FGFR1抑制剂Ponatinib对乳腺癌细胞增殖凋亡的影响

摘要

目的:研究FGFR1(成纤维生长因子受体1)抑制剂Ponatinib对乳腺癌细胞增殖和凋亡的影响以及机制.方法:依据前期Western Blot结果筛选出实验组和对照组,用不同浓度Ponatinib处理人乳腺癌细胞株实验组和对照组.CCK-8法检测乳腺癌细胞增殖抑制率;流式细胞术检测乳腺癌细胞凋亡的影响;WesternBlot法检测Poantinib对FGFR1及其下游通路及凋亡相关蛋白的影响.结果:依据前期Western Blot结果,选取不表达FGFR1的MCF-7为对照组细胞,高表达FGFR1的MDA-MB-231、BT-549为实验组细胞.CCK-8结果及Annexin V-APC/7-AAD法结合流式细胞仪检测结果显示:Ponatinib呈浓度依赖性地诱导MDA-MB-231、BT-549细胞的增殖抑制和凋亡,对MCF-7细胞也有一定的增殖抑制和诱导凋亡作用,实验组细胞的增殖抑制率及凋亡率较对照组细胞高.Western Blot结果显示:MDA-MB-231、BT-549细胞中,p-FGFR1、p-Stat5、p-PLCγ1蛋白表达量均明显下调;Bcl-2、Mcl-1表达量下调,Bak表达量上调(BT-549中Bak、Bax表达量均上调).MCF-7细胞中,p-Stat5、p-PLCγ1蛋白表达量下调.结论:Ponatinib抑制MDA-MB-231、BT-549细胞增殖可能与下调p-FGFR1,进而下调p-Stat5、p-PLCy1有关,促进细胞凋亡的机制可能与下调Bcl-2、Mcl-1,上调Bak有关.%Objective:To investigate the FGFR1 inhibitor Ponatinib's effect on proliferation,apoptosis,preliminary explore its mechanism at protein level of breast cancer cells.Methods:Divided the experimental group and control group on the basis of expression levels of FGFR1.CCK-8 assay was used to detect the influence of Ponatinib on cell proliferation rate and Annexin V-APC/7-AAD assay was used to detect on celluar apoptosis.Western Blot assay was used to detect the influence of Ponatinib on the proteins related to FGFR1 signal transduction pathways and apoptosis.Results:FGFR1 was highly expressed in MDA-MB-231,BT-549 cells,the expression level of MCF-7 was few.So MCF-7,MDA-MB-231,BT549 cells were screened for the next experiments.The result of CCK8 assay and Annexin V-APC/7-AAD:Ponatinib could inhibit the proliferation and increase celluar apoptosis rates of MCF -7,MDA-MB-231 and BT-549 as the increasement of Ponatinib concentration.The apoptosis rates of experimental group were higher than control group,and the inhibition of MCF-7 proliferation was lower than MDA-MB 231 and BT-549.The result of Western Blot assay:Western-Blot results showed that the expression of p-FGFR1,p-Stat5 and p-PLCγ1 were declining in MDA-MB-231 and BT-549 cells.In the two cells,the protein expression levels of Bcl-2 and Mcl-1 were declining,Bak was increasing(Bak and Bax increased in BT-549).The protein expression levels of p-StatS,p-PLCγ1 declined in MCF-7 cells.Conclusion:The mechanism of inhibiting proliferation may relate to the decreased expression levels of p-Stat5,p-PLCγ1,which may induced by down-regulation of p-FGFR1.The mechanism of promoting apoptosis may relate to down-regulation of Bcl-2,Mcl-1,up-regulation of Bak.