Drug Resistance, Multiple ; radiation effects ; Drug Resistance, Neoplasm ; radiation effects ; Hep G2 Cells ; Humans ; X-Rays

OBJECTIVETo investigate the mechanism of ethanol-induced calcium overload in hepatocytes and the related role of store-operated calcium channels (SOCs).

METHODSHepG2 cells were treated an ethanol concentration gradient with or without intervention treatment with the extracellular calcium chelator EGTA or the SOCs inhibitor 2-aminoethoxydiphenyl borate (2-APB). Effects on cell viability were assessed by the CCK8 assay. Effects on leakage of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by automatic biochemical analyzer measurements of the culture supernatants. Effects on cytoplasmic free Ca2+ concentration ([Ca2+]i) were accessed by detecting fluorescence intensity of the calcium indicator Fluo-3/AM with a flow cytometer. Effects on mRNA and protein expression levels of SOCs, stromal interacting factor 1 (STIM1), and calcium release-activated calcium channel protein 1 (Orai1) were evaluated by qPCR and western blotting.

RESULTSThe ethanol treatment produced dose-dependent reduction in cell viability (r = -0.985, P less than 0.01) and increases in leakage of ALT (F = 15.286, P less than 0.01) and AST (F = 39.674, P less than 0.01). Compared to untreated controls, the ethanol treatments of 25, 50, 100, 200 and 400 mM induced significant increases in [Ca2+]i level (1.25+/-0.36, 1.31+/-0.15, 1.41+/-0.18, 2.29+/-0.25, 2.58+/-0.19; F = 15.286, P less than 0.01). Both intervention treatments, EGTA and 2-APB, significantly reduced the 200 mM ethanol treatment-induced [Ca2+]i increase (2.32+/-0.08 reduced to 1.79+/-0.15 (t = 7.201, P less than 0.01) and 1.86+/-0.09 (t = 8.183, P less than 0.01) respectively). EGTA and 2-APB also increased the ethanol-treated cells' viability and reduced the ALT and AST leakage. The 200 mM ethanol treatment stimulated both gene and protein expression of STIM1 and Orai1, and the up-regulation effect lasted at least 72 h after treatment.

CONCLUSIONEthanol-induced dysregulation of SOCs may be an important molecular mechanism of ethanol-induced [Ca2+]i rise in hepatocytes and the related liver cell injury.

Calcium ; metabolism ; Calcium Channels ; metabolism ; Ethanol ; adverse effects ; Hep G2 Cells ; Hepatocytes ; drug effects ; metabolism ; Humans

Apoptosis ; drug effects ; Hep G2 Cells ; Humans ; Iron Compounds ; adverse effects ; pharmacology ; Nanostructures

DNA, Viral ; genetics ; Hep G2 Cells ; Hepatitis B virus ; drug effects ; Humans ; Oligonucleotides, Antisense ; pharmacology

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; pathology ; Cell Line ; Hep G2 Cells ; Humans ; Lactones ; pharmacology ; Sulfones ; pharmacology

OBJECTIVETo investigate the effect of luteolin on cell growth and apoptosis of HepG2 cells in vitro.

METHODSCultured HepG2,HL60,A549 and LO2 cells were treated with luteolin for different doses (0 μg/ml,2.5 μg/ml,10 μg/ml and 20 μg/ml) and varied times (0 h,24 h,48 h and 72 h). Cell viability was measured with MTT assay and IC50 was calculated. The reactive oxygen species (ROS) levels in HepG2 cells treated with luteolin for 6 h and 12 h were measured with flow cytometry. Cell apoptosis of HepG2 cells treated with luteolin for 24h was examined with flow cytometry and Annexin V-FITC/PI. Expression levels of apoptosis pathway proteins (p53,ASPP2 and iASPP) in HepG2 cells were detected with western blot and the dose and time-effect was analyzed.

RESULTSLuteolin effectively inhibited tumor cell proliferation in a dose-and time-dependent manner,and the inhibition rates of 20 μg/ml Luteolin for 72 h were 39.34%,62.90%,57.57% and 62.90% to LO2,HepG2, HL60 and A549 cells,respectively. The intracellular ROS level was decreased in HepG2 cells by 13.88% and 41.11% after being treated with luteolin for 6 h and 12 h,respectively. The apoptosis rate of HepG2 cells treated with luteolin for 24 h was 14.43%,and western blot showed that luteolin reduced the expression level of iASPP by 77.07% and up-regulated the expression of p53 by 179.37% and ASPP2 by 725.02% in HepG2 cells treated with luteolin for 12 h.

CONCLUSIONLuteolin has ant-proliferative and pro-apoptotic activity on hepatoma HepG2 cells, which is associated with the altered expression of pro-apoptotic factors and decreased ROS level in HepG2 cells.

Apoptosis ; drug effects ; Hep G2 Cells ; Humans ; Luteolin ; administration & dosage ; pharmacology ; Reactive Oxygen Species ; metabolism

OBJECTIVETo investigate the chemical constituents of Excoecaria acerifclia and their antitumor activities.

METHODThe constituents were isolated and purified by column chromatography. Their structures were identified by their physicochemical properties and spectral features. Cytotoxicities of the purified compounds were evaluated by MTI method against human cancer cell lines HepG2.

RESULTEight compounds were isolated and identified as: 7-hydroxy-6-methoxy-coumarin (1), 8-hydroxy-6,7-methoxy-coumarin (2), kaempferol (3), kaempferol-3-O-beta-D-glucoside (4), quercetin-3-O-beta-D galactoside (5), kaempferol-3-O-beta-D-glucoside-2"-gallate (6), beta-sitosterol (7), daucosterol (8).

CONCLUSIONAll compounds were isolated from this plant for the first time. Compounds 5 showed inhibitory activity towards HepG2 with IC50 values of 7.13 mol x L(-1).

Cell Proliferation ; drug effects ; Euphorbiaceae ; chemistry ; Hep G2 Cells ; Humans ; Plant Extracts ; analysis ; isolation & purification ; pharmacology

OBJECTIVESTo investigate the effect of hepatitis B virus X protein (HBx) on adriamycin-induced apoptosis of hepatocellular carcinoma cells.

METHODSHBx gene fragment was amplified from subtype adr HBV plasmid by PCR, and inserted into Hind III and Kpn I sites of green fluorescent protein (GFP) eukaryotic expression vector pEGFP-C1 to construct recombinant pGFP/HBx. The pEGFP-C1 and pGFP-HBx were introduced into HepG2 cells by Lipofectamine 2000 to obtain HepG2 cells expressing GFP. GFP-HBx fusion protein was selected using G418. The expression of HBx gene was demonstrated by RT-PCR analysis. HepG2, HepG2/GFP and HepG2/GFP-HBx cells were treated with adriamycin (2.5 microg/ml), and apoptosis of the cells was determined by their morphological changes, trypan blue exclusion, and flow cytometry analysis.

RESULTSUnder a fluorescence microscope, visible expression of GFP and GFP-HBx fusion proteins were observed in HepG2/GFP and HepG2/GFP-HBx cells, even after growing over 70 generations. RT-PCR analysis showed that HBx gene was expressed in HepG2/GFP-HBx cells. Trypan blue exclusion showed adriamycin induced time-dependent cell death in HepG2 and HepG2/GFP cells while no significant cell death was observed in HepG2/GFP-HBx cells. Flow cytometry analysis showed that apoptosis rates in HepG2/GFP-HBx (3.94%) cells were significantly lower than those in HepG2 (59.03%) and HepG2/GFP cells (61.38%) at 36 hours after the adriamycin treatment (P < 0.01). No significant differences of apoptosis rates of HepG2/GFP-HBx (3.94%) and of the untreated cells (2.12%, 2.78%, 2.55%) (P > 0.05) were observed.

CONCLUSIONA HepG2 cell line expressing GFP and GFP-HBx fusion proteins was successfully established. HBV X protein blocks adriamycin-induced apoptosis of these HepG2 cells.

Apoptosis ; drug effects ; Doxorubicin ; pharmacology ; Hep G2 Cells ; Humans ; Plasmids ; Trans-Activators ; genetics

OBJECTIVETo establish a convenient and efficient model for investigating the expression of CYP3A4 and drug metabolism in vitro.

METHODS1alpha,25-dihydroxyvitamin D3 was utilized as an inducer to enhance CYP3A4 expression in HepG2 cells. 0.1, 0.25, 0.35 micromol/L 1alpha,25-dihydroxyvitamin D3 were added to the cell culture media, and cells were harvested after 24, 48, 72 and 96 hours. Cell proliferation was determined with MTT assay. CYP3A4 mRNA level was analyzed with RT-PCR and expressions of CYP3A4 protein were measured by Western blot.

RESULTS1alpha,25-dihydroxyvitamin D3 in 3 concentrations, namely 0.10, 0.25 and 0.35 micromol/L, did not show obvious toxicity to HepG2 cells. At 24 h of the cultivation, the expression of CYP3A4 mRNA was not increased significantly, but CYP3A4 mRNA expression significantly increased by 120%, 134%, 200% at 48 h, by 174%, 254%, 420% at 72 h, and by 258%, 450%, 370% at 96 h, respectively under the three concentrations. Similar results were observed in the induction of CYP3A4 protein expression. At 48, 72 and 96 hours after treatment with 0.25 micromol/L and 0.35 micromol/L 1alpha,25-dihydroxyvitamin D3, CYP3A4 protein increased in various folds in the controls (1.2 and 2.2 after 48 h, 3.4 and 6.5 after 72 h, 6.1 and 7.2 after 96 h), while 0.10 micromol/L 1alpha,25-dihydroxyvitamin D3 only induced protein expression at 72 h and 96 h (1.8 and 4.1 folds, respectively).

CONCLUSION1alpha,25-dihydroxyvitamin D3 could induce the expression of CYP3A4 mRNA as well as CYP3A4 protein in HepG2, which provides a convenient and efficient in vitro system for investigation of CYP3A4 and drug interaction.

Calcitriol ; pharmacology ; Cytochrome P-450 CYP3A ; drug effects ; genetics ; metabolism ; Hep G2 Cells ; Humans ; Transcription, Genetic

OBJECTIVETo study the antiproliferation effect on HepG2 cells and its underlying mechanism of the active chemical composition of the Viburnum Odoratissimum.

METHODS3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay and trypan blue dye exclusion assay were used to assess the effect of vibsane-type diterpenoids on the proliferation of various tumor cells. Alterations in cell cycle and apoptosis were determined by flowcytometry. The enzymatic activity of caspase-3/7 was measured by Apo-ONE homogeneous Caspase-3/7 Assay kit.

RESULTSCompound 1 #, a vibsane-type diterpenoid, was found to significantly inhibit the growth of HepG2 cells by anticancer proliferation activity screening. It was demonstrated that the modified groups on side chain coupled to C11 site affected the cell growth-inhibition activity of compounds by structure-activity analysis. In addition, HepG2 cell line was most sensitive to compound 1 #, which induced growth arrest of HepG2 cells in a dose- and time-dependent manner. Study on the mechanisms underlying these effects indicated that compound 1 # induced significant G0/G1 phase arrest of HepG2 cells in a time- and concentration-dependent manner. Meanwhile, It was found that higher concentrations of compound (5-10 micromol/L) caused evident increase in the unmber of apoptotic cells and dose-dependent activation of caspase-3/7.

CONCLUSIONVibsane-type diterpenoids could significantly inhibit the growth of HCC HepG2 cells. Induction of cell cycle arrest and apoptosis may play important roles in their anticancer effects.

Apoptosis ; drug effects ; Cell Cycle Checkpoints ; drug effects ; Cell Proliferation ; drug effects ; Diterpenes ; pharmacology ; Hep G2 Cells ; Humans ; Viburnum ; chemistry