p14ARF增强顺铂诱导的骨肉瘤U2OS细胞凋亡的研究

The effects of p14ARF on cisplatin-induced apoptosis in human osteosarcoma U2OS cells

目的探讨p14ARF对顺铂诱导骨肉瘤U2OS细胞凋亡的影响及其机制,为提高骨肉瘤化疗敏感性提供依据。方法在不表达p14ARF的U2OS细胞中稳定转染pcDNA3.1-p14ARF质粒,构建稳定表达株;采用RT-PCR和Western blot法鉴定其表达;台盼蓝拒染法测定生长抑制作用;Hoechst33258荧光染色检测凋亡;Western blot法检测Caspase-3,9和PARP的表达和活化。结果 mRNA和蛋白水平,U2OS和U2OS-vec细胞未见p14ARF表达,U2OS-ARF细胞可见p14ARF的高表达;p14ARF单独对U2OS细胞的生长无影响,但顺铂(5μM)处理72小时后,U2OS-ARF细胞的生长抑制率明显高于U2OS和U2OS-vec细胞,相对于U2OS和U2OS-vec细胞,U2OS-ARF细胞不仅表现更为明显的凋亡形态学变化,还明显出现了Caspase-3,9和PARP的活化裂解。结论 p14ARF能够增强顺铂诱导的骨肉瘤U2OS细胞毒作用和凋亡,通过激活Caspase-PARP级联活化,提高骨肉瘤U2OS细胞对顺铂的敏感性。

Objective To study the effects of p14ARF on cisplatin-induced apoptosis in human osteosarcoma U2OS cells with its molecular mechanisms and to provide evidences for increasing chemosensitivity of osteosarcoma.Methods pcDNA3.1-p14ARF plasmid was stably transfected into U2OS cells that did not express p14ARF.Expression of p14ARF on mRNA and protein level was detected by RT-PCR and Western blot.Growth inhibition rate was evaluated using trypan blue exclusion assay.Apoptosis was studied using Hoechst 33258 staining.Activation of Caspase-3,9 and PARP was detected by Western blot.Results There was no expression of p14ARF in U2OS and U2OS-vec cells but obvious expression was observed in U2OS-ARF cells on mRNA and protein level.p14ARF alone had no influence on growth of U2OS cells.Compared with U2OS and U2OS-vec cells,there were higher cell inhibition rate,more obvious apoptotic morphological changes,and higher level of Caspase-3,9 and PARP activation in U2OS-ARF cells after treatment with cisplatin（5μM） for 72h.Conclusions p14ARF enhances cisplatin-induced growth inhibition and apoptosis in human osteosarcoma U2OS cells by activation of caspases-PARP cascade.This work can be exploited to increase the chemosensitivity of osteosarcoma.