p14ARF经p53凋亡通路增强骨肉瘤U2OS细胞对顺铂的敏感性

p14ARF enhances chemosensitivity to cisplatin in human osteosarcoma U2OS cells through p53 apoptotic pathway

目的 探讨抑癌基因p14ARF增强骨肉瘤U2OS细胞对顺铂的敏感性及其作用机制.方法 使用顺铂处理不表达p14ARF的U2OS细胞和稳定表达p14ARF的U2OS-ARF细胞,采用噻唑蓝（MTT）比色法测定细胞毒作用和半数抑制浓度值;流式细胞术和Hoechst 33258荧光染色检测细胞凋亡;免疫印迹法检测p53以及其下游基因Bax、p21、Mdm2、Fas的表达;比色法检测半胱氨酸天门冬氨酸蛋白酶（caspase）-3、caspase-8、caspase-9蛋白酶的活性.结果 顺铂处理72 h后,U2OS、U2OS-vec和U2OS-ARF组的细胞活力分别为（84.8％±4.4％）、（86.9％±5.0％）和（66.7％±4.6％）,相比U2OS-ARF的细胞活力明显降低,U2OS、U2OS-vec和U2OS-ARF组的IC50分别为（15.8±0.9）、（16.3±0.6）μmol/L和（8.9±0.8）μmol/L,相比U2OS-ARF的IC50明显降低（P＜0.05）.流式细胞术和形态学鉴定表明相对于U2OS和U2OS-vec细胞,U2OS-ARF细胞表现更为明显的凋亡比例和凋亡特异性形态学变化.在U2OS-ARF细胞中,p53、Mdm2和p21的基础表达水平稍稍高于U2OS-vec对照细胞,顺铂处理在U2OS-vec和U2OS-ARF细胞均明显激活p53、Mdm2和p21的表达,但在U2OS-ARF细胞中更加明显;另外,顺铂处理对U2OS-vec细胞中Bax和Fas的表达没有影响,却在U2OS-ARF细胞中明显增强了Bax的表达,但对Fas无影响.p14ARF还增强顺铂对caspase-9和caspase-3的活化.结论 p14ARF通过p53凋亡通路增强骨肉瘤U2OS细胞对顺铂的敏感性,并与内源性线粒体通路有关.

Objective To explore the effects of tumor suppressor p14ARF on chemosensitivity of human osteosarcoma U2OS cells to cisplatin and elucidate its molecular mechanism.Methods U2OS cells expressing no p14ARF and U2OS-ARF cells expressing p14ARF stably through stable transfection were treated with cisplatin.Cell viability and IC50 were assayed with methyl thiazolyl tetrazolium （MTT）.Apoptosis was examined by fluorescence-activated cell sorting and Hoechst33258 staining.The expressions of p53,Bax,p21,Mdm2 and Fas were detected by Western blot.And colorimetry was used to determine the activities of caspase-3,caspase-8 and caspase-9.Results The viability was 84.8% ± 4.4%,86.9% ± 5.0% and 66.7% ±4.6% respectively in U2OS,U2OS-vec and U2OS-ARF cells.The values of IC50 were （15.8 ± 0.9） μmol/L,（16.3 ± 0.6） and （8.9 ± 0.8） μmol/L respectively in U2OS,U2OS-vec and U2OS-ARF cells.The levels of viability and IC50 obviously decreased in U2OS-ARF cells in response to cisplatin （P 〈 0.05）.There were higher apoptotic rate and more obvious apoptotic morphological changes in U2OS-ARF cells than U2OS and U2OS-vec cells.The basal levels of p53,Mdm2 and p21 in U2OS-ARF cells were slightly higher than those in U2OS-vec cells.Cisplatin up-regulated p53,Mdm2 and p21 in both cell lines.However,the up-regulation was more pronounced in U2OS-ARF cells.Cisplatin did not change the levels of Bax and Fas in U2OS-vec cells.Bax protein was up-regulated in U2OS-ARF cells while the level of Fas remained constant,p14ARF also enhanced the activities of caspase-9 and caspase-3 in response to cisplatin.Conclusion p14ARF enhances the chemosensitivity to cisplatin in human osteosarcoma U2OS cells through p53 apoptotic pathway.And intrinsic mitochondrial apoptosis is involved.