葫芦素I(JSI-124)通过p53信号通路诱导HepG2细胞凋亡

Cucurbitacin I( JSI-124 )-induced apoptosis of HepG2 cells via p53 signaling pathway

目的研究葫芦素I(cucurbitacin I,JSI-124)诱导HepG2人肝癌细胞凋亡的机制。方法用(0.01、0.10、1.00、10.00)μmol/L JSI-124分别处理培养的HepG2细胞24、48、72 h,CCK-8法检测细胞增殖,Hoechst33258染色法观察细胞核形态变化,克隆形成实验观察集落形成能力变化,异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)双标记结合流式细胞术检测细胞凋亡情况,实时定量PCR检测p53及其下游Bax、Fas、鼠双微体基因2(MDM2)的mRNA表达变化,Western blot法检测P53及活化caspase-3的蛋白表达变化。结果JSI-124呈浓度和时间依赖性地抑制HepG2细胞的增殖。Hoechst33258染色法显示JSI-124呈剂量及时间依赖性引起HepG2细胞核染色质的凝集。与对照组相比,流式细胞术检测结果表明1μmol/L JSI-124处理的HepG2细胞凋亡率明显增加。JSI-124显著增强p53及受其调控的下游促凋亡因子Bax和死亡受体Fas mRNA的表达,而与p53结合并抑制p53功能的MDM2的mRNA表达变化不明显。JSI-124处理HepG2细胞48 h后,p53及活化的caspase-3蛋白显著增加。结论 JSI-124通过p53及其下游的促凋亡因子诱导HepG2人肝癌细胞发生凋亡。

Objective To study the mechanism underlying cucurbitacin I（ JSI-124） inducing cell apoptosis in human hepatoma HepG2 cells. Methods HepG2 cells were exposed to 0. 01,0. 10,1. 00 and 10. 00 μmol/L JSI-124 for 24,48 and72 hours. Cell proliferation was detected by CCK-8 assay; the nuclear morphological changes were observed by Hoechst33258 staining; the formation of tumor cell colonies was visualized by violet staining; and cell apoptosis was detected by annexin V-FITC/PI double staining combined with flow cytometry. Furthermore,the mRNA expression levels of p53 and its downstream Bax,Fas and MDM2 genes were measured by quantitative real-time PCR,and the protein levels of P53 and activated caspase-3 were evaluated by Western blotting. Results JSI-124 inhibited the proliferation and induced Hoechst33258-stained chromatin condensation in HepG2 cells in a concentration-and time-dependent manner. Flow cytometry revealed that 1. 00 μmol/L JSI-124 treatment increased the apoptotic rate significantly in HepG2 cells compared with the control cells. Furthermore,JSI-124 significantly enhanced the mRNA expressions of p53 and its downstream apoptotic factors,including Bax and Fas,but did not change the gene expression of the p53 tumor suppressor,MDM2. The 48-hour treatment of JSI-124 in HepG2 cel s significantly increased the levels of p53 and cleaved caspase-3 proteins. Conclusion JSI-124 induces the apoptosis of HepG2 cells through the activation of p53 and its downstream pro-apoptotic factors.