抑癌基因p14ARF对顺铂诱导的骨肉瘤细胞凋亡的影响

p14ARF enhances cisplatin-induced apoptosis in human osteosarcoma cells in p53-independent pathway

目的 探讨抑癌基因p14ARF对顺铂诱导的骨肉瘤细胞凋亡的影响及其作用机制.方法 在不表达p14ARF的MG63细胞（p53突变）中稳定转染pcDNA3.1-p14ARF质粒,构建稳定表达株MG63-ARF;经反向聚合酶链反应（RT-PCR）和免疫印迹法鉴定;使用顺铂处理MG63和MG63-ARF细胞,通过MTT法测定细胞生长抑制和IC50;采用流式细胞术和Hoechst 33258荧光染色检测细胞凋亡;采用免疫印迹检测p53、Bax、p21、Mdm2、Fas、Caspase-3、Caspase-9、PARP的表达;通过RNA干扰进行p53的表达沉默.采用Caspase-9抑制剂Z-LEHD-FMK预处理各组细胞,检测该作用是否依赖Caspase-9.结果 mRNA和蛋白水平,MG63和MG63-vec细胞未见p14ARF表达,MG63-ARF细胞可见p14ARF的高表达.经顺铂处理72 h后,细胞活力在MG63、MG63-vec和MG63-ARF组为84.2％±4.3％、80.8％±4.3％和58.9％±5.4％,IC50值为（11.1±0.6）、（10.7±0.9）、（7.2±0.7） μmol/L,（P＜0.05）.流式细胞术显示MG63、MG63-vec和MG63-ARF组经顺铂处理72 h的凋亡率为：13.6％、18.5％和35.9％.荧光染色表明MG63-ARF细胞相对出现更多更明显的凋亡,在MG63-ARF还出现了更明显的Caspase-3,9和PARP的活化裂解.在顺铂处理的MG63-vec和MG63-ARF细胞中,p53、Bax、p21、Mdm2、Fas的表达无任何改变.在U2OS-vec和U2OS-ARF细胞中转染p53-siRNA明显减弱了p53的表达,再经顺铂处理72 h后,U2OS-vec细胞的转染空白组、转染对照siRNA组和转染p53-siRNA组细胞活力差异无统计学意义（P＞0.05）,U2OS-ARF细胞的转染空白组、转染对照siRNA组和转染p53-siRNA组细胞活力差异无统计学意义（P＞0.05）.Caspase-9抑制剂Z-LEHD-FMK预处理后,Z-LEHD-FMK、顺铂、顺铂＋Z-LEHD-FMK组细胞活力为：96.8％±3.6％、54.1％±5.8％和89.5％±5.1％.结论 p14ARF可通过p53非依赖Caspase-9依赖性途径增强顺铂诱导的骨肉瘤MG63细胞凋亡,该作用与内源性线粒体凋亡通路有关.

Objective 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.Methods 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 RTPCR 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.Results 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.Conclusion 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.