ClC-3 chloride channel modulates the proliferation and migration of osteosarcoma cells via AKT/GSK3β signaling pathway

Abstract

In cultured human osteosarcoma (OS) cells, we recently demonstrated that insulin-like growth factors (IGF-1)-induced MG-63 and 143B human OS cells proliferation were consistent with increasing ClC-3 expression, and ClC-3 was up-regulated in cells with high metastatic potency. Blockade of ClC-3 greatly suppressed the phosphorylation activation of Akt/GSK3β. We also found that blockade of ClC-3 effectively down-regulated the expression of cyclin D1 and cyclin E, and caused activation of p27(KIP) and p21(CIP). The synthesized effects on these proteins which play a major role in cell cycle regulation bring about G0/G1 cell cycle arrest in MG-63 cells, and finally abrogate the cell proliferation. Besides, ClC-3 deletion attenuates OS cell migration via down-regulation the expression of MMP-2 and MMP-9. Such information suggests that ClC-3 might be a potential target for anti-OS.

Keywords: AKT/GSK3β signaling pathway; ClC-3 chloride channel; osteosarcoma; proliferation and migration.

Figure 1

ClC-3 expression parallels with OS cells proliferation. A. Effect of 30-300 ng/ml IGF-1 on the cell viability in MG-63 cells. B and C. Effect of 30-300 ng/ml IGF-1 on the expression of ClC-3 in MG-63 cells. (Data represent mean ± SD (n = 4), Con: control, *P < 0.05 vs. con; **P < 0.01 vs. con. ANOVA test was used). D. Effect of 30-300 ng/ml IGF-1 on the cell viability in 143B cells. E and F. Effect of 30-300 ng/ml IGF-1 on the expression of ClC-3 in 143B cells. (Data represent mean ± SD (n = 4), Con: control, *P < 0.05 vs. con; **P < 0.01 vs. con. ANOVA test was used).

Figure 2

Effect of ClC-3 siRNA on cell cycle in MG-63 cells. A and B. Effect of 20-80 nM ClC-3 siRNA on the expression of endogenous ClC-3 protein in MG-63 cells detected by western blot analysis. (Data represent mean ± SD (n = 4), con: control, Lipo: Liposomal, NS: Negative siRNA, **P < 0.01 vs. con. ANOVA test was used). C. Knock-down of ClC-3 reduces the IGF-1-induced cell viability in MG-63 cells. D. Knock-down of ClC-3 arrests cell cycle in G₀/G₁ phase in MG-63 cells. E and F. ClC-3 siRNA restrained cyclin D1, cyclin E and enhanced p21, p27 protein expression induced by 100 ng/ml IGF-1 respectively in MG-63 cells. (Data represent mean ± SD (n = 4), con: control, Lipo: Liposomal, NS: Negative siRNA, #P < 0.05 vs. con; **P < 0.01 vs. IGF-1 only. ANOVA test was used).

Figure 3

ClC-3 knockdown attenuate cell migration in OS cells. A and B. The endogenous expression of ClC-3 in cells with different metastatic potency (MG-63 and 143B) and effect of ClC-3 siRNA on the expression of ClC-3 protein in both cells detected by western blot analysis. (Data represent mean ± SD (n = 4), con: control, NS: Negative siRNA, *P < 0.05 vs. MG-63 con; #P < 0.05 vs. MG-63 con; *#P < 0.05 vs. 143B con. ANOVA test was used). C and D. ClC-3 knockdown attenuated the migration ability of MG-63 and 143B cells detected by transwell assay. (Data represent mean ± SD (n = 4), con: control, NS: Negative siRNA, *P < 0.05 vs. MG-63 con; #P < 0.05 vs. MG-63 con; *#P < 0.05 vs. 143B con. ANOVA test was used). E and F. ClC-3 knockdown suppressed expression of MMP-2 and MMP-9 in MG-63 and 143B cells. (Data represent mean ± SD (n = 4), con: control, NS: Negative siRNA, *P < 0.05 vs. MG-63 con; #P < 0.05 vs. MG-63 con; *#P < 0.05 vs. 143B con. ANOVA test was used).

Figure 4

ClC-3 knockdown attenuates Akt signaling. A and B. ClC-3 knockdown significantly attenuated the phosphorylations of GSK-3β and Akt detected by western blot analysis. (Data represent mean ± SD (n = 4), con: control, Lipo: Liposomal, NS: Negative siRNA, #P < 0.05 vs. con; *P < 0.05 vs. IGF-1 only. ANOVA test was used). C and D. Akt inhibitor LY294002 diminished the expression of ClC-3 and the phosphorylations of GSK-3β. (Data represent mean ± SD (n = 4), con: control, Lipo: Liposomal, NS: Negative siRNA, #P < 0.05 vs. con; *P < 0.05 vs. IGF-1 only. ANOVA test was used).