Induction Of Apoptosis In PC12 Cells By Homocysteine

Object: To decide whether homocysteine plus copper induce apoptosis in PC12 cells and the potential mechanism involved.Methods: Cells were divided into control group and treated groups at random. Control group were normal cultured cells; treated groups were exposed to different concentrations of Hcy with or without 10μmol/L CuSO₄. Cell viability was determined by MTT assay, cell morphological changes were observed by phase-contrast microscope and fluorescence microscopy after Hoechst 33258 staining, Agarose gel electrophoresis was used to detected DNA fragmentation. Flow cytometry (FCM) showed the apoptotic ratio. The mitochondrial membrane potential (MMP) was detected by flow cytometry through Rhodamine 123 staining. The expression of bcl-2 and bax mRNA was measured by reverse transcription polymerse chain reaction (RT-PCR).Results: Plus 10μmol/L copper, treatment of PC 12 cells with Hcy in the range of 0.125, 0.25, 0.5, 1.0 mmol/L, caused a dose-dependent decrease in cell viability: 81%, 79%, 69%, 57%, and induced typical morphological changes of apoptosis. Flow cytometry showed "apoptotic peak" and the apoptosis ratios were 8.00%, 10.37%, 17.26%, 20.19% respectively. After NAC intervention, the apoptosis ratios came down accordantly. MMP of PC 12 cells were significantly dropped in the early. The expression of bcl-2 was significantly decreased whereas bax was significantly increased. But we also observed that none of these detective indexes was changed when PC 12 cells were exposed to different concentrations of Hcy alone.Conclusion: homocysteine plus copper may induce apoptosis in PC 12 cells and this effect might be attributed to oxidation toxicity, decrease of MMP and its regulation of bcl-2 gene and bax gene expression.