双氢青蒿素诱导前列腺癌细胞焦亡及调控凋亡相关斑点样蛋白甲基化

Dihydroartemisinin induces pyroptosis and regulates apoptosis-associated speck-like protein methylation in prostate cancer cells

目的探讨双氢青蒿素(dihydroartemisinin,DHA)诱导前列腺癌(prostate cancer,PCa)细胞焦亡及其可能的机制。方法将PC-3与DU145细胞分为对照组与药物组(50μmol/L DHA)。采用CCK-8检测DHA作用前列腺癌PC-3以及DU145细胞的增殖情况;电镜观察细胞焦亡现象;Caspase-1比色检测和乳酸脱氢酶(lactate dehydrogenase,LDH)释放检测验证细胞膜完整性;ELISA法检测IL-18和IL-1β表达水平;Annexin V-PI双染流式细胞术测定细胞焦亡量。生物信息学分析寻找正常前列腺细胞与PCa细胞的表达差异基因。选用地西他滨(decitabine,DAC,2μmol/L)作为阳性对照,亚硫酸氢盐测序法检测凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)的甲基化状态。RT-qPCR及蛋白免疫印迹法检测焦亡相关蛋白的表达情况。构建PC-3裸鼠种植瘤模型,分为对照组与药物组(50μmol/L DHA),对比肿瘤生长状况。蛋白免疫印迹法检测组织蛋白以及透射电镜验证体外实验的实验结果。结果CCK-8结果显示DHA明显抑制PC-3以及DU145细胞增殖(P<0.01)。透射电镜和扫描电镜均观察到药物组细胞焦亡形态学改变。与对照组比较,药物组细胞外Caspase-1、LDH明显升高(P<0.01),显示细胞膜完整性缺失;炎症因子IL-18和IL-1β水平升高(P<0.01),提示产生焦亡。Annexin V-PI双染流式细胞术显示药物组PI~+细胞定量较对照组增多(P<0.01)。生物信息学分析显示ASC在PCa中表达明显受限;DHA对抑癌基因ASC的去甲基化效果明显优于DAC(P<0.01)。实时荧光定量PCR以及蛋白免疫印迹法检测结果表明DHA明显上调焦亡通路中黑色素瘤缺乏因子2(AIM2)、ASC、Caspase-1、Gasdermin D(GSDMD)和GSDMD-N端的表达水平。PC-3裸鼠种植瘤药物组肿瘤体积明显小于对照组(P<0.01),目的蛋白的表达上调情况及透射电镜观察肿瘤组织发生细胞焦亡形态学改变均与体外实验一致。结论DHA能抑制PCa细胞增殖,恢复焦亡相关基因表达以及恢复抑癌基因ASC的甲基化状态,诱导细胞焦亡。

Objective To investigate the effect of dihydroartemisinin(DHA)on pyroptosis in prostate cancer(PCa)cells and the underlying possible mechanism.Methods PCa cell lines PC-3 and DU145 were divided into control group and DHA treatment group(50μmol/L DHA).Cell counting kit-8(CCK-8)assay was used to detect the cell proliferation in the PC-3 and DU145 cells with and without DHA treatment.Pyroptosis was observed by electron microscopy.The integrity of cell membrane was detected by Caspase-1 activity and lactate dehydrogenase(LDH)release.The contents of interleukin(IL)-18 and IL-1βwere detected by ELISA.Annexin V-PI double-staining flow cytometry was used to measure pyroptosis.Bioinformatics analysis were performed to seek out the differentially expressed genes between normal prostate cells and PCa cells.Decitabine(DAC,2μmol/L)was used as a compared agent,the methylation level of apoptosis-associated speck-like protein containing CARD(ASC)was detected by bisulfite sequencing PCR.The mRNA and protein levels of pyroptosis-related molecules were detected by quantitative real-time PCR and Western blotting respectively.After xenograft nude mice were established with PC-3 cells,the tumor growth were observed in the mice with and without treatment of 50μmol/L DHA.Western blotting and transmission electron microscopy were performed to verify the results.Results CCK-8 assay showed that DHA significantly inhibited the proliferative activity of PC-3 and DU145 cells(P<0.01).Both transmission and scanning electron microscope indicated the morphology of pyroptosis.The extracellular contents of Caspase-1 and LDH were significantly higher in the treatment group than the control group(P<0.01),indicating loss of cell membrane integrity.Elevated levels of IL-18 and IL-1βsuggested pyroptosis.Annexin V-PI double-staining flow cytometry showed there were larger number of PIcells in the treatment group than the control group(P<0.01).Bioinformatics analysis indicated that ASC was the differentially expressed gene.DHA showed better demethylation effect on tumor suppressor gene ASC than DAC(P<0.01).Quantitative real-time PCR and Western blotting indicated that DHA up-regulated the mRNA and protein levels of the genes related to pyroptosis,including absent in melanoma 2(AIM2),ASC,Caspase-1,Gasdermin D(GSDMD)and GSDMD-N.Xenograft tumors in the treatment groups were obviously smaller than those in the control groups(P<0.01).The up-regulation of target proteins and the morphology of pyroptosis under transmission electron microscope in vivo were consistency with the results in vitro.Conclusion DHA inhibits the proliferation of PCa cells.Up-regulation of pyroptosis related genes and restoring the methylation level of tumor suppressor gene ASC can induce pyroptosis.