P14ARF enhances cisplatin-induced apoptosis in human osteosarcoma cells in p53-independent pathway

Abstract

To study effect of tumor suppressor p14ARF on cisplatin-induced apoptosis in human osteosarcoma cells with its molecular mechanisms to provide evidences for increasing chemosensitivity of osteosarcoma. pcDNA3.1-p14ARF plasmid was stable transfected into MG63 cells lack of p14ARF expression. Expression of p14ARF on mRNA and protein level was evaluated with RT-PCR and Western blot. MG63, MG63-vec and MG63-ARF cells were treated with cisplatin. Cell growth inhibition and IC50 were determined through MTT assay. Apoptosis was detected using fluorescence-activated cell sorting and Hoechst33258 staining. The expression of p53, Bax, p21, Mdm2, Fas, Caspase-3, caspase-9 and PARP was detected with Western blot. RNAi was used to silence p53. Cells were pre-treated with Caspase-9 specific inhibitor Z-LEHD-FMK to determine whether the effect was Caspase-9-dependent. There was no expression of p14ARF in MG63 and MG63-vec cells but obvious expression in MG63-ARF cells on mRNA and protein level. Cell viability was 84.2%±4.3%, 80.8%±4.3% and 58.9%±5.4% in MG63, MG63-vec, and MG63-ARF cells after treatment of cisplatin for 72 h. IC50 was (11.1±0.6), (10.7±0.9) and (7.2±0.7) µmol/L. The apoptotic rate was 13.6%, 18.5% and 35.9% in groups, There were more obvious apoptotic more changes in MG63-ARF cells than MG63 and MG63-vec cells, and activation of Caspase-3, 9 and PARP on higher level in U2OS-ARF cells after stimulation with cisplatin for 72 h. The expression of p53, Bax, p21, Mdm2 and Fas, in MG63-vec and MG63-ARF cells did not changed (P>0.05). The expression of p53 was effectively and continuously suppressed by p53-siRNA in U2OS-vec and U2OS-ARF cells. The p53 silencing did not alter the cytotoxicity mediated by cisplatin treatment for 72 h (P>0.05). Cell viability was 96.8%±3.6%, 54.1%±5.7% and 89.5%±5.1% in Z-LEHD-FMK, cisplatin and Z-LEHD-FMK+cisplatin groups. p14ARF enhances cisplatin-induced apoptosis in human osteosarcoma MG63 cells in p53-independent caspase-9-dependent pathway, in which the intrinsic mitochondrial apoptotic pathway is involved.