土槿皮乙酸通过PI3K/Akt信号通路诱导人肾癌细胞A498凋亡

Pseudolaric Acid B Induced the Apoptosis of Human Renal Cell Carcinoma A498 via PI3K/Akt Signaling Pathway

目的:研究土槿皮乙酸对人肾癌细胞A498凋亡的影响,并探讨其内在的分子机制,为肾癌治疗寻找有效的新靶点和新策略。方法:人肾癌细胞A498经10、15μmol/L土槿皮乙酸处理48 h后,用流式细胞仪检测细胞凋亡,同时通过Western印迹和实时荧光定量PCR检测凋亡相关蛋白和m RNA的表达;加入PI3K/Akt通路抑制剂LY294002或15μmol/L土槿皮乙酸处理A498细胞48 h后,Western印迹检测相关蛋白的表达。结果:经不同浓度土槿皮乙酸作用A498细胞48 h后,与未加土槿皮乙酸组细胞相比,细胞凋亡率显著上升,且呈剂量依赖性,比较差异有统计学意义(P<0.05);同时,Blc-2表达减少,Bax、caspase-9、caspase-3表达增多。Blc-2蛋白的表达量随LY294002或土槿皮乙酸的逐步加入而减少,而Bax、caspase-9、caspase-3的表达呈增加趋势;PI3K和Akt蛋白的表达量与是否加入LY294002或土槿皮乙酸无关,p-PI3K和Aktp-Ser473蛋白的表达量随LY294002或土槿皮乙酸的逐步加入而减少。结论:土槿皮乙酸可抑制A498细胞凋亡,其作用机制可能与PI3K/Akt通路相关。

Objective： To investigate the effect and molecular mechanism of pseudolaric acid B（PAB） on theapoptosis of human renal cell carcinoma A498 and to find a new treatment target for renal cell carcinoma. Methods： A498 cells were incubated with the indicated concentrations of PAB for 48 h and then cell apoptosis was de-termined by flow cytometry. Meanwhile Western blot and real-time quantitative PCR were applied to detect the ex-pression level of related protein and m RNA, respectively. Western blot was performed to determine the expressionof protein after adding PI3K/Akt signaling pathway inhibitor LY294002 and 15 μmol/L PAB. Results： The PAB-in-duced A498 cell apoptosis was significantly increased than that of the control cells. Furthermore, the expression ofBlc-2 was obviously decreased and the levels of Bax, caspase-9, caspase-3 were significantly increased in PAB-treated A498 cells. When A498 cells were traeted with LY294002 or 15 μmol/L PAB, the protein level of Blc-2was decreased and Bax, caspase-9, caspase-3 were significantly increased. Meanwhile, p-PI3 K, Aktp-Ser473 were dra-matically downregulated without PI3 K and Akt. Conclusion： PAB inhibits the apoptosis of human renal cell carci-noma A498 and its mechanism may be related to PI3K/Akt pathway.