高良姜素对非小细胞肺癌A549细胞SIRT1/mTOR通路及放射敏感性的影响

Effects of Galangin on SIRT1/mTOR Pathway and Radiosensitivity in Non-small Cell Lung Cancer A549 Cells

目的:探讨高良姜素对非小细胞肺癌(NSCLC)A549细胞沉默信息调节因子1(SIRT1)/哺乳动物雷帕霉素靶蛋白(mTOR)通路及放射敏感性的影响。方法:体外培养NSCLC A549细胞,细胞计数试剂盒(CCK-8)法检测不同浓度(10,20,40,60,80,100μmol·L^(-1))高良姜素处理的A549细胞的增殖抑制率;将A549细胞经不同剂量(0,1,2,4,6,8 Gy)放射处理,并设置各剂量与35μmol·L^(-1)高良姜素联用组,克隆形成实验观察高良姜素的放射增敏作用;将A549细胞分为高良姜素+放射线组(35μmol·L^(-1)+6 Gy)、高良姜素组(35μmol·L^(-1))、放射线组(6 Gy)及对照组,流式细胞术检测细胞凋亡率;蛋白免疫印迹法检测增殖蛋白细胞周期蛋白D1(CyclinD1)、细胞增殖核抗原(Ki67)、凋亡蛋白半胱氨酸蛋白酶(Caspase)-3、Caspase-9及STRT1/mTOR通路蛋白SIRT1、腺苷酸活化蛋白激酶(AMPK)、磷酸化AMPK(p-AMPK)、磷酸化mTOR(p-mTOR)、mTOR的表达。结果:与对照组比较,随着高良姜素浓度的增加,A549细胞增殖抑制率逐渐增加(P<0.05);与各剂量射线单独处理的A549细胞相比,35μmol·L^(-1)高良姜素+各剂量射线处理的A549细胞存活分数降低(P<0.05),辐射增敏比(SER)=1.522;与对照组比较,高良姜素组、放射线组、高良姜素+放射线组A549细胞凋亡率、Caspase-3、Caspase-9、p-AMPK/AMPK蛋白表达显著升高,CyclinD1、Ki67、SIRT1、p-mTOR/mTOR蛋白表达显著降低(P<0.05);与高良姜素组、放射线组比较,高良姜素+放射线组A549细胞凋亡率、Caspase-3、Caspase-9、p-AMPK/AMPK蛋白表达显著升高,高良姜素+放射线组CyclinD1、Ki67、SIRT1、p-mTOR/mTOR蛋白表达显著降低(P<0.05)。结论:高良姜素可能通过调控SIRT1/mTOR通路影响A549细胞的增殖凋亡及放射敏感性,可能是治疗NSCLC的潜在药物。

Objective: To investigate the effect of galangin on silent information regulator 1(SIRT1)/mammalian target of rapamycin(mTOR) pathway and radiosensitivity in non-small cell lung cancer(NSCLC) A549 cells. Methods: NSCLC A549 cells were cultured in vitro. The proliferation inhibition rate of A549 cells treated with different concentrations of galangin(10, 20, 40, 60, 80, 100 μmol·L^(-1)) was detected by cell counting kit-8(CCK-8) method;A549 cells were irradiated with different doses(0, 1, 2, 4, 6, 8 Gy) of radiation, the different doses of radiation + 35 μmol·L -1 galangin groups were set up, and the radiosensitization effect of galangin was observed by clone formation assay;A549 cells were divided into galangin + radiation group(35 μmol·L^(-1) +6 Gy), galangin group(35 μmol·L^(-1)), radiation group(6 Gy) and control group. The apoptosis rate was detected by flow cytometry;the expressions of CyclinD1, Ki67, Caspase-3, Caspase-9, SIRT1, AMP activated protein kinase(AMPK), phosphorylation-AMPK(p-AMPK), phosphorylation-mTOR(p-mTOR) and mTOR were detected by Western blot. Results: Compared with that in the control group, the proliferation inhibition rate of A549 cells increased gradually with the increase of galangin concentration(P<0.05);compared with that of A549 cells treated with different doses of radiation alone, the survival fraction of A549 cells treated with 35 μmol·L^(-1)galangin + different doses of radiation was decreased(P<0.05), and the radiosensitization ratio(SER) was 1.522;compared with those in the control group, the apoptosis rate of A549 cells, and the expressions of Caspase-3, Caspase-9 and p-AMPK/AMPK protein were significantly higher in galangin group, radiation group and galangin+radiation group, and the expressions of CyclinD1, Ki67, SIRT1 and p-mTOR/mTOR protein were significantly lower(P<0.05);compared with those in galangin group and radiation group, the apoptosis rate of A549 cells and the expressions of Caspase-3, Caspase-9 and p-AMPK/AMPK protein were significantly higher in galangin + radiation group, and the expressions of CyclinD1, Ki67, SIRT1 and p-mTOR/mTOR proteinwas significantly lower(P<0.05). Conclusion: Galangin may affect the proliferation, apoptosis and radiosensitivity of A549 cells by regulating SIRT1/mTOR pathway, which may be a potential drug for the treatment of NSCLC.