ABSTRACT
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.