华蟾素联合顺铂对人骨肉瘤U2OS细胞增殖和凋亡的影响

目的:研究华蟾素联合顺铂对人骨肉瘤U2OS细胞增殖和凋亡的影响,并探寻联合治疗增敏的可能分子机制。方法:采用不同浓度的华蟾素、顺铂单药和华蟾素联合顺铂处理人骨肉瘤U2OS细胞1-3天,通过CCK-8法检测其对U2OS细胞生长的抑制作用;平板克隆实验检测其对U2OS细胞集落形成能力的影响;流式细胞仪检测其对U2OS细胞凋亡的影响;RT-PCR及Western blotting检测Bax、Bcl-2、caspase-3、caspase-9等凋亡相关分子mRNA及蛋白水平的表达。结果:单用华蟾素或顺铂均可以浓度和时间依赖的方式抑制骨肉瘤U2OS细胞的增殖并诱导其凋亡,两药联合应用具有协同效应,并可以上调促凋亡基因Bax下调抑制凋亡基因Bcl-2的表达;联合用药组凋亡相关蛋白caspase-3、caspase-9的表达较单药组明显增加。结论:华蟾素联合顺铂与单一用药相比能够显著抑制人骨肉瘤细胞U2OS细胞的增殖,促进其凋亡,两药联合应用对骨肉瘤细胞的杀伤效应具有一定的协同效应,其机制可能与激活凋亡通路有关。

Effect of Cinobufacini plus Cisplatin on the Proliferation and Apoptosis of Osteosarcoma U2OS Cells

ABSTRACT Objective: To explore the effect of Cinobufacini plus Cisplatin (CDDP) on the proliferation and apoptosis of U2OS cell line, and explore the possible molecular mechanism of combination therapy. Methods: U2OS cells were treated with different concentrations of Cinobufacini and CDDP or in combination for one to three days. The proliferation of U2OS was tested by CCK-8 assay. The clonality of U2OS was assessed by clone formation assay. The cell apoptosis was detected by flow cytometer. The expression of apoptosis related genes and proteins was detected by RT-PCR and Western blot. Results: Cinobufacini or CDDP could inhibit the proliferation and induce the apoptosis of U2OS cells in a dose- and time-dependent manner. However, combination of two drugs could inhibit the cell clone formation and markedly promote the cell apoptosis. Combination of two drugs had synergistic effect on the cell proliferation and apoptosis. The pro-apoptosis gene Bax was up-regulated and suppression apoptosis Bcl-2 was significantly down regulated in the combination group. The expression of apoptosis associated proteins caspase-3 and caspase-9 in the combined administration group were obviously increased. Conclusion: Cinobufacini and cisplatin could significantly inhibit the proliferation and induce apoptosis of osteosarcoma cell, combination these two drugs had synergistic effect, its mechanism might be related to the activation of apoptosis pathway.