抑制黏着斑激酶经Akt/NF-κB信号通路逆转结肠癌多细胞耐药  

Reversing multi-cellular resistance of colon carcinoma cells by suppressing focal adhesion kinase through FAK/Akt/NF-κB signaling pathway

作　　者：陈玉英[1,2] 向浏欣[2] 潘凤[1] 蒋金妍[1] 杨黎[1] 李建军[1] 梁后杰[1] 

机构地区：[1]第三军医大学西南医院肿瘤科,重庆400038 [2]重庆邮电大学生物信息学院分子生物重点实验室,重庆400065

出　　处：《第三军医大学学报》2010年第16期1745-1749,共5页Journal of Third Military Medical University

基　　金：重庆市自然科学基金(CSTC2007BB5444);重庆邮电大学博士基金(A2008-65)~~

摘　　要：目的探讨抑制黏着斑激酶(focaladhesionkinase,FAK)在结肠癌HT29细胞中的表达能否逆转结肠癌多细胞耐药。方法采用免疫细胞化学和Westernblot检测单层细胞中FAK、FAKp的抑制情况,用MTT法检测单层细胞和多细胞球对5-FU敏感性的变化,采用流式细胞术检测抑制FAK对多细胞球5-FU诱导凋亡和自然凋亡的影响,免疫细胞化学检测单层细胞、Westernblot检测多细胞球中FAKshRNA干扰对Akt、NF-κB、Bcl-2、Caspase-3的影响。结果靶向干扰FAK能显著抑制单层细胞FAK、FAKp蛋白表达。MTT结果显示,FAKshRNA干扰增强了单层细胞和多细胞球对5-FU的敏感性,特别是后者,且二者差异无统计学意义(P>0.05)。流式细胞术结果显示,FAKshRNA干扰能够使多细胞球的自发凋亡率和5-FU诱导凋亡率显著增加(P=0.001,P=0.000),且两者之间差异显著(P=0.003)。在单层和多细胞球细胞中,FAKshRNA干扰后,Akt、NF-κB、Bcl-2的水平明显下调,而Caspase-3水平上调。结论抑制FAK表达能增强结肠癌多细胞球细胞对5-FU的敏感性,逆转其多细胞耐药性,其分子机制是通过FAK/Akt/NF-κB信号通路实现的。Objective To determine whether suppressing the expression of focal adhesion kinase （FAK） in colon carcinoma cell line HT29 will reverse colon carcinoma multicellular resistance.Methods The cells were divided into untreat HT29 cells,control shRNA cells,and FAK shRNA cells.The suppression of FAK and FAKp expression in monolayer culture cells of the above 3 groups were detected by immunocytochemistry and Western blotting analysis.Effects of suppressing FAK on the drug sensitivity to 5-FU of monolayer and multicellular spheroid culture cells were quantified by MTT assay.Effects of suppressing FAK on 5-FU-induced apoptosis and natural apoptosis of multicellular spheroids were determined by flow cytometry （FCM）.Effects of FAK shRNA interference on protein expressions of Akt,NF-κB,Bcl-2,and Caspase-3 of monolayer culured cells were detected by immunocytochemistry,and their protein expressions in multicellular spheroid culture cells were detected by Western blotting.Results RNA interference targeting FAK markedly suppressed FAK and FAKp protein expressions in monolayer culture cells.MTT assay indicated that FAK shRNA interference enhanced the drug sensitivity to 5-FU of monolayer and multicellular spheroid culture cells evidently,especial the latter,but the difference between the 2 was not markedly （P0.05）.FCM manifested that FAK shRNA interference stimulated natural apoptosis and 5-FU-induced apoptosis markedly in multicellular spheroid cells （P=0.001,P=0.000）,and the difference between the 2 was markedly （P=0.003）.Following FAK shRNA treatment,Akt,NF-κB and Bcl-2 levels were obviously suppressed,but the Caspase-3 level was obviously upregulated both in monolayer and in multicellular spheroid cells.Conclusion Suppressing FAK enhances the drug sensitivity of colon carcinoma multicellular spheroid cells to 5-FU through a FAK/Akt/NF-κB signaling pathway,and reverses multicellular resistance at the same tme.

关 键 词：黏着斑激酶 结肠癌 敏感性 信号通路 RNA干扰 

分 类 号：R735.35[医药卫生—肿瘤] R329.26[医药卫生—临床医学]