基于ERK/mTOR通路探讨蔓性千斤拔素A对非小细胞肺癌细胞自噬的影响

Effect of flemiphilippinin A on autophagy in non-small cell lung cancer cells based on ERK/mTOR pathway

目的探讨蔓性千斤拔素A通过细胞外调节蛋白激酶(ERK)/哺乳动物雷帕霉素靶蛋白(mTOR)通路对非小细胞肺癌细胞自噬的影响。方法将A549细胞分为对照组及蔓性千斤拔素A低、中、高浓度(20、25、30μmol·L^(-1))组或对照组、蔓性千斤拔素A 30μmol·L^(-1)、SCH77298410μmol·L^(-1)+蔓性千斤拔素A 30μmol·L^(-1)和SCH77298410μmol·L^(-1)浓度组,对照组细胞培养液为含1%胎牛血清的RMPI 1640培养基,其余各组分别加入蔓性千斤拔素A或SCH772984(预处理细胞1 h)使达到相应浓度,连续培养12 h。MTT法检测细胞活力;细胞集落形成实验检测细胞集落形成的情况;细胞自噬染色检测试剂盒检测细胞内自噬的情况;免疫荧光检测细胞内LC3B蛋白表达水平的变化;Western blotting实验检测细胞内磷酸化的细胞外调节蛋白激酶(p-ERK)、磷酸化的哺乳动物雷帕霉素靶标(p-mTOR)、自噬相关蛋白Beclin 1(Beclin 1)、螯合体1(P62)蛋白表达水平的变化。结果与对照组比较,MTT法检测结果显示各浓度(20、25、30μmol·L^(-1))蔓性千斤拔素A能够显著抑制A549细胞活力(P<0.05、0.001),SCH772948(10μmol·L^(-1))能够显著逆转蔓性千斤拔素A诱导的A549细胞死亡(P<0.01);细胞集落形成实验显示20、25、30μmol·L^(-1)蔓性千斤拔素A能显著抑制A549细胞集落的形成(P<0.001);细胞自噬染色检测试剂盒结果显示20、25、30μmol·L^(-1)蔓性千斤拔素A能显著抑制A549细胞自噬,SCH77294810μmol·L^(-1)能够显著逆转蔓性千斤拔素A介导的A549细胞自噬抑制;免疫荧光结果显示蔓性千斤拔素A 30μmol·L^(-1)能够显著减弱LC3B的荧光,SCH77294810μmol·L^(-1)能够显著逆转蔓性千斤拔素A导致的LC3B荧光减弱;Western blotting实验结果表明,20、25、30μmol·L^(-1)蔓性千斤拔素A能够使A549细胞内p-ERK、p-mTOR、P62蛋白表达水平显著升高及Beclin 1蛋白表达水平显著降低(P<0.001),SCH77294810μmol·L^(-1)能够显著逆转蔓性千斤拔素A导致的A549细胞内p-ERK、p-mTOR、P62蛋白表达水平升高及Beclin 1蛋白降低(P<0.001)。结论蔓性千斤拔素A可通过ERK/mTOR通路抑制非小细胞肺癌细胞自噬并导致细胞死亡。

Objective To investigate the effect of flemiphilippinin A on autophagy in non-small cell lung cancer(NSCLC)cells through the ERK/mTOR signaling pathway.Methods A549 cells were divided into control,flemiphilippinin A low,medium and high concentration(20,25 and 30μmol·L^(-1))groups or control,flemiphilippinin A 30μmol·L^(-1),SCH77298410μmol·L^(-1)+flemiphilippinin A 30μmol·L^(-1)and SCH77298410μmol·L^(-1)concentration groups.The cells in the control group were cultured in RMPI 1640 medium containing 1%fetal bovine serum,and the cells in the other groups were pretreated with flemiphilippinin A or SCH772984 for 1 h to achieve the corresponding concentration,and the cells were continuously cultured for 12 h.Cell viability was detected by the MTT assay.Cell colony formation assay was used to detect cell colony formation.Autophagy staining detection kit was used to detect autophagy in cells.The expression of the LC3B protein was detected by immunofluorescence.The protein expression levels of p-ERK,p-mTOR,Beclin 1,and P62 were detected by Western blotting.Results The results of MTT assay showed that each concentration of flemiphilippinin A could significantly inhibit the viability of A549 cells(P<0.05,0.001).SCH772948(10μmol·L^(-1))could reverse the inhibition of flemiphilippinin A-induced proliferation of A549 cells(P<0.01).The cell colony formation assay showed that flemiphilippinin A(20,25,and 30μmol·L^(-1))could inhibit the colony formation of A549 cells(P<0.01).The results of the autophagy staining assay kit showed that flemiphilippinin A(20,25,and 30μmol·L^(-1))could inhibit autophagy in A549 cells,and SCH77294810μmol·L^(-1)could reverse the flemiphilippinin A-mediated autophagy inhibition in A549 cells.Immunofluorescence results showed that the fluorescence of LC3B induced by flemiphilippinin A(30μmol·L^(-1))was decreased,and SCH772948(10μmol·L^(-1))could reverse the decrease of LC3B fluorescence induced by flemiphilippinin A.The results of Western blotting showed that the expression levels of p-ERK,p-mTOR,and P62 proteins in A549 cells were significantly increased,and the expression level of Beclin 1 protein was significantly decreased of flemiphilippinin A(20,25,and 30μmol·L^(-1))(P<0.001).10μmol·L^(-1)SCH772948 could significantly reverse the increase of p-ERK,p-mTOR,and P62 protein expression levels and the decrease of Beclin 1 protein in A549 cells induced by flemiphilippinin A(P<0.001).Conclusion Flemiphilippinin A can inhibit autophagy and cause cell death in non-small-cell lung cancer cells through the ERK/mTOR pathway.