TRAIL-Mu3蛋白的体内外抗肿瘤活性及其机制研究  

Antitumor Activity of TRAIL-Mu3 Protein in vitro and in vivo and the Mechanisms

作　　者：杨婷[1] 朱艾晶 汪靖松 金钊[4] YANG Ting;ZHU Ai-jing;WANG Jing-song;JIN Zhao(Cancer Research Institute,Chengdu University of Traditional Chinese Medicine,Chengdu 610037,China;Department of Emergency,West China Hospital,Sichuan University,Chengdu 610041,China;Department of Gynaecology and Obstetrics,North Sichuan Medical College,Nanchong 637100,China;School of Basic Medical Science,Chengdu University of Traditional Chinese Medicine,Chengdu 610037,China)

机构地区：[1]成都中医药大学肿瘤研究所,成都610037 [2]四川大学华西医院急诊科,成都610041 [3]川北医学院妇产科,成都637100 [4]成都中医药大学基础医学院,成都610037

出　　处：《四川大学学报（医学版）》2020年第3期304-311,共8页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(No.81372444);四川省科技厅重点研发项目(No.2018SZ0089)资助。

摘　　要：目的TRAIL-Mu3是通过将野生型肿瘤坏死因子相关凋亡诱导配体(TRAIL)的N端突变为8个连续的精氨酸(第114~122位氨基酸)得到的可溶性蛋白突变体。本研究在前期药物构建和制备已经完成的基础上进一步对TRAILMu3蛋白的体内外药效和机理进行初步探索。方法采用CCK-8法检测TRAIL-Mu3对肺癌细胞株NCI-H460、A549、NCIH1299、calu-1的增殖抑制作用,通过流式细胞术(FCM)检测TRAIL-Mu3对A549和NCI-H460细胞凋亡诱导作用,并通过Western blot法检测体外凋亡相关蛋白死亡受体4(death receptor 4,DR4)、死亡受体5(death receptor 5,DR5)、Caspase-3、Caspase-8、X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)的表达情况。建立肺癌细胞株NCI-H460荷瘤小鼠移植瘤模型,TRAIL-Mu3给药方式为尾静脉注射,给药频率分为每日注射1次、隔日注射1次、每周注射3次,观察不同给药频率对移植瘤模型的治疗效果,并进行免疫组织化学法检测肿瘤组织凋亡相关蛋白DR4、DR5、Caspase-3、Caspase-8、XIAP的体内表达情况。结果通过细胞增殖及凋亡检测等体外实验证实TRAIL-Mu3较野生型TRAIL表现出更强的体外肿瘤细胞毒性和凋亡诱导能力,并可在体外上调DR4、Caspase-3、Caspase-8的表达或活化(P<0.05)。动物实验结果显示,TRAIL-Mu3隔日给药与每周3次给药抑制作用相近,优于每日给药(P<0.05),但3种给药方案均可显著抑制荷瘤小鼠移植瘤的生长。免疫组化结果表明其可在体内上调DR4、Caspase-3的表达或活化,下调XIAP的活化(P<0.05)。结论突变体TRAIL-Mu3通过上调死亡受体DR4的表达、增加凋亡相关蛋白Caspase-3/-8的活化、减少XIAP的活化而展现出良好的体内外抑瘤效果。Objective TRAIL-Mu3 was obtained by mutating the N-terminus of human tumor necrosis factorrelated apoptosis-inducing ligand(TRAIL)gene to an eight continuous arginine sequence.The present study was designed to explore the antitumor effect of this soluble mutant protein and the underlying mechanisms.Methods The inhibitory effect of TRAIL-Mu3 on the proliferation of lung cancer cell lines NCI-H460,A549,NCI-H1299 and calu-1 was tested by CCK8 assay.The apoptotic rates of A549 and NCI-H460 treated by TRAIL-Mu3 were detected by flow cytometer(FCM).The expressions of apoptosis related proteins death receptor(DR)4,DR5,Caspase-3,Caspase-8 and X-linked inhibitor of apoptosis protein(XIAP)were detected by Western blot.Moreover,a subcutaneous xenograft tumor mouse model of NCI-H460 was established and treated with TRAIL-Mu3 daily or every other day or three times a week.The expressions of DR4,DR5,Caspase-3,Caspase-8 and XIAP were detected by immunohistochemical staining.Results The in vitro study demonstrated that as compared to the TRAIL,the TRAIL-Mu3 was more toxic and pro-apoptotic by upregulation of the expression and activity of DR4,Caspase-3 and Caspase-8.Also,the animal study showed a similar antitumor effect between treatment with TRAIL-Mu3 every other day and three time a week,which was better than daily use.All treatments significantly suppressed the growth of xenograft tumor,increased the expression or activity of DR4 and Caspase-3,and down-regulated the expression of XIAP(P<0.05).Conclusion TRAIL-Mu3 could improve antitumor activity in vivo and in vitro through elevating DR4 expression,activating Caspase-3/-8,and inhibiting XIAP activation.

关 键 词：肿瘤坏死因子相关凋亡诱导配体(TRAIL) 突变体 精氨酸 肺癌 细胞凋亡 

分 类 号：R965[医药卫生—药理学]