去氢毛钩藤碱抗突变型p53(R273H)结直肠癌的体外作用机制研究  

In vitro anticancer mechanism of hirsuteine on mutant p53(R273H)colorectal cancer

作　　者：任泽昊 张彦 刘东辉 孔德新[1] 邱玉玲[1] REN Zehao;ZHANG Yan;LIU Donghui;KONG Dexin;QIU Yuling(School of Pharmacy,Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]天津医科大学药学院,天津300070

出　　处：《中草药》2024年第10期3354-3362,共9页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(81973570)。

摘　　要：目的从抑制结直肠癌细胞HT29增殖角度研究去氢毛钩藤碱(hirsuteine,HST)对突变型p53(R273H)结直肠癌的体外抗癌作用机制。方法采用MTT和平板克隆形成实验评价HST对HT29细胞增殖的影响;流式细胞术和丹酰尸胺(dansylcadaverine,MDC)染色法检测细胞周期分布和自噬情况;Western blotting、qRT-PCR分析细胞中p53蛋白、mRNA表达水平和p53下游效应子的表达情况;放线菌酮(actidione,CHX)-chase实验检测p53蛋白半衰期;染色质免疫共沉淀(chromatin immunoprecipitation,ChIP)实验检测p53与其下游靶基因p21启动子区的结合情况。结果HST显著抑制突变型p53(R273H)结直肠癌HT29细胞增殖,引起细胞周期阻滞,诱导杀伤性细胞自噬,缩短突变型p53(R273H)蛋白半衰期,诱导其发生MDM2介导的泛素-蛋白酶体降解,增强其与p21启动子的结合,并上调p21的mRNA表达。结论HST能够促进HT29细胞中突变型p53(R273H)降解并恢复p53野生型转录激活功能,其体外抗结直肠癌活性可能与p53通路密切相关。Objective To study in vitro anticancer mechanism of hirsuteine(HST)on mutant p53(R273H)colorectal cancer from a perspective of inhibiting colorectal cancer cell(HT29)proliferation.Methods MTT and plate clone formation assay were used to evaluate the effect of HST on proliferation of HT29 cells;Flow cytometry and dansylcadaverine(MDC)staining were used to detect cell cycle distribution and cell autophagy;The protein and mRNA expression levels of p53 and the expression of p53 downstream effectors were analyzed by Western blotting and qRT-PCR;Actidione(CHX)-chase assay was used to detect the half-life of p53;Chromatin immunoprecipitation(ChIP)was used to detect the binding situation of p53 and its downstream target gene p21 promoter region.Results HST significantly inhibited cell proliferation,arrested cell cycle,and induced autophagy on mutant p53(R273H)colorectal cancer HT29 cells.Meanwhile,HST shortened mutp53(R273H)protein half-life,induced MDM2-mediated ubiquitin-proteasome degradation of mutp53(R273H),increased the DNA binding ability of mutp53(R273H)at the p21 promoter,and up-regulated p21 mRNA expression.Conclusion HST promotes mutp53(R273H)degradation and restoresits wild-type transcriptional activation function in HT29 cells.The in vitro anticancer effect of HST on mutant p53(R273H)colorectal cancer might be closely related with p53 pathway.

关 键 词：结直肠癌 去氢毛钩藤碱 突变型p53(R273H) 蛋白酶体降解 p53野生型转录特性恢复 

分 类 号：R285.5[医药卫生—中药学]