雷公藤红素在前列腺癌细胞中诱导自噬与凋亡关系的研究

发布时间：2018-05-29 21:24

本文选题：前列腺癌 + 雷公藤红素　；参考：《哈尔滨工业大学》2017年硕士论文

【摘要】：前列腺癌的发病率在西方国家位于榜首、其死亡率位于男性因癌症致死的第二位。雄激素剥夺是早期前列腺癌的首选治疗手段,虽然最初反应良好,但其中大部分会发展为去势抵抗型前列腺癌,表现为雄激素非依赖性。大量临床前研究显示雷公藤红素,传统中药雷公藤(Tripterygium wilfordii)的主要成分,对雄激素非依赖性前列腺癌的疗效显著。课题组前期研究结果表明,雷公藤红素在雄激素受体(Androgen receptor,AR)阳性的前列腺癌细胞中通过靶向AR诱导保护性细胞自噬。然而,自噬对细胞凋亡的影响因药物的不同以及相同药物与不同细胞相互作用的不同而发生改变。本课题以DU145与PC-3两种雄激素非依赖的AR阴性前列腺癌细胞系为研究对象,探讨雷公藤红素诱导的细胞自噬与细胞凋亡的关系,为雷公藤红素在雄激素非依赖性前列腺癌治疗的临床前研究提供实验依据。本课题通过Western Blotting实验明确了雷公藤红素在AR阴性前列腺癌细胞中诱导细胞自噬与细胞凋亡。为探究雷公藤红素诱导的细胞自噬与细胞凋亡的关系,分别采用细胞自噬与细胞凋亡抑制剂处理后检测细胞凋亡与细胞自噬的水平变化。抑制细胞自噬后,DU145与PC-3对雷公藤红素的敏感性增强,凋亡水平上调,表明雷公藤红素在AR阴性前列腺癌细胞中诱导保护性细胞自噬。抑制细胞凋亡后,Western Blotting结果显示在DU145与PC-3中,与雷公藤红素单独用药相比,LC3-II/LC3-I、Atg7、Beclin1的表达水平均有不同程度的上调,说明细胞自噬的启动过程被激活;p62蛋白在8-24 h均显著上调,说明抑制细胞凋亡阻滞自噬潮的进行,表明细胞凋亡促进细胞自噬。接下来探究在AR阴性前列腺癌细胞中,雷公藤红素诱导细胞自噬的机制。首先,Western Blotting显示雷公藤红素诱导的自噬与Bcl-2/Beclin1相关通路无关。其次,通过q RT-PCR证实在DU145与PC-3中,雷公藤红素抑制自噬抑制分子mi R-101的表达。HIF-1可结合在mi R-101转录起始位点上游,调控其表达。Western Blotting显示雷公藤红素处理引起HIF-1α蛋白上调。在DU145与PC-3中转染HIF-1α质粒,通过q RT-PCR证实HIF-1α抑制mi R-101的表达。综上,本课题的实验结果表明在AR阴性的前列腺癌细胞中,雷公藤红素促进HIF-1α的表达,造成mi R-101表达抑制,从而诱导细胞自噬,而抑制细胞自噬有利于增强雷公藤红素在前列腺癌细胞中诱导的细胞凋亡。
[Abstract]:Prostate cancer is at the top of the list in the West and is the second leading cause of cancer deaths among men. Androgen deprivation is the preferred treatment for early prostate cancer. Although the initial response is good, most of them develop castrated resistant prostate cancer, showing androgen independent. A large number of preclinical studies showed that tripterygium wilfordii, the main component of tripterygium wilfordii, a traditional Chinese medicine, has a significant effect on androgen independent prostate cancer. The previous study results showed that tripterine induced autophagy by targeting AR in androgen receptor AR-positive prostate cancer cells. However, the effect of autophagy on apoptosis changes with different drugs and different interactions between the same drug and different cells. In this study, two androgen independent AR negative prostate cancer cell lines, DU145 and PC-3, were used to investigate the relationship between autophagy induced by tripterine and apoptosis. To provide experimental evidence for the preclinical study of Tripterygium wilfordii in the treatment of androgen-independent prostate cancer. In this study, Western Blotting assay showed that tripterine induced autophagy and apoptosis in AR negative prostate cancer cells. In order to investigate the relationship between autophagy and apoptosis induced by tripterine, the level of apoptosis and autophagy were detected by the treatment of autophagy and apoptosis inhibitor respectively. After inhibition of autophagy, the sensitivity of DU145 and PC-3 to tripterin was enhanced, and the level of apoptosis was up-regulated, which indicated that tripterin induced autophagy in AR negative prostate cancer cells. After inhibiting apoptosis, the results of Western Blotting showed that in DU145 and PC-3, compared with tripterine alone, the expression level of LL-3-IILC3-Itg7BL-Beclin1 was up-regulated in different degrees, which indicated that the activation of p62 protein in autophagy was significantly up-regulated in 8-24 h. The inhibition of apoptosis blocked the development of autophagy, suggesting that apoptosis promoted autophagy. The mechanism of autophagy induced by tripterine in AR-negative prostate cancer cells was then explored. Firstly, Western Blotting showed that the autophagy induced by tripterine was independent of Bcl-2/Beclin1 related pathway. Secondly, it was confirmed by Q RT-PCR that tripterine inhibited the expression of autophagy inhibitor mi R-101 in DU145 and PC-3. HIF-1 could bind to the upstream of the transcription initiation site of miR-101. Western Blotting showed that tripterine treatment induced the up-regulation of HIF-1 伪 protein. After transfection of HIF-1 伪 plasmid into DU145 and PC-3, Q RT-PCR confirmed that HIF-1 伪 inhibited the expression of miR-101. In conclusion, our results show that in AR negative prostate cancer cells, tripterine promotes the expression of HIF-1 伪 and induces autophagy by inhibiting the expression of miR-101. Inhibition of autophagy can enhance the apoptosis induced by tripterine in prostate cancer cells.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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