摘要
目的研究MTX对RAS蛋白定位改变以及下游信号途径的影响,并探讨MTX抑制人胶质母细胞瘤细胞株U87生长的可能机制。方法不同浓度的MTX(75nmol/L、375nmol/L、750nmol/L、7,500nmol/L、15,000nmol/L)处理U87细胞后,采用MTT法和FCM法分别检测细胞活力,细胞凋亡率及细胞周期。RAS-GFP和蛋白质印迹法分别检测RAS蛋白的分布定位和其下游MAPK/ERK蛋白的表达水平。采用逆转录病毒法将RAS-GFP融合基因转导至U87细胞,共聚焦显微镜技术能检测到RAS活化后定位分布,与不同的MAKP/ERK信号途径特异性抑制剂(U0126和PD98059)共培养,采用蛋白质印迹法检测MTX对P-P42/P44 MARK蛋白磷酸化水平的影响以及其调控转录因子c-MYC表达水平发的影响。50nmol/L的MTX处理U87细胞后,进一步用FCM法检测细胞表面分子CD47的表达水平。结果不同浓度的MTX(75nmol/L、375nmol/L、750nmol/L、7,500nmol/L、15,000nmol/L)均能抑制人神经胶质母细胞瘤细胞株U87的增殖,呈剂量依赖性,不同浓度的MTX可将U87细胞阻滞于个G2/S期,并不能直接诱导细胞发生凋亡。转导RAS-GFP融合基因到U87细胞(100%GFP),同对照组相比,用MTX处理后发现RAS蛋白从细胞膜转移到细胞质,从而通过降低或者抑制P-p42/p44MARK蛋白磷酸化水平(P<0.05),以及抑制转录因子c-MYC的表达水平(P<0.05),导致U87细胞生长受到抑制。我们还近一步证明了MTX抑制与神经胶质母细胞瘤生长密切相关的细胞表面分子CD47在不同时间的表达水平。结论我们首次证明MTX通过改变RAS蛋白定位抑制MAPK/ERK/C-MYC信号导致神经胶质母细胞瘤U87细胞株的生长收到抑制,细胞停滞在G2/S期,提示MTX有可能作为复发胶质瘤患者可选的治疗药物。
Objective To investigate the effect of methotrexate(MTX)on the localization of RAS protein and the downstream signal pathway and to explore the mechanism of MTX to inhibit the growth of human glioblastoma cell line U87.Methods After the treatment of U87 cells with different concentrations of MTX(75nmol/L,375nmol/L,750nmol/L,7,500nmol/L,15,000nmol/L),the cell viability and apoptosis rate were detected by MTT and FCM respectively.The distribution of RAS protein and activation was observed by confocal microscopy and the expression level of the MAPK/ERK protein in the downstream was measured by immune blotting method after U87 cell transduced retrovirus carrying RAS-GFP fusion gene.Further,the effect of MTX on the level of phosphorylation of P-p42/p44 MARK protein and the level of its regulation of the transcription factor c-MYC expression using different MAKP/ERK signaling pathway specific inhibitors(U0126 and PD98059)was investigated.The expression level of CD47 on the surface of cells was further assayed by FCM on U87 cells treated with low dose of MTX at different time points.Results The different concentrations of MTX(75nmol/L,375nmol/L,750nmol/L,7,500nmol/L,15,000nmol/L)could inhibit the proliferation of human glioblastoma cell line U87 in a dose-dependent manner but can not directly induce apoptosis.The cell cycle of most U87 treated with the different concentrations of MTX was arrested at G2/S compared to untreated U87 cells.After U87 cells transduced RAS-GFP fusion gene(100%GFP)were treated with U87compared with the control group,the RAS protein was mislocation from the cell membrane to the cytoplasm;the P-p42/p44 MARK level was inhibited(P<0.05);the expression level of the transcription factor c-MYC was down-regulated(P<0.05).More important we addressed that CD47 related to glioblastoma growth and metastasis on the cell surface of U87 treated with MTX was decreased at different times,which confirmed by flow cytometry.Conclusions It is the first time that we have demonstrated that MTX can inhibit the growth
作者
白玥
钟晓松
李文斌
BAI Yue;ZHONG Xiao-song;LI Wen-bin(Department of Oncology,Beijing Shijitan Hospital,Capital Medical University,Beijing 100038,China;Clinical Center of Gene and Cell Engineering,Beijing Shijitan Hospital,Beijing 100038,China;Department of Glioma,Beijing Shijitan Hospital,Capital Medical University,Beijing 100038,China)
出处
《肿瘤代谢与营养电子杂志》
2018年第2期144-150,共7页
Electronic Journal of Metabolism and Nutrition of Cancer
基金
北京市科委脑科学研究基金(Z16110000021636)
