DAPT对耐雷帕霉素骨肉瘤细胞增殖、凋亡的影响

Effect of DAPT on the Proliferation and Apoptosis of Rapamycin-resistant Osteosarcoma Cells

目的通过研究γ-分泌酶抑制剂(DAPT)对耐雷帕霉素(RAPA)骨肉瘤细胞株增殖、凋亡的影响,探讨mTOR通路与Notch通路在骨肉瘤细胞增殖、凋亡中的关系。方法体外培养骨肉瘤MG63细胞株及建立耐雷帕霉素骨肉瘤MG63细胞株;噻唑蓝(MTT)比色法测定不同剂量雷帕霉素及DAPT对各细胞株增殖的影响;流式细胞仪检测不同剂量药物对各细胞株凋亡的影响;Western blot法检测各细胞株中p53的表达。结果 MTT法检测显示雷帕霉素及DAPT均能抑制各细胞株的增殖。流式细胞术检测结果表明雷帕霉素及DAPT均能诱导各细胞株发生凋亡,MG63细胞早、晚期凋亡率均与药物作用剂量呈正比,耐雷帕霉素MG63细胞仅在高浓度药物作用下,早、晚期凋亡率与细胞正常凋亡率相比差异有统计学意义。Western blot检测结果显示,不同浓度雷帕霉素、DAPT作用下,MG63细胞p53表达量随药物浓度升高而增加,而耐雷帕霉素MG63细胞p53表达量未随药物浓度升高而增加。结论雷帕霉素、DAPT对MG63细胞及耐雷帕霉素MG63细胞均能抑制增殖、促进凋亡。二者均可能是通过促进p53蛋白表达而发挥凋亡效应。凋亡过程中阻断mTOR后可能会降低骨肉瘤细胞对DAPT的敏感性,同时抑制Notch通路与p53的协同作用。

Objective To examine the effects of theγ-secretase inhibitor（DAPT）on the proliferation and apoptosis of rapamycin（RAPA）-resistant osteosarcoma cells,and probe into the relationship between the mTOR pathway and the Notch pathway in the proliferation and apoptosis of osteosarcoma cells.Methods Osteosarcoma cells MG63 were cultured in vitro and RAPAresistant cells were established.The methyl thiazolyl tetrazolium（MTT）assay and flow cytometry were used to detect the effects of different concentrations of RAPA and DAPT on the proliferation and apoptosis of normal and RAPA-resistant MG63 cells.The expression of P53 protein was detected by Western blotting.Results Both RAPA and DAPT could inhibit the proliferation and induce the apoptosis of the two cell populations in vitro.The early and late apoptosis rates of normal MG63 cells were proportionally increased with the doses of the two drugs.There were significant differences in the early and late apoptosis rates between RAPA-resistant MG63 cells and normal MG63 cells treated by high concentrations of drugs.Western blotting showed that the protein expression of p53 was significantly increased in a dose-dependent manner in RAPA-or DAPT-treated MG63 cells rather than in RAPA-resistant MG63 cells.Conclusion Both RAPA and DAPT could inhibit the proliferation and induce the apoptosis of MG63 cells and RAPA-resistant MG63 cells by up-regulating the expression of p53.During the process of apoptosis,blocking mTOR might reduce the susceptibility of RAPA-resistant MG63 cells to DAPT,and inhibit the synergy between Notch pathway and p53.