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[目的]探讨PI3K/Akt通路在肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor related apoptosis inducing ligand,TRAIL)诱导携带EGFR/KRAS不同基因表型的非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞凋亡中的作用。[方法 ]CCK8法检测TRAIL对A549和PC9细胞活性的影响;Western blot检测TRAIL作用后Akt、p-Akt蛋白表达的变化。使用PI3K特异性抑制剂LY294002处理细胞,CCK8法检测对TRAIL抗细胞增殖活力的影响;流式细胞术检测对TRAIL诱导细胞凋亡和细胞周期影响。[结果]当TRAIL浓度<100 ng/ml时,A549和PC9细胞的存活率与对照组无明显差异。当TRAIL浓度分别为100 ng/ml[(64.29±11.39)%vs(100±12.07)%,t=6.900,P=0.020]、[(74.57±9.70)%vs(100±11.20)%,t=4.786,P=0.041]和500ng/ml[(48.85±10.92)%vs(100±12.07)%,t=13.390,P=0.006][(46.34±8.99)%vs(100±11.20)%,t=18.419,P=0.003]时,A549和PC9细胞存活率明显低于对照组。TRAIL以时间依赖的方式上调A549和PC9细胞内的p-Akt的水平。使用LY294002抑制PI3K/Akt通路活性能够明显增加TRAIL抑制A549细胞[(40.74±2.53)%vs(64.29±9.30)%,t=6.092,P=0.026]和PC9细胞[(42.38±3.40)%)vs(74.57±7.92)%,t=12.689,P=0.006]增殖的能力,诱导更多的A549细胞[(44.98±8.99)%vs(23.07±2.92)%,t=7.836,P=0.016)]和PC9细胞[(46.32±7.42)%vs(3.44±1.46)%,t=40.727,P=0.001)]凋亡,并使细胞周期更多地阻滞在G0/G1期[(73.60±3.43)%vs(60.20±5.48)%;(70.51±3.86)%vs(42.37±4.55)%](P均<0.05)。[结论]TRAIL引起的PI3K/Akt通路活化,可拮抗TRAIL的诱导凋亡作用。抑制PI3K/Akt活性可明显增强EGFR-TKI敏感的和EGFR-TKI不敏感的NSCLC细胞对TRAIL诱导凋亡的敏感性。
[Objective] To investigate the effect of PI3K/Akt pathway on tumor necrosis factor related apoptosis inducing ligand (TRAIL)-induced non-small cell lung cancer with different phenotypes of EGFR/KRAS. Apoptosis in NSCLC). [Methods] CCK8 assay was used to detect the effect of TRAIL on the activity of A549 and PC9 cells. The expression of Akt and p-Akt protein was detected by Western blot. Cells were treated with PI3K specific inhibitor LY294002. The effect of TRAIL on cell proliferation was detected by CCK8 assay. The effect of TRAIL on cell apoptosis and cell cycle was detected by flow cytometry. [Results] When the TRAIL concentration was <100 ng/ml, the survival rates of A549 and PC9 cells were not significantly different from those of the control group. When TRAIL concentrations were 100 ng/ml [(64.29±11.39)% vs (100±12.07)%, t=6.900, P=0.020], [(74.57±9.70)% vs (100±11.20)%, t= 4.786, P=0.041] and 500 ng/ml [(48.85±10.92)% vs (100±12.07)%, t=13.390, P=0.006] [(46.34±8.99)% vs (100±11.20)%, t= At 18.419, P=0.003], the survival rate of A549 and PC9 cells was significantly lower than that of the control group. TRAIL upregulates p-Akt levels in A549 and PC9 cells in a time-dependent manner. Using LY294002 to inhibit PI3K/Akt pathway activity significantly increased TRAIL inhibition in A549 cells [(40.74±2.53)% vs (64.29±9.30)%, t=6.092, P=0.026] and PC9 cells [(42.38±3.40)%] vs (74.57±7.92)%, t=12.689, P=0.006] Ability to proliferate, induce more A549 cells [(44.98±8.99)% vs (23.07±2.92)%, t=7.836, P=0.016)] and PC9 cells [(46.32±7.42)% vs (3.44±1.46)%, t=40.727, P=0.001)] apoptosis and more arrested cell cycle in G0/G1 phase [(73.60±3.43)% (60.20±5.48)%; (70.51±3.86)% vs (42.37±4.55)%] (P all < 0.05). [Conclusion] The activation of PI3K/Akt pathway caused by TRAIL can antagonize the apoptosis-inducing effect of TRAIL. Inhibition of PI3K/Akt activity significantly enhanced the sensitivity of EGFR-TKI-sensitive and EGFR-TKI-insensitive NSCLC cells to TRAIL-induced apoptosis.