Effects of Ezrin and Heat Shock Protein 70 on Apoptosis and Proliferation of Human Osteosarcoma Cells.

Abstract

To investigate the influence of knocking down ezrin expression in combination with heat shock protein (HSP)-induced immune killing on the apoptosis and proliferation of mouse osteosarcoma cells. The HSP70 and ezrin-shRNA DNA fragments cloned into the expression vector pGFP-V-RS and the expression vectors pGFP-V-RS-shRNA and pGFP-V-RS-shRNA-HSP70 constructed and transfected into MG63 cell line, where their status was observed by fluorescent microscopy. Expression of ezrin and HSP70 was determined by RT-PCR and western blot. Changes in cell apoptosis and proliferation were assessed by flow cytometry and MTS and changes in expression of apoptosis and cell cycle-related proteins by western blot. Specific cytotoxic T lymphocytes (CTLs) were induced by HSP70 and its lethal effect on target MG63 tumor cells analyzed by MTS assay. The specific vector simultaneously downregulated ezrin and upregulated HSP70. Compared with ezrin knockdown alone, simultaneous HSP70 overexpression partially recovered the promoted cellular apoptosis and proliferation suppression by induced by ezrin knockdown; however, the apoptosis rate of MG63 cells was significantly greater than that of a negative control. In addition, ezrin-shRNA and ezrin-shRNA/HSP70 promoted expression of Bax. However, expression of these agents reduces Bcl-2 and Cyclin D1. The cytotoxic effects of CTLs on target MG63 tumor cells were significantly greater in the CTL + IL-2 + HSP70 group than the CTL + IL-2 group. Simultaneously knocking down ezrin and overexpressing HSP70 promotes apoptosis and inhibits proliferation of osteosarcoma cells and HSP70 induces CTL, enhancing the lethal effect on tumor cells.