厄洛替尼通过下调CD44表达抑制非小细胞肺癌转移的机制研究

Study on the action mechanism of erlotinib in inhibiting metastasis of non-small cell lung cancer through down-regulating the expression of CD44 in vitro

目的探讨表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)下调黏附分子CD44表达抑制非小细胞肺癌转移的机制。方法采用MTT法检测厄洛替尼对HCC827细胞株的增殖抑制效应及计算药物作用48 h的IC50;采用Transwell和划痕实验检测3组[control、EGF(50 ng/ml)刺激组、厄洛替尼(0.323μmol/L)处理组]细胞侵袭迁移能力的变化;采用流式细胞术及Western blot方法分别从细胞及蛋白水平检测3组[control、EGF(50 ng/ml)刺激组、厄洛替尼(0.323μmol/L)处理组]细胞CD44表达水平变化。结果 MTT结果显示,随着药物浓度升高,厄洛替尼对细胞的增殖抑制率也逐渐增大,差异有统计学意义(P<0.05);IC50为0.323μmol/L。Transwell及划痕实验结果显示,厄洛替尼阻断EGFR信号通路后可降低细胞的侵袭迁移能力(P<0.05)。流式细胞术及Western blot检测三组细胞CD44表达水平变化结果显示:当使用EGF刺激细胞后,CD44表达明显上调,而使用厄洛替尼阻断EGFR信号通路后CD44表达则被明显下调(P<0.05)。结果提示,厄洛替尼抑制肿瘤转移可能与改变CD44表达有关。结论使用厄洛替尼阻断EGFR信号通路后,可间接下调与肿瘤转移密切相关的黏附分子CD44的表达,进而发挥抑制肿瘤转移的作用。

Objective To investigate the action mechanism of erlotinib in inhibiting metastasis of non-small cell lung cancer （HCC827 cell line ） through down-regulating the expression of CD44 in vitro. Methods MTF method was used to detect the inhibitory effects of erlotinib on cell proliferation of HCC827 cell line and to calculate IC50 after 48-hour drug intervention. The changes of invasion and migration ability of lung cancer cells in thee three groups, control group, EGF （50ng/ml） stimulation group and erlotinib （0. 323μmol/L） intervention group, were detected by Transwell migration assay and scratch test. Moreover the expression levels of CD44 in the three groups were detected by flow cytometry and Western Blot, respectively. Results MTT assay showed that the inhibition rate of edotinib on cell proliferation was increased with the increase of drug concentration （ P 〈 0.05 ） , with IC50 being 0.323 μmol/L. Transwell migration assay and scratch test showed that edotinib could decrease invasion and migration ability of lung cancer cells after blocking EGFR signal pathway （ P 〈 0.05）. The results by flow cytometry and Western Blot showed thai after the ceils were stimulated by EGF, the expression levels of CD44 were obviously increased in three groups, however, after EGFR signal pathway was blocked by erlotinib, the expression levels of CD44 were significantly decreased （ P 〈 0.05 ） , which suggested that the inhibitory effects of edotinib on tumor metastasis might be correlated to regulating the expression levels of Cd44. Conclution The~ erlotinib can block EGFR signal pathway to down - regulate indirectly the expression levels of CD44 that is closely correlated with tumor metastasis, and then which can play an important role in inhibiting tumor metastasis .