Nutlin3和JQ1协同抗骨肉瘤的作用

Nutlin3 enhances JQ1-triggered apoptosis in osteosarcoma cancer cells

目的 探讨Nutlin3和JQ1在骨肉瘤细胞中是否具有协同的抗骨肉瘤作用.方法 采用1.0μmol/L Nutlin3单药,0.5μmol/L JQ1单药以及两药联合处理骨肉瘤细胞,通过细胞计数试剂盒（CCK-8）细胞活性检测、协同效应分析、流式细胞仪检测凋亡和Western blot实验,检测Nutlin3 和JQ1两种药物的协同抗骨肉瘤效应,并探讨其分子机制.结果 CCK-8结果显示,在SJSA-1细胞株中,Nutlin3和JQ1单药均能够一定程度的抑制骨肉瘤细胞活性,半数抑制浓度（IC50）值分别为3.7 μmol/L和1.0 μmol/L;但两药联合处理后抗癌效果显著提高,IC50值减低至0.12 μmol/L;采用联合作用指数研究表明在携带有野生型p53的SJSA-1骨肉瘤细胞中CI值为0.18,在p53缺失的Saos-2骨肉瘤细胞中CI值为0.94,表明两个抗癌制剂具有高度协同作用,且这两种新型抗癌制剂的协同作用依赖于p53的活性.流式细胞仪检测凋亡发现1μmol/L Nutlin3和0.5μmol/L JQ1单药分别诱导15％和37％的细胞发生凋亡,两者联合能够诱导80％的细胞发生凋亡,显著高于单药的凋亡作用（P=0.000）,表明两者联合能够诱导大量骨肉瘤细胞发生凋亡.Western blot结果显示Nutlin3和JQ1联合应用能够激发半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-3高度活化,导致PARP绝大部分裂解,促进了凋亡信号的活化;同时也发现,Nutlin3和JQ1联合应用能够促进p53的表达升高.结论 新型抗癌制剂Nutlin3和JQ1具有明显的协同抗骨肉瘤作用,这种协同作用具有p53依赖性.

Objective To explore whether Nutlin3 and JQ1 have a synergistic anti-ostcosarcoma effect in osteosarcoma cells.Methods Osteosarcoma cells were treated with 1.0 μmol/L Nutlin3 alone,0.5 μmol/L JQl alone or their combination.By counting kit-8 （CCK-8） assay,synergistic effect analysis,flow cytometry and Western blotting,synergistic anti-osteosarcoma effects of Nutlin3 and JQ1 and their molecular mechanisms were studied.Results The results of CCK-8 assay showed that both Nutlin3 and JQ1 could inhibit osteosarcoma cell activity in SJSA-1 cell line with half maximal inhibitory concentration （IC50） values of 3.7 μmol/L and 1.0 μmol/L,respectively.The effect was significantly improved with the IC50 value being reduced to 0.12 μmol/L.A combined effect index study showed a CI of 0.18 in SJSA-1 osteosarcoma cells harboring wild-type p53,and a CI of 0.94 in Saos-2 osteosarcoma cells that were deficient in p53,indicating that the two anticancer agents had a high degree of synergic effects,and that the synergic effects of the two novel anticancer agents depended on the activity of p53.Apoptosis detected by flow cytometry showed that 1 μmol/L of Nutlin3 and 0.5 μmol/L of JQ1 induced apoptosis in 15% and 37% of cells,respectively.The combination of them could induce apoptosis in 80% of cells,which was significantly higher than that of Nutlin3 and JQ1 used alone （P =0.001）,indicating that the combination of the two drugs could induce apoptosis of a large number of osteosarcoma cells.Western blotting results showed that combination of Nutlin3 and JQ1 could stimulate the high activation of cysteinyl aspartate-specific protease （Caspase）-3,resuhing in the majority of PARP cleavage and promoting the activation of apoptosis signals;meanwhile,the combination of Nutlin3 and JQ1 could also promote the expression of p53.Conclusion The novel anticancer agents Nutlin3 and JQ1 have a significantly synergistic anti-osteosarcoma effect,and this synergistic effect is p53 dependent.