Crizotinib诱导肺癌细胞保护性自噬及其机制研究
发布时间：2019-06-05 23:31
【摘要】：Crizotinib是靶向MET、ALK融合基因和ROS1的多靶点受体酪氨酸激酶抑制剂。Crizotinib作为MET抑制剂被研发,但首先被FDA批准用于EML4-ALK融合基因阳性的局部进展期及转移性肺腺癌的一线治疗,并同时建议用于存在MET基因扩增的肺癌患者。然而同大多数分子靶向治疗药物类似,半数以上患者在初始治疗1年内发生耐药,其原因包括原发性及获得性耐药突变,多种旁路癌基因的激活等。作为细胞对外界刺激的一种应激性存活机制,自噬参与了多种肿瘤细胞的耐药。常见的抗肿瘤治疗如化疗、放疗、生物靶向治疗等均可以引起肿瘤细胞自噬。Crizotinib能否诱导肺癌细胞自噬发生,从而导致其耐药尚不清楚。目的研究Crizotinib对肺癌细胞自噬水平的影响及自噬在肺癌细胞耐药中所起的作用；分析Crizotinib诱导自噬发生的具体分子机制；在此基础上观察抑制自噬是否增加肺癌细胞对Crizotinib的敏感性。方法通过Western blot.免疫荧光、透射电镜等方法观察Crizotinib处理后肺癌细胞SPC-A1、A549、H2228中自噬相关蛋白表达情况及自噬体的形成；Western blot分析Criztinib处理后肺癌细胞MET, STAT3, AKT/MTOR, ERK等信号通路关键分子的表达情况；采用MTS方法检测通过自噬抑制剂或下调自噬关键基因Beclinl抑制自噬对Crizotinib抗肿瘤作用的影响；采用流式细胞仪定量分析药物处理后细胞凋亡比率；建立肺癌裸鼠移植瘤模型评估自噬抑制剂对Crizotinib的增敏作用。结果Crizotinib能够抑制多种肺癌细胞株的活力,用Crizotinib处理表达MET的SPC-A1细胞,表达EML4-ALK的H2228细胞,和同时具有MET表达和KRAS突变的A549细胞,均可见细胞自噬水平的上升,表现为LC3 Ⅰ向LC3 Ⅱ转换增多；免疫荧光显示LC3Ⅱ puncta增多及电镜下自噬体的增加等。Crizotinib能够抑制MET及其下游STAT3、AKTYMTOR和ERK的磷酸化水平。进一步的研究发现Crizotinib诱导肺癌细胞自噬主要是通过分阶段的抑制胞质及胞核中的STAT3信号通路实现,胞质中STAT3的抑制导致EIF2AK2与其解离,继而磷酸化EIF2A而诱导自噬增加,而胞核中STAT3的抑制导致其转录激活的BCL2水平下降,进而促进自噬水平的持续增加。体外研究表明使用自噬抑制剂或下调自噬关键基因Beclinl抑制自噬促进Crizotinib对肺癌细胞的增殖抑制作用。不同浓度的Crizotinib联合CQ或3-MA作用于SPC-A1和A549细胞后对其抑制作用显著高于Crizotinib单药组,(p0.001)。以shRNA Beclinl转染SPC-A1和A549细胞,也能够显著提高其对Crizotinib的敏感性。流式细胞仪检测结果表明与Crizotinib单药处理相比,联合CQ和3-MA时,SPC-A1细胞凋亡率显著增加(p0.05)。裸鼠移植瘤模型表明自噬抑制剂HCQ增加Crizotinib对SPC-A1细胞移植瘤生长的抑制作用。Crizotinib联合HCQ组抑制肿瘤更为明显,而不显著增加毒性。结论多靶点酪氨酸激酶抑制剂Crizotinib能够诱导肺癌细胞保护性自噬,其机制是通过阶段性的抑制胞浆和胞核内的STAT3信号通路实现。抑制自噬促进Crizotinib在体内外对肺癌细胞的增殖抑制作用,提高细胞凋亡率。联合自噬抑制剂可以作为增敏Crizotinib治疗肺癌的一种新的策略。
[Abstract]:Crizotinib is a multi-target receptor tyrosine kinase inhibitor targeting MET, ALK fusion genes, and ROS1. Crizotinib is developed as a MET inhibitor, but is first approved by the FDA for first-line treatment for EML4-ALK fusion gene-positive local progression and metastatic lung adenocarcinoma, and is also recommended for patients with lung cancer with MET gene amplification. However, similar to most of the molecular targeted therapies, more than half of the patients experienced resistance in the initial treatment for 1 year, including primary and acquired resistance mutations, activation of multiple by-pass oncogene, and the like. As a stress-based survival mechanism for external stimulation of cells, autophagy is involved in the resistance of multiple tumor cells. Common anti-tumor treatments such as chemotherapy, radiotherapy, and biological targeted therapy can cause autophagy of the tumor cells. The ability of rizotinib to induce autophagy in lung cancer cells leads to a lack of clarity in its drug resistance. Objective To study the effect of crizotinib on autophagy of lung cancer cells and the role of autophagy in the drug resistance of lung cancer cells, and to analyze the specific molecular mechanism of the induction of autophagy by Crizotinib. Methods Western blot was used. The expression of autophagy in lung cancer cells SPC-A1, A549 and H2228 and the formation of self-phagocytosis were observed by immunofluorescence and transmission electron microscopy. The expression of MET, STAT3, AKT/ MOR, ERK and other signal pathways in lung cancer cells after treatment with criztepotinib was analyzed by Western blot. The effect of autophagy on the anti-tumor effect of Crizotinib by autophagy inhibitor or down-regulated autophagy is detected by MTS method, and the cell apoptosis ratio after drug treatment is quantitatively analyzed by flow cytometry; and the sensitization effect of the autophagy inhibitor on the Critzotinib is evaluated by establishing a nude mouse transplantation tumor model of the lung cancer. Results The activity of Critzotinib could inhibit the activity of multiple lung cancer cell lines. The SPC-A1 cells expressing MET were treated with Crizotinib, and the H2228 cells expressing EML4-ALK and A549 cells with MET expression and KRAS mutation were observed. Immunofluorescence showed the increase of LC3 鈪，
本文编号：2493894
[Abstract]:Crizotinib is a multi-target receptor tyrosine kinase inhibitor targeting MET, ALK fusion genes, and ROS1. Crizotinib is developed as a MET inhibitor, but is first approved by the FDA for first-line treatment for EML4-ALK fusion gene-positive local progression and metastatic lung adenocarcinoma, and is also recommended for patients with lung cancer with MET gene amplification. However, similar to most of the molecular targeted therapies, more than half of the patients experienced resistance in the initial treatment for 1 year, including primary and acquired resistance mutations, activation of multiple by-pass oncogene, and the like. As a stress-based survival mechanism for external stimulation of cells, autophagy is involved in the resistance of multiple tumor cells. Common anti-tumor treatments such as chemotherapy, radiotherapy, and biological targeted therapy can cause autophagy of the tumor cells. The ability of rizotinib to induce autophagy in lung cancer cells leads to a lack of clarity in its drug resistance. Objective To study the effect of crizotinib on autophagy of lung cancer cells and the role of autophagy in the drug resistance of lung cancer cells, and to analyze the specific molecular mechanism of the induction of autophagy by Crizotinib. Methods Western blot was used. The expression of autophagy in lung cancer cells SPC-A1, A549 and H2228 and the formation of self-phagocytosis were observed by immunofluorescence and transmission electron microscopy. The expression of MET, STAT3, AKT/ MOR, ERK and other signal pathways in lung cancer cells after treatment with criztepotinib was analyzed by Western blot. The effect of autophagy on the anti-tumor effect of Crizotinib by autophagy inhibitor or down-regulated autophagy is detected by MTS method, and the cell apoptosis ratio after drug treatment is quantitatively analyzed by flow cytometry; and the sensitization effect of the autophagy inhibitor on the Critzotinib is evaluated by establishing a nude mouse transplantation tumor model of the lung cancer. Results The activity of Critzotinib could inhibit the activity of multiple lung cancer cell lines. The SPC-A1 cells expressing MET were treated with Crizotinib, and the H2228 cells expressing EML4-ALK and A549 cells with MET expression and KRAS mutation were observed. Immunofluorescence showed the increase of LC3 鈪，
本文编号：2493894
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