穿心莲内酯恢复突变型p53野生型功能的作用及机制研究  

Study on The Mechanism of Reactivation of Mutant p53 to Wild-type-like p53 by Andrographolide

作　　者：宋斌 王嘉健 苏永南 王玉玲 杨帆 张继虹 SONG Bin;WANG Jia-Jian;SU Yong-Nan;WANG Yu-Ling;YANG Fan;ZHANG Ji-Hong(Medical School,Kunming University of Science and Technology,Kunming 650500,China)

机构地区：[1]昆明理工大学医学院,昆明650500

出　　处：《生物化学与生物物理进展》2022年第9期1763-1773,共11页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(81560601,81960670);云南省重点项目(202001AS070012)资助。

摘　　要：目的p53是人体内重要的肿瘤抑制因子,但在人类肿瘤中因高频突变而失去抑癌功能。突变型p53(mutant p53,mutp53)可促进肿瘤的发生、发展和转移。由于在肿瘤细胞中通常有较高表达,mutp53已成为区别于正常细胞的一个特异性抗肿瘤靶点。本研究旨在探索穿心莲内酯的抗肿瘤作用机制,为寻找靶向mutp53的抗肿瘤化合物提供理论依据。方法构建可以快速筛选具有恢复mutp53下游转录因子的荧光素酶系统,观察穿心莲内酯对H1299-p53 R273H-PUMAluciferase和H1299-p53R175H-PUMA-luciferase细胞中PUMA基因的表达情况;采用免疫荧光实验,检测穿心莲内酯对HT29(R273H)和SK-BR-3(R175H)细胞中mutp53的表达影响;采用免疫印迹实验进一步观察穿心莲内酯恢复了mutp53肿瘤细胞中p53下游靶蛋白PUMA、p21、Noxa的表达;随后采用MTT和流式细胞分析,检测穿心莲内酯对肿瘤细胞增殖和凋亡的影响;此外,还通过siRNA敲低Hsp70表达后,研究穿心莲内酯对mutp53下游基因的重激活作用。结果穿心莲内酯可以增加H1299-p53 R273H-PUMA-luciferase和H1299-p53R175H-PUMA-luciferase细胞中PUMA基因的表达;穿心莲内酯可以降低HT29(R273H)和SK-BR-3(R175H)细胞中mutp53的比例,同时增加野生型p53的比例;穿心莲内酯恢复了mutp53肿瘤细胞中p53下游靶蛋白PUMA、p21、Noxa的表达,进而抑制肿瘤细胞增殖和诱导凋亡;穿心莲内酯可以增加分子伴侣Hsp70的表达,通过siRNA敲低Hsp70后,穿心莲内酯对mutp53下游基因的重激活作用明显受到抑制。结论穿心莲内酯可能通过影响Hsp70的表达从而激活突变p53下游靶基因而发挥抗肿瘤作用。Objective TP53 is an important tumor suppressor gene,however,mutations of p53 occur in over50%of all cancers and are indicative of highly aggressive cancers that are hard to treat.Targeting mutant p53(mutp53)is one of the effective strategies in cancer therapy.Methods We constructed a high throughput screen system which contains the p53 targeted puma,H1299-p53 R273 H-PUMA-luciferase,and H1299-p53 R175 HPUMA-luciferase to screen compounds targeting mutp53.Using immunofluorescence assay to detect the effect of andrographolide on the expression of mutp53 in HT29(R273 H)and SK-BR-3(R175 H)cells.Western blotting experiment and qRT-PCR analysis were used to further observe the protein and mRNA expressions of andrographolide on p53 downstream target genes PUMA,p21,and Noxa in mutp53 tumor cells.Then MTT and flow cytometry were used to detect andrographis paniculata The effect of lactone on tumor cell proliferation and apoptosis.In addition,after knocking down Hsp70 expression by siRNA,the reactivation effect of andrographolide on mutp53 downstream genes was also studied.Results Andrographolide enhanced PUMAluciferase expressions in both cell lines.Andrographolide could reduce the expressions of mutp53 in HT29(R273 H)and SK-BR-3(R175 H)cells,while the expressions of wild-type p53 increased by immunofluorescence assay.Andrographolide can inhibit the cancer cell proliferation and induce apotposis in mutant p53 cancer cells.Andrographolide can enhance the p53 downstream target protein and mRNA expressions of PUMA,p21,and Noxa.Andrographolide increased the expression of molecular chaperone Hsp70 in HT29 and SK-BR-3 cells,as the chaperones play important role in p53 structure function,we knocked down Hsp70 by si RNA and found that the upregulation of p53 targeted genes was reversed.Conclusion Andrographolide restores the wild-type-like p53 function dependent on Hsp70.

关 键 词：突变型P53 穿心莲内酯 重激活 细胞凋亡 分子伴侣 

分 类 号：Q28[生物学—细胞生物学] Q291