Effect of demethylation on adenosine and homocysteine-induced apoptosis in HepG2 cells / 中国药理学通报

Aim To investigate the mechanism of demethylation on adenosine (ADO )and homocysteine (HCY)-induced apoptosis in human hepatoma HepG2 cells .Methods HepG 2 cells were treated with differ-ent concentrations of ADO (1.0、2.0、4.0 mol · L-1 ) alone or in combination with HCY for 6h,12h and 24h,5-aza-2-deoxycytidine (5-Aza-CdR)as a positive control.Cell viabilities were assessed by CCK8 assay. Cell apoptosis was observed by AnnexinV-FITC/PI staining.The mitochondrial membrane potentials(ΔΨ) were measured by flow cytometry.The mRNA and pro-tein expressions of caspase-3,caspase-8,caspase-9, MDM-2,p53,Cytochrome C,DNMT1,DNMT3a,DN-MT3 b were detected by RT-qPCR and Western blot re-spectively.Results ADO alone or in combination with HCY significantly reduced the viability of HepG2 cells in a dose and time-dependent manner.The apoptotic rates of HepG2 cells after combination treatment with ADO and HCY at 1 .0,2.0,4.0 mol · L-1 for 24 h were (1 8.63 ± 1.25 )%,(29.42 ±2.37 )% and (42.47 ±3.09 )%,compared with the control group (1.30 ±0.82 )%,P <0.01;and the mitochondrial membrane potentials were decreased from 674.15 ± 82.8%(black control group)to (428.38 ±54.5)%, (297.78 ±30.5)%,(74.45 ±5.73)%,P<0.01, respectively.The expressions of caspase-3,caspase-8, caspase-9,MDM-2,p53,Cytochrome C were up-regula-ted and MDM-2 were down-regulated after combination treatment of ADO and HCY.The mRNA expressions of DNMT1 ,DNMT3 a and DNMT3 b were down-regulated after combination treatment with ADO and HCY or 5-Aza-CdR alone.Conclusion Combination treatment of ADO and HCY can cause cellular methylation chan-ges.The effects of demethylation of ADO and HCY may activate p53 gene and mitochondrial pathway, which at last leads to HepG2 cell apoptosis.

Effects of tanshinone IIA on proliferation, apoptosis and expression of HIF-1α, VEGF and wild-type P53 in human hepatoma HepG2 cells under hypoxia / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the effects of tanshinone IIA ( Tan IIA) on proliferation, apoptosis and its mo-lecular mechanism in human hepatoma HepG2 cells under hypoxic condition.METHODS:Hypoxia model was established by treatment with cobalt chloride ( CoCl2 ) .The cells were divided into normoxia control group, hypoxia control group and hypoxia combined at different concentrations of Tan IIA groups.After HepG2 cells were incubated with different concentra-tions of Tan IIA (0.5, 1.0, 2.0, 5.0 and 10.0 mg/L) for 24 h, 48 h and 72 h under hypoxic condition, the cell prolifer-ation was determined by MTT assay.After Tan IIA was added to the media at different concentrations for 24 h and 48 h, the apoptotic cells were observed by Hoechst 33258 staining.The protein levels of hypoxia-inducible factor 1 alpha (HIF-1α) , vascular endothelial growth factor ( VEGF) and wild-type P53 were detected by Western blotting after cultured with different concentrations of Tan IIA for 48 h.RESULTS:Tan IIA inhibited the proliferation of HepG2 cells in a dose-and time-dependent manner.Tan IIA induced the typical morphology of apoptotic cells and increased the apoptotic rate in a dose-and time-dependent manner after treatment with 1.0 mg/L~5.0 mg/L for 24 h and 48 h under hypoxic condition. The protein levels of HIF-1αand VEGF were weakly expressed in HepG2 cells under normoxia but up-regulated after incu-bated under hypoxia for 48 h.The protein expression of HIF-1αand VEGF were decreased with the increase in the concen-tration of Tan IIA under hypoxia.The protein expression of wild-type P53 was increased with the increase in the concentra-tions of Tan IIA under hypoxia.CONCLUSION:Tan IIA significantly inhibits the proliferation and induces the apoptosis of human hepatoma HepG2 cells under hypoxia, which may be related to the down-regulation of HIF-1αand VEGF and up-regulation of wild-type P53.

Oxidative stress induces apoptosis in HepG2 cells / 中国病理生理杂志

AIM: Direct exposure of cells to reactive oxygen species can induce apoptosis. In this study we investigate how oxidative stress induces cell death in HepG2 cells and characterize the molecular events involved. METHODS: Oxidative stress was created by exposing HepG2 cells to 2 mmol/L H2O2. Apoptosis was determined by analysis of DNA fragmentation by agarose gel electorphoresis. The mitochondrial membrane potential was analyzed using DePsipher fluorescent staining and the expression of cytochrome c in the cytosolic fraction was measured by Western blotting analysis.The caspase activity was detected using fluorometric assay kit by a fluorescence microplate reader. RESULTS: When HepG2 cells were treated with 2 mmol/L H2O2, the cells displayed DNA fragmentation, a typical feature of apoptosis, after 12 h. The mitochondrial membrane potential appeared different in two group of cells. H2O2 -treated cells appeared green fluorescence as early as 4 h, which represents de - energized mitochondria, the untreated cells appeared red fluorescence,a feature of mitochondria with intact membrane potential. In treated cells, the expression of cytochrome c increased and accumulated in cytosolic fraction with treatment time, caspase - 3 activity increased by 6.7 - fold ( P ＜ 0.01 ) at 8 h and caspase -9 activity increased by 3.6 - fold (P ＜ 0.01 ) at 12 h, respectively, however, the activity of caspase - 8 remained unchanged. CONCLUSION: These findings suggest that oxidative stress can induce apoptotic cell death in HepG2 cells, and the mechanism is related to mitochondrial pathway, which activates caspase -9 and- 3, but not caspase -8.

The research of adenosine induced HepG2 cell apoptosis through endoplasmic reticulum stress pathway / 中国药理学通报

Aim To investigate the role of endoplasmic reticulum stress in adenosine induced HepG2 cell apoptosis.Methods HepG2 cells were treated with different concentrations of ADO for 36 h,and the effect of ADO on cell proliferation was measured by MTT assay.Cell nuclei DAPI staining was used to detect the nuclei change after being treated with different concentrations ADO for 36 h or 2.0 mmol?L-1 ADO for different time.The effect of ADO on HepG2 cell cycle was analysed by flow cytometry after being treated with 2.0 mmol?L-1 ADO for 12 h or 24 h.Translocation of CHOP and Caspase-3 were measured by immunofluorescence after being treated with 2.0 mmol?L-1 ADO for 36 h.The proteins expressions of CHOP,Caspase-4,Caspase-3 and JNK were assayed by western blot.Results The viability of HepG2 cell decreased in a dose-dependent manner;the relative cell viability of 0.5,1,2,4,6 mmol?L-1 decreased by 13.48%?0.12%,27.92%?0.25%,35.21%?0.42%,51.46%?0.24%,71.42%?0.58%,compared with the control group respectively.The nuclei of HepG2 cells treated with different ADO concentrations or 2.0 mmol?L-1 ADO showed condensation,rounding and shrinkage,then fragmentation,which demonstrated cell apoptosis.After being treated with 2.0 mmol?L-1 ADO for 12 h or 24 h,cell cycle analysis showed sub-G1 phase increased;the apoptotic ratio of control,12 h and 24 h was 1.55%?0.12%,10.96%?0.07% and 21.04%?0.26% respectively.Immunofluorescence assay showed the CHOP and Caspase-3 translocation to the nuclei after being treated with 2.0 mmol?L-1 ADO.The expressions of CHOP,Caspase-3 and Caspase-4 increased after being treated with different concentrations ADO for 36 h,while the expression of JNK did not change.Conclusion Endoplasmic reticulum stress is involved in adenosine-induced HepG2 cell apoptosis.