Inhibiting DNA methylation alleviates cigarette smoke extract-induced dysregulation of Bcl-2 and endothelial apoptosis.

INTRODUCTION There is evidence that cigarette smoking participates in disease progression through endothelial apoptosis. Bcl-2 family proteins are essential and critical regulators of apoptosis. We explored whether Bcl-2 plays a role in cigarette smoke extract induced (CSE-induced) endothelial apoptosis. Furthermore, given the involvement of epigenetics in apoptosis and Bcl-2 expression, we hypothesized that CSE-induced apoptosis might be caused by gene methylation. METHODS Human umbilical vascular endothelial cells (HUVECs) were treated with CSE, CSE plus 5-aza-2'-deoxycytidine (AZA, an inhibitor of DNA methylation), or AZA and phosphate-buffered saline (PBS). Endothelial apoptosis was determined by Annexin-V and propidium iodide staining. The expression levels of Bcl-2, Bax, and cytochrome C (cyt C) were assessed by immunoblotting and RT-PCR. The methylation status of the Bcl-2 promoter was observed by bisulfite sequencing PCR (BSP). RESULTS The apoptotic index of endothelial cells in the CSE-treated group increased. Decreased expression of Bcl-2 and high methylation of the Bcl-2 promoter were observed after CSE treatment. AZA alleviated the endothelial apoptosis caused by CSE. AZA treatment also increased Bcl-2 expression along with decreased Bcl-2 promoter methylation. CONCLUSIONS Inhibiting DNA methylation alleviates CSE-induced endothelial apoptosis and Bcl-2 promoter methylation. Bcl-2 promoter methylation might be involved in CES-induced endothelial apoptosis.