双氢青蒿素对前列腺癌PC-3细胞中UCHL1基因表达的调控机制研究

Medulative effect of dihydroartemisinin on UCHL 1 gene expression in human prostate cancer PC-3 cells and its mechanism

目的 :研究双氢青蒿素(dihydroartemisinin,DHA)对人前列腺癌PC-3细胞株中抑癌基因泛素羧基末端水解酶L1(ubiquitin carboxyl-terminal hydrolase L1,UCHL1)表达的影响,并探讨其可能的调节机制。方法 :PC-3细胞株经不同浓度(25、50和100μmol/L)的DHA处理48 h,并设空白对照组。采用FCM法检测各组细胞凋亡率和细胞周期的变化。采用免疫荧光染色法检测UCHL1和DNA甲基化转移酶1(DNA methyltransferase 1,DNMT1)在细胞内的蛋白定位及表达量。蛋白质印迹法检测UCHL1、DNMT1、磷酸化Akt(phospho-Akt,p-Akt)和Akt蛋白的表达;并且以1μmol/L磷脂酰肌醇3激酶(phosphoinositide-3-kinase,PI3K)家族抑制剂Wortmanin作为阳性对照,比较分析DHA处理组p-Akt蛋白的表达量。结果 :DHA能明显诱导PC-3细胞凋亡,并使细胞停滞在G2/M期,与空白对照组比较差异有统计学意义(P<0.05)。DHA处理组中DNMT1蛋白由细胞核转移到细胞质,与空白对照组比较,DNMT1蛋白在细胞核与细胞质中总的表达水平明显下调(P<0.05);而UCHL1蛋白表达在细胞质内明显上调(P<0.05)。与空白对照组比较,DHA处理组与阳性对照组的UCHL1蛋白表达均上调(P<0.05),DNMT1表达水平均下调(P<0.05),p-Akt蛋白表达水平下调(P<0.05),而Akt蛋白表达量无明显变化(P>0.05);其中,高浓度DHA处理组的p-Akt蛋白表达下调更为显著,更接近于阳性对照组。结论 :DHA能够抑制DNMT1的表达,恢复抑癌基因UCHL1的表达,从而诱导前列腺癌PC-3细胞凋亡,阻滞细胞周期进程;推测其作用机制可能与抑制PI3K/Akt信号通路的活性有关。

Objective: To investigate the effect of dihydroartemisinin(DHA) on expression of tumor suppressor gene ubiquitin carboxyl-terminal hydrolase L1(UCHL 1) in human prostate cancer cell line PC-3, and explore its regulative mechanism. Methods: PC-3 cells were treated with different concentrations(25, 50, and 100 μmol/L) of DHA for 48 h, while PC-3 cells without DHA treatment were used as the control. Then the apoptosis and cell cycle distribution were detected by flow cytometry. The expressions and cellular locations of DNA methyltransferase 1(DNMT1) and UCHL1 proteins were detected by immunofluorescence staining. The expression levels of UCHL1, DNMT1, phospho-Akt(p-Akt) and Akt proteins were detected by Western blotting. The 1 μmol/L phosphoinositide-3-kinase(PI3K) inhibitor Wortmanin was used as a positive control to analyze the expression of p-Akt protein in DHAtreated group. Results: DHA significantly induced the apoptosis of PC-3 cells and arrested the cell cycle distribution at phase G2/M as compared with those of the control group(both P < 0.05). After DHA treatment, DNMT1 proteins translocated from nuclei to cytoplasms, and the overall expression level in nuclei and cytoplasms was significantly decreased as compared with that of the control group(P < 0.05).However, the expression of UCHL1 protein was significantly increased in the cytoplasm of PC-3 cells treated with DHA(P < 0.05). As compared with the control group, the expressions of UCHL1 protein were up-regulated while the expressions of DNMT1 protein were down-regulated(both P < 0.05), and the expressions of p-Akt protein were reduced(P < 0.05), but the expressions of Akt protein were not significantly changed(P > 0.05) in the DHA-treated group and the positive control group. The downregulation of p-Akt expression was more obvious in high-concentration of DHA-treated group, much closer to that in the positive control group. Conclusion: DHA can inhibit the expression of DNMT1, restore the function of UCHL 1 gene, induce the apoptosis of PC-3 cells, and block the cell cycle progression. These mechanisms may be related to the suppressive activity of PI3K/Akt pathway.