钙激活中性蛋白酶-2对肝癌细胞阿霉素耐药性的影响  

Effect of calcium-activated neutral protease-2 on drug resistance of hepatocarcinoma cell to adriamycin

作　　者：徐国强[1] 唐佳艳[1,2] 向敏[1,3] 王艳[1] 洪阳[1] 胡晓霞[1] 王旭东[1] 陈腾祥[1] 

机构地区：[1]贵阳医学院生理学教研室,贵州贵阳550081 [2]贵阳医学院附属医院消化内科,贵州贵阳550081 [3]贵阳医学院附属医院儿科,贵州贵阳550004

出　　处：《中国药理学通报》2011年第8期1108-1111,共4页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No81060176);贵州省科技厅自然科学基金资助项目(No黔科合J字[2008]2280);贵州省教育厅自然科学研究资助项目(NoF2008-3)

摘　　要：目的观察阿霉素作用下,钙激活中性蛋白酶-2(cal-pain-2)在肝癌细胞(HepG2)中的表达及其对细胞存活率的影响,评价其作为抗癌药靶的可能性。方法以倍比稀释的ADM作用于HepG2细胞,用四甲基偶氮唑盐比色(MTT)实验检测阿霉素(Adriamycin,ADM)72 h的半数致死量(LD50);用蛋白质印迹(Western blot)检测的ADM(LD50)作用下,calpain-2和cyclin E的表达和酶解情况;给予calpain-2抑制剂(calpastatin peptide)干预后,用MTT法观察ADM对HepG2细胞存活率的影响。结果 MTT实验结果显示,ADM对HepG2的LD50为2.5μmol.L-1;Western blot发现,随ADM作用时间的延长,calpain-2的表达水平增高(P<0.01),而且被剪切的程度增加(P<0.05);MTT检测ADM对HepG2的作用发现,calpastatin peptide干预的细胞存活率明显低于对照组(P<0.01),Cyclin E被剪切的程度减轻(P<0.01)。结论肝癌细胞HepG2在ADM作用下发生凋亡的同时,也上调calpain-2的表达水平和活性,并通过其信号通路降低对ADM的敏感性,而针对calpain-2的活性抑制可以提高HepG2对ADM敏感性,提示calpain-2可以作为降低肿瘤化疗耐药性的潜在靶点。Aim To explore the expression of calcium-activated neutral protease-2（calpain-2） in hepatocarcinoma cell（HepG2）,the effect on survival rate of HepG2 for evaluating potential value of calpain-2 as drug target.Methods Median lethal dose（LD50） of HepG2 treated with different dosage adriamycin（ADM） for 72 h was evaluated with tetrazolium salt colorimetric method（MTT）.The expression and zymohydrolysis of calpain-2 and cyclin E in HepG2 treated with ADM with LD50 were detected with Western blot.The survival rates of HepG2 treated with ADM（LD50） or companied with calpastatin peptide,inhibitor for calpain-2,were tested with MTT.Results The LD50 of ADM to HepG2 was 2.5 μmol·L-1 as MTT result showing.Western blot showed that Calpain-2 was up-regulated（P0.05）and cut into low molecular weight forms（LMW）（P0.01） when ADM administrated.The survival rates of HepG2 treated with ADM and calpastatin peptide were lower than those of control group（P0.01）,companied with lower level of zymohydrolysis of cyclin E（P0.01 vs control group）.Conclusions As an explanation of drug resistance mechanism,calpain-2 is up-regulated and activated by digested to LMW responding to ADM stress,which in turn to increases the resistance to ADM induced apoptosis of HepG2.The inhibition of calpain-2 increase the sensitivity of HepG2 to ADM,and calpain-2 can be selected as a potential target to impose the effects of anti-cancer.

关 键 词：肝癌细胞 钙激活中性蛋白酶-2 阿霉素 周期蛋白E 化疗 耐药性 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学] R345.79[医药卫生—基础医学]