EGFR突变型非小细胞肺癌STAT3活化对盐酸埃克替尼敏感性的影响

The Effect of STAT3 Activation on Icotinib Sensitivity in Mon-small Cell Lung Cancer Cells with EGFR Mutation

目的本研究拟利用表皮生长因子受体(epidermal growth factor receptor,EGFR)突变型非小细胞肺癌细胞系,探讨影响盐酸埃克替敏感性的因素及其机制。方法应用噻唑蓝比色法(MTT)法检测盐酸埃克替尼对肺癌细胞增殖的影响;蛋白免疫印迹法(Western Blot)检测相关信号通路蛋白的表达;实时荧光定量PCR(real-time PCR)检测盐酸埃克替尼对白细胞介素-6(interleukin-6,IL-6)信使RNA(messenger RNA,mRNA)表达水平影响。每次实验重复3次,数据以均数±标准差表示,采用SPSS 17.0软件分别对两组数据之间的差异进行t检验。结果(1)盐酸埃克替尼对EGFR突变的肺腺癌细胞PC9和HCC827有明显的抑制作用;(2)盐酸埃克替尼能明显抑制PC9和HCC827细胞的EGFR、蛋白激酶B(AKT)、细胞外调节激酶(extracellular regulatory kinases,ERK)磷酸化水平;(3)盐酸埃克替尼可使PC9细胞信号转导子和转录激活子(signal transducers and activators of transcription 3,STAT3)磷酸化水平显著提高,而对HCC827细胞STAT3磷酸化水平无影响;(4)盐酸埃克替尼可上调PC9细胞IL-6 mRNA表达水平,而对HCC827细胞无影响;(5)敲除STAT3并不影响AKT和ERK磷酸化水平;(6)抑制STAT3活化可增强PC9细胞对盐酸埃克替尼的敏感性。结论盐酸埃克替尼可通过抑制EGFR突变型非小细胞肺癌的EGFR及其下游磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/AKT和丝裂原激活蛋白激酶(mitogen-activated protein kinase,MAPK)/ERK信号通路的活化抑制肿瘤细胞的增殖;然而,在部分非小细胞肺癌中,盐酸埃克替尼可能通过上调IL-6 mRNA的表达,激活STAT3信号通路,降低肿瘤细胞对盐酸埃克替尼的敏感性。

Objective This study aims to use epidermal growth factor receptor(EGFR)mutant non-small cell lung cancer cell lines to explore the factors affecting the icotinib sensitivity and their mechanisms.Methods The effect of icotinib on the proliferation of PC9,HCC827 and A549 cells was measured using MTT assay;protein expression was determined by Western Blot;real-time fluorescence quatifative PCR(real-time PCR)was used to dctect the ectinib hydrochloride on the expression level of interleakin-6(IL-6)messenger RNA(mRNA).The experiment was repeated at least three times.The data were expressed as mean±standard deviation.SPSS 17.0 software was used to conduct t test for the difference between the two groups of data.Results(1)Icotinib significantly inhibited the proliferation of EGFR-mutated lung cancer cells PC9 and HCC 827;(2)icotinib could inhibit the phosphorylation level of EGFR,AKT,ERK in PC9 and HCC827 cell;(3)the phosphorylation level of STAT3 was significantly increased with icotinib treatment in PC9 cell,but not in HCC827 cells;(4)icotinib up-regulated the expression of IL-6 mRNA in PC9 cell,but had no effect on HCC827 cells;(5)STAT3 inhibitor or STAT3 siRNA could not affect the activation of AKT and ERK;(6)inhibition of STAT3 activation could enhance sensitivity of icotinib PC9 cells.Conclusion Icotinib can inhibit the proliferation of tumor cells by inhibiting the activation of EGFR and its downstream phosphatidylinositol 3-kinase/AKT and MAPK/ERK signaling pathways in EGFR mutant non-small cell lung cancer;however,in some non-small cell lung cancers,icotinib may reduce the sensitivity of tumor cells to icotinib by up-regulating the expression of IL-6 mRNA and activating STAT3 signaling pathway.