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

Abstract

Objective: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Apoptosis; Ezrin; Heat shock protein 70; Osteosarcoma; Proliferation.

Figure 1

Sketch map of construction of pGFP‐V‐RS‐shRNA‐HSP70 vector.

Figure 2

Light and fluorescence microscopy images of MG63 transfected with ezrin‐shRNA and ezrin‐shRNA/HSP70. Magnification ×100 for all images. (A) Fluorescence microscopy image of MG63 transfected with ezrin‐shRNA. (B) Light microscopy images of MG63 transfected with ezrin‐shRNA. (C) Fluorescence microscopy image of MG63 transfected with ezrin‐shRNA/HSP70. (D) Light microscopy image of MG63 transfected with ezrin‐shRNA/HSP70.

Figure 3

(A) Real‐time PCR and (B) western blot determination of ezrin and HSP70 expression. Ezrin expression was downregulated in cells transfected with ezrin‐shRNA or ezrin‐shRNA/HSP70 vector compared with negative control. HSP70 expression was upregulated in cells transfected with ezrin‐shRNA/HSP70 compared with cells transfected with ezrin‐shRNA or NC. NC, normal control; **, P < 0.01.

Figure 4

Apoptosis as assessed by flow cytometry. The number of MG63 transfected with ezrin‐shRNA was significantly fewer than for NC. The number of apoptotic cells was significantly greater, especially the number in late apoptosis. Apoptosis of MG63 transfected with Ezrin‐shRNA was also greater than for NC. NC, normal control; **, P < 0.01.

Figure 5

MTS analysis cell proliferation. Compared to negative control, the proliferation of MG63 transfected with ezrin‐shRNA or ezrin‐shRNA/HSP70 significantly reduced. The proliferation of MG63 transfected with ezrin‐shRNA/HSP70 slightly increased on Days 5–7 but was still significantly less than for the NC. NC, normal control.

Figure 6

MTS analysis of cell inhibition rate. The inhibition rate of MG63 transfected with ezrin‐shRNA and ezrin‐shRNA/HSP70 significantly increased with prolongation of the culture time and the inhibition rate of MG63 transfected with ezrin‐shRNA/HSP70 was less than that of MG63 transfected with ezrin‐shRNA. However, they still inhibited cellular proliferation compared with NC. NC, normal control.

Figure 7

Western blot determined expression of cell cycle and apoptosis association proteins. Knockdown of ezrin promoted proapoptotic Bax expression, and inhibited Bcl‐2 and cyclin D1 expression. Simultaneous knockdown of ezrin and overexpression of HSP70 in MG63 inhibited proapoptotic Bax expression and promoted Bcl‐2 and Cyclin D1 expression, compared with MG63 downregulated by ezrin alone. However, it still differed significantly from that of NC. NC, normal control.

Figure 8

HSP70 promotes CTL activity. CTL activity significantly increased when MG63 was transected with ezrin‐shRNA/HSP70 compared with MG63 transfected with ezrin‐shRNA or NC. (**, P < 0.01 for MG63 transected with ezrin‐shRNA/HSP70 and NC). NC, normal control.