替莫唑胺联合高压氧通过调控AKT/mTOR信号通路影响人肺癌A549细胞周期及凋亡

Temozolomide combined with hyperbaric oxygen can inhibit lung cancer A549 cell cycle and apoptosis by regulating the AKT/mTOR signaling pathway

目的探讨高压氧(HBO)联合替莫唑胺(TMZ)对肺癌A549细胞周期和凋亡的影响及其作用机制。方法人肺癌A549细胞培养完成后, 按照实验设计分成对照组、HBO组、TMZ组和HBO+TMZ组, 分别予以不作任何处处理、0.2 MPa HBO处理3 h、50 μmol/L TMZ处理24 h及0.2 MPa HBO预处理3 h后加入50 μmol/L TMZ处理24 h。CCK-8实验检测A549细胞增殖情况, 流式细胞术检测A549细胞凋亡及细胞周期情况, Western blotting实验检测A549细胞AKT/mTOR信号通路蛋白的表达水平。结果 HBO+TMZ组A549细胞存活率明显低于其他3组(P<0.05或P<0.01)。对照组、HBO组、TMZ组和HBO+TMZ组细胞凋亡率分别(6.73±1.47)%、(8.52±0.87)%、(32.78±3.49)%、(49.61±5.74)%, HBO+TMZ组细胞凋亡率明显高于其他3组, 差异均有统计学意义(P<0.05)。细胞周期实验结果显示, HBO+TMZ组中G2/M期A549细胞数量增加, 细胞比例为35.81%, 明显高于其他3组(P<0.05)。HBO+TMZ组A549细胞p-AKT和mTOR蛋白表达量明显低于其他3组(P<0.01)。结论 HBO与TMZ联用能够明显提高对肺癌A549细胞的增殖抑制作用, 并诱导细胞凋亡, 其作用机制是通过调控AKT/mTOR信号通路, 将A549细胞阻滞在细胞的G2/M期实现的。

Objective To investigate the effects of hyperbaric oxygen(HBO)combined with Temozolomide(TMZ)on the cell cycle and apoptosis of lung cancer A549 cells,and to study its mechanisms.Methods After the cell culture,the human lung cancer A549 cells were divided into control group,HBO group,TMZ group,and HBO+TMZ group according to the experimental design,and were given,respectively,no treatment,50μmol/L TMZ for 24 h,0.2 MPa HBO for 3 h,and 0.2 MPa HBO pretreatment for 3 h followed by 50μmol/L TMZ for 24 h.The cell proliferation was detected by Cell Counting Kit-8(CCK-8)experiment;the cell apoptosis and cell cycle were detected by flow cytometry;the expression of AKT/mTOR signaling pathway proteins in A549 cells was detected by Western blotting.Results The survival rate of A549 cells in the HBO+TMZ group was significantly lower than those of the other three groups(P<0.05 or P<0.01).The apoptosis rates in the control group,HBO group,TMZ group,and HBO+TMZ group were(6.73±1.47)%,(8.52±0.87)%,(32.78±3.49)%,and(49.61±5.74)%,respectively;the apoptosis rate in the HBO+TMZ group was significantly higher than those in the other three groups,and the differences were statistically significant(P<0.05).The results of cell cycle experiment showed that the number of A549 cells in G2/M phase was increased in the HBO+TMZ group with a cell percentage of 35.81%,which was significantly higher than those of the other three groups(P<0.05).The expression levels of p-AKT and mTOR proteins in A549 cells in the HBO+TMZ group were significantly lower than those in the other three groups(P<0.01).Conclusion The combination of HBO and TMZ can greatly improve the inhibition on the proliferation and induce apoptosis of lung cancer A549 cells,which is achieved by regulating the AKT/mTOR signaling pathway to block the A549 cells in the G2/M phase.