coexisting carnitine deficiency. © 2015 American Society for Nutrition.

  15. Alisol A 24-Acetate Prevents Hepatic Steatosis and Metabolic Disorders in HepG2 Cells

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    Lu Zeng

    2016-11-01

    Full Text Available Background: Non-alcoholic fatty liver disease (NAFLD is closely associated with metabolic disorders including hepatic lipid accumulation and inflammation. Alisol A 24-acetate, a triterpene from Alismatis rhizome, has multiple biologic activities such as hypolipidemic, anti-inflammatory and anti-diabetic. Thus we hypothesized that Alisol A 24 -acetate would have effect on NAFLD. The present study was conducted to investigate the therapeutic effects and potential mechanisms of Alisol A 24-acetate against hepatic steatosis in a free fatty acids (FFAs induced NAFLD cell model. Methods: This study was divided into four groups including Control group, Model group (FFA group, Alisol A 24-acetate (FFA+A group, Fenofibrate (FFA+F group. Preventive role of Alisol A 24-acetate was evaluated using 10µM Alisol A 24-acetate plus 1 mM FFA (oleate:palmitate=2:1 incubated with HepG2 cells for 24 h, which was determined by Oil Red O Staining, Oil Red O based colorimetric assay and intracellular triglyceride (TG content. Besides, the inflammatory cytokines tumor necrosis factor (TNF- α, interleukin (IL-6 levels as well as the protein and mRNA expressions that were involved in fatty acid synthesis and oxidation including Adiponectin, AMP-activated protein kinase (AMPK α, peroxisome proliferator-activated receptor (PPAR α, sterol regulatory element binding protein 1c (SREBP-1c, acetyl-CoA carboxylase (ACC, fatty acid synthase (FAS, carnitine palmitoyltransferase 1 (CPT1 and acyl coenzyme A oxidase 1 (ACOX1 were detected. Results: Alisol A 24-acetate significantly decreased the numbers of lipid droplets, Oil Red O lipid content, and intracellular TG content. Besides, inflammatory cytokines TNF-α, IL-6 levels were markedly inhibited by Alisol A 24-acetate. Furthermore, Alisol A 24-acetate effectively increased the protein and mRNA expressions of Adiponectin, the phosphorylation of AMPKα, CPT1 and ACOX1, whereas decreased SREBP-1c, the phosphorylation of ACC and FAS at both protein and mRNA levels. However, there was no significant effect on the protein and mRNA expressions of PPARα by Alisol A 24-acetate. Conclusions: These results demonstrated that Alisol A 24-acetate effectively ameliorated hepatic steatosis likely through Adiponectin, which activated AMPKα signaling pathways via down-regulating SREBP-1c, ACC, FAS and up-regulating CPT1 and ACOX1, and inhibited inflammation. Thereby, Alisol A 24-acetate could be a promising candidate for the treatment of NAFLD.

  16. Expression of albumin and cytochrome P450 enzymes in HepG2 cells cultured with a nanotechnology-based culture plate with microfabricated scaffold.

    Science.gov (United States)

    Nakamura, Kazuaki; Kato, Natsuko; Aizawa, Kazuko; Mizutani, Reiko; Yamauchi, Junji; Tanoue, Akito

    2011-10-01

    The Nanoculture plate (NCP) is a recently developed plate which essentially consists of a textured surface with specific characteristics that induce spheroid formation: microfabrications with a micro-square pattern on the culture surface. The NCP can be used to generate uniform adhesive spheroids of cancer cell lines using conventional techniques without the need of any animal compounds. In this study, we assessed the performance of human hepatoma cell line HepG2 cells cultured with an NCP to evaluate the effects of the NCP on their hepatocyte-specific functions. The NCP facilitated the formation of three-dimensional (3D) HepG2 cell architecture. HepG2 cells cultured with an NCP exhibited enhanced mRNA expression levels of albumin and cytochrome P450 (CYP) enzymes compared to those cultured with a two-dimensional (2D) conventional plate. The expression levels of two specific liver-enriched transcription factors, hepatocyte nuclear factor 4α (HNF4α) and CCAAT/enhancer binding protein α (C/EBPα), were higher in HepG2 cells grown with the NCP than those in HepG2 cells grown with conventional plates before albumin and CYP enzymes expression levels were increased. The inducibility of CYP1A2 and CYP3A4 mRNA following exposure to inducers in HepG2 cells cultured with an NCP was comparable to that in HepG2 cells cultured with conventional plates, while the expression levels of CYP1A2 and CYP3A4 mRNA following exposure to inducers were higher when using an NCP than when using conventional plates. These results suggest that the use of an NCP enhances the hepatocyte-specific functions of HepG2 cells, such as drug-metabolizing enzyme expression, making the NCP/HepG2 system a useful tool for evaluating drug metabolism in vitro.

  17. LXR agonist increases apoE secretion from HepG2 spheroid, together with an increased production of VLDL and apoE-rich large HDL

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    Koike Kazuhiko

    2011-08-01

    Full Text Available Abstract Background The physiological regulation of hepatic apoE gene has not been clarified, although the expression of apoE in adipocytes and macrophages has been known to be regulated by LXR. Methods and Results We investigated the effect of TO901317, a LXR agonist, on hepatic apoE production utilizing HepG2 cells cultured in spheroid form, known to be more differentiated than HepG2 cells in monolayer culture. Spheroid HepG2 cells were prepared in alginate-beads. The secretions of albumin, apoE and apoA-I from spheroid HepG2 cells were significantly increased compared to those from monolayer HepG2 cells, and these increases were accompanied by increased mRNA levels of apoE and apoA-I. Several nuclear receptors including LXRα also became abundant in nuclear fractions in spheroid HepG2 cells. Treatment with TO901317 significantly increased apoE protein secretion from spheroid HepG2 cells, which was also associated with the increased expression of apoE mRNA. Separation of the media with FPLC revealed that the production of apoE-rich large HDL particles were enhanced even at low concentration of TO901317, and at higher concentration of TO901317, production of VLDL particles increased as well. Conclusions LXR activation enhanced the expression of hepatic apoE, together with the alteration of lipoprotein particles produced from the differentiated hepatocyte-derived cells. HepG2 spheroids might serve as a good model of well-differentiated human hepatocytes for future investigations of hepatic lipid metabolism.

  18. Chemosensitization of HepG2 cells by suppression of NF-κB/p65 gene transcription with specific-siRNA.

    Science.gov (United States)

    Shi, Yun; Wang, Si-Ye; Yao, Min; Sai, Wen-Li; Wu, Wei; Yang, Jun-Ling; Cai, Yin; Zheng, Wen-Jie; Yao, Deng-Fu

    2015-12-07

    To investigate small interfering RNA (siRNA)-mediated inhibition of nuclear factor-kappa B (NF-κB) activation and multidrug-resistant (MDR) phenotype formation in human HepG2 cells. Total RNA was extracted from human HepG2 or LO2 cells. NF-κB/p65 mRNA was amplified by nested reverse transcription polymerase chain reaction and confirmed by sequencing. NF-κB/p65 was analyzed by immunohistochemistry. Specific-siRNA was transfected to HepG2 cells to knock down NF-κB/p65 expression. The effects on cell proliferation, survival, and apoptosis were assessed, and the level of NF-κB/p65 or P-glycoprotein (P-gp) was quantitatively analyzed by enzyme-linked immunosorbent assay. HepG2 cells express NF-κB/p65 and express relatively less phosphorylated p65 (P-p65) and little P-gp. After treatment of HepG2 cells with different doses of doxorubicin, the expression of NF-κB/p65, P-p65, and especially P-gp were dose-dependently upregulated. After HepG2 cells were transfected with NF-κB/p65 siRNA (100 nmol/L), the expression of NF-κB/p65, P-p65, and P-gp were downregulated significantly and dose-dependently. The viability of HepG2 cells was decreased to 23% in the combination NF-κB/p65 siRNA (100 nmol/L) and doxorubicin (0.5 μmol/L) group and 47% in the doxorubicin (0.5 μmol/L) group (t = 7.043, P HepG2 cell growth by downregulating P-gp expression associated chemosensitization and apoptosis induction.

  19. Akbu-LAAO exhibits potent anti-tumor activity to HepG2 cells partially through produced H2O2 via TGF-β signal pathway.

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    Guo, Chunmei; Liu, Shuqing; Dong, Panpan; Zhao, Dongting; Wang, Chengyi; Tao, Zhiwei; Sun, Ming-Zhong

    2015-12-14

    Previously, we characterized the biological properties of Akbu-LAAO, a novel L-amino acid oxidase from Agkistrodon blomhoffii ussurensis snake venom (SV). Current work investigated its in vitro anti-tumor activity and underlying mechanism on HepG2 cells. Akbu-LAAO inhibited HepG2 growth time and dose-dependently with an IC50 of ~38.82 μg/mL. It could induce the apoptosis of HepG2 cells. Akbu-LAAO exhibited cytotoxicity by inhibiting growth and inducing apoptosis of HepG2 as it showed no effect on its cell cycle. The inhibition of Akbu-LAAO to HepG2 growth partially relied on enzymatic-released H2O2 as catalase only partially antagonized this effect. cDNA microarray results indicated TGF-β signaling pathway was linked to the cytotoxicity of Akbu-LAAO on HepG2. TGF-β pathway related molecules CYR61, p53, GDF15, TOB1, BTG2, BMP2, BMP6, SMAD9, JUN, JUNB, LOX, CCND1, CDK6, GADD45A, CDKN1A were deregulated in HepG2 following Akbu-LAAO stimulation. The presence of catalase only slightly restored the mRNA changes induced by Akbu-LAAO for differentially expressed genes. Meanwhile, LDN-193189, a TGF-β pathway inhibitor reduced Akbu-LAAO cytotoxicity on HepG2. Collectively, we reported, for the first time, SV-LAAO showed anti-tumor cell activity via TGF-β pathway. It provides new insight of SV-LAAO exhibiting anti-tumor effect via a novel signaling pathway.

  20. Sorafenib-irinotecan sequential therapy augmented the anti-tumor efficacy of monotherapy in hepatocellular carcinoma cells HepG2.

    Science.gov (United States)

    Wang, Z; Zhao, Z; Wu, T; Song, L; Zhang, Y

    2015-01-01

    The current study aimed to evaluate the efficacy of sorafenib-based combined therapy against hepatocellular carcinoma (HCC). HepG2 cells were exposed to sorafenib, irinotecan, and oxaliplatin and then subjected to MTT assay to determine chemosensitivity. Flow cytometry was used to examine cell cycle distribution and cell apoptosis. Levels of cleaved caspase-8, -3, and PARP were determined by Western blot. Real-time PCR and Western blot were used to determine p53 expression, respectively. The efficacy of combined therapy were verified in nude mice bearing HepG2 xenografts. HepG2 cells used in the current study were sensitive to sorafenib, irinotecan, and oxaliplatin. Sorafenib arrested cell cycle in S phase and the peak effect appeared at 30 h post treatment. Sorafenib exposure for 30 h followed by irinotecan exposure for 48 h synergistically induced cell apoptosis in HepG2 cells. On the other hand, sorafenib-oxaliplatin sequential exposure for the same time only acted an additive effect in soliciting cell apoptosis. Sorafenib and irinotecan sequential treatment significantly increased the levels of cleaved caspase-8, -3, and PARP in HepG2 cells. Sorafenib suppressed p53 expression at both mRNA and protein levels, which might contribute to cell cycle arrest and sensitize tumor cells to irinotecan. Sorafenib and irinotecan sequential therapy was obviously superior to monotherapy in suppressing the growth of HepG2 xenografts. Sorafenib-irinotecan sequential treatment augmented the efficacy of either drug used alone in soliciting HepG2 cells apoptosis in vitro and in suppressing the growth of HepG2 xenografts in vivo. hepatocellular carcinoma, irinotecan, sorafenib, synergistic effect.

  1. β3-Adrenoceptor activation upregulates apolipoprotein A-I expression in HepG2 cells, which might further promote cholesterol efflux from macrophage foam cells.

    Science.gov (United States)

    Gao, Xia-Qing; Li, Yan-Fang; Jiang, Zhi-Li

    2017-01-01

    The aim of this study was to explore the effects of β3-adrenoceptor (β3-AR) activation on HepG2 cells and its influence on cholesterol efflux from macrophage foam cells. HepG2 cells were cultured and treated with the β3-AR agonist, BRL37344, and antagonist, SR52390A, and the expression of apolipoprotein (Apo) A-I, ApoA-II, ApoB, and β3-AR in the supernatants and cells was determined. The expression of peroxisome proliferator-activated receptor (PPAR) γ and PPARα in the HepG2 cells was also assessed. Next, using the RAW264.7 macrophage foam cell model, we also assessed the influence of the HepG2 cell supernatants on lipid efflux. The cholesterol content of the foam cells was also measured, and the cholesterol efflux from the macrophages was examined by determining (3)H-labeled cholesterol levels. Expression of ATP-binding cassette transporter (ABC) A1 and ABCG1 of the macrophage foam cells was also assessed. β3-AR activation increased ApoA-I expression in both the HepG2 cells and the supernatants; PPARγ expression was upregulated, but PPARα expression was not. Treatment with GW9662 abolished the increased expression of ApoA-I induced by the β3-AR agonist. The HepG2 cell supernatants decreased the lipid accumulation and increased the cholesterol efflux from the macrophage foam cells. ABCA1 expression, but not ABCG1 expression, increased in the macrophage foam cells treated with BRL37344-treated HepG2 cell supernatants. Activation of β3-AR in HepG2 cells upregulates ApoA-I expression, which might further promote cholesterol efflux from macrophage foam cells. PPARγ might be required for the induction of ApoA-I expression.

  2. 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, *Pcomponents in TCM and SSb2 may be a valuable novel drug agent for the treatment of atherosclerosis.

  3. Exosomes derived from SW480 colorectal cancer cells promote cell migration in HepG2 hepatocellular cancer cells via the mitogen-activated protein kinase pathway.

    Science.gov (United States)

    Chiba, Mitsuru; Watanabe, Narumi; Watanabe, Miki; Sakamoto, Maki; Sato, Akika; Fujisaki, Mizuki; Kubota, Shiori; Monzen, Satoru; Maruyama, Atsushi; Nanashima, Naoki; Kashiwakura, Ikuo; Nakamura, Toshiya

    2016-01-01

    Exosomes are membrane-derived extracellular vesicles that have recently been recognized as important mediators of intercellular communication. In the present study, we investigated the effects of exosomes derived from SW480 colorectal cancer cells in recipient HepG2 hepatocellular cancer cells. We demonstrated that SW480-derived exosomes were taken up by the recipient HepG2 cells via dynamin-dependent endocytosis and were localized to the HepG2 lysosomes. In addition, SW480-derived exosomes induced the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 following their uptake into HepG2 cells. Of note, these changes occurred during the early phase after exosome treatment. Furthermore, SW480-derived exosomes promoted the migration of recipient HepG2 cells in a wound-healing assay, which was suppressed by pretreatment with U0126, an upstream inhibitor of ERK1/2. These results indicated that SW480-derived exosomes activated a classical mitogen-activated protein kinase pathway in recipient HepG2 cells via dynamin-dependent endocytosis and subsequently enhanced cell migration by ERK1/2 activation. Our results provide new insights into the regulation of cellular functions by exosomes.

  4. Cinnamomum osmophloeum Kanehira ethanol extracts prevents human liver-derived HepG2 cell death from oxidation stress by induction of ghrelin gene expression

    Indian Academy of Sciences (India)

    SHU-YING LIU; CHIH-HAO HUANG; JIA-CHING SHIEH; TAI-LIN LEE

    2017-09-01

    Diabetes patients associated with liver disease carry a significant risk of morbidity and mortality. Cinnamon has beenreported to reduce fructose-induced oxidative stress in the rat liver. However, the mechanism by which cinnamon protectsthe liver in a high-saccharide environment remains to be investigated. HepG2 cells were cultured with 30 mM D-ribose tomimic the high-oxidative-stress environment, typical of a liver in a diabetic patient. Three different chemical types of C.osmophloeum ethanol extracts (CEEs) were added in HepG2 culture media and the administration of all three CEEsprotected HepG2 cells from D-ribose damage and increased cell survival by approximately 20%. Exclusively, the transcriptvariant 1 of the ghrelin gene, but not variant 3, was 2–3 times induced by the addition of these CEEs. Moreover, themRNAs of ghrelin processing enzyme, furin, and mboat4 were detected in HepG2 cells. The ghrelin hormones in theculture media were increased 4–9 times by the addition of CEEs. The protective effects of ghrelin on HepG2 cells inD-ribose environment were further confirmed by recombinant ghrelin transfection. We conclude that the CEEs induceghrelin gene expression and protect HepG2 cells from D-ribose-induced oxidative damage through ghrelin signalling.

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

    Science.gov (United States)

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

    2016-07-07

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

  6. Tat-DJ-1 enhances cell survival by inhibition of oxidative stress, NF-κB and MAPK activation in HepG2 cells.

    Science.gov (United States)

    Jo, Hyo Sang; Yeo, Eun Ji; Shin, Min Jea; Choi, Yeon Joo; Yeo, Hyeon Ji; Cho, Su Bin; Park, Jung Hwan; Lee, Chi Hern; Eum, Won Sik; Choi, Soo Young

    2017-04-01

    To identify the protective effect of DJ-1 protein against oxidative stress-induced HepG2 cell death, we used cell-permeable wild type (WT) and a mutant (C106A Tat-DJ-1) protein. By using western blotting and fluorescence microscopy, we observed WT and C106A Tat-DJ-1 proteins were efficiently transduced into HepG2 cells. Transduced WT Tat-DJ-1 proteins increased cell survival and protected against DNA fragmentation and intracellular ROS generation levels in H2O2-exposed HepG2 cells. At the same time, transduced WT Tat-DJ-1 protein significantly inhibited NF-κB and MAPK (JNK and p38) activation as well as regulated the Bcl-2 and Bax expression levels. However, C106A Tat-DJ-1 protein did not show any protective effect against cell death responses in H2O2-exposed HepG2 cells. Oxidative stress-induced HepG2 cell death was significantly reduced by transduced WT Tat-DJ-1 protein, not by C106A Tat-DJ-1 protein. Thus, transduction of WT Tat-DJ-1 protein could be a novel strategy for promoting cell survival in situations of oxidative stress-induced HepG2 cell death.

  7. 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 interaction with the toxifying agent and an induction of repair mechanisms.O objetivo deste estudo foi a investigação de uma potente influência do extrato das folhas da alcachofra (ALE na fisiologia celular e na expressão gênica de enzimas de fase I/II de células hepáticas humanas HepG2 e investigação no potencial efeito protetor celular em células HepG2 contra toxicidade celular induzida por etanol. Ensaios biológicos de células em condições in vitro usando células de fígado HepG2 e investigação da atividade mitocondrial (teste MTT, ensaio de proliferação, LDH como marcador de toxicidade, análise de expressão gênica por RT-PCR e atividade da enzima glutationa transferase. O extrato da alcachofra, contendo 27% de ácidos cafeoilquínico e 7% de flavonóides, induzem a atividade mitocondrial, proliferação e o teor de proteína total em condições in vitro em células hepáticas humanas HepG2. Estes efeitos não podem ser correlacionados aos compostos secundários conhecidos da alcachofra, cinarina, ácido cafeico, ácido clorogênico, luteolina e luteolin-7-O-glicosídeo. As flavonas luteolina e luteolin-7-O-glicosídeo possuem efeitos inibitórios em nível de 100 µg/mL em células HepG2, com a luteonina sendo uma inibidora significativamente mais forte comparada com o respectivo glicosídeo. O extrato das folhas de alcachofra possui um efeito mínimo da estimulação na expressão gênica de CYP1A2, enquanto CYP3A4, GGT, GPX2, GSR e GST foram sutilmente inibidos. A inibição de GST em condições in vitro também foi mostrada pela quantificação da atividade da enzima GST. Indução da expressão gênica de CYP1A2 mostrou-se supraaditiva após aplicação simultânea do etanol mais o extrato de alcachofra. O extrato das folhas de alcachofra exibiu efeitos protetores celulares frente à toxicidade induzida por etanol em co-tratamento em condições in vitro. Além disso, danos por H2O2 foram significativamente inibidos pela incubação simultânea do extrato de alcachofra. Pré e pós-tratamento não exerceram efeitos protetores. Toxicidade induzida por DMSO foi significativamente reduzida por pré, pós e co-tratamento com extrato de alcachofra e especialmente com luteína-7-O-glicosídeo, indicando uma interação direta com o agente toxicante e a indução dos mecanismos de reparo.

  8. Solid lipid nanoparticles for hydrophilic biotech drugs: optimization and cell viability studies (Caco-2 & HEPG-2 cell lines).

    Science.gov (United States)

    Severino, Patrícia; Andreani, Tatiana; Jäger, Alessandro; Chaud, Marco V; Santana, Maria Helena A; Silva, Amélia M; Souto, Eliana B

    2014-06-23

    Insulin was used as model protein to developed innovative Solid Lipid Nanoparticles (SLNs) for the delivery of hydrophilic biotech drugs, with potential use in medicinal chemistry. SLNs were prepared by double emulsion with the purpose of promoting stability and enhancing the protein bioavailability. Softisan(®)100 was selected as solid lipid matrix. The surfactants (Tween(®)80, Span(®)80 and Lipoid(®)S75) and insulin were chosen applying a 2(2) factorial design with triplicate of central point, evaluating the influence of dependents variables as polydispersity index (PI), mean particle size (z-AVE), zeta potential (ZP) and encapsulation efficiency (EE) by factorial design using the ANOVA test. Therefore, thermodynamic stability, polymorphism and matrix crystallinity were checked by Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD), whereas the effect of toxicity of SLNs was check in HepG2 and Caco-2 cells. Results showed a mean particle size (z-AVE) width between 294.6 nm and 627.0 nm, a PI in the range of 0.425-0.750, ZP about -3 mV, and the EE between 38.39% and 81.20%. After tempering the bulk lipid (mimicking the end process of production), the lipid showed amorphous characteristics, with a melting point of ca. 30 °C. The toxicity of SLNs was evaluated in two distinct cell lines (HEPG-2 and Caco-2), showing to be dependent on the concentration of particles in HEPG-2 cells, while no toxicity in was reported in Caco-2 cells. SLNs were stable for 24 h in in vitro human serum albumin (HSA) solution. The resulting SLNs fabricated by double emulsion may provide a promising approach for administration of protein therapeutics and antigens.

  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. Safrole-2',3'-oxide induces cytotoxic and genotoxic effects in HepG2 cells and in mice.

    Science.gov (United States)

    Chiang, Su-yin; Lee, Pei-yi; Lai, Ming-tsung; Shen, Li-ching; Chung, Wen-sheng; Huang, Hui-fen; Wu, Kuen-yuh; Wu, Hsiu-ching

    2011-12-24

    Safrole-2',3'-oxide (SAFO) is a reactive electrophilic metabolite of the hepatocarcinogen safrole, the main component of sassafras oil. Safrole occurs naturally in a variety of spices and herbs, including the commonly used Chinese medicine Xi xin (Asari Radix et Rhizoma) and Dong quai (Angelica sinensis). SAFO is the most mutagenic metabolite of safrole tested in the Ames test. However, little or no data are available on the genotoxicity of SAFO in mammalian systems. In this study, we investigated the cytotoxicity and genotoxicity of SAFO in human HepG2 cells and male FVB mice. Using MTT assay, SAFO exhibited a dose- and time-dependent cytotoxic effect in HepG2 cells with TC(50) values of 361.9μM and 193.2μM after 24 and 48h exposure, respectively. In addition, treatment with SAFO at doses of 125μM and higher for 24h in HepG2 cells resulted in a 5.1-79.6-fold increase in mean Comet tail moment by the alkaline Comet assay and a 2.6-7.8-fold increase in the frequency of micronucleated binucleated cells by the cytokinesis-block micronucleus assay. Furthermore, repeated intraperitoneal administration of SAFO (15, 30, 45, and 60mg/kg) to mice every other day for a total of twelve doses caused a significant dose-dependent increase in mean Comet tail moment in peripheral blood leukocytes (13.3-43.4-fold) and in the frequency of micronucleated reticulocytes (1.5-5.8-fold). Repeated administration of SAFO (60mg/kg) to mice caused liver lesions manifested as a rim of ballooning degeneration of hepatocytes immediately surrounding the central vein. Our data clearly demonstrate that SAFO significantly induced cytotoxicity, DNA strand breaks, micronuclei formation both in human cells in vitro and in mice. More studies are needed to explore the role SAFO plays in safrole-induced genotoxicity.

  11. 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; Choe, Myeon

    2017-06-01

    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. 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. 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. 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.

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

    Science.gov (United States)

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

    2009-08-01

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

  13. Restoration of miR-20a expression suppresses cell proliferation, migration, and invasion in HepG2 cells.

    Science.gov (United States)

    Chen, Guang Shun; Zhou, Ning; Li, Jie-Qun; Li, Ting; Zhang, Zhong-Qiang; Si, Zhong-Zhou

    2016-01-01

    To study microRNA (miR)-20a expression in hepatocellular carcinoma (HCC) and its effects on the proliferation, migration, and invasion of HepG2. The real-time polymerase chain reaction was used to detect the expression of miR-20a in HCC tissue and normal tissue, as well as in HCC cell lines and normal liver cells. miR-20a mimic and miR negative control (NC) were transfected into HepG2 cells. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay was used to detect cell proliferation. Annexin fluorescein isothiocyanate/propidium iodide assay was run to examine the early apoptosis of cells. Transwell chamber assay was carried out to investigate the cell invasion and migration abilities. miR-20a was lowly expressed both in HCC tissues and HCC cell lines. After transfection of exogenous miR-20 mimics, miR-20a expression in HepG2 cells was significantly increased by 61.29% compared to the blank group (PHepG2 cells in the miR-20a mimics group was significantly inhibited, and optical density values during the 36-96 hour time period were dramatically decreased compared to the blank group (PHepG2 cells, the number of cell migration and invasion in the small interfering (si)-CCND1 group were 0.444 and 0.435 times that of the si-NC group (PHepG2 cells, and is therefore promising as a new molecular target for diagnosis and therapy of HCC.

  14. Low concentration of ethanol induce apoptosis in HepG2 cells: role of various signal transduction pathways

    Directory of Open Access Journals (Sweden)

    Francisco Castaneda, Sigrid Rosin-Steiner

    2006-01-01

    Full Text Available As we previously demonstrated in human hepatocellular carcinoma (HepG2 cells, ethanol at low concentration triggers the Fas apoptotic pathway. However, its role in other intracellular signaling pathways remains unknown. Therefore, the aim of the present study was to evaluate the role of low concentration of ethanol on different intracellular signaling pathways. For this purpose, HepG2 cells were treated with 1 mM ethanol for 10 min and the phosphorylation state of protein kinases was determined. In addition, the mRNA levels of transcription factors and genes associated with the Fas apoptotic pathway were determined. Our data demonstrated that ethanol-induced phosphorylation of protein kinases modulates both anti-apoptotic and pro-apoptotic mechanisms in HepG2 cells. Pro-apoptosis resulted mainly from the strong inhibition of the G-protein couple receptor signaling pathway. Moreover, the signal transduction initiated by ethanol-induced protein kinases phosphorylation lead to increased expression of the transcription factors with subsequent expression of genes associated with the Fas apoptotic pathway (Fas receptor, Fas ligand, FADD and caspase 8. These results indicate that low concentration of ethanol exert their effect by predominant activation of pro-apoptotic events that can be divided in two phases. An early phase characterized by a rapid transient effect on protein kinases phosphorylation, after 10 min exposure, with subsequent increased expression of transcription factors for up to 6 hr. This early phase is followed by a second phase associated with increased gene expression that began after 6 hr and persisted for more than 24 hr. This information provided a novel insight into the mechanisms of action of ethanol (1mM in human hepatocellular carcinoma cells.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. 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.

  17. Ursolic acid sensitizes cisplatin-resistant HepG2/DDP cells to cisplatin via inhibiting Nrf2/ARE pathway

    Directory of Open Access Journals (Sweden)

    Wu S

    2016-10-01

    Full Text Available Shouhai Wu,1,2 Tianpeng Zhang,1 Jingsheng Du3 1School of Life Sciences, Sun Yat-sen University, 2Center for Regenerative and Translational Medicine, 3Department of Pharmacy, The Second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, People’s Republic of China Background: Combinations of adjuvant sensitizers with anticancer drugs is a promising new strategy to reverse chemoresistance. Ursolic acid (UA is one of the natural pentacyclic triterpene compounds known to have many pharmacological characteristics such as anti-inflammatory and anticancer properties. This study investigates whether UA can sensitize hepatocellular carcinoma cells to cisplatin.Materials and methods: Cells were transfected with nuclear factor erythroid-2-related factor 2 (Nrf2 small interfering RNA and Nrf2 complementary DNA by using Lipofectin 2000. The cytotoxicity of cells was investigated by Cell Counting Kit 8 assay. Cell apoptosis, cell cycle, reactive oxygen species, and mitochondrial membrane potential were detected by flow cytometry fluorescence-activated cell sorting. The protein level of Nrf2, NAD(PH quinone oxidoreductase 1 (NQO1, glutathione S-transferase (GST, and heme oxygenase-1 (HO-1 was detected by Western blot analysis.Results: The results showed that the reverse index was 2.9- and 9.69-fold by UA of 1.125 µg/mL and 2.25 µg/mL, respectively, for cisplatin to HepG2/DDP cells. UA–cisplatin combination induced cell apoptosis and reactive oxygen species, blocked the cell cycle in G0/G1 phase, and reduced the mitochondrial membrane potential. Mechanistically, UA–cisplatin dramatically decreased the expression of Nrf2 and its downstream genes. The sensibilization of UA–cisplatin combination was diminished in Nrf2 small interfering RNA-transfected HepG2/DDP cells, as well as in Nrf2 complementary DNA-transfected HepG2/DDP cells.Conclusion: The results confirmed the sensibilization of UA on HepG2/DDP cells to cisplatin, which was possibly mediated via the Nrf2/antioxidant response element pathway. Keywords: ursolic acid, chemoresistance, cisplatin, liver cancer, Nrf2/ARE

  18. Galangin Induces Autophagy via Deacetylation of LC3 by SIRT1 in HepG2 Cells

    Science.gov (United States)

    Li, Xv; Wang, Yajun; Xiong, Yuzhen; Wu, Jun; Ding, Hang; Chen, Xiaoyi; Lan, Liubo; Zhang, Haitao

    2016-01-01

    Galangin suppresses proliferation and induces apoptosis and autophagy in hepatocellular carcinoma (HCC) cells, but the precise mechanism is not clear. In this study, we demonstrated that galangin induced autophagy, enhanced the binding of SIRT1-LC3 and reduced the acetylation of endogenous LC3 in HepG2 cells. But this autophagy was inhibited by inactivation of SIRT1 meanwhile, galangin failed to reduce the acetylation of endogenous LC3 after SIRT1 was knocked-down. Collectively, these findings demonstrate a new mechanism by which galangin induces autophagy via the deacetylation of endogenous LC3 by SIRT1. PMID:27460655

  19. Comparative cytotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells in culture.

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    Sahu, Saura C; Zheng, Jiwen; Graham, Lesley; Chen, Lynn; Ihrie, John; Yourick, Jeffrey J; Sprando, Robert L

    2014-11-01

    The use of silver nanoparticles in food, food contact materials, dietary supplements and cosmetics has increased significantly owing to their antibacterial and antifungal properties. As a consequence, the need for validated rapid screening methods to assess their toxicity is necessary to ensure consumer safety. This study evaluated two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, as tools for assessing the potential cytotoxicity of food- and cosmetic-related nanoparticles. The two cell culture models were utilized to compare the potential cytotoxicity of 20-nm silver. The average size of the silver nanoparticle determined by our transmission electron microscopy (TEM) analysis was 20.4 nm. The dynamic light scattering (DLS) analysis showed no large agglomeration of the silver nanoparticles. The concentration of the 20-nm silver solution determined by our inductively coupled plasma-mass spectrometry (ICP-MS) analysis was 0.962 mg ml(-1) . Our ICP-MS and TEM analysis demonstrated the uptake of 20-nm silver by both HepG2 and Caco2 cells. Cytotoxicity, determined by the Alamar Blue reduction assay, was evaluated in the nanosilver concentration range of 0.1 to 20 µg ml(-1) . Significant concentration-dependent cytotoxicity of the nanosilver in HepG2 cells was observed in the concentration range of 1 to 20 µg ml(-1) and at a higher concentration range of 10 to 20 µg ml(-1) in Caco2 cells compared with the vehicle control. A concentration-dependent decrease in dsDNA content was observed in both cell types exposed to nanosilver but not controls, suggesting an increase in DNA damage. The DNA damage was observed in the concentration range of 1 to 20 µg ml(-1) . Nanosilver-exposed HepG2 and Caco2 cells showed no cellular oxidative stress, determined by the dichlorofluorescein assay, compared with the vehicle control in the concentration range used in this study. A concentration-dependent decrease in mitochondria membrane potential in both nanosilver exposed cell types suggested increased mitochondria injury compared with the vehicle control. The mitochondrial injury in HepG2 cells was significant in the concentration range of 1 to 20 µg ml(-1) , but in Caco2 cells it was significant at a higher concentration range of 10 to 20 µg ml(-1) . These results indicated that HepG2 cells were more sensitive to nanosilver exposure than Caco2 cells. It is generally believed that cellular oxidative stress induces cytotoxicity of nanoparticles. However, in this study we did not detect any nanosilver-induced oxidative stress in either cell type at the concentration range used in this study. Our results suggest that cellular oxidative stress did not play a major role in the observed cytotoxicity of nanosilver in HepG2 and Caco2 cells and that a different mechanism of nanosilver-induced mitochondrial injury leads to the cytotoxicity. The HepG2 and Caco2 cells used this study appear to be targets for silver nanoparticles. The results of this study suggest that the differences in the mechanisms of toxicity induced by nanosilver may be largely as a consequence of the type of cells used. This differential rather than universal response of different cell types exposed to nanoparticles may play an important role in the mechanism of their toxicity. In summary, the results of this study indicate that the widely used in vitro models, HepG2 and Caco2 cells in culture, are excellent systems for screening cytotoxicity of silver nanoparticles. These long established cell culture models and simple assays used in this study can provide useful toxicity and mechanistic information that can help to better inform safety assessments of food- and cosmetic-related silver nanoparticles. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

  20. 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.

  1. TALEN-induced disruption of Nanog expression results in reduced proliferation, invasiveness and migration, increased chemosensitivity and reversal of EMT in HepG2 cells.

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    Yu, Ai Qing; Ding, Yan; Li, Cheng Lin; Yang, Yi; Yan, Shi Rong; Li, Dong Sheng

    2016-03-01

    Accumulating evidence indicates that Nanog plays a central role in modulating the biological behaviors of human hepatocellular carcinoma (HCC). However, the underlying mechanisms remain unclear. In the present study, we employed transcription activator-like effector nucleases (TALEN) to disrupt Nanog expression in HepG2 cells and obtained subcloned cells with diallelic Nanog mutations. Significantly, we found that the expression of pluripotency factors Sox2, Oct4 and Klf4, as well as expression of cancer stem cell (CSC) marker CD133, in the Nanog-targeted HepG2 cells was markedly downregulated. This finding suggests that Nanog may play an important role in maintaining the pluripotency and malignancy of HepG2 cells. We also revealed that Nanog regulated cell proliferation by modulating the expression of cyclin D1/D3/E1 and CDK2, respectively. Additionally, the disruption of Nanog resulted in the downregulation of epithelial-mesenchymal transition (EMT) regulators Snail and Twist, which contributed to the elevated level of epithelial marker E-cadherin, and to the decreased level of mesenchymal markers N-cadherin and vimentin in the HepG2 cells. In addition, the Nanog-targeted HepG2 cells exhibited reduced ability of invasion, migration and chemoresistance in vitro. In conclusion, the disruption of Nanog expression results in less proliferation, invasiveness, migration, more chemosensitivity and reversal of EMT in HepG2 cells, by which Nanog plays crucial roles in influencing the malignant phenotype of HepG2 cells.

  2. Activation of AMP-activated protein kinase and phosphorylation of glycogen synthase kinase3 β mediate ursolic acid induced apoptosis in HepG2 liver cancer cells.

    Science.gov (United States)

    Son, Hyun-Soo; Kwon, Hee Young; Sohn, Eun Jung; Lee, Jang-Hoon; Woo, Hong-Jung; Yun, Miyong; Kim, Sung-Hoon; Kim, Young-Chul

    2013-11-01

    Despite the antitumour effect of ursolic acid observed in several cancers, the underlying mechanism remains unclear. Thus, in the present study, the roles of AMP-activated protein kinase (AMPK) and glycogen synthase kinase 3 beta (GSK3β) were examined in ursolic acid induced apoptosis in HepG2 hepatocellular carcinoma cells. Ursolic acid significantly exerted cytotoxicity, increased the sub-G1 population and the number of ethidium homodimer and terminal deoxynucleotidyl transferase(TdT) mediated dUTP nick end labeling positive cells in HepG2 cells. Also, ursolic acid enhanced the cleavages of poly-ADP-ribose polymerase (PARP) and caspase3, attenuated the expression of astrocyte elevated gene (AEG1) and survivin in HepG2 cells. Interestingly, ursolic acid increased the phosphorylation of AMPK and coenzyme A carboxylase and also enhanced phosphorylation of GSK3β at inactive form serine 9, whereas ursolic acid attenuated the phosphorylation of AKT and mTOR in HepG2 cells. Conversely, AMPK inhibitor compound C or GSK3β inhibitor SB216763 blocked the cleavages of PARP and caspase 3 induced by ursolic acid in HepG2 cells. Furthermore, proteosomal inhibitor MG132 suppressed AMPK activation, GSK3β phosphorylation, cleaved PARP and deceased AEG-1 induced by ursolic acid in HepG2 cells. Overall, our findings suggest that ursolic acid induced apoptosis in HepG2 cells via AMPK activation and GSK3β phosphorylation as a potent chemopreventive agent. Copyright © 2013 John Wiley & Sons, Ltd.

  3. Differential effects of c-myc and ABCB1 silencing on reversing drug resistance in HepG2/Dox cells.

    Science.gov (United States)

    Yahya, Shaymaa M M; Hamed, Ahmed R; Emara, Mohamed; Soltan, Maha M; Abd-Ellatef, Gamal Eldein F; Abdelnasser, Salma M

    2016-05-01

    Multidrug resistance (MDR) in various kinds of cancers represents a true obstacle which hinders the successes of most of current available chemotherapies. ATP-binding cassette (ABC) trasporter proteins have been shown to contribute to the majority of MDR in various types of malignancies. c-myc has recently been reported to participate, at least partly, in MDR to some types of cancers. This study aimed to test whether c-myc could play a role, solely or with coordination with other ABCs, in the resistance of HepG2 cells to doxorubicin (Dox). MDR has been induced in wild-type HepG2 and has been verified both on gene and protein levels. Various assays including efflux assays as well as siRNA targeting ABCB1 and c-myc have been employed to explore the role of both candidate molecules in MDR in HepG2. Results obtained, with regard to ABCB1 silencing on HepG2/Dox cells, have shown that ABCB1-deficient cells exhibited a significant reduction in ABCC1 expression as compared to ABCB1-sufficient cells. However, these cells did not show a significant reduction in other tested ABCs (ABCC5 and ABCC10) while c-myc silencing had no significant effect on any of the studied ABCs. Moreover, silencing of ABCB1 on HepG2 significantly increased fluorescent calcein retention in HepG2 cells as compared to the control cells while downregulation of c-myc did not have any effect on fluorescent calcein retention. Altogether, this work clearly demonstrates that c-myc has no role in MDR of HepG2 to Dox which has been shown to be ABCB1-mediated in a mechanism which might involve ABCC1.

  4. Dose-Dependent Cytotoxic Effects of Boldine in HepG-2 Cells—Telomerase Inhibition and Apoptosis Induction

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    Sakineh Kazemi Noureini

    2015-02-01

    Full Text Available Plant metabolites are valuable sources of novel therapeutic compounds. In an anti-telomerase screening study of plant secondary metabolites, the aporphine alkaloid boldine (1,10-dimethoxy-2,9-dihydroxyaporphine exhibited a dose and time dependent cytotoxicity against hepatocarcinoma HepG-2 cells. Here we focus on the modes and mechanisms of the growth-limiting effects of this compound. Telomerase activity and expression level of some related genes were estimated by real-time PCR. Modes of cell death also were examined by microscopic inspection, staining methods and by evaluating the expression level of some critically relevant genes. The growth inhibition was correlated with down-regulation of the catalytic subunit of telomerase (hTERT gene (p < 0.01 and the corresponding reduction of telomerase activity in sub-cytotoxic concentrations of boldine (p < 0.002. However, various modes of cell death were stimulated, depending on the concentration of boldine. Very low concentrations of boldine over a few passages resulted in an accumulation of senescent cells so that HepG-2 cells lost their immortality. Moreover, boldine induced apoptosis concomitantly with increasing the expression of bax/bcl2 (p < 0.02 and p21 (p < 0.01 genes. Boldine might thus be an interesting candidate as a potential natural compound that suppresses telomerase activity in non-toxic concentrations.

  5. Apoptosis-inducing effects of extracts from desert plants in HepG2 human hepatocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Deepak; Bhatia; Animesh; Mandal; Eviatar; Nevo; Anupam; Bishayee

    2015-01-01

    Objective:To investigat the mechanism of antitumor efficacy of Origanum clayi(O.clayi) and Ochradenus baccatus(O.baccatus) extracts by exploring apoptosis-inducing potential.Methods:The aqueous extracts of aerial parts of aforementioned plants were prepared and used for this study.HepG2 cells were treated with varying concentrations(0,2 and 5 mg/mL)of each plant extract for 24 or 48 h.Cell apoptosis was measured by annexin V-fluorescein isothiocyanate binding assay and flow cytometry.The expression levels of various apoptosisrelated genes were determined by semi-quantitative reverse transcription-polymerase chain reaction.Results:O.clayi and O.baccatus extracts exerted apoptotic effects on HepG2 cells for 48 h following treatment.O.clayi extract was found to be a better apoptosis-inducing agent than O.baccatus extract as the former delivered greater efficacy at a lower concentration.Both extracts manifested upregulation of Bax,Bad.cytochrome c.caspase-3,caspase-7.caspase-9 and poly(adenosine diphosphate-ribose) polymerase.Conclusions:The aqueous extracts of O.clayi and O.baccatus are capable of inducing apoptosis in HepG2 cells through modulation of mitochondrial pathway which explains their antitumor activities.These desert plants may serve as useful resources to develop effective remedies for hepatocellular carcinoma and other human malignancies.

  6. Interaction between caspase-8 activation and endoplasmic reticulum stress in glycochenodeoxycholic acid-induced apoptotic HepG2 cells.

    Science.gov (United States)

    Iizaka, Toru; Tsuji, Mayumi; Oyamada, Hideto; Morio, Yuri; Oguchi, Katsuji

    2007-11-30

    The accumulation of hydrophobic bile acid, such as glycochenodeoxycholic acid (GCDCA), in the liver has been thought to induce hepatocellular damage in human chronic cholestatic liver diseases. We previously reported that GCDCA-induced apoptosis was promoted by both mitochondria-mediated and endoplasmic reticulum (ER) stress-associated pathways in rat hepatocytes. In this study, we elucidated the relationship between these pathways in GCDCA-induced apoptotic HepG2 cells. HepG2 cells were treated with GCDCA (100-500microM) with or without a caspase-8 inhibitor, Z-IETD-fluoromethyl ketone (Z-IETD-FMK) (30microM) for 3-24h. We demonstrated the presence of both apoptotic pathways in these cells; that is, we showed increases in cleaved caspase-3 proteins, the release of cytochrome c from mitochondria, and the expression of ER resident molecular chaperone Bip mRNA and ER stress response-associated transcription factor Chop mRNA. On the other hand, pretreatment with Z-IETD-FMK significantly reduced the increases, compared with treatment with GCDCA alone. Immunofluorescence microscopic analysis showed that treatment with GCDCA increased the cleavage of BAP31, an integral membrane protein of ER, and pretreatment with Z-IETD-FMK suppressed the increase of caspase-8 and BAP31 cleavage. In conclusion, these results suggest that intact activated caspase-8 may promote and amplify the ER stress response by cleaving BAP31 in GCDCA-induced apoptotic cells.

  7. Synthesis and anticancer activity of novel benzimidazole and benzothiazole derivatives against HepG2 liver cancer cells.

    Science.gov (United States)

    Youssef, Amal M; Malki, Ahmed; Badr, Mona H; Elbayaa, Rasha Y; Sultan, Ahmed S

    2012-03-01

    Most of cancer chemotherapeutics and chemopreventives exert their effects by triggering apoptotic cell death. In this study, novel benzimidazole and benzothiazole derivatives have been synthesized to investigate their effects on HepG2 liver cancer cell lines after initial screening study. A dose response curve was constructed and the most active derivatives were further studied for apoptotic analysis. Six active benzimidazole derivatives (8, 9, 10, 12, 13 and 14) significantly induced apoptosis compared to control group. Two compounds 10 and 12 induced apoptosis by arresting cells in G1 phase of cell cycle which is confirmed by increased expression level of p21. The activity of caspase-3 which is well known as one of the key executioners of apoptosis was determined in the presence and absence of the tested derivatives. Our results indicated that compounds 10 and 12 significantly increased caspase-3 activity compared to control group. Moreover, a docked pose of compounds 10 and 12 was obtained bound to caspase-3 active site using Molecular Operating Environment module. This study demonstrated that benzimidazole derivatives 10 and 12 provoke cytotoxicity and induced apoptosis in liver cancer cells HepG2.

  8. Walnut-enriched diet increases the association of LDL from hypercholesterolemic men with human HepG2 cells.

    Science.gov (United States)

    Muñoz, S; Merlos, M; Zambón, D; Rodríguez, C; Sabaté, J; Ros, E; Laguna, J C

    2001-12-01

    In a randomized, cross-over feeding trial involving 10 men with polygenic hypercholesterolemia, a control, Mediterranean-type cholesterol-lowering diet, and a diet of similar composition in which walnuts replaced approximately 35% of energy from unsaturated fat, were given for 6 weeks each. Compared with the control diet, the walnut diet reduced serum total and LDL cholesterol by 4.2% (P = 0.176), and 6.0% (P = 0.087), respectively. No changes were observed in HDL cholesterol, triglycerides, and apolipoprotein A-I levels or in the relative proportion of protein, triglycerides, phospholipids, and cholesteryl esters in LDL particles. The apolipoprotein B level declined in parallel with LDL cholesterol (6.0% reduction). Whole LDL, particularly the triglyceride fraction, was enriched in polyunsaturated fatty acids from walnuts (linoleic and alpha-linolenic acids). In comparison with LDL obtained during the control diet, LDL obtained during the walnut diet showed a 50% increase in association rates to the LDL receptor in human hepatoma HepG2 cells. LDL uptake by HepG2 cells was correlated with alpha-linolenic acid content of the triglyceride plus cholesteryl ester fractions of LDL particles (r(2) = 0.42, P < 0.05). Changes in the quantity and quality of LDL lipid fatty acids after a walnut-enriched diet facilitate receptor-mediated LDL clearance and may contribute to the cholesterol-lowering effect of walnut consumption.

  9. Secretory Transactivating Transcription-apoptin fusion protein induces apoptosis in hepatocellular carcinoma HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Su-Xia Han; Jin-Lu Ma; Yi Lv; Chen Huang; Hai-Hua Liang; Kang-Min Duan

    2008-01-01

    AIM: To determine whether SP-TAT-apoptin induces apoptosis and also maintains its tumor cell specificity.METHODS: In this study, we designed a secretory protein by adding a secretory signal peptide (SP) to the N terminus of Transactivating Transcription (TAT)-apoptin (SP-TAT-apoptin), to test the hypothesis that it gains an additive bystander effect as an anti-cancer therapy. We used an artificial human secretory SP whose amino acid sequence and corresponding cDNA sequence were generated by the SP hidden Markov model.RESULTS: In human liver carcinoma HepG2 cells, SP-TAT-apoptin expression showed a diffuse pattern in the early phase after transfection. After 48h, however, it translocated into the nuclear compartment and caused massive apoptotic cell death, as determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and annexin-V binding assay. SP-TAT-apoptin did not, however, cause any cell death in non-malignant human umbilical vein endothelial cells (HUVECs). Most importantly, the conditioned medium from Chinese hamster ovary (CHO) cells transfected with SP-TAT-apoptin also induced significant cell death in HepG2 cells, but not in HUVECs.CONCLUSION: The data demonstrated that SP-TAT-apoptin induces apoptosis only in malignant cells, and its secretory property might greatly increase its potency once it is delivered in vivo for cancer therapy.

  10. Cichoric Acid Reverses Insulin Resistance and Suppresses Inflammatory Responses in the Glucosamine-Induced HepG2 Cells.

    Science.gov (United States)

    Zhu, Di; Wang, Yutang; Du, Qingwei; Liu, Zhigang; Liu, Xuebo

    2015-12-30

    Cichoric acid, a caffeic acid derivative found in Echinacea purpurea, basil, and chicory, has been reported to have bioactive effects, such as anti-inflammatory, antioxidant, and preventing insulin resistance. In this study, to explore the effects of CA on regulating insulin resistance and chronic inflammatory responses, the insulin resistance model was constructed by glucosamine in HepG2 cells. CA stimulated glucosamine-mediated glucose uptake by stimulating translocation of the glucose transporter 2. Moreover, the production of reactive oxygen, the expression of COX-2 and iNOS, and the mRNA levels of TNF-α and IL-6 were attenuated. Furthermore, CA was verified to promote glucosamine-mediated glucose uptake and inhibited inflammation through PI3K/Akt, NF-κB, and MAPK signaling pathways in HepG2 cells. These results implied that CA could increase glucose uptake, improve insulin resistance, and attenuate glucosamine-induced inflammation, suggesting that CA is a potential natural nutraceutical with antidiabetic properties and anti-inflammatory effects.

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

    Science.gov (United States)

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

    2007-12-01

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

  12. 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.

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

    Science.gov (United States)

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

    2013-01-01

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

  14. Effect of ethanolic extracts of Ananas comosus L. leaves on insulin sensitivity in rats and HepG2.

    Science.gov (United States)

    Xie, Weidong; Wang, Wei; Su, Hui; Xing, Dongming; Pan, Yang; Du, Lijun

    2006-08-01

    Ethanolic extracts of Ananas comosus L. leaves (AC) enriched with phenols have hypoglycemic activity in diabetic rats. Here, we investigated the effect of AC on insulin sensitivity in rats and HepG2. In high-fat diet-fed and low-dose streptozotozin-treated diabetic Wistar rats subjected to challenge with exogenous human insulin, AC treatment at an oral dose of 0.40 g/kg could significantly improve sensitivity to exogenous insulin. After a sub-acute treatment, AC also could inhibit the development of insulin resistance in high-fat diet-fed and low-dose streptozotozin-treated diabetic rats following the test of loss of tolbutamide-induced blood glucose lowering action. For intravenous insulin/glucose infusion test, high-fat diet-fed and low-dose alloxan-treated Wistar rats were associated with insulin resistance, which was improved after AC or fenofibrate treatment. AC application inhibited the development of insulin resistance in HepG2 cells. The above animal models were well developed to simulate type 2 diabetes. Taken together, our results suggest that AC may improve insulin sensitivity in type 2 diabetes and could be developed into a new potential natural product for handling of insulin resistance in diabetic patients.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Cytoprotective and antigenotoxic potential of Mangiferin, a glucosylxanthone against cadmium chloride induced toxicity in HepG2 cells.

    Science.gov (United States)

    Satish Rao, B S; Sreedevi, M V; Nageshwar Rao, B

    2009-03-01

    Mangiferin (MGN), a glucosylxanthone present in large amounts in the leaves and edible mango fruits of Mangifera indica. Here, we report about MGN's potential for mitigating cadmium chloride (CdCl(2)) induced cytotoxic and genotoxic effects in HepG2 cells growing in vitro. The cytoprotective potential was assessed by MTT, clonogenic and apoptotic assays, while antigenotoxic effect by micronucleus and comet assay. The established cytotoxic and genotoxic effects were well indicated after CdCl(2) treatment and was mitigated by pretreatment with MGN. MGN prior to CdCl(2) treatment increased the cell survival (MTT), surviving fraction (clonogenic assay) and inhibited sub-G(1) population (flow cytometric analysis). Further, inhibition of CdCl(2) induced apoptotic cell death by MGN was confirmed by microscopic and DNA fragmentation assays. A significant (pinhibited significantly (p<0.001) by MGN. Taken together, our study revealed that MGN has potent cytoprotective and antigenotoxic effect against CdCl(2) induced toxicity in HepG2 cell line and which may be attributed to decrease in CdCl(2) induced reactive oxygen species levels and resultant oxidative stress.

  17. 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.

  18. Pfaffosidic Fraction from Hebanthe paniculata Induces Cell Cycle Arrest and Caspase-3-Induced Apoptosis in HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Tereza Cristina da Silva

    2015-01-01

    Full Text Available Hebanthe paniculata roots (formerly Pfaffia paniculata and popularly known as Brazilian ginseng show antineoplastic, chemopreventive, and antiproliferative properties. Functional properties of these roots and their extracts are usually attributed to the pfaffosidic fraction, which is composed mainly by pfaffosides A–F. However, the therapeutic potential of this fraction in cancer cells is not yet entirely understood. This study aimed to analyze the antitumoral effects of the purified pfaffosidic fraction or saponinic fraction on the human hepatocellular carcinoma HepG2 cell line. Cellular viability, proliferation, and apoptosis were evaluated, respectively, by MTT assay, BrdU incorporation, activated caspase-3 immunocytochemistry, and DNA fragmentation assay. Cell cycle was analyzed by flow cytometry and the cell cycle-related proteins were analyzed by quantitative PCR and Western blot. The cells exposed to pfaffosidic fraction had reduced viability and cellular growth, induced G2/M at 48 h or S at 72 h arrest, and increased sub-G1 cell population via cyclin E downregulation, p27KIP1 overexpression, and caspase-3-induced apoptosis, without affecting the DNA integrity. Antitumoral effects of pfaffosidic fraction from H. paniculata in HepG2 cells originated by multimechanisms of action might be associated with cell cycle arrest in the S phase, by CDK2 and cyclin E downregulation and p27KIP1 overexpression, besides induction of apoptosis through caspase-3 activation.

  19. Alkaloids from beach spider lily (Hymenocallis littoralis) induce apoptosis of HepG-2 cells by the fas-signaling pathway.

    Science.gov (United States)

    Ji, Yu-Bin; Chen, Ning; Zhu, Hong-Wei; Ling, Na; Li, Wen-Lan; Song, Dong-Xue; Gao, Shi-Yong; Zhang, Wang-Cheng; Ma, Nan-Nan

    2014-01-01

    Alkaloids are the most extensively featured compounds of natural anti-tumor herbs, which have attracted much attention in pharmaceutical research. In our previous studies, a mixture of major three alkaloid components (5, 6-dihydrobicolorine, 7-deoxy-trans-dihydronarciclasine, littoraline) from Hymenocallis littoralis were extracted, analyzed and designated as AHL. In this paper, AHL extracts were added to human liver hepatocellular cells HepG-2, human gastric cancer cell SGC-7901, human breast adenocarcinoma cell MCF-7 and human umbilical vein endothelial cell EVC-304, to screen one or more AHL-sensitive tumor cell. Among these cells, HepG-2 was the most sensitive to AHL treatment, a very low dose (0.8μg/ml) significantly inhibiting proliferation . The non- tumor cell EVC-304, however, was not apparently affected. Effect of AHL on HepG-2 cells was then explored. We found that the AHL could cause HepG-2 cycle arrest at G2/M checkpoint, induce apoptosis, and interrupt polymerization of microtubules. In addition, expression of two cell cycle-regulated proteins, CyclinB1 and CDK1, was up-regulated upon AHL treatment. Up-regulation of the Fas, Fas ligand, Caspase-8 and Caspase-3 was observed as well, which might imply roles for the Fas/FsaL signaling pathway in the AHL-induced apoptosis of HepG-2 cells.

  20. Physical supports from liver cancer cells are essential for differentiation and remodeling of endothelial cells in a HepG2-HUVEC co-culture model.

    Science.gov (United States)

    Chiew, Geraldine Giap Ying; Fu, Afu; Low, Kar Perng; Luo, Kathy Qian

    2015-06-08

    Blood vessel remodeling is crucial in tumor growth. Growth factors released by tumor cells and endothelium-extracellular matrix interactions are highlighted in tumor angiogenesis, however the physical tumor-endothelium interactions are highly neglected. Here, we report that the physical supports from hepatocellular carcinoma, HepG2 cells, are essential for the differentiation and remodeling of endothelial cells. In a HepG2-HUVEC co-culture model, endothelial cells in direct contact with HepG2 cells could differentiate and form tubular structures similar to those plated on matrigel. By employing HepG2 cell sheet as a supportive layer, endothelial cells formed protrusions and sprouts above it. In separate experiments, fixed HepG2 cells could stimulate endothelial cells differentiation while the conditioned media could not, indicating that physical interactions between tumor and endothelial cells were indispensable. To further investigate the endothelium-remodeling mechanisms, the co-culture model was treated with inhibitors targeting different angiogenic signaling pathways. Inhibitors targeting focal adhesions effectively inhibited the differentiation of endothelial cells, while the growth factor receptor inhibitor displayed little effect. In conclusion, the co-culture model has provided evidences of the essential role of cancer cells in the differentiation and remodeling of endothelial cells, and is a potential platform for the discovery of new anti-angiogenic agents for liver cancer therapy.

  1. Simultaneous detection of MCF-7 and HepG2 cells in blood by ICP-MS with gold nanoparticles and quantum dots as elemental tags.

    Science.gov (United States)

    Li, Xiaoting; Chen, Beibei; He, Man; Wang, Han; Xiao, Guangyang; Yang, Bin; Hu, Bin

    2017-04-15

    In this work, we demonstrate a novel method based on inductively coupled plasma mass spectrometry (ICP-MS) detection with gold nanoparticles (Au NPs) and quantum dots (QDs) labeling for the simultaneous counting of two circulating tumor cell lines (MCF-7 and HepG2 cells) in human blood. MCF-7 and HepG2 cells were captured by magnetic beads coupled with anti-EpCAM and then specifically labeled by CdSe QDs-anti-ASGPR and Au NPs-anti-MUC1, respectively, which were used as signal probes for ICP-MS measurement. Under the optimal experimental conditions, the limits of detection of 50 MCF-7, 89 HepG2 cells and the linear ranges of 200-40000 MCF-7, 300-30000 HepG2 cells were obtained, and the relative standard deviations for seven replicate detections of 800 MCF-7 and HepG2 cells were 4.6% and 5.7%, respectively. This method has the advantages of high sensitivity, low sample consumption, wide linear range and can be extended to the simultaneous detection of multiple CTC lines in human peripheral blood. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Anti-proliferative effects of Atractylis lancea (Thunb.) DC. via down-regulation of the c-myc/hTERT/telomerase pathway in Hep-G2 cells.

    Science.gov (United States)

    Guo, Wei-Qiang; Li, Liang-Zhi; He, Zhuo-Yang; Zhang, Qi; Liu, Jia; Hu, Cui-Ying; Qin, Fen-Ju; Wang, Tao-Yun

    2013-01-01

    Atractylis lancea (Thunb.) DC. (AL), an important medicinal herb in Asia, has been shown to have anti-tumor effects on cancer cells, but the involved mechanisms are poorly understood. This study focused on potential effects and molecular mechanisms of AL on the proliferation of the Hep-G2 liver cancer cell line in vitro. Cell viability was assessed by MTT test in Hep-G2 cells incubated with an ethanol extract of AL. Then, the effects of AL on apoptosis and cell cycle progression were determined by flow cytometry. Telomeric repeat amplification protocol (TRAP) assays was performed to investigate telomerase activity. The mRNA and protein expression of human telomerase reverse transcriptase (hTERT) and c-myc were determined by real-time RT-PCR and Western blotting. Our results show that AL effectively inhibits proliferation in Hep-G2 cells in a concentration- and time-dependent manner. When Hep-G2 cells were treated with AL after 48h,the IC50 was about 72.1 μg/ mL. Apoptosis was induced by AL via arresting the cells in the G1 phase. Furthermore, AL effectively reduced telomerase activity through inhibition of mRNA and protein expression of hTERT and c-myc. Hence, these data demonstrate that AL exerts anti-proliferative effects in Hep-G2 cells via down-regulation of the c-myc/hTERT/ telomerase pathway.

  3. PUMA and survivin are involved in the apoptosis of HepG2 cells induced by microcystin-LR via mitochondria-mediated pathway.

    Science.gov (United States)

    Ma, Junguo; Feng, Yiyi; Liu, Yang; Li, Xiaoyu

    2016-08-01

    The present study aimed to determine the cytotoxicity of microcystin-LR (MC-LR) on the human hepatocellular carcinoma (HepG2) cells in order to elucidate the mechanism of apoptosis induced by MC-LR. Morphological evaluation results showed that MC-LR induced time- and concentration-dependent apoptosis in HepG2 cells. The biochemical assays revealed that MC-LR-exposure caused overproduction of reactive oxygen species (ROS), cyclooxygenase-2 activity alteration, cytochrome c release, and remarkable activation of caspase-3 and caspase-9 in HepG2 cells, indicating that MC-LR-induced apoptosis is mediated by mitochondrial pathway. Moreover, we also found that p53 and Bax might play an important role in MC-LR-induced apoptosis in HepG2 cells in which PUMA and survivin were involved. However, further studies are necessary to elucidate the possible functions of PUMA and survivin in MC-LR-induced apoptosis in HepG2 cells. Copyright © 2016. Published by Elsevier Ltd.

  4. Physical supports from liver cancer cells are essential for differentiation and remodeling of endothelial cells in a HepG2-HUVEC co-culture model

    Science.gov (United States)

    Chiew, Geraldine Giap Ying; Fu, Afu; Perng Low, Kar; Qian Luo, Kathy

    2015-01-01

    Blood vessel remodeling is crucial in tumor growth. Growth factors released by tumor cells and endothelium-extracellular matrix interactions are highlighted in tumor angiogenesis, however the physical tumor-endothelium interactions are highly neglected. Here, we report that the physical supports from hepatocellular carcinoma, HepG2 cells, are essential for the differentiation and remodeling of endothelial cells. In a HepG2-HUVEC co-culture model, endothelial cells in direct contact with HepG2 cells could differentiate and form tubular structures similar to those plated on matrigel. By employing HepG2 cell sheet as a supportive layer, endothelial cells formed protrusions and sprouts above it. In separate experiments, fixed HepG2 cells could stimulate endothelial cells differentiation while the conditioned media could not, indicating that physical interactions between tumor and endothelial cells were indispensable. To further investigate the endothelium-remodeling mechanisms, the co-culture model was treated with inhibitors targeting different angiogenic signaling pathways. Inhibitors targeting focal adhesions effectively inhibited the differentiation of endothelial cells, while the growth factor receptor inhibitor displayed little effect. In conclusion, the co-culture model has provided evidences of the essential role of cancer cells in the differentiation and remodeling of endothelial cells, and is a potential platform for the discovery of new anti-angiogenic agents for liver cancer therapy. PMID:26053957

  5. In Vivo and in Vitro Study on Drug-Drug Interaction of Lovastatin and Berberine from Pharmacokinetic and HepG2 Cell Metabolism Studies.

    Science.gov (United States)

    Cui, Hanming; Wang, Jialong; Zhang, Qiuyan; Dang, Mengmeng; Liu, Hui; Dong, Yu; Zhang, Lu; Yang, Fang; Wu, Jianhua; Tong, Xiaolin

    2016-04-08

    We assumed that the pharmacokinetics of lovastatin could be changed by the induction effect of berberine. An UPLC-MS/MS method was developed and validated for the pharmacokinetics tudy of lovastatin to investigate the in vivo drug-drug interactions between lovastatin and berberine. SD male rats were random divided into lovastatin group and berberine induced prior to lovastatin group for the in vivo pharmacokinetic studies. Meanwhile HepG2 cells were induced by berberine for three days to study the metabolism of lovastatin. The AUC (p HepG2 cell ccould be increased by berberine induction in vitro. The metabolism parameters of lovastatin such as CL, Vmax and Km were increased after the induction of berberine. From the pharmacokinetic study of lovastatin induced with berberine, we obtained pharmacokinetic parameters which are compliance with the metabolic parameters of lovastatin in HepG2 cells with berberine induction in vitro. From the in vivo pharmacokinetics study and the HepG2 cell metabolism study in vitro, berberine could be an inducer for the metabolism of lovastatin according to our previous research on berberine induction effects on HepG2 cells, which may be relevant to the fact that berberine possesses induction effects through the CYP 450 3A4 enzyme.

  6. The protective effects of carvacrol and thymol against paracetamol-induced toxicity on human hepatocellular carcinoma cell lines (HepG2).

    Science.gov (United States)

    Palabiyik, S S; Karakus, E; Halici, Z; Cadirci, E; Bayir, Y; Ayaz, G; Cinar, I

    2016-12-01

    Acetaminophen (APAP) overdose could induce liver damage and lead to acute liver failure. The treatment of APAP overdoses could be improved by new therapeutic strategies. Thymus spp., which has many beneficial effects and has been used in folk medicine, is one such potential strategy. In the present study, the hepatoprotective activity of the main constituents of Thymus spp., carvacrol and thymol, were evaluated in light of APAP-induced hepatotoxicity. We hoped to understand the hepatoprotective mechanism of these agents on the antioxidant system and pro-inflammatory cytokines in vitro. Dose-dependent effects of thymol and carvacrol (25, 50, and 100 µM) were tested on cultured HepG2 cells. N-Acetylcysteine (NAC) was tested as positive control. We showed that APAP inhibited HepG2 cell growth by inducing inflammation and oxidative stress. Incubating APAP-exposed HepG2 cells with carvacrol and thymol for 24 h ameliorated this inflammation and oxidative stress. We also evaluated alanine transaminase and lactate dehydrogenase levels of HepG2 cells. We found that thymol and carvacrol protected against APAP-induced toxicity in HepG2 cells by increasing antioxidant activity and reducing pro-inflammatory cytokines, such as tumor necrosis factor α and interleukin 1β. Taking together high-dose thymol and carvacrol treatment has an effect close to NAC treatment in APAP toxicity, but thymol has better treatment effect than carvacrol. © The Author(s) 2016.

  7. 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.

  8. Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

    Science.gov (United States)

    Valdameri, Glaucio; Rocha, Maria Eliane Merlin; Martinez, Glaucia Regina; Noleto, Guilhermina Rodrigues; Acco, Alexandra; Alves de Souza, Carlos Eduardo; Echevarria, Aurea; Moretto dos Reis, Camilla; Di Pietro, Attilio; Suter Correia Cadena, Sílvia Maria

    2015-01-01

    In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO2, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma. PMID:26083249

  9. Mechanisms of dysregulation of low-density lipoprotein receptor expression in HepG2 cells induced by inflammatory cytokines

    Institute of Scientific and Technical Information of China (English)

    CHEN Ya-xi; RUAN Xiong-zhong; HUANG Ai-long; LI Qiu; John F. Moorhead; Zac Varghese

    2007-01-01

    Background Low-density lipoprotein(LDL)receptor is normally regulated via a feedback system that is dependent on intracellular cholesterol levels.We have demonstrated that cytokines disrupt cholesterol-mediated LDL receptor feedback regulation causing intracellular accumulation of unmodified LDL in peripheral cells.Liver is the centraI organ for lipid homeostasis.The aim of this study was to investigate the regulation of cholesterol exogenous uptake via LDL receptor and its underlying mechanisms in human hepatic cell line(HepG2)cells under physiological and inflammatory conditions.Methods Intracellular total cholesterol(TC),free cholesterol(FC)and cholesterol ester(CE)were measured by an enzymic assay.Oil Red O staining was used to visualize lipid droplet accumulation in cells.Total cellular RNA was isolated from cells for detecting LDL receptor,sterol regulatory element binding protein (SREBP)-2 and SREBP cleavage-activating protein(SCAP)mRNA levels using real-time quantitative PCR.LDL receptor and SREBP-2 protein expression were examined by Western blotting.Confocal microscopy was used to investigate the translocation of SCAP-SREBP complex from the endoplasmic reticulum(ER)to the Golgi by dual staining with anti-human SCAP and anti-Golgin antibodies.Results LDL loading increased intracellular cholesterol level,thereby reduced LDL receptor mRNA and protein expression in HepG2 cells under physiological conditions.However,interleukin 1β(IL-1β)further increased intracellular cholesterol level in the presence of LDL by increasing both LDL receptor mRNA and protein expression in HepG2.LDL also reduced the SREBP and SCAP mRNA level under physiological conditions.Exposure to IL-1β caused Over-expression of SREBP-2 and also disrupted normal distribution of SCAP-SREBP complex in HepG2 by enhancing translocation of SCAP-SREBP from the ER to the Golgi despite a high concentration of LDL in the culture medium.Conclusions IL-1β disrupts cholesterol-mediated LDL receptor feedback regulation by enhancing SCAP-SREBP complex translocation from the ER to the Golgi,thereby increasing SREBP-2 mediated LDL receptor expression even in the presence of high concentration of LDL.This results in LDL cholesterol accumulation in hepatic cells via LDL receptor pathway under inflammatory stress.

  10. Comparative effects of fructose and glucose on lipogenic gene expression and intermediary metabolism in HepG2 liver cells.

    Directory of Open Access Journals (Sweden)

    Kristin M Hirahatake

    Full Text Available Consumption of large amounts of fructose or sucrose increases lipogenesis and circulating triglycerides in humans. Although the underlying molecular mechanisms responsible for this effect are not completely understood, it is possible that as reported for rodents, high fructose exposure increases expression of the lipogenic enzymes fatty acid synthase (FAS and acetyl-CoA carboxylase (ACC-1 in human liver. Since activation of the hexosamine biosynthesis pathway (HBP is associated with increases in the expression of FAS and ACC-1, it raises the possibility that HBP-related metabolites would contribute to any increase in hepatic expression of these enzymes following fructose exposure. Thus, we compared lipogenic gene expression in human-derived HepG2 cells after incubation in culture medium containing glucose alone or glucose plus 5 mM fructose, using the HBP precursor 10 mM glucosamine (GlcN as a positive control. Cellular metabolite profiling was conducted to analyze differences between glucose and fructose metabolism. Despite evidence for the active uptake and metabolism of fructose by HepG2 cells, expression of FAS or ACC-1 did not increase in these cells compared with those incubated with glucose alone. Levels of UDP-N-acetylglucosamine (UDP-GlcNAc, the end-product of the HBP, did not differ significantly between the glucose and fructose conditions. Exposure to 10 mM GlcN for 10 minutes to 24 hours resulted in 8-fold elevated levels of intracellular UDP-GlcNAc (P<0.001, as well as a 74-126% increase in FAS (P<0.05 and 49-95% increase in ACC-1 (P<0.01 expression above controls. It is concluded that in HepG2 liver cells cultured under standard conditions, sustained exposure to fructose does not result in an activation of the HBP or increased lipogenic gene expression. Should this scenario manifest in human liver in vivo, it would suggest that high fructose consumption promotes triglyceride synthesis primarily through its action to provide lipid precursor carbon and not by activating lipogenic gene expression.

  11. Black Soybean Seed Coat Extract Prevents Hydrogen Peroxide-Mediated Cell Death via Extracellular Signal-Related Kinase Signalling in HepG2 Cells.

    Science.gov (United States)

    Hashimoto, Naoto; Oki, Tomoyuki; Sasaki, Kazunori; Suda, Ikuo; Okuno, Shigenori

    2015-01-01

    Oxidative stress reduces cell viability and contributes to disease processes. Flavonoids including anthocyanins and proanthocyanidins reportedly induce intracellular antioxidant defence systems. Thus, in this study, we examined the antioxidant effects of a commercial extract from black soybean seed coats (BE), which are rich in anthocyanin and proanthocyanidin, and investigated the associated intracellular mechanisms in HepG2 cells. HepG2 cells treated with hydrogen peroxide (HPO) showed 60% viability, whereas pretreatment with BE-containing media for 2 h ameliorated HPO-mediated cell death by up to 90%. Pretreatment with BE for 2 h partially blocked HPO-mediated activation of ERK in HepG2 cells, and that for 1 h led to a 20% increase in intracellular total protein phosphatase (PP) activity, which is known to deactivate protein kinases. These results indicate that BE prevents HPO-mediated cell damage by inhibiting ERK signalling, potentially via PPs.

  12. Effect of Human Hepatocellular Carcinoma HepG2 Cell-derived Exosome on the Differentiation of Mesenchymal Stem Cells and Their Interaction.

    Science.gov (United States)

    Luo, Fei; Sun, Zhao; Han, Qin; Xue, Chunling; Bai, Chunmei

    2017-06-20

    Objective To investigate the effect of human hepatocellular carcinoma HepG2 cell-derived Exosome on the differentiation of mesenchymal stem cells(MSC)into cancer-associated myofibroblasts(CAF)and the impacts of CAF on liver cancer cell proliferation,migration,and invasion. Methods The protein expression of HepG2 cell-derived Exosome was detected by Western blotting. MSCs were separated from human adipose tissue and cultured with HepG2 cell-derived Exosome(100 ng/nl)to initiate differentiation. The expressions of mesenchymal markers and several interleukins were also detected by Western blotting. HepG2 cells were co-cultured with the conditioned media(CM),in which HepG2 Exosome induced the differentiation of MSC into CAF. The expressions of epithelial and mesenchymal markers were detected by real-time polymerase chain reaction(PCR)and Western blotting. Cell proliferation was assessed using MTS assay. Transwell chambers were used in the in vitro migration and invasion assay. Results HepG2 cell-derived particles expressed CD63,70 kilodalton heat shock proteins,and 90 kilodalton heat shock proteins. With the treatment of HepG2 cell-derived Exosome,the expressions of mesenchymal marker α-smooth muscle actin,fibroblast activation protein α,interleukin(IL)-6,IL-8,and IL-1β were up-regulated,while vascular endothelial growth factor had no significant change. The conditioned media which HepG2 Exosome induced MSC differentiation CAF(CAF-CM)could significantly promote HepG2 cells proliferation(1.075±0.104),compared to BSA control(0.874±0.066,P=0.023)and MSC-CM(0.649±0.034,P=0.0005). CAF-CM could significantly enhance cell migration [(42.5±9.1) cells vs.(18.5±3.1) cells,P=0.001] and invasion [(29.0±3.5) cells vs.(13.1±3.7) cells,P=0.009] compared to its control group. Moreover the conditioned medium which HepG2 Exosome induced MSC to differentiate into CAF could also promote the expressions of mesenchyme-related genes Smad interacting protein 1(P=0.040),β-catenin(P=0.038),fibronectin(P=0.029),and Vimentin(P=0.013)and inhibit the expression of epithelial related genes zonula ocdudens-1(P=0.010).Conclusions Exosome extracted from HepG2 cells can induce human adipose-derived MSC to differentiate into cancer-associated myofibroblasts. CAF-like cells can promote the migration of the liver cancer cell line HepG2.

  13. 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.

  14. Inhibition of adhesion and metastasis of HepG2 hepatocellular carcinoma cells in vitro by DNA aptamer against sialyl Lewis X.

    Science.gov (United States)

    Wang, Xiao-Kang; Peng, Yan; Tao, Hao-Ran; Zhou, Fen-Fang; Zhang, Chi; Su, Fei; Wang, Shi-Pei; Liu, Qing; Xu, Li-Hua; Pan, Xue-Kai; Xie, Wei; Feng, Mao-Hui

    2017-06-01

    The sialyl Lewis X (SLe(x)) antigen encoded by the FUT7 gene is the ligand of endotheliam-selectin (E-selectin). The combination of SLe(x) antigen and E-selectin represents an important way for malignant tumor metastasis. In the present study, the effect of the SLe(x)-binding DNA aptamer on the adhesion and metastasis of hepatocellular carcinoma HepG2 cells in vitro was investigated. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence staining were conducted to detect the expression of FUT7 at both transcriptional and translational levels. The SLe(x) expression in HepG2 cells treated with different concentrations of SLe(x)-binding DNA aptamer was detected by flow cytometry. Besides, the adhesion, migration, and invasion of HepG2 cells were measured by cell adhesion assay, and the Transwell migration and invasion assay. The results showed that the FUT7 expression was up-regulated at both mRNA and protein levels in HepG2 cells. SLe(x)-binding DNA aptamer could significantly decrease the expression of SLe(x) in HepG2 cells. The cell adhesion assay revealed that the SLe(x)-binding DNA aptamer could effectively inhibit the interactions between E-selectin and SLe(x) in the HepG2 cells. Additionally, SLe(x)-binding DNA aptamers at 20 nmol/L were found to have the similar effect to the monoclonal antibody CSLEX-1. The Transwell migration and invasion assay revealed that the number of penetrating cells on the down-side of Transwell membrane was significantly less in cells treated with 5, 10, 20 nmol/L SLe(x)-binding DNA aptamer than those in the negative control group (PHepG2 cells, suggesting that the SLe(x)-binding DNA aptamer may be used as a potential molecular targeted drug against metastatic hepatocellular carcinoma.

  15. Inhibition of Aurora A Kinase by Alisertib Induces Autophagy and Cell Cycle Arrest and Increases Chemosensitivity in Human Hepatocellular Carcinoma HepG2 Cells.

    Science.gov (United States)

    Zhu, Qiaohua; Yu, Xinfa; Zhou, Zhi-Wei; Zhou, Chengyu; Chen, Xiao-Wu; Zhou, Shu-Feng

    2017-01-01

    Aurora A kinase represent a feasible target in cancer therapy. To evaluate the proteomic response of human liver carcinoma cells to alisertib (ALS) and identify the molecular targets of ALS, we examined the effects of ALS on the proliferation, cell cycle, autophagy, apoptosis, and chemosensitivity in HepG2 cells. The stable-isotope labeling by amino acids in cell culture (SILAC) based quantitative proteomic study was performed to evaluate the proteomic response to ALS. Cell cycle distribution and apoptosis were assessed using flow cytometry and autophagy was determined using flow cytometry and confocal microscopy. Our SILAC proteomic study showed that ALS regulated the expression of 914 proteins, with 407 molecules being up-regulated and 507 molecules being down-regulated in HepG2 cells. Ingenuity pathway analysis (IPA) and KEGG pathway analysis identified 146 and 32 signaling pathways were regulated by ALS, respectively, which were associated with cell survival, programmed cell death, and nutrition-energy metabolism. Subsequently, the verification experiments showed that ALS remarkably arrested HepG2 cells in G2/M phase and led to an accumulation of aneuploidy via regulating the expression of key cell cycle regulators. ALS induced a marked autophagy in a concentration- and time-dependent manner via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Autophagy inhibition promoted the pro-apoptotic effect of ALS, indicating a cyto-protective role of ALS-induced autophagy. ALS increased the chemosensitivity of HepG2 cells to cisplatin and doxorubicin. Taken together, ALS induces autophagy and cell cycle arrest in HepG2 cells via PI3K/Akt/mTOR-mediated pathway. Autophagy inhibition may promote the anticancer effect of ALS and sensitize the chemotherapy in HepG2 cells. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Specific COX-2 inhibitor NS398 induces apoptosis in human liver cancer cell line HepG2 through BCL-2

    Institute of Scientific and Technical Information of China (English)

    Dong-Sheng Huang; Ke-Zhen Shen; Jian-Feng Wei; Ting-Bo Liang; Shu-Sen Zheng; Hai-Yang Xie

    2005-01-01

    AIM: To evaluate the effects of NS-398, a cyclooxygenase2 (COX-2) inhibitor, on the proliferation and apoptosis of HepG2 cells.METHODS: The effects of NS-398 on the proliferation of HepG2 cells were evaluated by MTT. DNA fragmentation gel analysis was used to analyze the apoptotic cells. DNA ploidy and apoptotic cell percentage were calculated by flow cytometry.The expression of COX-2 and Bcl-2 mRNA was identified by competitive RT-PCR. Furthermore, expression level of Bcl-2 was detected using Western blot in HepG2 after treated with NS-398.RESULTS: NS-398 inhibited cell proliferation and induced apoptosis of HepG2 cells in a concentration-dependent manner. DNA ploidy analysis showed that S phase cells were significantly decreased with increase of NS-398 concentration.The quiescent G0/G1 phase was accumulated with decrease of Bcl-2 mRNA. Whereas NS-398 had no effect on the expression of COX-2 mRNA, and no correlations were found between COX-2 mRNA and HepG2 cell proliferation and apoptosis induced by NS-398 (r = 0.056 and r= 0.119,respectively). Bcl-2 protein level was inhibited after treated with NS-398.CONCLUSION: NS-398 significantly inhibits the proliferation and induces apoptosis of HepG2 cells. Mechanisms involved may be accumulation of quiescent G0/G1 phase and decrease of Bcl-2 expression.

  17. Hesperidin from Citrus seed induces human hepatocellular carcinoma HepG2 cell apoptosis via both mitochondrial and death receptor pathways.

    Science.gov (United States)

    Banjerdpongchai, Ratana; Wudtiwai, Benjawan; Khaw-On, Patompong; Rachakhom, Wasitta; Duangnil, Natthachai; Kongtawelert, Prachya

    2016-01-01

    Citrus seeds are full of phenolic compounds, such as flavonoids. The aims of this study were to identify the types of flavonoids in Citrus seed extracts, the cytotoxic effect, mode of cell death, and signaling pathway in human hepatic cancer HepG2 cells. The flavonoids contain anticancer, free radical scavenging, and antioxidant activities. Neohesperidin, hesperidin, and naringin, active flavanone glycosides, were identified in Citrus seed extract. The cytotoxic effect of three compounds was in a dose-dependent manner, and IC50 levels were determined. The sensitivity of human HepG2 cells was as follows: hesperidin > naringin > neohesperidin > naringenin. Hesperidin induced HepG2 cells to undergo apoptosis in a dose-dependent manner as evidenced by the externalization of phosphatidylserine and determined by annexin V-fluorescein isothiocyanate and propidium iodide staining using flow cytometry. Hesperidin did not induce the generation of reactive oxygen species, which was determined by using 2',7'-dichlorohydrofluorescein diacetate and flow cytometry method. The number of hesperidin-treated HepG2 cells with the loss of mitochondrial transmembrane potential increased concentration dependently, using 3,3'-dihexyloxacarbocyanine iodide employing flow cytometry. Caspase-9, -8, and -3 activities were activated and increased in hesperidin-treated HepG2 cells. Bcl-xL protein was downregulated whereas Bax, Bak, and tBid protein levels were upregulated after treatment with hesperidin in a dose-dependent manner. In conclusion, the bioflavanone from Citrus seeds, hesperidin, induced human HepG2 cell apoptosis via mitochondrial pathway and death receptor pathway. Citrus seed flavonoids are beneficial and can be developed as anticancer drug or food supplement, which still needs further in vivo investigation in animals and human beings.

  18. Spectroscopic characterization and antiproliferative activity on HepG2 human hepatoblastoma cells of flavonoid C-glycosides from Petrorhagia velutina.

    Science.gov (United States)

    Pacifico, Severina; Scognamiglio, Monica; D'Abrosca, Brigida; Piccolella, Simona; Tsafantakis, Nikolaos; Gallicchio, Marialuisa; Ricci, Andreina; Fiorentino, Antonio

    2010-12-27

    Eight flavonoid C-glycosides, including three new analogues, have been isolated from leaf and root methanolic extracts of Petrorhagia velutina, a Mediterranean herbaceous plant. The antiproliferative activity against human hepatoblastoma cancer cell line HepG2 has been analyzed by the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) test. Isoorientin (4) significantly reduces the proliferation of HepG2 cells as determined by the complete conversion of the tetrazolium probe into formazan after 48 h of exposure.

  19. 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 ± 0.05%, 23.32 ± 0.03%, and 34.51 ± 0.01% for 0, 1, 2, and 3 μg/mL of mercury respectively, also indicating a gradual increase in Caspase positive cells with increasing doses of mercury.

  20. Metabolites profiling of 10 bufadienolides in human liver microsomes and their cytotoxicity variation in HepG2 cell.

    Science.gov (United States)

    Han, Lingyu; Wang, Hongjie; Si, Nan; Ren, Wei; Gao, Bo; Li, Yan; Yang, Jian; Xu, Miao; Zhao, Haiyu; Bian, Baolin

    2016-04-01

    Bufadienolides, a class of polyhydroxy steroids, exhibit significant antitumor activity. In this study, a total of 39 metabolites from 10 bufadienolides were detected and identified by ultrahigh-performance liquid chromatography (UHPLC) coupled with an LTQ Orbitrap mass spectrometer. The results showed that hydroxylation and dehydrogenation were the major metabolic pathways of bufadienolides in human liver microsomes (HLMs). CYP3A4 was found to be the major metabolic enzyme and CYP2D6 only mediated the dehydrogenation reaction. A systematic validated cytotoxicity evaluation method for bufadienolide metabolites at equal equivalents was established. Hellebrigenin (1), hellebrigenol (2), arenobufagin (3), bufotalin (5), and bufalin (6) were selected to determine their cytotoxicity against HepG2 cells before and after incubation in HLMs. All the test samples were enriched by a validated solid-phase extraction (SPE) method. Although the cytotoxicities of metabolites were weaker than those of the parent compounds to different degrees, their effects were still strong.

  1. 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

    STARD4, a member of the evolutionarily conserved START gene family, has been implicated in the non-vesicular 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...

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

    Directory of Open Access Journals (Sweden)

    Yuanyuan Zhou

    2015-08-01

    Full Text Available 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.

  3. 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

    2015-01-01

    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.

  4. De novo LINE-1 retrotransposition in HepG2 cells preferentially targets gene poor regions of chromosome 13.

    Science.gov (United States)

    Bojang, Pasano; Anderton, Mark J; Roberts, Ruth A; Ramos, Kenneth S

    2014-08-01

    Long interspersed nuclear elements (Line-1 or L1s) account for ~17% of the human genome. While the majority of human L1s are inactive, ~80-100 elements remain retrotransposition competent and mobilize through RNA intermediates to different locations within the genome. De novo insertions of L1s account for polymorphic variation of the human genome and disruption of target loci at their new location. In the present study, fluorescence in situ hybridization and DNA sequencing were used to characterize retrotransposition profiles of L1(RP) in cultured human HepG2 cells. While expression of synthetic L1(RP) was associated with full-length and truncated insertions throughout the entire genome, a strong preference for gene-poor regions, such as those found in chromosome 13 was observed for full-length insertions. These findings shed light into L1 targeting mechanisms within the human genome and question the putative randomness of L1 retrotransposition.

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

    Science.gov (United States)

    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.

  6. Streptozotocin-Induced Cytotoxicity, Oxidative Stress and Mitochondrial Dysfunction in Human Hepatoma HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Haider Raza

    2012-05-01

    Full Text Available Streptozotocin (STZ is an antibiotic often used in the treatment of different types of cancers. It is also highly cytotoxic to the pancreatic beta-cells and therefore is commonly used to induce experimental type 1 diabetes in rodents. Resistance towards STZ-induced cytotoxicity in cancer cells has also been reported. Our previous studies have reported organ-specific toxicity and metabolic alterations in STZ-induced diabetic rats. STZ induces oxidative stress and metabolic complications. The precise molecular mechanism of STZ-induced toxicity in different tissues and carcinomas is, however, unclear. We have, therefore, investigated the mechanism of cytotoxicity of STZ in HepG2 hepatoma cells in culture. Cells were treated with different doses of STZ for various time intervals and the cytotoxicity was studied by observing the alterations in oxidative stress, mitochondrial redox and metabolic functions. STZ induced ROS and RNS formation and oxidative stress as measured by an increase in the lipid peroxidation as well as alterations in the GSH-dependent antioxidant metabolism. The mitochondria appear to be a highly sensitive target for STZ toxicity. The mitochondrial membrane potential and enzyme activities were altered in STZ treated cells resulting in the inhibition of ATP synthesis. ROS-sensitive mitochondrial aconitase activity was markedly inhibited suggesting increased oxidative stress in STZ-induced mitochondrial toxicity. These results suggest that STZ-induced cytotoxicity in HepG2 cells is mediated, at least in part, by the increase in ROS/RNS production, oxidative stress and mitochondrial dysfunction. Our study may be significant for better understanding the mechanisms of STZ action in chemotherapy and drug induced toxicity.

  7. Ethyl acetate extract of Squilla oratoria suppresses growth of HepG2 cells by inducing S phase arrest

    Directory of Open Access Journals (Sweden)

    Xiangwei Qi

    2013-08-01

    Full Text Available Objective: The oceans and seas are a rich source of organisms from which anti-cancer drugs can be isolated and developed. Marine organisms have been screened in our laboratory, and organic solvent extracts of Squilla oratoria (ESO have been shown to possess cytostatic effects on cancer cell lines of diverse origins. To explore the underlying mechanisms, the growth inhibition by ESO was investigated in the present study. Methods: Human hepatocellular carcinoma (HCC derived cells (HepG2 were used. The cells were challenged with ESO, cell cycle profile was assayed, and level of proliferating cell nuclear antigen (PCNA expression and that of cyclin D1 and cyclin A were evaluated with flow cytomtery. The in vivo antitumor effect of ESO was tested in nude mouse xenografts. PCNA expression was evaluated immunohistochemically in nude mouse xenograft tissues. Results: With the increase in dose of injected ESO, expression of PCNA by human HCC xenografts increased. ESO inhibited the growth of human HCC HepG2 cells both in vitro and in vivo. The effect was correlated with arrest of the cell cycle in S phase. Expression of PCNA, which is a cell-cycle regulator that promotes S phase entry, was elevated in both cell lines and xenografts whereas that of cyclins that promote M phase entry was down-regulated by exposure to ESO. Conclusion: Growth inhibition was explained by arrest of the cell cycle in S phase and down-regulation of molecules that promote cells to enter S phase. [J Exp Integr Med 2013; 3(4.000: 313-322

  8. 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.

  9. In vitro treatment of HepG2 cells with saturated fatty acids reproduces mitochondrial dysfunction found in nonalcoholic steatohepatitis.

    Science.gov (United States)

    García-Ruiz, Inmaculada; Solís-Muñoz, Pablo; Fernández-Moreira, Daniel; Muñoz-Yagüe, Teresa; Solís-Herruzo, José A

    2015-02-01

    Activity of the oxidative phosphorylation system (OXPHOS) is decreased in humans and mice with nonalcoholic steatohepatitis. Nitro-oxidative stress seems to be involved in its pathogenesis. The aim of this study was to determine whether fatty acids are implicated in the pathogenesis of this mitochondrial defect. In HepG2 cells, we analyzed the effect of saturated (palmitic and stearic acids) and monounsaturated (oleic acid) fatty acids on: OXPHOS activity; levels of protein expression of OXPHOS complexes and their subunits; gene expression and half-life of OXPHOS complexes; nitro-oxidative stress; and NADPH oxidase gene expression and activity. We also studied the effects of inhibiting or silencing NADPH oxidase on the palmitic-acid-induced nitro-oxidative stress and subsequent OXPHOS inhibition. Exposure of cultured HepG2 cells to saturated fatty acids resulted in a significant decrease in the OXPHOS activity. This effect was prevented in the presence of a mimic of manganese superoxide dismutase. Palmitic acid reduced the amount of both fully-assembled OXPHOS complexes and of complex subunits. This reduction was due mainly to an accelerated degradation of these subunits, which was associated with a 3-tyrosine nitration of mitochondrial proteins. Pretreatment of cells with uric acid, an antiperoxynitrite agent, prevented protein degradation induced by palmitic acid. A reduced gene expression also contributed to decrease mitochondrial DNA (mtDNA)-encoded subunits. Saturated fatty acids induced oxidative stress and caused mtDNA oxidative damage. This effect was prevented by inhibiting NADPH oxidase. These acids activated NADPH oxidase gene expression and increased NADPH oxidase activity. Silencing this oxidase abrogated totally the inhibitory effect of palmitic acid on OXPHOS complex activity. We conclude that saturated fatty acids caused nitro-oxidative stress, reduced OXPHOS complex half-life and activity, and decreased gene expression of mtDNA-encoded subunits. These effects were mediated by activation of NADPH oxidase. That is, these acids reproduced mitochondrial dysfunction found in humans and animals with nonalcoholic steatohepatitis.

  10. ERK1/2 contributes negative regulation to STAT3 activity in HSS-transfected HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    Signal transducer and activator of transcription 3 (STAT3) is a recently characterized transcription factor which is essential to liver regeneration. We have previously reported that hepatic stimulator substance (HSS), a novel growthpromoting substance, phosphorylated the epidermal growth factor (EGF) receptors and activated downstream RasMAP kinase (extracellular signal-regulated kinases, ERK1/2) cascade. However, whether HSS signal is related to STAT3pathway remains unclear. The present study is aiming to explore the regulatory effect of activation of ERK1/2 evoked by HSS on STAT3 phosphorylation and STAT3 signaling. Human hepatoma cell line HepG2 was stably transfected with HSS cDNA and HSS expression was measured by Northern blot. The results showed that the transfection of HSS into HepG2 resulted in remarkable increase in cellular proliferation as compared with the non-transfected cells, and it was further proved that the cellular proliferation in the HSS-transfected cells was related to ERK1/2 activation. Treatment of the cells with 50 μM of PD98059, an ERK1/2 specific upstream inhibitor, resulted in ERK1/2 inactivation completely.Inhibition of ERK1/2 allowed the tyrosine of STAT3 to be phosphorylated in a dose-dependent manner to PD98059.Furthermore, transient transfection of STAT3 mutant (STAT3S727A) into HSS-bearing cells could remarkably reverse the inhibitory effect of ERK1/2 on STAT3 phosphorylation. Based upon these results, it is concluded that ERK1/2negatively modulates STAT3 phosphorylation and this function is dependent on residual serine-727 (S727) of STAT3.

  11. Downregulation of hepcidin and haemojuvelin expression in the hepatocyte cell-line HepG2 induced by thalassaemic sera.

    Science.gov (United States)

    Weizer-Stern, Orly; Adamsky, Konstantin; Amariglio, Ninette; Levin, Carina; Koren, Ariel; Breuer, William; Rachmilewitz, Eliezer; Breda, Laura; Rivella, Stefano; Cabantchik, Z Ioav; Rechavi, Gideon

    2006-10-01

    Beta-thalassaemia represents a group of diseases, in which ineffective erythropoiesis is accompanied by iron overload. In a mouse model of beta-thalassaemia, we observed that the liver expressed relatively low levels of hepcidin, which is a key factor in the regulation of iron absorption by the gut and of iron recycling by the reticuloendothelial system. It was hypothesised that, despite the overt iron overload, a putative plasma factor found in beta-thalassaemia might suppress liver hepcidin expression. Sera from beta-thalassaemia and haemochromatosis (C282Y mutation) patients were compared with those of healthy individuals regarding their capacity to induce changes the expression of key genes of iron metabolism in human HepG2 hepatoma cells. Sera from beta-thalassaemia major patients induced a major decrease in hepcidin (HAMP) and lipocalin2 (oncogene 24p3) (LCN2) expression, as well as a moderate decrease in haemojuvelin (HFE2) expression, compared with sera from healthy individuals. A significant correlation was found between the degree of downregulation of HAMP and HFE2 induced by beta-thalassaemia major sera (r = 0.852, P < 0.0009). Decreased HAMP expression was also found in HepG2 cells treated with sera from beta-thalassaemia intermedia patients. In contrast, the majority of sera from hereditary haemochromatosis patients induced an increase in HAMP expression, which correlated with transferrin (Tf) saturation (r = 0.765, P < 0.0099). Our results suggest that, in beta-thalassaemia, serum factors might override the potential effect of iron overload on HAMP expression, thereby providing an explanation for the failure to arrest excessive intestinal iron absorption in these patients.

  12. 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.

  13. Comparative Effects of Fructose and Glucose on Lipogenic Gene Expression and Intermediary Metabolism in HepG2 Liver Cells

    Science.gov (United States)

    Fiehn, Oliver; Adams, Sean H.

    2011-01-01

    Consumption of large amounts of fructose or sucrose increases lipogenesis and circulating triglycerides in humans. Although the underlying molecular mechanisms responsible for this effect are not completely understood, it is possible that as reported for rodents, high fructose exposure increases expression of the lipogenic enzymes fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC-1) in human liver. Since activation of the hexosamine biosynthesis pathway (HBP) is associated with increases in the expression of FAS and ACC-1, it raises the possibility that HBP-related metabolites would contribute to any increase in hepatic expression of these enzymes following fructose exposure. Thus, we compared lipogenic gene expression in human-derived HepG2 cells after incubation in culture medium containing glucose alone or glucose plus 5 mM fructose, using the HBP precursor 10 mM glucosamine (GlcN) as a positive control. Cellular metabolite profiling was conducted to analyze differences between glucose and fructose metabolism. Despite evidence for the active uptake and metabolism of fructose by HepG2 cells, expression of FAS or ACC-1 did not increase in these cells compared with those incubated with glucose alone. Levels of UDP-N-acetylglucosamine (UDP-GlcNAc), the end-product of the HBP, did not differ significantly between the glucose and fructose conditions. Exposure to 10 mM GlcN for 10 minutes to 24 hours resulted in 8-fold elevated levels of intracellular UDP-GlcNAc (Pfructose does not result in an activation of the HBP or increased lipogenic gene expression. Should this scenario manifest in human liver in vivo, it would suggest that high fructose consumption promotes triglyceride synthesis primarily through its action to provide lipid precursor carbon and not by activating lipogenic gene expression. PMID:22096489

  14. Unsaturated fatty acids and phytosterols regulate cholesterol transporter genes in Caco-2 and HepG2 cell lines.

    Science.gov (United States)

    Park, Youngki; Carr, Timothy P

    2013-02-01

    Dietary consumption of phytosterols and certain fatty acids has been shown to reduce cholesterol absorption and plasma cholesterol concentrations. However, it has not been fully elucidated whether phytosterols or fatty acids can alter the expression of cholesterol transporters by functioning as signaling molecules. This study tested the hypothesis that various fatty acids and phytosterols commonly found in the food supply can modulate the expression of transporters including Niemann-Pick C1-like 1, low-density lipoprotein receptor, and scavenger receptor class B type I and 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the intestine and liver. Caco-2 cells were used as models of enterocytes, and HepG2 cells were used as a model of hepatocytes. The cells were treated for 18 hours with 100 μmol/L of a fatty acid, or for 24 hours with 10 μmol/L of 25α-hydroxycholesterol, or 100 μmol/L of cholesterol, sitosterol, and stigmasterol to measure expression of genes involved in cholesterol transport using quantitative real-time polymerase chain reaction. Polyunsaturated fatty acids in Caco-2 cells and sterols in HepG2 cells significantly reduced the messenger RNA expression levels of Niemann-Pick C1-like 1, scavenger receptor class B type I, low-density lipoprotein receptor, and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Importantly, sitosterol and stigmasterol reduced the messenger RNA levels of genes to a similar extent as cholesterol. The data support the hypothesis that unsaturated fatty acid and phytosterols can act as signaling molecules and alter the expression of genes involved in cholesterol transport and metabolism.

  15. 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.

  16. Evaluating the extent of LINE-1 mobility following exposure to heavy metals in HepG2 cells.

    Science.gov (United States)

    Karimi, Abbas; Madjd, Zahra; Habibi, Laleh; Akrami, Seyed Mohammad

    2014-07-01

    The long interspersed elements-1 (LINE1 or L1 retrotransposon) constitute 17% of the human genome and retain mobility properties within the genome. At present, 80-100 human L1 elements are thought to be active in the genome. The mobilization of these active elements may be influenced upon exposure to the heavy metals. In the present study, we evaluated the association of aluminum, lead, and copper exposure with L1 retrotransposition in human hepatocellular carcinoma (HepG2) cell line. An in vitro retrotransposition assay using an enhanced green fluorescent protein (EGFP)-tagged L1RP cassette was established to track EGFP shining as the mark of retrotransposition. Following determination of noncytotoxic concentrations of these metals, pL1RP-EGFP-transfected HepG2 cells were subjected to long-term treatment. Flow cytometry analysis of cells treated with various concentrations of these metals along with quantitative real-time PCR was used to quantify L1 retrotransposition frequencies. Aluminum significantly increased L1 retrotransposition frequency, while no significant association was found concerning lead exposure and L1 retrotransposition. Copper treatment downregulated L1 retrotransposition as a result of EGFP-tagged L1RP expression. Our findings suggest that aluminum might have the potential to cause genomic instability by the enhancement of L1 mobilization. Thus, the risk of induced L1 retrotransposition should be considered during drug safety evaluation and risk assessments of exposure to toxic environmental agents. Further studies are needed for a more robust assay to evaluate any associations between long-term lead exposure and L1 mobility in cell culture assay.

  17. Effects of Tamarindus indica fruit pulp extract on abundance of HepG2 cell lysate proteins and their possible consequential impact on metabolism and inflammation.

    Science.gov (United States)

    Chong, Ursula R W; Abdul-Rahman, Puteri S; Abdul-Aziz, Azlina; Hashim, Onn H; Mat-Junit, Sarni

    2013-01-01

    The fruit pulp extract of Tamarindus indica has been reported for its antioxidant and hypolipidemic properties. In this study, the methanol extract of T. indica fruit pulp was investigated for its effects on the abundance of HepG2 cell lysate proteins. Cell lysate was extracted from HepG2 cells grown in the absence and presence of the methanol extract of T. indica fruit pulp. Approximately 2500 spots were resolved using two-dimensional gel electrophoresis and the abundance of 20 cellular proteins was found to be significantly reduced. Among the proteins of reduced abundance, fourteen, including six proteins involved in metabolism (including ethanolamine phosphate cytidylyltransferase), four mitochondrial proteins (including prohibitin and respiratory chain proteins), and four proteins involved in translation and splicing, were positively identified by mass spectrometry and database search. The identified HepG2 altered abundance proteins, when taken together and analyzed by Ingenuity Pathways Analysis (IPA) software, are suggestive of the effects of T. indica fruit pulp extract on metabolism and inflammation, which are modulated by LXR/RXR. In conclusion, the methanol fruit pulp extract of T. indica was shown to cause reduced abundance of HepG2 mitochondrial, metabolic, and regulatory proteins involved in oxidative phosphorylation, protein synthesis, and cellular metabolism.

  18. Effects of Tamarindus indica Fruit Pulp Extract on Abundance of HepG2 Cell Lysate Proteins and Their Possible Consequential Impact on Metabolism and Inflammation

    Directory of Open Access Journals (Sweden)

    Ursula R. W. Chong

    2013-01-01

    Full Text Available The fruit pulp extract of Tamarindus indica has been reported for its antioxidant and hypolipidemic properties. In this study, the methanol extract of T. indica fruit pulp was investigated for its effects on the abundance of HepG2 cell lysate proteins. Cell lysate was extracted from HepG2 cells grown in the absence and presence of the methanol extract of T. indica fruit pulp. Approximately 2500 spots were resolved using two-dimensional gel electrophoresis and the abundance of 20 cellular proteins was found to be significantly reduced. Among the proteins of reduced abundance, fourteen, including six proteins involved in metabolism (including ethanolamine phosphate cytidylyltransferase, four mitochondrial proteins (including prohibitin and respiratory chain proteins, and four proteins involved in translation and splicing, were positively identified by mass spectrometry and database search. The identified HepG2 altered abundance proteins, when taken together and analyzed by Ingenuity Pathways Analysis (IPA software, are suggestive of the effects of T. indica fruit pulp extract on metabolism and inflammation, which are modulated by LXR/RXR. In conclusion, the methanol fruit pulp extract of T. indica was shown to cause reduced abundance of HepG2 mitochondrial, metabolic, and regulatory proteins involved in oxidative phosphorylation, protein synthesis, and cellular metabolism.

  19. Rho kinase, myosin-II, and p42/44 MAPK control extracellular matrix-mediated apical bile canalicular lumen morphogenesis in HepG2 cells

    NARCIS (Netherlands)

    Herrema, H; Czajkowska, D; Theard, D; van der Wouden, JM; Kalicharan, D; Zolghadr, B; Hoekstra, D; Ijzendoorn, SCD

    The molecular mechanisms that regulate multicellular architecture and the development of extended apical bile canalicular lumens in hepatocytes are poorly understood. Here, we show that hepatic HepG2 cells cultured on glass coverslips first develop intercellular apical lumens typically formed by a

  20. Metabolomic effects in HepG2 cells exposed to four TiO2 amd two CeO2 naomaterials

    Science.gov (United States)

    Abstract It is difficult to evaluate nanomaterials potential toxicity and to make science-based societal choices. To better assess potential hepatotoxicity issues, human liver HepG2 cells were exposed to four Ti02 and two Ce02 nanomaterials at 30 ug m1-1 for t...

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

    Science.gov (United States)

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

    2014-10-01

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

  2. Apoptotic and inhibitory effects on cell proliferation of hepatocellular carcinoma HepG2 cells by methanol leaf extract of Costus speciosus.

    Science.gov (United States)

    Nair, Sandhya V G; Hettihewa, Menik; Rupasinghe, H P Vasantha

    2014-01-01

    Costus speciosus is a medicinal plant commonly known as wild ginger distributed in South and Southeast Asian countries. Leaves of this plant are used for ayurvedic treatment regimes in malignancies and mental illness. Rhizome extract from the plant is used to treat malignancies, pneumonia, urinary disorders, jaundice, rheumatism, and diabetes. The goal of this study was to investigate the effects of methanol extract of leaves of C. speciosus on the growth of human hepatocellular carcinoma (HepG2) cells and understand possible mechanisms of its action. Viability of HepG2 cells were measured by MTS assay after 24 h and 48 h treatment with extracts of 1, 10, 50, 100, and 200 μg/mL concentrations. Cell cycle analysis and apoptosis were evaluated by flow cytometry and caspase-3 induction. HepG2 cells treated with 100 μg/mL methanol leaf extract for 24 h displayed a significant reduction in cell viability (P ≤ 0.05). The methanol extract perturbed cell cycle progression, modulated cell cycle and regulated, signal molecules were involved in induction of apoptosis in HepG2 cells. Our findings indicate that phytochemicals of leaves of C. speciosus shows potential for natural therapeutic product development for hepatocellular carcinoma. This is the first report to demonstrate in vitro anticancer activity of leaf extract of C. speciosus in relation to liver cancer.

  3. Apoptotic and Inhibitory Effects on Cell Proliferation of Hepatocellular Carcinoma HepG2 Cells by Methanol Leaf Extract of Costus speciosus

    Directory of Open Access Journals (Sweden)

    Sandhya V. G. Nair

    2014-01-01

    Full Text Available Costus speciosus is a medicinal plant commonly known as wild ginger distributed in South and Southeast Asian countries. Leaves of this plant are used for ayurvedic treatment regimes in malignancies and mental illness. Rhizome extract from the plant is used to treat malignancies, pneumonia, urinary disorders, jaundice, rheumatism, and diabetes. The goal of this study was to investigate the effects of methanol extract of leaves of C. speciosus on the growth of human hepatocellular carcinoma (HepG2 cells and understand possible mechanisms of its action. Viability of HepG2 cells were measured by MTS assay after 24 h and 48 h treatment with extracts of 1, 10, 50, 100, and 200 μg/mL concentrations. Cell cycle analysis and apoptosis were evaluated by flow cytometry and caspase-3 induction. HepG2 cells treated with 100 μg/mL methanol leaf extract for 24 h displayed a significant reduction in cell viability (P≤0.05. The methanol extract perturbed cell cycle progression, modulated cell cycle and regulated, signal molecules were involved in induction of apoptosis in HepG2 cells. Our findings indicate that phytochemicals of leaves of C. speciosus shows potential for natural therapeutic product development for hepatocellular carcinoma. This is the first report to demonstrate in vitro anticancer activity of leaf extract of C. speciosus in relation to liver cancer.

  4. Biochemical effects of six TiO2 and four CeO2 nanomaterials in HepG2 cells

    Science.gov (United States)

    Biochemical effects of six TiO2 and four CeO2 nanomaterials in HepG2 cellsBecause of their growing number of uses, nanoparticles composed of CeO2 (cosmetics, polishing materials and automotive fuel additives) and TiO2 (pigments, sunscreens and photocatalysts) are of particular to...

  5. Pinus densiflora Sieb. et Zucc. Alleviates Lipogenesis and Oxidative Stress during Oleic Acid-Induced Steatosis in HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Yu-Jin Hwang

    2014-07-01

    Full Text Available Excess accumulation of lipids and oxidative stress in the liver contribute to nonalcoholic fatty liver disease (NAFLD. We hypothesized that Pinus densiflora Sieb. et Zucc. (PSZ can protect against NAFLD by regulating lipid accumulation and oxidative stress in the liver. To investigate the effect of PSZ upon NAFLD, we used an established cellular model: HepG2 cells treated with oleic acid. Then, the extent of hepatic steatosis and oxidative stress was assessed and levels of inflammatory markers measured. Oleic acid-treated HepG2 cells, compared with controls, had greater lipid accumulation. PSZ decreased lipid accumulation by 63% in oleic acid-treated HepG2 cells. Additionally, PSZ decreased the target gene expression of lipogenesis such as sterol regulatory element binding protein-1c, fatty acid synthase, stearoyl-CoA desaturase-1, diacylglycerol O-acyltransferase-1, and acetyl-CoA carboxylase-1 by 1.75, 6.0, 2.32, 1.93 and 1.81 fold, respectively. In addition, Oleic acid-treated HepG2 cells elicited extensive accumulation of tumor necrosis factor-α (TNFα by 4.53 fold, whereas PSZ-treated cells decreased the expression of TNFα mRNA by 1.76 fold. PSZ significantly inhibited oxidative stress induced by reactive oxygen species. These results suggest that PSZ has effects on steatosis in vitro and further studies are needed in vivo to verify the current observations.

  6. Pinus densiflora Sieb. et Zucc. alleviates lipogenesis and oxidative stress during oleic acid-induced steatosis in HepG2 cells.

    Science.gov (United States)

    Hwang, Yu-Jin; Wi, Hae-Ri; Kim, Haeng-Ran; Park, Kye Won; Hwang, Kyung-A

    2014-07-23

    Excess accumulation of lipids and oxidative stress in the liver contribute to nonalcoholic fatty liver disease (NAFLD). We hypothesized that Pinus densiflora Sieb. et Zucc. (PSZ) can protect against NAFLD by regulating lipid accumulation and oxidative stress in the liver. To investigate the effect of PSZ upon NAFLD, we used an established cellular model: HepG2 cells treated with oleic acid. Then, the extent of hepatic steatosis and oxidative stress was assessed and levels of inflammatory markers measured. Oleic acid-treated HepG2 cells, compared with controls, had greater lipid accumulation. PSZ decreased lipid accumulation by 63% in oleic acid-treated HepG2 cells. Additionally, PSZ decreased the target gene expression of lipogenesis such as sterol regulatory element binding protein-1c, fatty acid synthase, stearoyl-CoA desaturase-1, diacylglycerol O-acyltransferase-1, and acetyl-CoA carboxylase-1 by 1.75, 6.0, 2.32, 1.93 and 1.81 fold, respectively. In addition, Oleic acid-treated HepG2 cells elicited extensive accumulation of tumor necrosis factor-α (TNFα) by 4.53 fold, whereas PSZ-treated cells decreased the expression of TNFα mRNA by 1.76 fold. PSZ significantly inhibited oxidative stress induced by reactive oxygen species. These results suggest that PSZ has effects on steatosis in vitro and further studies are needed in vivo to verify the current observations.

  7. Screening and identification of a novel target specific for hepatoma cell line HepG2 from the FliTrx bacterial peptide library

    Institute of Scientific and Technical Information of China (English)

    Wenhan Li; Ping Lei; Bing Yu; Sha Wu; Jilin Peng; Xiaoping Zhao; Huffen Zhu; Michael Kirschfink; Guanxin Shen

    2008-01-01

    To explore new targets for hepatoma research, we used a surface display library to screen novel tumor cell-specific peptides. The bacterial FliTrx system was screened with living normal liver cell line L02 and hepatoma cell line HepG2 successively to search for hepatoma-specific peptides. Three clones (Hep1, Hep2, and Hep3) were identified to be specific to HepG2 compared with L02 and other cancer cell lines.Three-dimensional structural prediction proved that peptides inserted into the active site of Escherichia coli thioredoxin (TrxA) formed certain loop structures protruding out of the surface. Western blot analysis showed that FliC/TrxA-pepfide fusion proteins could be directly used to detect HepG2 cells.Three different FliC/TrxA-peptide fusion proteins targeted the same molecule, at approximately 140 kDa, on HepG2 cells.This work presented for the first time the application of the FliTrx library in screening living cells. Three peptides were obtained that could be potential candidates for targeted liver cancer therapy.

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

    Science.gov (United States)

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

    2013-01-01

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

  9. A Novel Exploration of a Combination of Gambogic Acid with TiO2 Nanofibers: The Photodynamic Effect for HepG2 Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Jingyuan Li

    2014-09-01

    Full Text Available As a good photosensitizer, TiO2 nanomaterials show potential biomedical applications, such as drug carriers or enhancers in photodynamic therapy. In this contribution, novel nanocomposites through the blending of TiO2 nanofibers with the active compound, gambogic acid (GA, were explored, and the results showed that GA could inhibit cancer cell proliferation in a time-dependent and dose-dependent manner, inducing apoptosis and cell cycle arrest at the G0/G1 phase in HepG2 cells. It is evident that after the GA-TiO2 nanocomposites were cultured with the cancer cells, the cooperation effect could effectively enhance the cytotoxicity of GA for HepG2 cells. Meanwhile, if activated by UV irradiation, under the presence of GA-TiO2 nanocomposites, this would lead to significant apoptosis and necrosis for HepG2 cells with a photodynamic therapy (PDT effect. Associated with the controlled drug-release from these nanocomposites, TiO2 nanofibers could readily cut down the drug consumption in HepG2 cells and reduce the side-effect for the normal cells and tissue, which may be further utilized in the therapeutic alliance for cancer therapy.

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

    Science.gov (United States)

    Differential genomic effects on signaling pathways by two different CeO2 nanoparticles in HepG2 cells. Sheau-Fung Thai1, Kathleen A. Wallace1, Carlton P. Jones1, Hongzu Ren2, Benjamin T. Castellon1, James Crooks2, Kirk T. Kitchin1. 1Integrated Systems Toxicology Divison, 2Resea...

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

    Science.gov (United States)

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

    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.

  12. Cellular interactions and biological responses to titanium dioxide nanoparticles in HepG2 and BEAS-2B cells: role of cell culture media.

    Science.gov (United States)

    Prasad, Raju Y; Simmons, Steven O; Killius, Micaela G; Zucker, Robert M; Kligerman, Andrew D; Blackman, Carl F; Fry, Rebecca C; Demarini, David M

    2014-05-01

    We showed previously that exposure of human lung cells (BEAS-2B) to TiO2 nanoparticles (nano-TiO2 ) produced micronuclei (MN) only when the final concentration of protein in the cell-culture medium was at least 1%. Nanoparticles localize in the liver; thus, we exposed human liver cells (HepG2) to nano-TiO2 and found the same requirement for MN induction. Nano-TiO2 also formed small agglomerates in medium containing as little as 1% protein and caused cellular interaction as measured by side scatter by flow cytometry and DNA damage (comet assay) in HepG2 cells. Nano-TiO2 also increased the activity of the inflammatory factor NFkB but not of AP1 in a reporter-gene HepG2 cell line. Suspension of nano-TiO2 in medium containing 0.1% protein was sufficient for induction of MN by the nanoparticles in either BEAS-2B or HepG2 cells as long the final concentration of protein in the cell-culture medium was at least 1%.

  13. Metabolomic effects in HepG2 cells exposed to four TiO2 amd two CeO2 naomaterials

    Science.gov (United States)

    Abstract It is difficult to evaluate nanomaterials potential toxicity and to make science-based societal choices. To better assess potential hepatotoxicity issues, human liver HepG2 cells were exposed to four Ti02 and two Ce02 nanomaterials at 30 ug m1-1 for t...

  14. Functional involvement of proteins, interacting with sphingolipids, in sphingolipid transport to the canalicular membrane in the human hepatocytic cell line, HepG2?

    NARCIS (Netherlands)

    Zegers, MMP; Zaal, KJM; Hoekstra, D

    A photoreactive sphingolipid precursor was used to investigate the potential involvement of protein-lipid interactions that may convey specificity to sphingolipid transport in the human hepatoma cell line, HepG2, A I-125-labeled, photoreactive ceramide, I-125-N-3-Cer, was incubated with the cells

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

    Science.gov (United States)

    Differential genomic effects on signaling pathways by two different CeO2 nanoparticles in HepG2 cells. Sheau-Fung Thai1, Kathleen A. Wallace1, Carlton P. Jones1, Hongzu Ren2, Benjamin T. Castellon1, James Crooks2, Kirk T. Kitchin1. 1Integrated Systems Toxicology Divison, 2Resea...

  16. DIFFERENTIAL-EFFECTS OF EICOSAPENTAENOIC ACID ON GLYCEROLIPID AND APOLIPOPROTEIN-B METABOLISM IN PRIMARY HUMAN HEPATOCYTES COMPARED TO HEPG2 CELLS AND PRIMARY RAT HEPATOCYTES

    NARCIS (Netherlands)

    LIN, YG; SMIT, MJ; HAVINGA, R; VERKADE, HJ; VONK, RJ; KUIPERS, F

    1995-01-01

    We compared the effects of eicosapentaenoic acid (EPA) and oleic acid (OA) on glycerolipid and apolipoprotein B (apoB) metabolism in primary human hepatocytes, HepG2 cells and primary rat hepatocytes. Cells were incubated for 1 to 5 h with 0.25 mM bovine serum albumin in the absence (control) or

  17. Biochemical effects of six TiO2 and four CeO2 nanomaterials in HepG2 cells

    Science.gov (United States)

    Biochemical effects of six TiO2 and four CeO2 nanomaterials in HepG2 cellsBecause of their growing number of uses, nanoparticles composed of CeO2 (cosmetics, polishing materials and automotive fuel additives) and TiO2 (pigments, sunscreens and photocatalysts) are of particular to...

  18. Amygdalin isolated from Semen Persicae (Tao Ren) extracts induces the expression of follistatin in HepG2 and C2C12 cell lines

    National Research Council Canada - National Science Library

    Yang, Chuanbin; Li, Xuechen; Rong, Jianhui

    2014-01-01

    .... The aqueous extracts of seven ISF-1 ingredients including Semen Persicae (Tao Ren) and the S. Persicae-derived fractions were assayed for the induction of follistatin mRNA expression in human hepatocarcinoma HepG2 cells by RT-PCR...

  19. Oleanolic Acid Attenuates Insulin Resistance via NF-κB to Regulate the IRS1-GLUT4 Pathway in HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Ming Li

    2015-01-01

    Full Text Available The aim of our study is to elucidate the mechanisms of oleanolic acid (OA on insulin resistance (IR in HepG2 cells. HepG2 cells were induced with FFA as the insulin resistance model and were treated with OA. Then the glucose content and the levels of tumor necrosis factor-α (TNF-α and interleukin-6 (IL-6 were analyzed. Moreover, protein expression of nuclear factor kappa B (NF-κB, insulin receptor substrate 1(IRS1, and glucose transporter 4 (GLUT4 in cells treated with OA were measured by Western blot analysis. Additionally, IRS1 protein expression exposed to OA was detected after using pyrrolidine dithiocarbamate (PDTC.Our results revealed that OA decreased the glucose content in HepG2 cells in vitro. Moreover, OA reduced the levels of TNF-α and IL-6 and upregulated IRS1 and GLUT4 protein expression. Furthermore, OA also reduced NF-κB protein expression in insulin-resistant HepG2 cells. After blocking NF-κB, the expression of IRS1 protein had no obvious changes when treated with OA. OA attenuated insulin resistance and decreased the levels of TNF-α and IL-6. Meanwhile, OA decreased NF-κB protein expression and upregulated IRS1 and GLUT4 protein expression. Therefore, regulating the IRS1-GLUT4 pathway via NF-κB was the underlying mechanism of OA on insulin resistance.

  20. Functional genomics analysis of low concentration of ethanol in human hepatocellular carcinoma (HepG2 cells. Role of genes involved in transcriptional and translational processes

    Directory of Open Access Journals (Sweden)

    Francisco Castaneda, Sigrid Rosin-Steiner, Klaus Jung

    2007-01-01

    Full Text Available We previously found that ethanol at millimolar level (1 mM activates the expression of transcription factors with subsequent regulation of apoptotic genes in human hepatocellular carcinoma (HCC HepG2 cells. However, the role of ethanol on the expression of genes implicated in transcriptional and translational processes remains unknown. Therefore, the aim of this study was to characterize the effect of low concentration of ethanol on gene expression profiling in HepG2 cells using cDNA microarrays with especial interest in genes with transcriptional and translational function. The gene expression pattern observed in the ethanol-treated HepG2 cells revealed a relatively similar pattern to that found in the untreated control cells. The pairwise comparison analysis demonstrated four significantly up-regulated (COBRA1, ITGB4, STAU2, and HMGN3 genes and one down-regulated (ANK3 gene. All these genes exert their function on transcriptional and translational processes and until now none of these genes have been associated with ethanol. This functional genomic analysis demonstrates the reported interaction between ethanol and ethanol-regulated genes. Moreover, it confirms the relationship between ethanol-regulated genes and various signaling pathways associated with ethanol-induced apoptosis. The data presented in this study represents an important contribution toward the understanding of the molecular mechanisms of ethanol at low concentration in HepG2 cells, a HCC-derived cell line.

  1. 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.

  2. Simultaneous recovery of dual pathways for ammonia metabolism do not improve further detoxification of ammonia in HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Fei-Yuan Zhang; Nan-Hong Tang; Xiao-Qian Wang; Xiu-Jin Li and Yan-Ling Chen

    2013-01-01

    BACKGROUND: Key  enzyme  deficiency  in  the  dual-pathway of  ammonia  metabolism  leads  to  low  detoxification  capacity of  HepG2  cells.  Previously,  we  established  a  HepG2/AFhGS cell  line  with  overexpression  of  human  glutamine  synthetase (hGS)  in  pathway    1  and  a  HepG2/(hArgI+hOTC)4  cell  line with overexpression of human arginase I (hArgI) and human ornithine transcarbamylase (hOTC) in pathway 2. The present study  aimed  to  investigate  whether  simultaneous  recovery  of the  two  pathways  contributes  to  the  further  improvement  of ammonia detoxification in HepG2 cells. METHODS: We  adopted  a  recombinant  retrovirus  carrying the  hGS  gene  to  infect  HepG2/(hArgI+hOTC)4  cells  and selected a new recombinant HepG2 cell line. The capacities of ammonia tolerance and detoxification in cells were detected by biochemical methods. Cell cycle PCR chip was used to assess the changes of gene expression. RESULTS: Introducing  hGS  into  HepG2/(hArgI+hOTC)4 cells did not lead to hGS overexpression, but inhibited hArgI expression.  The  levels  of  synthetic  glutamine  and  urea  in HepG2/(hArgI+hOTC+AFhGS)1 cells were significantly lower than  those  in  HepG2/(hArgI+hOTC)4  cells  when  cultured in  the  medium  with  10  and  15  mmol/L  glutamate  (Glu)  and with 60 and 180 mmol/L NH4Cl, respectively. In addition, the comparison of different cell growth showed that HepG2/AFhGS cells significantly lagged behind the other cells by the 5th and 7th  day,  indicating  that  introduction  of  hGS  impedes  HepG2 cell proliferation. Analysis of the mechanism suggested that the decreased expression of BCL2 played an important role. CONCLUSIONS: This study demonstrated that the recovery of two ammonia metabolic pathways in HepG2 cells is not helpful in increasing ammonia metabolism. The reinforcement of the pathway of urea metabolism is more important and valuable in improving the ammonia metabolism capacity in HepG2 cells.

  3. [Inhibitory effect of lentivirus-mediated hTERTp-TK combined with hTERTp-tumstatin on human hepatocarcinoma HepG2 cells].

    Science.gov (United States)

    Meng, Yu-Xi; Niu, Xin; Deng, Zhi-Hua

    2015-11-01

    To observe targeted expression of recombinant lentivirus-mediated (Lv)-hTERTp-TK and Lv-hTERTp-tumstatin in HepG2 cells, and explore the inhibitory effect of their combination on HepG2 cells both in vitro and in vivo. Lv-hTERTp-TK and Lv-hTERTptumstatin were used to infect HepG2 and L02 cells at different MOIs. Transfection efficiency was observed by fluorescence microscopy. Expression of TK and tumstatin mRNA was detected by reverse-transcriptase PCR. Proliferation and apoptosis were detected by MTT and flow cytometry, respectively. The HepG2 cells were examined by real time-PCR and western blotting to determine expression level of bcl-2 and VEGF mRNA and protein.A murine hepatocellular carcinoma model was established by injecting 1 * 10(7) HepG2 cells into 30 BALB/c nude mice. The modeled mice were randomly divided into a control group, mock group, Lv-hTERTp-tumstatin group, Lv-hTERTp-TK group, and combination group for four weeks of injections at regular intervals of PBS, Lv-hTERTp-null, Lv-hTERTp-tumstatin, Lv-hTERTp-TK, and Lv-hTERTp-tumstatin plus Lv-hTERTp-TK, respectively.Changes in tumor volume and weight, and cell morphology of tumor and major organs, were assessed by hematoxylin-eosin staining.Microvascular density of tumor tissue and cell apoptosis were assessed by immunohistochemical and TUNEL staining, respectively. The Lv-infected HepG2 cells, and not the Lv-infected L02 cells, expressed TK and tumstatin. Lv-hTERTp-TK and Lv-hTERTp-tumstatin, alone or in combination, inhibited proliferation and increased apoptosis of the HepG2 cells, but the combination was more effective than either alone (P less than 0.05). None of the treatments affected proliferation or apoptosis of the L02 cells (P more than 0.05). The combination also led to a greater reduction of bcl-2 and VEGF than either alone (all, P less than 0.05). Tumor growth was significantly inhibited by the combination (P less than 0.05). In vivo, the combination treatment induced the greatest amount of apoptosis of the HepG2 cells. Cell morphology of major organs such as liver, spleen and kidney were similar to the control group. The combination also produced the most significant effect on tumor microvascular density (P less than 0.05) and the highest apoptosis index (P less than 0.05). The HTERT promoter can induce targeted expression of TK and tumstatin in HepG2 cells. Lv-hTERTp-TK combined with Lv-hTERTp-tumstatin can significantly inhibit proliferation and induce apoptosis of human HepG2 cells in vitro and in vivo, which may be related to down-regulation ofbcl-2 and VEGF.

  4. Optimization of Albumin Secretion and Metabolic Activity of Cytochrome P450 1A1 of Human Hepatoblastoma HepG2 Cells in Multicellular Spheroids by Controlling Spheroid Size.

    Science.gov (United States)

    Nishikawa, Tomoko; Tanaka, Yutaro; Nishikawa, Makiya; Ogino, Yuka; Kusamori, Kosuke; Mizuno, Narumi; Mizukami, Yuya; Shimizu, Kazunori; Konishi, Satoshi; Takahashi, Yuki; Takakura, Yoshinobu

    2017-01-01

    Multicellular spheroids are useful as three-dimensional cell culture systems and for cell-based therapies. Their successful application requires an understanding of the consequences of spheroid size for cellular functions. In the present study, we prepared multicellular spheroids of different sizes using the human hepatoblastoma HepG2 cells, as hepatocytes are frequently used for in vitro drug screening and cell-based therapy. Precise polydimethylsiloxane-based microwells with widths of 360, 450, 560, and 770 µm were fabricated using a micromolding technique. Incubation of HepG2 cells in cell culture plates containing the microwells resulted in the formation of HepG2 spheroids with average diameters of 195, 320, 493, and 548 µm. The cell number per spheroid positively correlated with its diameter, and the viability of HepG2 cells was 94% or above for all samples. The smallest HepG2 spheroids showed the highest albumin secretion. On the other hand, the metabolic activity of 7-ethoxyresorufin, a fluorometric substrate for CYP1A1, increased with increasing spheroid size. These results indicate that controlling spheroid size is important when preparing HepG2 spheroids and that the size of HepG2 spheroids greatly influences the cellular function of HepG2 cells in the spheroids.

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

    Science.gov (United States)

    Motoyama, Keiichi; Hirai, Yumi; Nishiyama, Rena; Maeda, Yuki; Higashi, Taishi; Ishitsuka, Yoichi; Kondo, Yuki; Irie, Tetsumi; Era, Takumi

    2015-01-01

    Summary 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. PMID:26664628

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  7. Combination of small interfering RNA and lamivudine on inhibition of human B virus replication in HepG2.2.15 cells

    Institute of Scientific and Technical Information of China (English)

    Gui-Qiu Li; Wei-Zhen Xu; Jing-Xia Wang; Wen-Wei Deng; Di Li; Hong-Xi Gu

    2007-01-01

    AIM: To observe the inhibition of hepatitis B virus (HBV) replication and expression by combination of siRNA and lamivudine in HepG2.2.15 cells.METHODS: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. The transfected cells were cultured in lamivudine-containing medium (0.05 μmol/L) and harvested at 48, 72 and 96 h. The concentration of HBeAg and HBsAg was determined using ELISA. HBV DMA replication was examined by realtime PCR and the level of HBV mRNA was measured by RT-PCR.RESULTS: In HepG2.2.15 cells treated with combination of siRNA and lamivudine, the secretion of HBeAg and HBsAg into the supernatant was found to be inhibited by 91.80% and 82.40% (2.89 ± 0.48 vs 11.73±0.38,P<0.05;4.59±0.57 vs 16.25±O.48,P<0.05) at 96 h,respectively;the number of HBV DNA copies within culture medium was also significantly decreased at 96 h (1.04±0.26 vs 8.35±0.33,P<0.05).Moreover,mRNA concentration in HepG2.2.15 cells treated with combination of siRNA and lamivudine was obviously lower compared to those treated either with siRNA or lamivudine (19.44±0.17 vs 33.27±0.21 or 79.9±0.13,P<O.05).CONCLUSION:Combination of siRNA and lamivudine is more effective in inhibiting HBV replication as compared to the single use of siRNA or lamivudine in HepG2.2.15 cells.

  8. 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.

  9. HBsAg inhibits the translocation of JTB into mitochondria in HepG2 cells and potentially plays a role in HCC progression.

    Directory of Open Access Journals (Sweden)

    Yun-Peng Liu

    Full Text Available BACKGROUND AND AIMS: The expression of the jumping translocation breakpoint (JTB gene is upregulated in malignant liver tissues; however, JTB is associated with unbalanced translocations in many other types of cancer that suppress JTB expression. No comprehensive analysis on its function in human hepatocellular carcinoma (HCC has been performed to date. We aimed to define the biological consequences for interaction between JTB and HBsAg in HCC cell lines. METHODS: We employed the stable transfection to establish small HBsAg expressing HepG2 cell line, and stably silenced the JTB expression using short hairpin RNA in HepG2 cell line. The effects of JTB and small HBsAg in vitro were determined by assessing cell apoptosis and motility. RESULTS: Silencing of JTB expression promoted cancer cell motility and reduced cell apoptosis, which was significantly enhanced by HBs expression. Expression of HBsAg inhibited the translocation of JTB to the mitochondria. Furthermore, silencing of the JTB resulted in an increase in the phosphorylation of p65 in HepG2 cells and HepG2-HBs cells, whereas HBsAg expression decreased the phosphorylation of p65. The silencing of JTB in HepG2-HBs cells conferred increased advantages in cell motility and anti-apoptosis. CONCLUSION: HBsAg inhibited the translocation of JTB to the mitochondria and decreased the phosphorylation of p65 through the interaction with JTB, After JTB knockdown, HBsAg exhibited a stronger potential to promote tumor progression. Our data suggested that JTB act as a tumor suppressor gene in regards to HBV infection and its activation might be applied as a therapeutic strategy for in control of HBV related HCC development.

  10. 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.

  11. Protective effects of an ethanol extract of Angelica keiskei against acetaminophen-induced hepatotoxicity in HepG2 and HepaRG cells

    Science.gov (United States)

    Choi, Yoon-Hee; Lee, Hyun Sook; Chung, Cha-Kwon

    2017-01-01

    BACKGROUND/OBJECTIVE Although Angelica keiskei (AK) has widely been utilized for the purpose of general health improvement among Asian, its functionality and mechanism of action. The aim of this study was to determine the protective effect of ethanol extract of AK (AK-Ex) on acute hepatotoxicity induced by acetaminophen (AAP) in HepG2 human hepatocellular liver carcinoma cells and HepaRG human hepatic progenitor cells. MATERIALS/METHODS AK-Ex was prepared HepG2 and HepaRG cells were cultured with various concentrations and 30 mM AAP. The protective effects of AK-Ex against AAP-induced hepatotoxicity in HepG2 and HepaRG cells were evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, lactate dehydrogenase (LDH) assay, flow cytometry, and Western blotting. RESULTS AK-Ex, when administered prior to AAP, increased cell growth and decreased leakage of LDH in a dose-dependent manner in HepG2 and HepaRG cells against AAP-induced hepatotoxicity. AK-Ex increased the level of Bcl-2 and decreased the levels of Bax, Bok and Bik decreased the permeability of the mitochondrial membrane in HepG2 cells intoxicated with AAP. AK-Ex decreased the cleavage of poly (ADP-ribose) polymerase (PARP) and the activation of caspase-9, -7, and -3. CONCLUSIONS These results demonstrate that AK-Ex downregulates apoptosis via intrinsic and extrinsic pathways against AAP-induced hepatotoxicity. We suggest that AK could be a useful preventive agent against AAP-induced apoptosis in hepatocytes. PMID:28386382

  12. Effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca2+]i in the cells

    Institute of Scientific and Technical Information of China (English)

    Shi-Yong Gao; Qiu-Juan Wang; Yu-Bin Ji

    2006-01-01

    AIM: To observe the effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca2+]i in the cells, and to uncover the mechanism by which solanine induces apoptosis.METHODS: HepG2 cells were double stained with AO/EB, and morphological changes of the cells were observed using laser confocal scanning microscopy (LCSM). HepG2 cells were stained with TMRE, and change in the membrane potential of mitochondria in the cells were observed using LCSM. HepG2 cells were double stained with Fluo-3/AM, and change of [Ca2+]i in the cells were observed using LCSM. HepG2 cells were double stained with TMRE and Fluo-3/AM, and both the change in membrane potential of mitochondria and that of [Ca2+]i in the cells were observed using LCSM.RESULTS: Cells in treated groups showed typical signs of apoptosis. Staining with TMRE showed that solanine could lower membrane potential; staining with Fluo-3/AM showed that solanine could increase the concentration of Ca2+ in tumor cells; and those of double staining with TMRE and Fluo-3/AM showed that solanine could increase the concentration of Ca2+ in the cells at the same time as it lowered the membrane potential of mitochondria.CONCLUSION: Solanine opens up the PT channels in the membrane by lowering the membrane potential, leading to Ca2+ being transported down its concentration gradient, which in turn leads to the rise of the concentration of Ca2+ in the cell, turning on the mechanism for apoptosis.

  13. LRD-22, a novel dual dithiocarbamatic acid ester, inhibits Aurora-A kinase and induces apoptosis and cell cycle arrest in HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Huiling; Li, Ridong [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing (China); Li, Li [Department of Cell Biology, School of Basic Medical Sciences, Peking University, Beijing (China); Ge, Zemei [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing (China); Zhou, Rouli, E-mail: rlzhou@bjmu.edu.cn [Department of Cell Biology, School of Basic Medical Sciences, Peking University, Beijing (China); Li, Runtao, E-mail: lirt@bjmu.edu.cn [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing (China)

    2015-02-27

    In this study we investigated the antitumor activity of the novel dual dithiocarbamatic acid ester LRD-22 in vitro and in vivo. Several cancer cell lines were employed to determine the effect of LRD-22 on cell growth, and the MTT assay showed there was a significant decrease in viable tumor cell numbers in the presence of LRD-22, especially in the HepG2 cell line. Colony formation assay also showed LRD-22 strongly inhibits HepG2 cell growth. Evaluation of the mechanism involved showed that inhibitory effects of LRD-22 on cell growth are due to induction of apoptosis and G2/M arrest. LRD-22 inhibited Aurora-A phosphorylation at Thr{sub 288} and subsequently impaired p53 phosphorylation at Ser{sub 315} which was associated with the proteasome degradation pathway. Tumor suppressor protein p53 is stabilized by this mechanism and accumulates through inhibition of Aurora-A kinase activity via treatment with LRD-22. In vivo study of HepG2 xenograft in nude mice also shows LRD-22 suppresses tumor growth at a concentration of 5 mg/kg without animals suffering loss of body weight. In conclusion, our results demonstrate LRD-22 acts as an Aurora-A kinase inhibitor to induce apoptosis and inhibit proliferation in HepG2 cells, and should be considered as a promising targeting agent for HCC therapy. - Highlights: • LRD-22 significantly inhibits cancer cell growth, especially in the HepG2 cell line. • The inhibitory effect of LRD-22 is due to induction of apoptosis and cell cycle arrest. • LRD-22 inhibits Aurora-A phosphorylation which results in subsequent impairment of the p53 pathway. • LRD-22 suppresses tumor growth in xenograft mice without body weight loss.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Raza, Haider; John, Annie

    2015-01-01

    Non-steroidal anti-inflammatory drugs (NSAIDs), including acetaminophen (APAP), have been reported to induce cytotoxicity in cancer and non-cancerous cells. Overdose of acetaminophen (APAP) causes liver injury in humans and animals. Hepatic glutathione (GSH) depletion followed by oxidative stress and mitochondrial dysfunction are believed to be the main causes of APAP toxicity. The precise molecular mechanism of APAP toxicity in different cellular systems is, however, not clearly understood. Our previous studies on mouse macrophage J774.2 cells treated with APAP strongly suggest induction of apoptosis associated with mitochondrial dysfunction and oxidative stress. In the present study, using human hepatoma HepG2 cells, we have further demonstrated that macrophages are a more sensitive target for APAP-induced toxicity than HepG2 cells. Using similar dose- and time-point studies, a marked increase in apoptosis and DNA fragmentation were seen in macrophages compared to HepG2 cells. Differential effects of APAP on mitochondrial respiratory functions and oxidative stress were observed in the two cell lines which are presumably dependent on the varying degree of drug metabolism by the different cytochrome P450s and detoxification by glutathione S-transferase enzyme systems. Our results demonstrate a marked increase in the activity and expression of glutathione transferase (GST) and multidrug resistance (MDR1) proteins in APAP-treated HepG2 cells compared to macrophages. This may explain the apparent resistance of HepG2 cells to APAP toxicity. However, treatment of these cells with diallyl sulfide (DAS, 200 μM), a known chemopreventive agent from garlic extract, 24 h prior to APAP (10 μmol/ml for 18h) exhibited comparable cytoprotective effects in the two cell lines. These results may help in better understanding the mechanism of cytotoxicity caused by APAP and cytoprotection by chemopreventive agents in cancer and non-cancerous cellular systems.

  16. Alcohol dehydrogenase and cytochrome P450 2E1 can be induced by long-term exposure to ethanol in cultured liver HEP-G2 cells.

    Science.gov (United States)

    Balusikova, Kamila; Kovar, Jan

    2013-09-01

    It has been shown in previous studies that liver HEP-G2 cells (human hepatocellular carcinoma) lose their ability to express active alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1). Although both are ethanol-inducible enzymes, short-term exposure to ethanol does not cause any changes in expression or activity in cultured HEP-G2 cells. Therefore, we tested the effect of long-term exposure to ethanol on the expression and activity of both ADH and CYP2E1 in these cells. The expression of ADH and CYP2E1 was assessed at the mRNA and/or protein level using real-time PCR and Western blot analysis. Specific colorimetric assays were used for the measurement of ADH and CYP2E1 enzymatic activities. Caco-2 cells (active CYP2E1 and inactive ADH) were used as control cells. Significantly increased protein expression of ADH (about 2.5-fold) as well as CYP2E1 (about 1.6-fold) was found in HEP-G2 cells after long-term (12 mo) exposure to ethanol. The activity of ADH and CYP2E1 was also significantly increased from 12 ± 3 and 6 ± 1 nmol/h/mg of total protein to 191 ± 9 and 57 ± 9 nmol/h/mg of total protein, respectively. We suggest that the loss of activity of ethanol-metabolizing enzymes in cultured HEP-G2 cells is reversible and can be induced by prolonged exposure to ethanol. We are therefore able to reactivate HEP-G2 cells metabolic functions concerning ethanol oxidation just by modification of in vitro culture conditions without necessity of transfection with its side effect - enzyme overexpression.

  17. Diosmetin inhibits cell proliferation and induces apoptosis by regulating autophagy via the mammalian target of rapamycin pathway in hepatocellular carcinoma HepG2 cells.

    Science.gov (United States)

    Liu, Jie; Ren, Hao; Liu, Bin; Zhang, Qingyu; Li, Mingyi; Zhu, Runzhi

    2016-12-01

    Hepatocellular carcinoma (HCC), which is a type of malignant tumor, is the fifth most common cancer in men and ninth in women worldwide. The aim of the present study was to investigate the antitumor effect of diosmetin (DIOS) in hepatocellular carcinoma HepG2 cells. The proliferation, apoptosis and autophagy rates of HepG2 cells were measured following treatment with DIOS. The effects of DIOS treatment on HepG2 cell proliferation and apoptosis rates were analyzed using MTT assays and Annexin V staining, respectively. The effect of DIOS treatment on autophagy levels was assessed using transmission electron microscopy, green fluorescent protein (GFP)-microtubule-associated protein 1 light chain (LC3) transfection and LysoTracker Red staining. Furthermore, bafilomycin A1 (BA1), an autophagy inhibitor, was used to assess the association between DIOS and cell autophagy, proliferation and apoptosis. In addition, the expression of autophagy-related proteins [mammalian target of rapamycin (mTOR), phosphatidylinositol 3-kinase, P70S6K, phosphoinositide-dependent kinase-1, extracellular signal-regulated kinase, 5'-AMP-activated protein kinase and Akt] and apoptosis-related proteins [B-cell lymphoma (Bcl)-2-associated X protein, Bak, p53, Bcl-2 and caspase-3] were analyzed by western blotting. The results revealed that DIOS significantly inhibited proliferation (PHepG2 cells. It was also demonstrated that DIOS triggered autophagy by regulating the mTOR pathway in HepG2 cells. Notably, following treatment of HepG2 cells with the autophagy inhibitor, BA1, the expression of apoptosis-related proteins, including Bax, Bak and p53, were significantly decreased (PHepG2 cells via regulation of the mTOR pathway. Thus, the results of the current study indicate that DIOS may present a potential therapeutic agent for HCC treatment.

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

    Science.gov (United States)

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

    2017-03-15

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

  19. NaHCO3 enhances the antitumor activities of cytokine-induced killer cells against hepatocellular carcinoma HepG2 cells.

    Science.gov (United States)

    Yuan, Ya Hong; Zhou, Chun Fang; Yuan, Jiang; Liu, Li; Guo, Xing Rong; Wang, Xiao Li; Ding, Yan; Wang, Xiao Nan; Li, Dong Sheng; Tu, Han Jun

    2016-11-01

    The extracellular pH is lower inside solid tumors than in normal tissue. The acidic environment inhibits the cytotoxicity of lymphocytes in vitro and promotes tumor cell invasion. In the present study, both in vitro and in vivo experiments were conducted to investigate how NaHCO3 would affect the antitumor activities of cytokine-induced killer (CIK) cells against hepatocellular carcinoma (HCC) cells. For the in vitro experiments, HepG2 cells were cultured at pH 6.5 and 7.4 in the presence of CIK cells or CIK cell-conditioned medium (CMCIK). For the in vivo experiments, nude mice were xenografted with HepG2-luc cells and divided into four groups: i) CIK cells injection plus NaHCO3 feeding; ii) CIK cells injection plus drinking water feeding; iii) normal saline injection plus NaHCO3 feeding; and iv) normal saline injection plus drinking water feeding. The results indicated that the viability and growth rate of HepG2 cells were remarkably suppressed when co-cultured with CIK cells or CMCIK at pH 7.4 compared with those of HepG2 cells cultured under the same conditions but at pH 6.5. In the xenograft study, a marked synergistic antitumor effect of the combined therapy was observed. NaHCO3 feeding augmented the infiltration of cluster of differentiation 3-positive T lymphocytes into the tumor mass. Taken together, these data strongly suggest that the antitumor activities of CIK cells against HepG2 cells were negatively affected by the acidic environment inside the tumors, and neutralizing the pH (for example, via NaHCO3 administration), could therefore reduce or eliminate this influence. In addition, it should be recommended that oncologists routinely prescribe soda water to their patients, particularly during CIK cell therapy.

  20. [Extracellular Y-box binding protein-1 promotes proliferation and metastasis of HepG2 cells through Notch3 receptor].

    Science.gov (United States)

    Shi, J; Li, P; Zou, L; Chen, P; Zhang, L P

    2016-03-20

    To clarify whether HepG2 cells actively secrete Y-box binding protein-1 (YB-1) under stress conditions, and to investigate the pathological significance and mechanism of action of extracellular YB-1. HepG2 cells were stimulated and treated by gradient concentrations of lipopolysaccharide (LPS) and adriamycin, the supernatant of the culture solution was collected by centrifugation, and the established chemiluminescence immunoassay (CLIA) was used for real-time quantitative determination of YB-1 level in the supernatant. The co-immunoprecipitation assay was used to detect whether extracellular YB-1 specifically bound to Notch3 receptor, and Western blot was used to measure the expression of Notch-NICD. The gradient concentrations of recombinant YB-1 were co-cultured with HepG2 cells, and MTT and migration assays were used to analyze the proliferation and invasion/metastasis of HepG2 cells. One-way analysis of variance was used for comparison of data between multiple groups. The results of CLIA confirmed that the level of extracellular YB-1 in the supernatant was significantly higher than that in the control group (F= 10.54,PHepG2 cells (F= 9.405,PHepG2 cells can actively secrete YB-1 via non-classical pathways. Extracellular YB-1 can specifically bind to Notch3 receptor and further up-regulate its expression, and then promote the proliferation and invasion/metastasis of HepG2 cells. This study lays a foundation for further clarifying the pathogenesis of hepatocellular carcinoma and investigating the biological relationship between extracellular YB-1 and malignant tumors.

  1. Simultaneous induction of apoptosis and necroptosis by Tanshinone IIA in human hepatocellular carcinoma HepG2 cells

    Science.gov (United States)

    Lin, C-Y; Chang, T-W; Hsieh, W-H; Hung, M-C; Lin, I-H; Lai, S-C; Tzeng, Y-J

    2016-01-01

    Tanshinone IIA (Tan IIA), a constituent of the traditional medicinal plant Salvia miltiorrhiza BUNGE, has been reported to possess anticancer activity through induction of apoptosis in many cancer cells. Surprisingly, the present study finds that Tan IIA simultaneously causes apoptosis and necroptosis in human hepatocellular carcinoma HepG2 cells. We further find that apoptosis can be converted to necroptosis by pan-caspase inhibitor Z-VAD-fmk, and the two death modes can be blocked by necroptotic inhibitor necrostatin-1. The underlying mechanisms are revealed by analysis of the signaling molecules using western blotting. In control cells, FLICE inhibitory protein in short form (FLIPS) is expressed in relatively high levels and binds to caspase 8 in ripoptosome, which supposedly sustains cell survival. However, in Tan IIA-treated cells, FLIPS is down-regulated and may thus cause homodimer formation of cleaved caspase 8, cleavage of receptor-interacting serine/threonine-protein kinases 1, 3 (RIP1, RIP3), and mixed-lineage kinase domain-like (MLKL), in turn leads to cell apoptosis. In parallel, Tan IIA causes necroptosis by forming a suggested necrosomal complex composed of RIP1/RIP3. Regarding the inhibitors, z-VAD-fmk diminishes the cleaved caspase 8, RIP1, RIP3, and MLKL induced by Tan IIA, and reconstructs the ripoptosome complex, which marks cells moving from apoptosis to necroptosis. Nec-1 recovers the Tan IIA down-regulated FLIPS, consequently causes FLIPS to form heterodimer with caspase 8 and thus block apoptosis. Meanwhile, cleaved forms of RIP1 and RIP3 were observed preventing necroptosis. Intriguingly, the cytotoxicity of tumor necrosis factor-related apoptosis-inducing ligand to HepG2 cells is enhanced by Tan IIA in a pilot study, which may be attributed to low FLIPS levels induced by Tan IIA. In short, Tan IIA simultaneously induces both Nec-1 inhibition and FLIPS regulation-mediated apoptosis/necroptosis, which has not been previously documented. Moreover, the involvement of the cleavage type of MLKL in executing necroptosis warrants further investigation.

  2. In vitro treatment of HepG2 cells with saturated fatty acids reproduces mitochondrial dysfunction found in nonalcoholic steatohepatitis

    Directory of Open Access Journals (Sweden)

    Inmaculada García-Ruiz

    2015-02-01

    Full Text Available Activity of the oxidative phosphorylation system (OXPHOS is decreased in humans and mice with nonalcoholic steatohepatitis. Nitro-oxidative stress seems to be involved in its pathogenesis. The aim of this study was to determine whether fatty acids are implicated in the pathogenesis of this mitochondrial defect. In HepG2 cells, we analyzed the effect of saturated (palmitic and stearic acids and monounsaturated (oleic acid fatty acids on: OXPHOS activity; levels of protein expression of OXPHOS complexes and their subunits; gene expression and half-life of OXPHOS complexes; nitro-oxidative stress; and NADPH oxidase gene expression and activity. We also studied the effects of inhibiting or silencing NADPH oxidase on the palmitic-acid-induced nitro-oxidative stress and subsequent OXPHOS inhibition. Exposure of cultured HepG2 cells to saturated fatty acids resulted in a significant decrease in the OXPHOS activity. This effect was prevented in the presence of a mimic of manganese superoxide dismutase. Palmitic acid reduced the amount of both fully-assembled OXPHOS complexes and of complex subunits. This reduction was due mainly to an accelerated degradation of these subunits, which was associated with a 3-tyrosine nitration of mitochondrial proteins. Pretreatment of cells with uric acid, an antiperoxynitrite agent, prevented protein degradation induced by palmitic acid. A reduced gene expression also contributed to decrease mitochondrial DNA (mtDNA-encoded subunits. Saturated fatty acids induced oxidative stress and caused mtDNA oxidative damage. This effect was prevented by inhibiting NADPH oxidase. These acids activated NADPH oxidase gene expression and increased NADPH oxidase activity. Silencing this oxidase abrogated totally the inhibitory effect of palmitic acid on OXPHOS complex activity. We conclude that saturated fatty acids caused nitro-oxidative stress, reduced OXPHOS complex half-life and activity, and decreased gene expression of mtDNA-encoded subunits. These effects were mediated by activation of NADPH oxidase. That is, these acids reproduced mitochondrial dysfunction found in humans and animals with nonalcoholic steatohepatitis.

  3. Salvianolic acid B modulates the expression of drug-metabolizing enzymes in HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Qing-LanWang; QuocWu; Yan-Yan Tao; Cheng-Hai Liu; Hani El-Nezami

    2011-01-01

    BACKGROUND: Enzymes involved in drug and xenobiotic metabolism have been considered to exist in two groups: phase I and phase II enzymes. Cytochrome P450 isoenzymes (CYPs) are the most important phase I enzymes in the metabolism of xenobiotics. The products of phase I metabolism are then acted upon by phase II enzymes, including glutathione S-transferases (GSTs). Herbs that inhibit CYPs such as CYP3A4 or that induce GSTs may have the potential to protect against chemical carcinogenesis since the mutagenic effects of carcinogens are often mediated through an excess of CYP-generated reactive intermediates. This study was designed to investigate the effects of salvianolic acid B (Sal B), a pure compound extracted from Radix Salviae Miltiorrhizae, a Chinese herb, on cell proliferation and CYP1A2 and CYP3A4 mRNA expression in the presence or absence of rifampicin, a potent inducer of CYPs and GST protein expression in HepG2 cells. METHODS: HepG2 cells were incubated with different concentrations of Sal B. Cell proliferation was determined by SYTOX-Green nucleic acid staining. CYP3A4 and CYP1A2 mRNA expression was assayed by real-time PCR. GST protein expression was analyzed by Western blotting. RESULTS: Low concentrations of Sal B (0-20 μmol/L) had no significant effects on cell proliferation, while higher concentrations (100-250 μmol/L) significantly inhibited proliferation in a concentration-dependent manner. Tenμmol/L Sal B, but not 1 μmol/L, down-regulated CYP3A4 and CYP1A2 mRNA expression after 24 hours of incubation, whereas both 1 and 10 μmol/L Sal B down-regulated CYP3A4 mRNA expression after 96 hours of incubation; moreover, 1 and 10 μmol/L Sal B inhibited CYP3A4 mRNA expression induced by rifampicin. Both 1 μmol/L and 10 μmol/L Sal B increased GST expression. CONCLUSION: Sal B inhibits CYP3A4 and CYP1A2 mRNA expression and induces GST expression in HepG2 cells.

  4. 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 OTA in vitro.

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

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    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 sensitive to Se@PEI@siRNA. Moreover, Se@PEI@siRNA exhibited enhanced cytotoxic effects on cancer cells and triggered intracellular reactive oxygen species, and the signaling pathways of p53 and AKT were activated to advance cell apoptosis. Taken together, this study provides a strategy for the design of an anticancer nanosystem as a carrier of HSP70 siRNA to achieve synergistic cancer therapy. Keywords: selenium nanoparticles, siRNA, polyethylenimine, reactive oxygen species, apoptosis, anticancer

  6. Gene expression profiles in human HepG2 cells treated with extracts of the Tamarindus indica fruit pulp.

    Science.gov (United States)

    Razali, Nurhanani; Aziz, Azlina A; Junit, Sarni M

    2010-12-01

    Tamarindus indicaL. (T. indica) or locally known as asam jawa belongs to the family of Leguminosae. The fruit pulp had been reported to have antioxidant activities and possess hypolipidaemic effects. In this study, we attempted to investigate the gene expression patterns in human hepatoma HepG2 cell line in response to treatment with low concentration of the fruit pulp extracts. Microarray analysis using Affymetrix Human Genome 1.0 S.T arrays was used in the study. Microarray data were validated using semi-quantitative RT-PCR and real-time RT-PCR. Amongst the significantly up-regulated genes were those that code for the metallothioneins (MT1M, MT1F, MT1X) and glutathione S-transferases (GSTA1, GSTA2, GST02) that are involved in stress response. APOA4, APOA5, ABCG5 and MTTP genes were also significantly regulated that could be linked to hypolipidaemic activities of the T. indica fruit pulp.

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

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    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.

  8. 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.

  9. Pyrroloquinoline quinone increases the expression and activity of Sirt1 and -3 genes in HepG2 cells.

    Science.gov (United States)

    Zhang, Jian; Meruvu, Sunitha; Bedi, Yudhishtar Singh; Chau, Jason; Arguelles, Andrix; Rucker, Robert; Choudhury, Mahua

    2015-09-01

    Sirtuin (Sirt) 1 and Sirt 3 are nicotinamide adenine dinucleotide ((+))-dependent protein deacetylases that are important to a number of mitochondrial-related functions; thus, identification of sirtuin activators is important. Herein, we hypothesize that pyrroloquinoline quinone (PQQ) can act as a Sirt1/Sirt3 activator. In HepG2 cell cultures, PQQ increased the expression of Sirt1 and Sirt3 gene, protein, and activity levels (P < .05). We also observed a significant increase in nicotinamide phosphoribosyltransferase gene expression (as early as 18 hours) and increased NAD(+) activity at 24 hours. In addition, targets of Sirt1 and Sirt3 (peroxisome proliferator-activated receptor γ coactivator 1α, nuclear respiratory factor 1 and 2, and mitochondrial transcription factor A) were increased at 48 hours. This is the first report that demonstrates PQQ as an activator of Sirt1 and Sirt3 expression and activity, making it an attractive therapeutic agent for the treatment of metabolic diseases and for healthy aging. Based on our study and the available data in vivo, PQQ has the potential to serve as a therapeutic nutraceutical, when enhancing mitochondrial function.

  10. Anti-hepatocarcinoma Effects of a Food Additive Resveratrol Nanosuspension Against Human HepG2 Cells

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    Shi-Qiong Luo

    2015-05-01

    Full Text Available 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, a major symbol ingredient in red grapes and peanuts, 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, Res-nanosuspension (Res-NS composed of Res and poloxamer 188 was prepared by high pressure homogenization technique. The in vitro anti-hepatocarcinoma effects of Res-NS relative to efficacy of bulk Res were evaluated. The particle size and zeta potential of Res-NS were 159.4 nm and -2.1 mV, respectively. MTT assay showed that Res-NS effectively inhibited the proliferation of HepG2 cells and the corresponding IC50 values of Res-NS and bulk Res were 2.91 and 7.13 &mug/mL. These results suggest that the delivery of Res-NS is a promising approach for treating tumors.

  11. Anti-Hepatocarcinoma Effects of a Food Additive Resveratrol Nanosuspension against Human HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Shi-Qiong Luo

    2015-05-01

    Full Text Available 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, a major symbol ingredient in red grapes and peanuts, 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, Res-nanosuspension (Res-NS composed of Res and poloxamer 188 was prepared by high pressure homogenization technique. The in vitro anti-hepatocarcinoma effects of Res-NS relative to efficacy of bulk Res were evaluated. The particle size and zeta potential of Res-NS were 159.4 nm and -22.1 mV, respectively. MTT assay showed that Res-NS effectively inhibited the proliferation of HepG2 cells and the corresponding IC50 values of Res-NS and bulk Res were 2.91 and 7.13 &mug/mL. These results suggest that the delivery of Res-NS is a promising approach for treating tumors.

  12. Anti-hepatocarcinoma Effects of a Food Additive Chrysin Nanosuspension against Human HepG2 Cells

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    Zhi-ping Wang

    2015-03-01

    Full Text Available 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. Chrysin (Chr, a major symbol ingredient in Chinese Propolis, 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, Chr-Nanosuspension (Chr-NS composed of Chr and poloxamer 188 was prepared by high pressure homogenization technique. The in vitro anti-hepatocarcinoma effects of Chr-NS relative to efficacy of bulk Chr were evaluated. The particle size and zeta potential of Chr-NS were 291.1 nm and -28.7 mV, respectively. MTT assay showed that Chr-NS effectively inhibited the proliferation of HepG2 cells and the corresponding IC50 values of Chr-NS and bulk Chr were 1.55 and 3.76 &mug/mL. These results suggest that the delivery of Chr-NS is a promising approach for treating tumors.

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

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    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.

  14. 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.

  15. The protective effects of hydroxytyrosol against ortho-phenylphenol-induced DNA damage in HepG2 cells.

    Science.gov (United States)

    Li, Jianqing; Yang, Guang; Wang, Shaopeng; Jiang, Liping; Liu, Xiaofang; Geng, Chengyan; Zhong, Laifu; Chen, Min

    2012-07-01

    Ortho-phenylphenol (OPP) has been found to cause carcinomas in the urinary tract of rats. Since OPP is a potent genotoxic compound, and used as fungicides and antibacterial agents in fruits and fruit products, search for newer, better agents for protection against toxicity of OPP is required. In this study, the chemoprotective effect of hydroxytyrosol (HT) against OPP-induced DNA damage in HepG2 cells was investigated. Comet assay was used to detect the DNA damage induced by OPP. To elucidate the possible mechanisms, we tested lysosomal membrane stability, mitochondrial membrane potential, intracellular generation of reactive oxygen species (ROS), and reduced glutathione (GSH). Results showed that HT significantly reduced the DNA strand breaks caused by OPP. Moreover, HT effectively suppressed OPP-induced ROS formation, and increased the GSH level. Lysosomal membrane and mitochondrial membrane were also protected when cells were pretreated with HT. These results suggested that the disruption of lysosomal membrane integrity and the oxidative stress, leading to DNA fragmentation, may be the mechanism of DNA damage induced by OPP. The antioxidant activity of HT may play an important part in attenuating the DNA damage of OPP.

  16. 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

    2015-01-01

    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.

  17. 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.

  18. 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

    2014-01-01

    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. PMID:26190955

  19. Identification of protein tyrosine phosphatase SHP-2 as a new target of perfluoroalkyl acids in HepG2 cells.

    Science.gov (United States)

    Yang, Yu; Lv, Qi-Yan; Guo, Liang-Hong; Wan, Bin; Ren, Xiao-Min; Shi, Ya-Li; Cai, Ya-Qi

    2016-08-29

    Perfluoroalkyl acids (PFAAs) are widespread environmental contaminants which have been detected in humans and linked to adverse health effects. Previous toxicological studies mostly focused on nuclear receptor-mediated pathways and did not support the observed toxic effects. In this study, we aimed to investigate the molecular mechanisms of PFAA toxicities by identifying their biological targets in cells. Using a novel electrochemical biosensor, 16 PFAAs were evaluated for inhibition of protein tyrosine phosphatase SHP-2 activity. Their potency increased with PFAA chain length, with perfluorooctadecanoic acid (PFODA) showing the strongest inhibition. Three selected PFAAs, 25 μM perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid, and PFODA, also inhibited SHP-2 activity in HepG2 cells and increased paxillin phosphorylation level. PFOA was detected in the immunoprecipitated SHP-2 from the cells exposed to 250 μM PFOA, providing unequivocal evidence for the direct binding of PFOA with SHP-2 in the cell. Molecular docking rationalized the formation of PFAA/SHP-2 complex and chain length-dependent inhibition potency. Our results have established SHP-2 as a new cellular target of PFAAs.

  20. Gomisin A alters substrate interaction and reverses P-glycoprotein-mediated multidrug resistance in HepG2-DR cells.

    Science.gov (United States)

    Wan, Chi-Keung; Zhu, Guo-Yuan; Shen, Xiao-Ling; Chattopadhyay, Apurba; Dey, Saibal; Fong, Wang-Fun

    2006-09-28

    Through an extensive herbal drug screening program, we found that gomisin A, a dibenzocyclooctadiene compound isolated from Schisandra chinensis, reversed multidrug resistance (MDR) in Pgp-overexpressing HepG2-DR cells. Gomisin A was relatively non-toxic but without altering Pgp expression, it restored the cytotoxic actions of anticancer drugs such as vinblastine and doxorubicin that are Pgp substrates but may act by different mechanisms. Several lines of evidence suggest that gomisin A alters Pgp-substrate interaction but itself is neither a Pgp substrate nor competitive inhibitor. (1) First unlike Pgp substrates gomisin A inhibited the basal Pgp-associated ATPase (Pgp-ATPase) activity. (2) The cytotoxicity of gomisin A was not affected by Pgp competitive inhibitors such as verapamil. (3) Gomisin A acted as an uncompetitive inhibitor for Pgp-ATPase activity stimulated by the transport substrates verapamil and progesterone. (4) On the inhibition of rhodamine-123 efflux the effects of gomisin A and the competitive inhibitor verapamil were additive, so were the effects of gomisin A and the ATPase inhibitor vanadate. (5) Binding of transport substrates with Pgp would result in a Pgp conformational change favoring UIC-2 antibody reactivity but gomisin A impeded UIC-2 binding. (6) Photocrosslinking of Pgp with its transport substrate [125I]iodoarylazidoprazosin was inhibited by gomisin A in a concentration-dependent manner. Taken together our results suggest that gomisin A may bind to Pgp simultaneously with substrates and alters Pgp-substrate interaction.

  1. Double Strand Breaks and Cell-Cycle Arrest Induced by the Cyanobacterial Toxin Cylindrospermopsin in HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Bojana Žegura

    2013-08-01

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

  2. Hexadecylphosphocholine disrupts cholesterol homeostasis and induces the accumulation of free cholesterol in HepG2 tumour cells.

    Science.gov (United States)

    Jiménez-López, José M; Carrasco, María P; Marco, Carmen; Segovia, Josefa L

    2006-04-14

    Hexadecylphosphocholine (HePC) is a synthetic lipid belonging to the alkylphosphocholines (APC), a new group of antiproliferative agents that are proving to be promising candidates in anticancer therapy. We reported in a previous study that HePC interferes with phosphatidylcholine (PC) synthesis in HepG2 cells via both CDP-choline and phosphatidylethanolamine (PE) methylation. We have subsequently extended our studies to show that HePC interferes with sphingolipid metabolism by hindering the formation of sphingomyelin (SM), an effect accompanied by a substantial increase in the incorporation of the exogenous lipogenic precursors into ceramides. Interestingly, we demonstrate for the first time that HePC strongly inhibits the esterification of free cholesterol (FC) by acting at the level of acyl CoA:cholesterol acyltransferase (ACAT) (EC 2.3.1.26) activity. This effect is accompanied by a considerable increase in the synthesis of cholesterol, which leads to a rise in the levels of FC in cells. We are left in no doubt that the imbalance in the metabolism of membrane-lipid components vital to cell survival may well be responsible for the observed DNA fragmentation and activation of caspase-3, an enzyme involved in the cell apoptosis found in this study.

  3. Palmitic acid suppresses apolipoprotein M gene expression via the pathway of PPARβ/δ in HepG2 cells.

    Science.gov (United States)

    Luo, Guanghua; Shi, Yuanping; Zhang, Jun; Mu, Qinfeng; Qin, Li; Zheng, Lu; Feng, Yuehua; Berggren-Söderlund, Maria; Nilsson-Ehle, Peter; Zhang, Xiaoying; Xu, Ning

    2014-02-28

    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 (PPARβ/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 PPARβ/δ pathway.

  4. Determination of glutathione in single HepG2 cells by capillary electrophoresis with reduced graphene oxide modified microelectrode.

    Science.gov (United States)

    Wang, Xiaolei; Wang, Jun; Fu, Hongyan; Liu, Dongju; Chen, Zhenzhen

    2014-12-01

    Determination of intracellular bioactive species will afford beneficial information related to cell metabolism, signal transduction, cell function, and disease treatment. In this study, the electrochemically reduced graphene oxide modified carbon fiber microdisk electrode (ER-GOME) was used as a detector of CZE-electrochemical detection and developed to detect glutathione (GSH). The electrocatalytic activity of the modified microelectrode was characterized by cyclic voltammetry. Under optimized experimental conditions, the concentration linear range of GSH was from 1 to 60 μM. When the S/N ratio was 3, the concentration detection limit was 1 μM. Compared with the unmodified carbon fiber microdisk electrode, the sensitivity was enhanced more than five times. With the use of this method, the average contents of GSH in single HepG2 cells were found to be 7.13 ± 1.11 fmol (n = 10). Compared with gold/mercury amalgam microelectrode, which was usually used in determining GSH, the electrochemically reduced graphene oxide modified carbon fiber microdisk electrode was friendly to environment for free mercury. Furthermore, there were several merits of the novel electrochemical detector coupled with CE, such as comparative repeatability, easy fabrication, and high sensitivity, hold great potential for the single-cell assay.

  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. Multitargeting and antimetastatic potentials of silibinin in human HepG-2 and PLC/PRF/5 hepatoma cells.

    Science.gov (United States)

    Ghasemi, Reza; Ghaffari, Seyed H; Momeny, Majid; Pirouzpanah, Saeed; Yousefi, Mehdi; Malehmir, Mohsen; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir

    2013-01-01

    Hepatocellular carcinoma (HCC) is the most common sort of primary liver malignancy with poor prognosis. This study aimed at examining the effects of silibinin (a putative antimetastatic agent) on some transcriptional markers mechanistically related to HCC recurrence and metastasis in HepG-2 [hepatitis B virus (HBV)-negative and P53 intact) and PLC/PRF/5 (HBV-positive and P53 mutated) cells. The expression of 27 genes in response to silibinin was evaluated by real-time RT-PCR. The MMP gelatinolytic assay and microculture tetrazolium test (MTT) were tested. Silibinin was capable of suppressing the transcriptional levels of ANGPT2, ATP6L, CAP2, CCR6, CCR7, CLDN-10, cortactin, CXCR4, GLI2, HK2, ID1, KIAA0101, mortalin, PAK1, RHOA, SPINK1, and STMN1 as well as the enzymatic activity of MMP-2 but promoted the transcripts of CREB3L3, DDX3X, and PROX1 in both cells. Some significant differences between the cells in response to silibinin were detected that might be related to the differences of the cells in terms of HBV infection and/or P53 mutation, suggesting the possible influence of silibinin on HCC through biological functions of these 2 prognostic factors. In conclusion, our findings suggest that silibinin could potentially function as a multitargeting antimetastatic agent and might provide new insights for HCC therapy particularly for HBV-related and/or P53-mutated HCCs.

  7. Inhibitory effects of Citrus flavonoids on starch digestion and antihyperglycemic effects in HepG2 cells.

    Science.gov (United States)

    Shen, Wei; Xu, Ying; Lu, Yan-Hua

    2012-09-26

    Flavonoids are a class of important bioactive natural products and are being extensively used in functional foods. In the present study, the effects of four Citrus flavonoids (i.e., hesperidin, naringin, neohesperidin, and nobiletin) on amylase-catalyzed starch digestion, major digestive enzyme activities (e.g., pancreatic α-amylase and α-glucosidase), and glucose use in HepG2 cells were investigated. The results showed that all of the tested Citrus flavonoids significantly inhibited amylase-catalyzed starch digestion. Moreover, naringin and neohesperidin mainly inhibited amylose digestion, whereas hesperidin and nobiletin inhibited both amylose and amylopectin digestion. However, these flavonoids showed weak inhibitory activities against digestive enzymes. Furthermore, glucose consumption, glycogen concentration, and glucokinase activity were significantly elevated, and glucose-6-phosphatase activity was markedly decreased by Citrus flavonoids. These results demonstrate that Citrus flavonoids play important roles in preventing the progression of hyperglycemia, partly by binding to starch, increasing hepatic glycolysis and the glycogen concentration, and lowering hepatic gluconeogenesis. This work suggests that Citrus flavonoids might be potentially used for the prevention of postprandial hyperglycemia.

  8. 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

    2017-01-01

    Background/Aims 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. Methods 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 liver-specific markers was quantified on days 1, 7, 14, and 21. Results 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. Conclusions 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. PMID:27559001

  9. 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.

  10. Flow cytometric evaluation of the contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells.

    Science.gov (United States)

    Sahu, Saura C; Njoroge, Joyce; Bryce, Steven M; Zheng, Jiwen; Ihrie, John

    2016-04-01

    Exposure to nanosilver found in food- and cosmetics-related consumer products is of public concern because of the lack of information about its safety. In this study, two widely used in vitro cell culture models, human liver HepG2 and colon Caco2 cells, and the flow cytometric micronucleus (FCMN) assay were evaluated as tools for rapid predictive screening of the potential genotoxicity of nanosilver. Recently, we reported the genotoxicity of 20 nm nanosilver using these systems. In the current study presented here, we tested the hypothesis that the nanoparticle size and cell types were critical determinants of its genotoxicity. To test this hypothesis, we used the FCMN assay to evaluate the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge and obtained from the same commercial source using the same experimental conditions and in vitro models (HepG2 and Caco2) as previously tested for the 20 nm silver. Results of our study show that up to the concentrations tested in these cultured cell test systems, the smaller (20 nm) nanoparticle is genotoxic to both the cell types by inducing micronucleus (MN). However, the larger (50 nm) nanosilver induces MN only in HepG2 cells, but not in Caco2 cells. Also in this study, we evaluated the contribution of ionic silver to the genotoxic potential of nanosilver using silver acetate as the representative ionic silver. The MN frequencies in HepG2 and Caco2 cells exposed to the ionic silver in the concentration range tested are not statistically significant from the control values except at the top concentrations for both the cell types. Therefore, our results indicate that the ionic silver may not contribute to the MN-forming ability of nanosilver in HepG2 and Caco2 cells. Also our results suggest that the HepG2 and Caco2 cell cultures and the FCMN assay are useful tools for rapid predictive screening of a genotoxic potential of food- and cosmetics-related chemicals including nanosilver.

  11. Orexin A affects HepG2 human hepatocellular carcinoma cells glucose metabolism via HIF-1α-dependent and -independent mechanism.

    Science.gov (United States)

    Wan, Xing; Liu, Yuanyuan; Zhao, Yuyan; Sun, Xiaoqi; Fan, Dongxiao; Guo, Lei

    2017-01-01

    Orexins are hypothalamic neuropeptides that regulate feeding, reward, wakefulness and energy homeostasis. The present study sought to characterize the involvement of orexin A in glucose metabolism in HepG2 human hepatocellular carcinoma cells, and investigated the role of hypoxia-inducible factor-1α (HIF-1α) in the response. HepG2 cells were exposed to different concentrations of orexin A (10-9 to 10-7 M) in vitro, without or with the orexin receptor 1 (OX1R) inhibitor (SB334867), HIF-1α inhibitor (YC-1) or a combination of both inhibitors. Subsequently, OX1R, HIF-1α expression and localization, glucose uptake, glucose transporter 1 (GLUT1) expression and ATP content were measured. We further investigated the intracellular fate of glucose by measuring the gene expression of pyruvate dehydrogenase kinase 1 (PDK1), lactate dehydrogenase (LDHA) and pyruvate dehydrogenase B (PDHB), as well as metabolite levels including lactate generation and mitochondrial pyruvate dehydrogenase (PDH) activity. The activity of phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway was also assessed. Our results showed that the expression of OX1R was predominantly located in the nucleus in HepG2 cells. Orexin A oxygen-independently promoted the mRNA and protein expression of HIF-1α as well as its nuclear accumulation in HepG2 cells and the elevated HIF-1α protein was associated, at least partly, with the activation of the PI3K/Akt/mTOR pathway. Orexin A stimulated GLUT1 expression, glucose uptake as well as ATP generation in HepG2 cells via OX1R acting through the HIF-1α pathway. Moreover, orexin A inhibited LDHA, PDK1 expression and lactate production, stimulated PDHB expression and PDH enzyme activity independent of HIF-1α. Our results indicated that orexin signaling facilitated the glucose flux into mitochondrial oxidative metabolism rather than glycolysis in HepG2 cells. These findings provide new insight into the regulation of glucose metabolism by orexin A in hepatocellular carcinoma cells.

  12. 当归酰天芥菜定对 L1210,HepG2和HCC细胞的抑制作用%Inhibitory effects of angeloyl-heliotridine on L1210, HepG2 and HCC cells

    Institute of Scientific and Technical Information of China (English)

    王跃虎; 王建华

    2003-01-01

    目的: 研究当归酰天芥菜定(AH)的抗肿瘤活性及机制. 方法: 通过细胞生长曲线的绘制,分析AH对L1210细胞的抑制作用;通过MTT比色法,测定AH对L1210,HepG2和HCC细胞的抑制率;通过流式细胞术(FCM)检测AH对L1210细胞周期的影响. 结果: 在AH作用下,L1210细胞生长曲线斜率和最大生长密度降低. AH 80 mg*L-1对L1210细胞的抑制率为18.18%;AH 320 mg*L-1对HepG2和HCC细胞抑制率分别为12.28%和10.24%. FCM检测表明,AH 80 mg*L-1作用24 h后,L1210细胞的G2-M期细胞明显增加(P<0.01). 结论: AH对L1210细胞有一定的抑制作用,对HepG2和HCC细胞抑制作用较差. AH对L1210细胞的抑制作用发生在细胞周期的G2-M期.

  13. The effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca2+]i in the cells

    Science.gov (United States)

    Ji, YuBin; Gao, ShiYong; Ji, ChenFeng; Zou, Xiang

    2008-12-01

    To observe the effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca2+]i in the cells, and to uncover the mechanism by which solanine induces apoptosis. HepG2 cells are double stained with and Fluo-3/AM, and both the change in membrane potential of mitochondria and that of [Ca2+]i in the cells are observed using LCSM. The results of double staining with TMRE and Fluo-3/AM show that solanine can lower membrane potential and increase the concentration of Ca2+ in the cells Solanine opens up the PT channels in the membrane by lowering the membrane potential, leading to Ca2+ being transported down its concentration gradient, which in turn leads to the rise of the concentration of Ca2+ in the cell, turning on the mechanism for apoptosis.

  14. Potential role of novel hepatocellular carcinoma-associated gene IDD01 in promoting tumorigenesis of HepG2 cell line

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiang-yu; LI Jian-sheng; MA Jun; DUAN Fang-ling; ZHONG Peng

    2006-01-01

    Background We have used suppression subtractive hybridization to construct a subtracted cDNA library of hepatocellular carcinoma (HCC) and isolated a panel of differential expression sequence tag (ESTs). By using bioinformatics and rapid amplification of cDNA ends (RACE), we found a novel HCC-associated gene IDD01.To further investigate its function, a recombinant eukaryotic vector pEGFP/ORF was constructed and transfected into the HepG2 cell line.Methods The open reading frame (ORF) of IDD01 was amplified by RT-PCR, digested with Bamh I and Hind Ⅲ, and subcloned into the pEGFP-C 1 vector. The ligation reaction was conducted with T4 DNA ligase, and the recombinant vector was named pEGFP/ORF. Untransfer control (control group), pEGFP-C 1 (HepG2/C 1 group)and pEGFP/ORF (HepG2/ORF group) transfer groups were designed. Gene transfer was conducted with lipofectamine. To obtain stable transfection in HepG2 cells, selection was initiated with 500μg/ml G418. Cellular IDD01 mRNA levels were assayed by semi-quantitative RT-PCR. The MTT colorimetric method and flow cytometry were used to determine the cell proliferation. The tumorigenic potential of transformed cells was determined from their ability to grow as anchorage-independent colonies on soft agar. Transient transfections were performed to observe subcellular location of GFP-IDD01 fusion protein.Results A 778 bp specific band of ORF was obtained by RT-PCR, and the positive clone of recombinant plasmid pEGFP/ORF (5.5 Kb) was identified by restriction endonuclease cleavage and sequence. The brighmess ratio of IDD01 mRNA was not obvious between control and pEGFP/C1 groups, whereas the ratio of pEGFP/ORF was higher than that in the other two groups. After culture for 24-72 hours, the A490 values in pEGFP/ORF were higher than those in the other two groups (P<0.01). On histograms of flow cytometry, the S phase ratio of HepG2/ORF cells was significantly higher than that of the control and HepG2/C1 groups. The HepG2/ORF cells were able to form more colonies in soft agar compared with other HepG2 cell lines (P<0.01). GFP-IDD01 fusion protein predominantly localized in the plasma, whereas EGFP protein diffused all over the cell.Conclusion The IDD01 gene is a positive effector in cell proliferation and contributes to the carcinogenesis and progression of HCC. This gene may serve as a potential target for pharmaceutical intervention of HCC.

  15. Inhibition of NF-κB transcriptional activation in HepG2 cells by diterpenoids from the soft coral Sinularia maxima.

    Science.gov (United States)

    Thao, Nguyen Phuong; Nam, Nguyen Hoai; Cuong, Nguyen Xuan; Luyen, Bui Thi Thuy; Tai, Bui Huu; Kim, Ji Eun; Song, Seok Bean; Kiem, Phan Van; Minh, Chau Van; Kim, Young Ho

    2014-06-01

    Anti-inflammatory transcriptional effects of nineteen compounds (1-19) from the soft coral Sinularia maxima were evaluated using NF-κB luciferase and reverse transcriptase polymerase chain reaction. Compounds 1, 2, 4, 8, 15, 17, and 18 significantly inhibited TNFα-induced NF-κB transcriptional activity in HepG2 cells in a dose-dependent manner, with IC50 values ranging from 15.81 ± 2.29 to 29.10 ± 1.54 μM. Furthermore, the transcriptional inhibitory function of these compounds was confirmed by a decrease in intercellular adhesion molecule-1 and inducible nitric oxide synthase gene expression levels in HepG2 cells. These results provide a scientific rationale for the use of the soft coral S. maxima warrant further studies to develop new agents for the prevention and treatment of inflammatory.

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

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    A. Guzdek

    1997-01-01

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

  17. A study of Nigella sativa induced growth inhibition of MCF and HepG2 cell lines: An anti-neoplastic study along with its mechanism of action

    Directory of Open Access Journals (Sweden)

    Y Padmanabha Reddy

    2015-01-01

    Full Text Available Objective: To evaluate the anticancer potential of seeds of Nigella sativa using MCF and HepG2 cell lines along with its mechanism of action. Materials and Methods: (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay and acridine orange/ethidium bromide nuclear staining technique were selected to evaluate anticancer potential and mechanism of action of test extract. Results: Aqueous extract of N.sativa at a test dose of 180 mg and 300 mg was identified to be the best as anticancer agent against MCF and HepG2 cell lines among different solvent test extract where doxorubicin and cisplatin were employed as standard references. Discussion: Further study including separation and characterization of active principles in the aqueous extract shall prove beneficial.

  18. A New HPLC-MS Method for Measuring Maslinic Acid and Oleanolic Acid in HT29 and HepG2 Human Cancer Cells

    Science.gov (United States)

    Peragón, Juan; Rufino-Palomares, Eva E.; Muñoz-Espada, Irene; Reyes-Zurita, Fernando J.; Lupiáñez, José A.

    2015-01-01

    Maslinic acid (MA) and oleanolic acid (OA), the main triterpenic acids present in olive, have important properties for health and disease prevention. MA selectively inhibits cell proliferation of the HT29 human colon-cancer cell line by inducing selective apoptosis. For measuring the MA and OA concentration inside the cell and in the culture medium, a new HPLC-MS procedure has been developed. With this method, a determination of the amount of MA and OA incorporated into HT29 and HepG2 human cancer-cell lines incubated with different concentrations of MA corresponding to 50% growth inhibitory concentration (IC50), IC50/2, IC50/4, and IC50/8 has been made. The results demonstrate that this method is appropriate for determining the MA and OA concentration in different types of cultured cells and reveals the specific dynamics of entry of MA into HT29 and HepG2 cells. PMID:26370984

  19. 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.

  20. Increased oxidative stress and cytotoxicity by hydrogen sulfide in HepG2 cells overexpressing cytochrome P450 2E1.

    Science.gov (United States)

    Caro, Andres A; Thompson, Sarah; Tackett, Jonathan

    2011-12-01

    The main objectives of this work were to evaluate the effects of hydrogen sulfide on oxidative stress and cytotoxicity parameters in HepG2 cells and to assess the extent to which cytochrome P450 2E1 (CYP2E1) activity modulates the effects of hydrogen sulfide on oxidative stress and cytotoxicity. Sodium hydrosulfide (NaHS) caused time- and concentration-dependent cytotoxicity in both non-P450-expressing HepG2 cells (C34 cells) and CYP2E1-overexpressing HepG2 cells (E47 cells); however, NaHS-dependent cytotoxicity was higher in E47 than C34 cells. Cytotoxicity by NaHS in C34 and E47 cells was mainly necrotic in nature and associated with an early decrease in mitochondrial membrane potential. NaHS caused increased oxidation of lipophilic (C11-BODIPY(581/591)) and hydrophilic (DCFH-DA) probes only in E47 cells, at a time point prior to overt cytotoxicity. Trolox, an amphipathic antioxidant, partially inhibited both the cytotoxicity and the increased oxidative stress detected in E47 cells exposed to NaHS. Cell-permeable iron chelators and CYP2E1 inhibitors significantly inhibited the oxidation of C11-BODIPY(581/591) in E47 cells in the presence of NaHS. NaHS produced lipid peroxidation and cytotoxicity in E47 cells supplemented with a representative polyunsaturated fatty acid (docosahexaenoic acid) but not in C34 cells; these effects were inhibited by α-tocopherol, a lipophilic antioxidant. These data suggest that CYP2E1 enhances H(2)S-dependent cytotoxicity in HepG2 cells through the generation of iron-dependent oxidative stress and lipid peroxidation.

  1. Betalain and betaine composition of greenhouse- or field-produced beetroot ( Beta vulgaris L.) and inhibition of HepG2 cell proliferation.

    Science.gov (United States)

    Lee, Eun Jin; An, Dami; Nguyen, Chau T T; Patil, Bhimanagouda S; Kim, Jeongyun; Yoo, Kil Sun

    2014-02-12

    The composition of betalain, red or yellow pigments, and betaine (trimethylglycine or glycinebetaine) of nine beetroot ( Beta vulgaris L.) cultivars produced in the greenhouse or field was studied. Inhibition of HepG2 cell proliferation by betanin and betaine was also tested. Four predominant betalains, two betacyanins (betanin and isobetanin) and two betaxanthins (vulgaxanthin I and miraxanthin V), were isolated and quantified. Betanin and vulgaxanthin I were the major compounds in red and yellow beetroot extracts, respectively, and they comprised >90% of the betalain content in the tested cultivars. The total betalain content of beetroots produced from the field was between 650 and 800 μg/g fresh weight, approximately 25% higher than those from the greenhouse. The betaine content of the beetroot grown in the field was between 3.0 and 4.8 mg/g fresh weight, approximately 20% higher than in plants from the greenhouse. There was great variation among the cultivars with respect to their contents of betalains and betaine. In vitro cancer cell cytotoxicity was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay on HepG2 cells after exposure to betanin and betaine at concentrations ranging from 0 to 400 μg/mL and from 0 to 800 μg/mL for 48 h, respectively. Betanin resulted in a 49% inhibition of HepG2 cell proliferation at 200 μg/mL, and betaine yielded a 25% inhibition at 800 μg/mL, implying a higher cytotoxicity of betanin compared with betaine. The results indicated that the contents of health-beneficial compounds in beetroots, betalains and betaine, could be increased by modifying the growing conditions and that betanin and betaine extracted from beetroots had some anticancer effects against HepG2 cells.

  2. Thymosin beta 4 may translocate from the cytoplasm in to the nucleus in HepG2 cells following serum starvation. An ultrastructural study.

    Science.gov (United States)

    Piludu, Marco; Piras, Monica; Pichiri, Giuseppina; Coni, Pierpaolo; Orrù, Germano; Cabras, Tiziana; Messana, Irene; Faa, Gavino; Castagnola, Massimo

    2015-01-01

    Due to its actin-sequestering properties, thymosin beta-4 (Tβ4) is considered to play a significant role in the cellular metabolism. Several physiological properties of Tβ4 have been reported;, however, many questions concerning its cellular function remain to be ascertained. To better understand the role of this small peptide we have analyzed by means of transmission immunoelectron microscopy techniques the ultrastructural localization of Tβ4 in HepG2 cells. Samples of HepG2 cells were fixed in a mixture of 3% formaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer and processed for standard electron microscopic techniques. The samples were dehydrated in a cold graded methanol series and embedded in LR gold resin. Ultrathin sections were labeled with rabbit antibodies to Tβ4, followed by gold-labeled goat anti-rabbit, stained with uranyl acetate and bismuth subnitrate, observed and photographed in a JEOL 100S transmission electron microscope. High-resolution electron microscopy showed that Tβ4 was mainly restricted to the cytoplasm of HepG2 growing in complete medium. A strong Tβ4 reactivity was detected in the perinuclear region of the cytoplasmic compartment where gold particles appeared strictly associated to the nuclear membrane. In the nucleus specific Tβ4 labeling was observed in the nucleolus. The above electron microscopic results confirm and extend previous observations at light microscopic level, highlighting the subcellular distribution of Tβ4 in both cytoplasmic and nuclear compartments of HepG2 cells. The meaning of Tβ4 presence in the nucleolus is not on the best of our knowledge clarified yet. It could account for the interaction of Tβ4 with nucleolar actin and according with this hypothesis, Tβ4 could contribute together with the other nucleolar acting binding proteins to modulate the transcription activity of the RNA polymerases.

  3. 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.

  4. 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...

  5. Juglanthraquinone C, a novel natural compound derived from Juglans mandshurica Maxim, induces S phase arrest and apoptosis in HepG2 cells.

    Science.gov (United States)

    Yao, Yao; Zhang, Yu-Wei; Sun, Lu-Guo; Liu, Biao; Bao, Yong-Li; Lin, Hua; Zhang, Yu; Zheng, Li-Hua; Sun, Ying; Yu, Chun-Lei; Wu, Yin; Wang, Guan-Nan; Li, Yu-Xin

    2012-08-01

    Juglanthraquinone C (1,5-dihydroxy-9,10-anthraquinone-3-carboxylic acid, JC), a naturally occurring anthraquinone isolated from the stem bark of Juglans mandshurica, shows strong cytotoxicity in various human cancer cells in vitro. Here, we first performed a structure-activity relationship study of six anthraquinone compounds (JC, rhein, emodin, aloe-emodin, physcion and chrysophanol) to exploit the relationship between their structural features and activity. The results showed that JC exhibited the strongest cytotoxicity of all compounds evaluated. Next, we used JC to treat several human cancer cell lines and found that JC showed an inhibitory effect on cell viability in dose-dependent (2.5-10 μg/ml JC) and time-dependent (24-48 h) manners. Importantly, the inhibitory effect of JC on HepG2 (human hepatocellular carcinoma) cells was more significant as shown by an IC(50) value of 9 ± 1.4 μg/ml, and 36 ± 1.2 μg/ml in L02 (human normal liver) cells. Further study suggested that JC-induced inhibition HepG2 cell proliferation was associated with S phase arrest, decreased protein expression of proliferation marker Ki67, cyclin A and cyclin-dependent kinase (CDK) 2, and increased expression of cyclin E and CDK inhibitory protein Cip1/p21. In addition, JC significantly triggered apoptosis in HepG2 cells, which was characterized by increased chromatin condensation and DNA fragmentation, activation of caspase-9 and -3, and induction of a higher Bax/Bcl2 ratio. Collectively, our study demonstrated that JC can efficiently inhibit proliferation and induce apoptosis in HepG2 cells.

  6. 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.

  7. Oroxylin A reverses CAM-DR of HepG2 cells by suppressing Integrinβ1 and its related pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Binbin; Zhao, Li; Zhu, Litao; Wang, Hu; Sha, Yunying; Yao, Jing [State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009 (China); Li, Zhiyu [Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009 (China); You, Qidong, E-mail: youqidong@gmail.com [Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009 (China); Guo, Qinglong, E-mail: anticancer_drug@yahoo.com.cn [State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009 (China)

    2012-03-15

    Oroxylin A, a naturally occurring monoflavonoid extracted from Scutellariae radix, shows effective anticancer activities and low toxicities both in vivo and in vitro in previous studies. In this study, we investigated whether the CAM-DR model of HepG2 cells showed resistance to cytotoxic agents compared with normally cultured HepG2 cells. Furthermore, after the treatment of Paclitaxel, less inhibitory effects and decreased apoptosis rate were detected in the model. Data also revealed increased expression of Integrinβ1 might be responsible for the resistance ability. Moreover, Integrinβ1-siRNA-transfected CAM-DR HepG2 cells exhibited more inhibitory effects and higher levels of apoptosis than the non-transfected CAM-DR cells. The data corroborated that Integrinβ1 played a significant role in CAM-DR. After the treatment of weakly-toxic concentrations of Oroxylin A, the apoptosis induced by Paclitaxel in the CAM-DR model increased dramatically. Western blot assay revealed Oroxylin A markedly down-regulated the expression of Integrinβ1 and the activity of related pathway. As a conclusion, Oroxylin A can reverse the resistance of CAM-DR via inhibition of Integrinβ1 and its related pathway. Oroxylin A may be a potential candidate of a CAM-DR reversal agent. Highlights: ► Adhesion of HepG2 cells to fibronectin exhibited resistance to Paclitaxel. ► The resistance was associated with the increased expression of Integrinβ1. ► Knocking down Integrinβ1 can increase the toxicity of Paclitaxel on CAM-DR model. ► Oroxylin A reversed the resistance by suppressing Integrinβ1 and related pathway.

  8. Synthetic peptides from two Pf sporozoite invasion-associated proteins specifically interact with HeLa and HepG2 cells.

    Science.gov (United States)

    Arévalo-Pinzón, Gabriela; Curtidor, Hernando; Muñoz, Marina; Patarroyo, Manuel A; Patarroyo, Manuel E

    2011-09-01

    Two recently described molecules have been associated with sporozoite traversal ability and hepatocyte entry: sporozoite invasion-associated proteins (SIAP)-1 and -2. The HeLa and HepG2 cell binding ability of synthetic peptides spanning the whole SIAP-1 and -2 sequences has been studied in the search for identifying these proteins' functionally active specific regions. Twelve HepG-2 and seventeen HeLa cell high-activity binding peptides (HABPs) have been identified in SIAP-1, 8 of them having high specific binding affinity for both cell lines. Four HepG2 HABPs and two HeLa HABPs have been identified in SIAP-2, one of them interacting with both HeLa and HepG2 cells. SIAP-1 and SIAP-2 HABPs bound specifically and saturably to heparin sulfate and chondroitin sulfate-type membrane receptors on host cells. Circular dichroism assays have shown high α-helix content in SIAP-1 and SIAP-2 HABP secondary structure. Immunofluorescence analysis has revealed that specific peptides against SIAP proteins are highly immunogenic in mice and that anti-SIAP-1 and -2 antibodies recognize the native protein in Plasmodium falciparum sporozoites. Polymorphism studies have shown that a most SIAP-1 and -2 HABPs are conserved among P. falciparum strains. Our results have suggested that SIAP-1 and -2 participate in host-pathogen interactions during cell-traversal and hepatocyte invasion and highlighted the relevance of the ongoing identification and study of potentially new molecules when designing a fully protective antimalarial vaccine.

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

    Science.gov (United States)

    König, Bettina; Eder, Klaus

    2006-06-01

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

  10. Transcriptome Analysis of HepG2 Cells Expressing ORF3 from Swine Hepatitis E Virus to Determine the Effects of ORF3 on Host Cells

    Directory of Open Access Journals (Sweden)

    Kailian Xu

    2016-01-01

    Full Text Available Hepatitis E virus- (HEV- mediated hepatitis has become a global public health problem. An important regulatory protein of HEV, ORF3, influences multiple signal pathways in host cells. In this study, to investigate the function of ORF3 from the swine form of HEV (SHEV, high-throughput RNA-Seq-based screening was performed to identify the differentially expressed genes in ORF3-expressing HepG2 cells. The results were validated with quantitative real-time PCR and gene ontology was employed to assign differentially expressed genes to functional categories. The results indicated that, in the established ORF3-expressing HepG2 cells, the mRNA levels of CLDN6, YLPM1, APOC3, NLRP1, SCARA3, FGA, FGG, FGB, and FREM1 were upregulated, whereas the mRNA levels of SLC2A3, DKK1, BPIFB2, and PTGR1 were downregulated. The deregulated expression of CLDN6 and FREM1 might contribute to changes in integral membrane protein and basement membrane protein expression, expression changes for NLRP1 might affect the apoptosis of HepG2 cells, and the altered expression of APOC3, SCARA3, and DKK1 may affect lipid metabolism in HepG2 cells. In conclusion, ORF3 plays a functional role in virus-cell interactions by affecting the expression of integral membrane protein and basement membrane proteins and by altering the process of apoptosis and lipid metabolism in host cells. These findings provide important insight into the pathogenic mechanism of HEV.

  11. Transcriptome Analysis of HepG2 Cells Expressing ORF3 from Swine Hepatitis E Virus to Determine the Effects of ORF3 on Host Cells

    Science.gov (United States)

    Guo, Shiyu; Zhao, Tianjing; Zhu, Huapei; Jiao, Hanwei; Shi, Qiaoyun; Pang, Feng; Li, Yaying; Li, Guohua; Peng, Dongmei; Nie, Xin; Wu, Kebang; Du, Li; Cui, Ke

    2016-01-01

    Hepatitis E virus- (HEV-) mediated hepatitis has become a global public health problem. An important regulatory protein of HEV, ORF3, influences multiple signal pathways in host cells. In this study, to investigate the function of ORF3 from the swine form of HEV (SHEV), high-throughput RNA-Seq-based screening was performed to identify the differentially expressed genes in ORF3-expressing HepG2 cells. The results were validated with quantitative real-time PCR and gene ontology was employed to assign differentially expressed genes to functional categories. The results indicated that, in the established ORF3-expressing HepG2 cells, the mRNA levels of CLDN6, YLPM1, APOC3, NLRP1, SCARA3, FGA, FGG, FGB, and FREM1 were upregulated, whereas the mRNA levels of SLC2A3, DKK1, BPIFB2, and PTGR1 were downregulated. The deregulated expression of CLDN6 and FREM1 might contribute to changes in integral membrane protein and basement membrane protein expression, expression changes for NLRP1 might affect the apoptosis of HepG2 cells, and the altered expression of APOC3, SCARA3, and DKK1 may affect lipid metabolism in HepG2 cells. In conclusion, ORF3 plays a functional role in virus-cell interactions by affecting the expression of integral membrane protein and basement membrane proteins and by altering the process of apoptosis and lipid metabolism in host cells. These findings provide important insight into the pathogenic mechanism of HEV. PMID:27648443

  12. XRCC1 deficiency increased the DNA damage induced by γ-ray in HepG2 cell: Involvement of DSB repair and cell cycle arrest.

    Science.gov (United States)

    Niu, Yujie; Zhang, Xing; Zheng, Yuxin; Zhang, Rong

    2013-09-01

    γ-ray irradiation can induce DNA damages which include base damages, single-strand breaks and double-strand breaks in various type cells. The DNA repair protein XRCC1, as a part of the BER pathway, forms complexes with DNA polymerase beta, DNA ligase III and poly-ADP-ribose polymerase (PARP) in the repair of DNA single strand breaks and also affects the repair of double strand breaks. However, it is still not known well whether XRCC1 contributes to affect the irradiation sensitivity and DNA damage in HepG2 cell and the potential mechanism. Hence, the purpose of this study was to explore whether abrogation of XRCC1 gene expression by shRNA could reduce DNA repair and thus sensitize HepG2 cells to γ-ray. Cell viability was measured by Trypan blue staining and cloning efficiency assay. The DNA damage was detected by Comet assay. Apoptosis and cell cycle were detected by flow cytometry. The DNA-PKcs and gadd153 mRNA expression were determined by Real-time PCR. Our results showed that abrogation of XRCC 1 could sensitize HepG2 cells to γ-ray. This enhanced sensitivity could be attributed to the increased DNA damage and increased cell cycle arrest, which might be related with the increasing of DNA-PKcs and gadd153 mRNA expression. Therefore, our results suggested that the γ-ray irradiation sensitivity could be increased by targeting inhibition of XRCC1 in HepG2 cell.

  13. 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...

  14. Thymosin Beta 4 May Translocate from the Cytoplasm in to the Nucleus in HepG2 Cells following Serum Starvation. An Ultrastructural Study

    Science.gov (United States)

    Piludu, Marco; Piras, Monica; Pichiri, Giuseppina; Coni, Pierpaolo; Orrù, Germano; Cabras, Tiziana; Messana, Irene; Faa, Gavino; Castagnola, Massimo

    2015-01-01

    Due to its actin-sequestering properties, thymosin beta-4 (Tβ4) is considered to play a significant role in the cellular metabolism. Several physiological properties of Tβ4 have been reported;, however, many questions concerning its cellular function remain to be ascertained. To better understand the role of this small peptide we have analyzed by means of transmission immunoelectron microscopy techniques the ultrastructural localization of Tβ4 in HepG2 cells. Samples of HepG2 cells were fixed in a mixture of 3% formaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer and processed for standard electron microscopic techniques. The samples were dehydrated in a cold graded methanol series and embedded in LR gold resin. Ultrathin sections were labeled with rabbit antibodies to Tβ4, followed by gold-labeled goat anti-rabbit, stained with uranyl acetate and bismuth subnitrate, observed and photographed in a JEOL 100S transmission electron microscope. High-resolution electron microscopy showed that Tβ4 was mainly restricted to the cytoplasm of HepG2 growing in complete medium. A strong Tβ4 reactivity was detected in the perinuclear region of the cytoplasmic compartment where gold particles appeared strictly associated to the nuclear membrane. In the nucleus specific Tβ4 labeling was observed in the nucleolus. The above electron microscopic results confirm and extend previous observations at light microscopic level, highlighting the subcellular distribution of Tβ4 in both cytoplasmic and nuclear compartments of HepG2 cells. The meaning of Tβ4 presence in the nucleolus is not on the best of our knowledge clarified yet. It could account for the interaction of Tβ4 with nucleolar actin and according with this hypothesis, Tβ4 could contribute together with the other nucleolar acting binding proteins to modulate the transcription activity of the RNA polymerases. PMID:25835495

  15. Hesperidin from Citrus seed induces human hepatocellular carcinoma HepG2 cell apoptosis via both mitochondrial and death receptor pathways

    OpenAIRE

    2015-01-01

    Citrus seeds are full of phenolic compounds, such as flavonoids. The aims of this study were to identify the types of flavonoids in Citrus seed extracts, the cytotoxic effect, mode of cell death, and signaling pathway in human hepatic cancer HepG2 cells. The flavonoids contain anticancer, free radical scavenging, and antioxidant activities. Neohesperidin, hesperidin, and naringin, active flavanone glycosides, were identified in Citrus seed extract. The cytotoxic effect of three compounds was ...

  16. 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.

  17. Pro-oxidant effect of ALA is implicated in mitochondrial dysfunction of HepG2 cells.

    Science.gov (United States)

    Laafi, Jihane; Homedan, Chadi; Jacques, Caroline; Gueguen, Naig; Schmitt, Caroline; Puy, Hervé; Reynier, Pascal; Carmen Martinez, Maria; Malthièry, Yves

    2014-11-01

    Heme biosynthesis begins in the mitochondrion with the formation of delta-aminolevulinic acid (ALA). In acute intermittent porphyria, hereditary tyrosinemia type I and lead poisoning patients, ALA is accumulated in plasma and in organs, especially the liver. These diseases are also associated with neuromuscular dysfunction and increased incidence of hepatocellular carcinoma. Many studies suggest that this damage may originate from ALA-induced oxidative stress following its accumulation. Using the MnSOD as an oxidative stress marker, we showed here that ALA treatment of cultured cells induced ROS production, increasing with ALA concentration. The mitochondrial energetic function of ALA-treated HepG2 cells was further explored. Mitochondrial respiration and ATP content were reduced compared to control cells. For the 300 μM treatment, ALA induced a mitochondrial mass decrease and a mitochondrial network imbalance although neither necrosis nor apoptosis were observed. The up regulation of PGC-1, Tfam and ND5 genes was also found; these genes encode mitochondrial proteins involved in mitochondrial biogenesis activation and OXPHOS function. We propose that ALA may constitute an internal bioenergetic signal, which initiates a coordinated upregulation of respiratory genes, which ultimately drives mitochondrial metabolic adaptation within cells. The addition of an antioxidant, Manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), resulted in improvement of maximal respiratory chain capacity with 300 μM ALA. Our results suggest that mitochondria, an ALA-production site, are more sensitive to pro-oxidant effect of ALA, and may be directly involved in pathophysiology of patients with inherited or acquired porphyria. Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

  18. Glycoalkaloids and metabolites inhibit the growth of human colon (HT29) and liver (HepG2) cancer cells.

    Science.gov (United States)

    Lee, Kap-Rang; Kozukue, Nobuyuki; Han, Jae-Sook; Park, Joon-Hong; Chang, Eun-Young; Baek, Eun-Jung; Chang, Jong-Sun; Friedman, Mendel

    2004-05-19

    As part of an effort to improve plant-derived foods such as potatoes, eggplants, and tomatoes, the antiproliferative activities against human colon (HT29) and liver (HepG2) cancer cells of a series of structurally related individual compounds were examined using a microculture tetrazolium (MTT) assay. The objective was to assess the roles of the carbohydrate side chain and aglycon part of Solanum glycosides in influencing inhibitory activities of these compounds. Evaluations were carried out with four concentrations each (0.1, 1, 10, and 100 microg/mL) of the the potato trisaccharide glycoalkaloids alpha-chaconine and alpha-solanine; the disaccharides beta(1)-chaconine, beta(2)-chaconine, and beta(2)-solanine; the monosaccharide gamma-chaconine and their common aglycon solanidine; the tetrasaccharide potato glycoalkaloid dehydrocommersonine; the potato aglycon demissidine; the tetrasaccharide tomato glycoalkaloid alpha-tomatine, the trisaccharide beta(1)-tomatine, the disaccharide gamma-tomatine, the monosaccharide delta-tomatine, and their common aglycon tomatidine; the eggplant glycoalkaloids solamargine and solasonine and their common aglycon solasodine; and the nonsteroidal alkaloid jervine. All compounds were active in the assay, with the glycoalkaloids being the most active and the hydrolysis products less so. The effectiveness against the liver cells was greater than against the colon cells. Potencies of alpha-tomatine and alpha-chaconine at a concentration of 1 microg/mL against the liver carcinoma cells were higher than those observed with the anticancer drugs doxorubicin and camptothecin. Because alpha-chaconine, alpha-solanine, and alpha-tomatine also inhibited normal human liver HeLa (Chang) cells, safety considerations should guide the use of these compounds as preventative or therapeutic treatments against carcinomas.

  19. Momordin Ic induces HepG2 cell apoptosis through MAPK and PI3K/Akt-mediated mitochondrial pathways.

    Science.gov (United States)

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

    2013-06-01

    Momordin Ic is a natural triterpenoid saponin enriched in various Chinese and Japanese natural medicines such as the fruit of Kochia scoparia (L.) Schrad. So far, there is little scientific evidence for momordin Ic with regard to the anti-tumor activities. The aim of this work was to elucidate the anti-tumor effect of momordin Ic and the signal transduction pathways involved. We found that momordin Ic induced apoptosis in human hepatocellular carcinoma HepG2 cells, which were supported by DNA fragmentation, caspase-3 activation and PARP cleavage. Meanwhile, momordin Ic triggered reactive oxygen species (ROS) production together with collapse of mitochondrial membrane potential, cytochrome c release, down-regulation of Bcl-2 and up-regulation of Bax expression. The activation of p38 and JNK, inactivation of Erk1/2 and Akt were also demonstrated. Although ROS production rather than NO was stimulated, the expression of iNOS and HO-1 were altered after momordin Ic treatment for 4 h. Furthermore, the cytochrome c release, caspase-3 activation, Bax/Bcl-2 expression and PARP cleavage were promoted with LY294002 and U0126 intervention but were blocked by SB203580, SP600125, PI3K activator, NAC and 1,400 W pretreatment, demonstrating the mitochondrial disruption. Furthermore, momordin Ic combination with NAC influenced MAPK, PI3K/Akt and HO-1, iNOS pathways, MAPK and PI3K/Akt pathways also regulated the expression of HO-1 and iNOS. These results indicated that momordin Ic induced apoptosis through oxidative stress-regulated mitochondrial dysfunction involving the MAPK and PI3K-mediated iNOS and HO-1 pathways. Thus, momordin Ic might represent a potential source of anticancer candidate.

  20. INTEGRATION OF CELLULAR AND MOLECULAR ENDPOINTS TO ASSESS THE TOXICITY OF POLYCYCLIC AROMATIC HYDROCARBONS IN HEPG2 CELL LINE.

    Science.gov (United States)

    Morgado, Patrícia I; Jose, Sílvia; Wanke, Riccardo; Antunes, Alexandra M M; Sofia Cardoso, Ana; Jordao, Luisa

    2017-07-21

    Polycyclic aromatic hydrocarbons (PAHs) are persistent pollutants present in the environment with known mutagenic and carcinogenic properties. In the present study the effect of exposure to single or multiple doses of benzo[a]anthracene (BaA), pyrene (Pyr) and three halogenated derivatives of these compounds (1-ClPyr; 1-BrPyr and 7-ClBaA) were evaluated in a liver-derived human cell line (HepG2). Cytotoxicity as assessed by the classic MTT and neutral red assays showed a mild toxic effect in response to single or multiple dose exposure for up to 72h; except for multiple dose exposure to BaA and 7-ClBaA (1μM per day during 4 days) and single exposure to 10 μM BaA. Furthermore, selective mitochondrial and lysosomal toxicity was observed for Pyr and BaA series, respectively. In order to understand the underlying molecular mechanisms responsible for this effect ROS production, mitochondrial membrane depolarization, lysosomal pH, DNA fragmentation, early and late apoptosis mediators were evaluated after exposure to single doses of the compounds. All compounds were able to trigger oxidative stress after 24h as measured by catalase activity and a good correlation was found between mitochondrial membrane depolarization, lysosomal pH increase and MTT and neutral red assays, respectively. The evaluation of cell death mediators showed that caspase-3/7 but not annexin-V pathways were involved in toxicity triggered by the studied compounds. The integration of all results showed that 1-BrPyr and BaA have a higher toxicity potential. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.