重楼皂苷Ⅰ对去势抵抗性前列腺癌DU145细胞增殖及凋亡的影响  

Effect of PolyphyllinⅠon growth and apoptosis of DU145 cells and its mechanism

作　　者：邹佩良 邹嘉茹 蔡家丽 何启雄 周建甫[2] 向松涛[2] ZOU Peiliang;ZOU Jiaru;CAI Jiali;HE Qixiong;ZHOU Jianfu;XIANG Songtao(School of Clinical Medicine,Zhaoqing Medical College,Zhaoqing 526020,China)

机构地区：[1]肇庆医学高等专科学校临床医学院,526020 [2]广东省中医院泌尿外科

出　　处：《现代泌尿生殖肿瘤杂志》2023年第2期98-103,共6页Journal of Contemporary Urologic and Reproductive Oncology

基　　金：广东大学生科技创新培育专项资金资助项目(pdjh2021b0992);肇庆市科技计划项目(202004030302);肇庆市科技计划项目(2022040303005);肇庆医学高等专科学校学生科技活动基金项目(2021XS09)。

摘　　要：目的 探讨重楼皂苷Ⅰ(PPⅠ)对去势抵抗性前列腺癌(CRPC)细胞株DU145细胞增殖的影响及其诱导细胞凋亡的分子机制。方法采用MTT法检测PPⅠ对CRPC细胞株DU145细胞增殖能力的影响,使用流式细胞仪检测DU145细胞的凋亡率,同时应用Western blot检测PPⅠ对DU145细胞p-ERK1/2、ERK1/2、特异性蛋白1(SP1)及Zeste基因增强子同源物2(EZH2)蛋白表达的影响;通过加入ERK1/2抑制剂(PD98059)检测PPⅠ对SP1表达的影响,通过转染SP1、EZH2过表达质粒分别检测PPⅠ对SP1、EZH2表达的影响;采用双荧光素酶报告基因检测EZH2启动子活性,并探讨ERK1/2、SP1和EZH2之间的关系。设未加药组为空白组。结果MTT法检测结果显示,PPⅠ能抑制DU145细胞体外生长,与空白组相比,细胞存活率从给药浓度0.4μmol·L^(-1)开始明显下降,且呈时间和剂量依赖性,差异有统计学意义(P<0.01)。流式细胞仪检测结果显示,PPⅠ能诱导DU145细胞早期凋亡,与空白组相比,细胞早期凋亡率从给药浓度0.4μmol·L^(-1)开始明显增加,差异有统计学意义(P<0.05)。Western blot结果显示,PPⅠ以时间依赖性方式促进p-ERK1/2和ERK1/2蛋白的激活和表达(P<0.05),以剂量依赖性方式抑制SP1、EZH2蛋白的表达(P<0.05);抑制ERK1/2磷酸化能逆转PPⅠ对SP1表达的下调作用,SP1过表达可逆转PPⅠ对EZH2蛋白表达的下调作用,然而EZH2过表达不能逆转PPⅠ对SP1表达的下调作用,与PPⅠ组相比,ERK1/2抑制剂+PPⅠ组SP1表达上调(P<0.05),PPⅠ+SP1过表达质粒组EZH2蛋白表达上调(P<0.05),而PPⅠ+EZH2过表达质粒组SP1表达差异无统计学意义(P>0.05)。双荧光素酶报告基因检测结果显示,SP1过表达可逆转PPⅠ对EZH2启动子活性的下调,与PPⅠ组相比,PPⅠ+SP1过表达质粒组EZH2启动子活性上调(P<0.05)。结论PPⅠ可通过介导ERK1/2通路抑制SP1和EZH2蛋白表达,从而诱导CRPC DU145细胞早期凋亡,进而抑制细胞生长。Objective To explore the effect of polyphyllinⅠ(PPⅠ)on growth and apoptosis of castration-resistant prostate cancer(CRPC)DU145 cells and the its molecular mechanism.Methods Cell viability was measured using MTT assay to detect the human prostate cancer cells(DU145 cells)treated with PPⅠ.The cell apoptosis was detected by flow cytometry analysis.Effects of PPⅠon protein expression levels of p-ERK1/2,ERK1/2,specificity protein 1(SP1)and enhancer of zeste homolog 2(EZH2)were detected by Western blot assay.Then we explored the relationship among ERK1/2,SP1 and EZH2 in the cells additionally treated with ERK1/2 inhibitor(PD98059),transfected with pcMV6-SP1 and pcMV6-EZH2 by Western blot and measured the EZH2 promoter activity by Secrete-Pair Dual Luminescence Assay Kit.Results Compared with the control group,DU145 cells treated with PPⅠshowed a dose and time-dependent reduction of survival rate at 0.4μmol·L^(-1)(P<0.01),dose-dependent early apoptosis at 0.4μmol·L^(-1)(P<0.05),increase of p-ERK1/2,ERK1/2 protein expression from 2 h,reduction of SP1,EZH2 protein expression from 1.2μmol·L^(-1)(P<0.05).Compared with PPⅠgroup,inhibition of ERK1/2 phosphorylation reversed the PPⅠ-decreased protein expression of SP1(P<0.05),exogenously expressed SP1 resisted the PPⅠ-inhibited expression and promoter activity of EZH2(P<0.05),but overexpression of EZH2 exert no effect on SP1 protein expression(P>0.05).Conclusions PPⅠinduces early apoptosis and suppresses the growth of CRPC DU145 cells by activating the ERK1/2 pathway and inhibiting the transcription factor SP1 protein expression,thus reducing the expression of EZH2.

关 键 词：重楼皂苷Ⅰ DU145 生长 凋亡 ERK1/2 

分 类 号：R285[医药卫生—中药学]