Induction of apoptosis and accumulation of p27~(Kip1) protein by proteasome inhibitor MG132 in human osteosarcoma MG-63 cells

Objective To investigate the apoptosis-inducing effect of proteasome inhibitor Z-LLL-CHO (MG132) on human osteosarcoma MG-63 cells and study the altered expression of p27Kip1 protein. Methods p53 mutation type human osteosarcoma MG-63 cells, normal diploid fibroblast WI-38 cells and p53 wild type human osteosarcoma U2OS cells were cultured with different concentrations of proteasome inhibitor MG132. Cell viability was determined by MTT assay at different cultured period. Agarose gel electrophoresis was used to detect cell apoptosis and cell apoptotic rate was quantitatively analyzed at different cultured period by flow cytometry in MG-63 and WI-38 cells. Western blot was performed to study the altered expression of p27Kip1 protein after treatment. Results MG132 selectively reduced the viability of human osteosarcoma MG-63 and U2OS cells. The IC50 value was (0.92±0.06) μmol/L and (0.33±0.05) μmol/L, respectively. Moreover, the inhibitory effect was higher on MG-63 or U2OS cells than on diploid fibroblastic WI-38 cells whose IC50 was (9.13±0.12) μmol/L (P0.01). After treatment with 1 μmol/L MG132 for 24 h, the ladder bands characteristic of internucleosomal DNA fragmentation were detected in MG-63 cells but not in WI-38 cells. Apoptotic sub-G1 DNA content was detected by flow cytometry in MG-63 cells incubated with 1 μmol/L MG132 for 12 h and displayed a time-dependent manner. By Western blot, exposure to MG132 led to an accumulation of p27Kip1 protein in MG-63 cells. Conclusion Proteasome inhibitor MG132 had a selective apoptosis-inducing effect on human osteosarcoma MG-63 cells in vitro, and altered expression of p27Kip1 protein possibly playing an important role in induction of apoptosis.