Effects of Ezrin and heat shock protein 70 on apoptosis and proliferation of human osteosarcoma

Abstract

Objective To investigate the influence of knocking down Ezrin expression in combination with HSP70-induced immune killing on the apoptosis and proliferation of mouse osteosarcoma cells. Methods Human osteosarcoma cell line MG63 was cultured, the HSP70 and Ezrin-shRNA DNA fragments were cloned into the expression vector pGFP-V-RS containing CMV and pU6 promoters, and constructed the expression vector pGFP-V-RS-shRNA and pGFP-V-RS-shRNA-HSP70. The vectors were transfected into MG63 cell line, respectively. The status of transfected MG63 cells was observed by fluorescent microscope. The expressions of Ezrin and HSP70 were examined by real time RT-PCR and Western blot. Flow cytometry、MTS test was used to detect the changes of cell apoptosis and proliferation, and changes of the expression of apoptosis and cell cycle related proteins was detected by Western blot. The specific cytotoxic T lymphocytes (CTLs) were induced by HSP70, and its killing effect on target MG63 tumor cells was analyzed by MTT assay. Results The specific vector simultaneously knocking down Ezrin and overexpressing HSP70 was constructed for the first time in China and confirmed by fluorescence microscope and gene/protein expression analysis. Compared to Ezrin knock-down alone, simultaneous HSP70 overexpression partially recovered the promoted cellular apoptosis and proliferation suppression by Ezrin knock-down, however, when compared to the normal control, the apoptosis rate of LM8 cells was still significantly increased from 11.01%±0.22% to 24.28%±0.50%, while the proliferation rate decreased from 395.14%±2.24% to 310%± 2.83%. In addition, Western detected that Ezrin-shRNA could promote the expression of Bax. However, the expression of Ezrin-shRNA could reduce the Bcl-2 and Cyclin D1. Ezrin-shRNA/HSP70 also has the same effect. MTT assays revealed that the CTL cytotoxic effect on target MG63 tumor cells at all concentrations were significantly higher in CT+IL-2+HSP70 group compared with that in CT+ IL-2 group, with a cytotoxicity as high as 56.33%±1.95%. Conclusion Simultaneous knocking down Ezrin and overexpressing HSP70 promotes the apoptosis and inhibits the proliferation of osteosarcoma cell. And the HSP70 can induce CTL which enhances the killing effect on tumor cell. And the HSP70 can induce CTL which enhance the killing effect on tumor cell. Key words: Osteosarcoma; HSP70 heat-shock proteins; Apoptosis; Cell proliferation