CAJ | 학술논문

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.
목적 탐토억암기인p14ARF증강골육류U2OS세포대순박적민감성급기작용궤제.방법 사용순박처리불표체p14ARF적U2OS세포화은정표체p14ARF적U2OS-ARF세포,채용새서람(MTT)비색법측정세포독작용화반수억제농도치;류식세포술화Hoechst 33258형광염색검측세포조망;면역인적법검측p53이급기하유기인Bax、p21、Mdm2、Fas적표체;비색법검측반광안산천문동안산단백매(caspase)-3、caspase-8、caspase-9단백매적활성.결과 순박처리72 h후,U2OS、U2OS-vec화U2OS-ARF조적세포활력분별위(84.8％±4.4％)、(86.9％±5.0％)화(66.7％±4.6％),상비U2OS-ARF적세포활력명현강저,U2OS、U2OS-vec화U2OS-ARF조적IC50분별위(15.8±0.9)、(16.3±0.6)μmol/L화(8.9±0.8)μmol/L,상비U2OS-ARF적IC50명현강저(P＜0.05).류식세포술화형태학감정표명상대우U2OS화U2OS-vec세포,U2OS-ARF세포표현경위명현적조망비례화조망특이성형태학변화.재U2OS-ARF세포중,p53、Mdm2화p21적기출표체수평초초고우U2OS-vec대조세포,순박처리재U2OS-vec화U2OS-ARF세포균명현격활p53、Mdm2화p21적표체,단재U2OS-ARF세포중경가명현;령외,순박처리대U2OS-vec세포중Bax화Fas적표체몰유영향,각재U2OS-ARF세포중명현증강료Bax적표체,단대Fas무영향.p14ARF환증강순박대caspase-9화caspase-3적활화.결론 p14ARF통과p53조망통로증강골육류U2OS세포대순박적민감성,병여내원성선립체통로유관.
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 ± O.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.