死亡相关蛋白激酶在结肠癌耐药中的作用  

Function of death associated protein kinase （DAPK） in colorectal cancer chemoresistance

作　　者：付强[1] 张永磊[1] 成静[2] 陈小兵[3] 谢建国[1] 罗素霞[3] 

机构地区：[1]河南省肿瘤医院普通外科,郑州450008 [2]郑州大学附属郑州中心医院急诊内科,450008 [3]河南省肿瘤医院内科,郑州450008

出　　处：《中华胃肠外科杂志》2014年第11期1101-1105,共5页Chinese Journal of Gastrointestinal Surgery

基　　金：河南省科技创新杰人才计划-杰出人才（201301014）

摘　　要：目的 探讨死亡相关蛋白激酶（DAPK）在结肠癌耐药中的作用.方法 应用免疫组织化学（免疫组化）SP法检测61例结肠癌组织及32例癌旁组织中DAPK的表达.以氟尿嘧啶（5-FU）诱导建立的结肠癌耐药细胞系HCT116/5-FU为模型.通过转染DAPK-siRNA下调DAPK的表达（DAPK-siRNA组）,转染FAM-siRNA（FAM-siRNA组）作为对照;通过过表达载体上调DAPK的表达（DAPK过表达组）.采用实时定量荧光PCR及蛋白质印迹法检测3组的DAPK、多药耐药蛋白（MRP）和P-糖蛋白（P-gp）的mRNA及蛋白表达水平;MTT法及流式细胞法分别测定3组细胞在未经5-FU处理及浓度为8 μg/ml的5-FU处理下的细胞增殖和凋亡情况.结果 DAPK在结肠癌组织中的阳性表达率明显低于癌旁组织[18.0％（11/61）比90.6％（29/32）,P＜0.05].与FAM-siRNA组比较,DAPK-siRNA组细胞中DAPK mRNA水平及蛋白表达水平均明显降低,DAPK过表达组则均显著升高（均P＜0.05）.在5-FU处理下,相比FAM-siRNA组,DAPK过表达组细胞增殖受到明显抑制,细胞凋亡率明显升高（均P＜0.05）;DAPK-siRNA组细胞增殖和细胞凋亡率均无明显变化（均P＞0.05）.与FAM-siRNA组比较,DAPK过表达组两种耐药蛋白的mRNA和蛋白表达水平均明显降低（P＜0.05）,而DAPK-siRNA组与FAM-siRNA组间差异无统计学意义（P＞0.05）.结论 DAPK能够抑制结肠癌耐药细胞的增殖,促进其凋亡,并可能通过抑制MRP和P-gp的mRNA和蛋白表达,来增强结肠癌细胞对药物的敏感性。Objective To investigate the role of death associated protein kinase （DAPK） in colon cancer drug-resistance.Methods Immunohistochemistry was used to detect DAPK expression in colon carcinoma tissues of 61 cases and adjacent tissues of 32 cases.5-fluorouracil （5-FU）-induced drug-resistant colon cancer cell lines HCT116/5-FU model was established.DAPK-siRNA was transfected into cells to down-regulate the DAPK gene expression （DAPK-siRNA grouyp）,FAM-siRNA was transfected as control group,and DAPK over-expression plasmid vectors were constructed to up-regulate the DAPK gene expression （DAPK over-expression group）.Real-time quantitative PCR and Western blotting were used to examine the mRNA and protein expression levels of DAPK,multidrug resistance protein （MRP） and P-glycoprotein （P-gp）.MTT and flow cytometry were used to detect cell proliferation and apoptosis for cells treated with 5-FU（8 mg/L） and cells without treatment of 5-FU in 3 groups respectively.Results Positive expression rate of DAPK in colon cancer tissues was significantly lower than that in adjacent normal tissues [18.0% （11/61） vs.90.6% （29/32）,P 〈0.05].Compared with FAM-siRNA group,DAPK mRNA and protein expression levels were significantly lower in DAPK-siRNA group,but significantly higher in DAPK over-expression group （P 〈0.05）.After treatment of 5-FU,cell proliferation was significantly inhibited,but cell apoptosis was significantly increased in DAPK over-expression group compared to FAM-siRNA group （P〈0.05）.Cell proliferation and apoptosis were not significantly different between DAPK siRNA and FAM-siRNA groups （all P〈0.05）.Compared with FAM-siRNA group,DAPK over-expression could significantly reduce the mRNA and protein levels of MRP and P-gp,whereas DAPK siRNA had no obvious such effects.Conclusion DAPK can inhibit the proliferation and promote the apoptosis in drug-resistant colon cancer cells,and it probably enhances the sensitivity of cancer cells to drugs by down-regulating the mRNA an

关 键 词：结肠肿瘤 死亡相关蛋白 耐药 多药耐药蛋白 P-糖蛋白 

分 类 号：R735.35[医药卫生—肿瘤]