南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (06): 884-892.doi: 10.12122/j.issn.1673-4254.2020.06.17

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吉非替尼抑制糖酵解诱导非小细胞肺癌的程序性死亡

周 巧，李佳会，庞金龙，范方田，李姗姗，刘 浩   

  • 出版日期:2020-06-20 发布日期:2020-06-20
  • 基金资助:

Gefitinib inhibits glycolysis and induces programmed cell death in non-small cell lung cancer cells

  

  • Online:2020-06-20 Published:2020-06-20

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摘要： 目的 探究吉非替尼对非小细胞肺癌A549和H1975细胞死亡形式的影响，并从糖酵解方面探讨其可能机制。方法 A549细胞加入浓度分别为0、20、30、40 µmol/L的吉非替尼，H1975细胞加入浓度分别为0、20、40、80 µmol/L的吉非替尼。采用MTT法检测吉非替尼对非小细胞肺癌细胞的增殖抑制作用。用乳酸试剂盒检测细胞内乳酸的变化，Western blot法检测细胞内糖酵解相关蛋白（PKM2、HK2）和PI3K-Akt-mTOR信号通路中蛋白的表达水平；2-NBDG检测细胞葡萄糖摄取能力，ATP试剂盒检测胞内ATP水平；JC-1试剂盒检测细胞线粒体膜电位，Annexin V-FITC/PI双染法检测细胞凋亡，Western blot法检测凋亡蛋白（Bax、Bcl-2）以及自噬标志蛋白LC3B的相对表达水平。结果 MTT结果显示吉非替尼可呈时间剂量依赖性的抑制A549和H1975细胞增殖（P<0.05），A549细胞24、48、72 h的IC50值分别为48.6、28.6和19.7 µmol/L，H1975细胞24、48、72 h的IC50值分别为321.6、49.1和14.6 µmol/L。乳酸检测结果显示，吉非替尼抑制胞内乳酸水平（P<0.05）。Western blot结果显示，糖酵解相关蛋白PKM2、HK2表达下调（P<0.05），PI3K-Akt-mTOR信号通路中相关蛋白表达下调（P<0.05）。吉非替尼也可以抑制A549和H1975细胞葡萄糖摄取、ATP水平（P<0.05）。JC-1试剂盒和Annexin V-FITC/PI双染法检测出吉非替尼能够诱导A549和H1975细胞发生凋亡，A549细胞中0、20、30、40 µmol/L的凋亡率分别为（10.77±1.0）%、（14.5±0.4）%、（17.4±0.2）%、（32.1±0.6）%，差异有统计学意义（P<0.05）；而H1975细胞0、20、40、80 µmol/L的凋亡率分别为（10.5±0.6）%、（13.2±0.92）%、（18.9±0.98）%、（35.1±1.4）%，差异有统计学意义（P<0.05）。促凋亡蛋白Bax表达增加和抑凋亡蛋白Bcl-2表达下调（P<0.05）。同时LC3B表达增加证明吉非替尼能够诱导A549和H1975细胞自噬增加（P<0.05）。结论 吉非替尼对A549和H1975细胞具有增殖抑制、诱导凋亡和增加自噬作用，凋亡机制可能为吉非替尼影响A549和H1975细胞糖酵解功能和PI3K-Akt-mTOR信号通路。

Abstract: Objective To observe the cell death pattern induced by gefitinib in non-small cell lung cancer A549 and H1975 cells and explore the possible mechanism in light of glycolysis. Methods The inhibitory effects of gefitinib at 20, 30, or 40 µmol/L in A549 cells and at 20, 40, or 80 µmol/L in H1975 cells were examined using MTT assay. The changes of lactic acid level in the cells were determined with a lactic acid kit, and the expression levels of glycolysis-related proteins (PKM2 and HK2) and the proteins in PI3K-Akt-mTOR signaling pathway were detected using Western blotting. 2-NBDG was used for detecting glucose uptake capacity of the cells, and ATP kit was used to detect the intracellular ATP level. The mitochondrial membrane potential of the cells was examined with the JC-1 kit, and cell apoptosis was analyzed with Annexin V-FITC/PI double staining. The relative expression levels of the apoptotic proteins Bax and Bcl-2 and the autophagy marker protein LC3B were detected with Western blotting. Results MTT assay showed that gefitinib inhibited the proliferation of A549 and H1975 cells in a time- and dose-dependent manner (P<0.05). The IC50 of gefitinib at 24, 48 and 72 h was 48.6, 28.6 and 19.7 µmol/L in A549 cells and was 321.6, 49.1 and 14.6 µmol/L in H1975 cells, respectively. Gefitinib significantly lowered intracellular lactic acid level of the cells (P<0.05) and down-regulated the expressions of PKM2 and HK2 proteins (P<0.05) and PI3K-Akt-mTOR signaling pathway-associated proteins (P<0.05). Gefitinib obviously inhibited glucose uptake and ATP levels in both A549 and H1975 cells (P<0.05). Treatment with gefitinib induced obviously enhanced apoptosis in the cells, resulting in apoptosis rates of (10.77± 1.0)%, (14.5±0.4)%, (17.4±0.2)% and (32.1±0.6)% at 0, 20, 30 and 40 µmol/L in A549 cells (P<0.05) and of (10.5±0.6)%, (13.2± 0.92)%, (18.9±0.98)% and (35.1±1.4)% at 0, 20, 40 and 80 µmol/L in H1975 cells, respectively (P<0.05). The protein expression of Bax increased and that of Bcl-2 decreased following gefitinib treatment in the cells (P<0.05). Gefitinib significantly increased autophagy in A549 and H1975 cells as shown by increased LC3B expressions following the treatment (P<0.05). Conclusion Gefitinib can inhibit the proliferation, induce apoptosis and increase autophagy in A549 and H1975 cells. Gefitinib induces apoptosis of the cells possibly by affecting glycolysis and PI3K-Akt-mTOR signaling pathway.