摘要
目的:本实验探讨抑制PI3K/AKT/mTOR通路对顺铂诱导人皮肤黑素瘤细胞株A375细胞凋亡作用及其机制。方法:用顺铂处理人皮肤黑素瘤细胞株A375细胞后Western blot检测细胞凋亡、PI3K/AKT/mTOR通路的活化情况以及细胞增殖-毒性检测试剂盒(Cell Counting Kit-8,CCK-8)。用PI3K的抑制剂LY294002(LY)和mTOR的抑制剂雷帕霉素(Rapamycin,Rap)分别预处理以研究其对顺铂处理后人皮肤黑素瘤细胞株A375细胞凋亡的协同作用。为进一步探索阻断PI3K/AKT/mTOR通路对顺铂处理后人皮肤黑素瘤细胞株A375细胞凋亡的协同作用机制,采用Western blot检测阻断PI3K/AKT/mTOR后联用顺铂处理人皮肤黑素瘤细胞株A375细胞中Bcl-2、Bcl-xl蛋白表达。结果:顺铂处理后的A375黑素瘤细胞中PARP的活化剪切体表达量水平呈浓度和时间依赖性增加,细胞活力呈浓度和时间依赖性降低(P<0.05)。顺铂处理A375黑素瘤细胞后PI3K/AKT/mTOR通路的活化。阻断PI3K/AKT/mTOR通路再用顺铂联合处理A375黑素瘤细胞后PARP的活化剪切体表达量明显上调以及细胞活力明显降低(P<0.05)。阻断PI3K/AKT/mTOR通路后再用顺铂联合处理A375黑素瘤细胞发现Bcl-2蛋白和Bcl-xl表达水平下调。结论:阻断PI3K/AKT/mTOR通路对顺铂诱导的细胞凋亡具有协同作用,该协同作用可能与Bcl-2和Bcl-xl蛋白下调有关。
Objective This study was designed to investigate the effects and mechanisms of inhibiting PI3K/AKT/mTOR pathway on cisplatin-induced human skin melanoma cell line A375 apoptosis.Methods Western blot analyses were used to detect apoptosis and activation of PI3K/AKT/mTOR pathway,CCK-8 was used to detect cell viability in human skin melanoma cell line A375 treated with cisplatin.The synergistic effects of PI3K inhibitor LY294002(LY)and mTOR inhibitor Rapamycin(Rap)on cisplatin human skin melanoma cell line A375 apoptosis were investigated.To further explore the synergistic mechanism of blocking PI3K/AKT/mTOR pathway in cisplatin-induced human skin melanoma cell line A375 apoptosis,Western blot analyses were used to detect Bcl-2 protein expression of blocking PI3K/AKT/mTOR Pathway in human skin melanoma cell line A375.Results The expression level of PARP activated-cleavage increased in a concentration-and timedependent manner in A375 melanoma cells cisplatin treatment.Cell viability decreased in a concentration and time-dependent manner(P<0.05).Activation of the PI3K/AKT/mTOR pathway following treatment of A375 melanoma cells with cisplatin.After blocking PI3K/AKT/mTOR pathway combined with cisplatin in A375 melanoma cells,the expression of PARP activatedcleavage increased significantly and cell viability decreased significantly(P<0.05).Bcl-2 protein and Bcl-xl protein expression was down-regulated after blocking PI3K/AKT/mTOR pathway combined with cisplatin in A375 melanoma cells.Conclusion Blocking PI3K/AKT/mTOR pathway has synergistic effect in cisplatin-induced apoptosis,it may be related to the down regulation of Bcl-2 protein and Bcl-xl protein.
作者
张斌斌
王湘琦
赵超然
熊爱兵
ZHANG Bin-bin;WANG Xiang-qi;ZHAO Chao-ran;XIONG Ai-bing(Department of Plastic Surgery and Burns,Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan,China)
出处
《中国美容医学》
CAS
2019年第8期72-76,共5页
Chinese Journal of Aesthetic Medicine
