非小细胞肺癌奥希替尼获得性耐药细胞株的建立及其耐药机制的探讨

Establishment of an Osimertinib-resistant Human Non-small Cell Lung Cancer Cell Line and Study of the Resistance Mechanism

耐药的产生是肺癌死亡率居高不下的主要原因。因此,建立非小细胞肺癌(NSCLC)第三代分子靶向药物奥希替尼获得性耐药细胞株,并探讨其耐药机制对肺癌耐药的研究具有重要意义。本研究采用间歇性大剂量冲击和梯度递增法,以HCC827为模型,在体外建立奥希替尼耐药细胞株HCC827OR。利用细胞计数(CCK8)法检测细胞增殖,通过耐药指数(RI)评估细胞对奥希替尼的敏感度;利用荧光染色法比较耐药前后细胞的形态学变化;利用Western blot法检测表皮生长因子受体(EGFR)及其下游信号蛋白的表达;第二代测序(NGS)检测EGFR的基因突变;细胞周期和划痕试验检测细胞的增殖和迁移能力。试验结果显示,HCC827OR的RI为18.65,表明HCC827OR为奥希替尼耐药细胞株。与野生型HCC827细胞相比,奥希替尼耐药细胞的核质比显著增大,关键蛋白p-EGFR表达显著下降,而细胞增殖相关的p-AKT显著增加,这表明其可能通过非EGFR依赖信号通路产生耐药。同时,流式细胞术和划痕试验结果表明,HCC827OR细胞株出现了明显的G2/M周期阻滞,但其迁移能力得到了提高。本研究成功构建了NSCLC奥希替尼耐药细胞株HCC827OR,初步证明了其可通过非EGFR依赖的旁路产生耐药,为研究肿瘤细胞耐药提供了重要的体外模型。

Drug resistance remains the key factor that results in the high mortality of lung cancer patients. Therefore, it is very important to explore the mechanisms underlying the lung cancer drug resistance by establishing a third-generation molecular targeting drug-resistant cell line of non-small cell lung cancer(NSCLC). This study successfully established an Osimertinib-resistant cell line, HCC827OR, based on HCC827 cell line through combining the methods of intermittent high-dose selection and concentration-increasing. Cytotoxicity of Osimertinib against cancer cells was firstly evaluated through using the CCK8 assay kit, and resistance index(RI) was used to evaluate the sensitivity of cancer cells to Osimertinib;morphological changes of the cancers cells before and after acquiring drug resistance were stained and imaged using an optical microscopy. Western blot was used to detect the expression level of epidermal growth factor receptor(EGFR) and its downstream signaling proteins;mutation of EGFR was measured through using the next generation sequencing(NGS);cell cycle and scratch assays were performed to evaluate cell proliferation and migration ability. The RI of HCC827OR was calculated to be 18.65, demonstrating that HCC827OR was Osimertinib-resistant cells. Compared with the wild-type cells, Osimertinib-resistant cells showed a greater karyoplasmic ratio. Additionally, reduced expression of the p-EGFR and increased expression of cell proliferation-associated p-AKT indicated that the HCC827OR probably acquired the Osimertinib-resistant character through a non-EGFR-dependent signaling pathway. Meanwhile, flow cytometry and scratch assay results demonstrated that HCC827OR cells have significant G2/M cycle arrest and enhanced migration ability in comparison with the HCC827. This study successfully established an Osimertinib-resistant NSCLC cell line, and tentatively demonstrated the acquisition of drug-resistant characteristics by the HCC827OR through a non-EGFR-dependent pathway, thus providing an important in vitro model for lung cancer drug-resistance research.