天然化合物维康醇通过活性氧依赖途径诱导前列腺癌细胞凋亡的研究

发布时间：2018-03-12 09:33

本文选题：维康醇　切入点：前列腺癌　出处：《中国人民解放军医学院》2014年博士论文　论文类型：学位论文

【摘要】：目的：对于去势抵抗性前列腺癌目前尚无有效的治疗手段,亟待开发一种新型的化疗药物。本研究主要通过体外实验来评估新型天然化合物维康醇对于恶性前列腺细胞的抗肿瘤作用,并进一步探讨其药物作用机制。材料与方法：通过蛋白免疫印迹及流式细胞计数等实验方法评估维康醇在不同前列腺癌细胞系及正常前列腺上皮诱导凋亡的作用,然后通过反应活性氧(Reactive oxygen species,ROS)荧光染料检测三株具有代表性的细胞系PC3,DU145,BPH1添加不同种类ROS清除剂后细胞内ROS水平的变化,探讨其作用途径。最后为了证明维康醇诱导的细胞凋亡是Bax蛋白依赖性的,我们通过对PC3细胞系进行RNA干扰及外源性导入Bax蛋白天然缺失的细胞系DU145,评估维康醇处理细胞后的凋亡指标。最后通过透射电镜及应用不同种类Caspase蛋白酶抑制剂探讨了Bax蛋白可能的下游途径。结果：通过对凋亡标志物的检测,如Caspase-3及PARP确认了维康醇诱导的细胞死亡方式为凋亡。ROS荧光染色结果发现维康醇诱导了前列腺癌细胞内广泛的氧化应激并活化凋亡途径最终导致细胞死亡。然而,维康醇诱导的这种细胞凋亡可被ROS清除剂N-乙酰-L-半胱氨酸(NAC)及二氢硫辛酸(DHLA)完全阻断。我们同时证明了Bax蛋白在维康醇诱导的细胞凋亡中激活并发挥着重要的作用,维康醇诱导的细胞凋亡均伴有Bax蛋白的激活。对于Bax缺失的前列腺癌细胞系DU145在外源性导入Bax质粒后,其对维康醇的抗药性便会消失,再次证明维康醇引起的凋亡是Bax依赖途径。激活的Bax蛋白最终导致线粒体损伤,最后通过Caspase依赖及非依赖途径诱导前列腺癌细胞的凋亡。这些结果表明为维康醇将来可能作为一种新型治疗晚期前列腺癌化疗药物奠定了理论基础。结论：维康醇可诱导前列腺癌细胞内广泛的氧化应激反应最终导致Bax依赖性的细胞凋亡。
[Abstract]:Objective:. There is no effective treatment for ovariectomized prostate cancer. It is urgent to develop a new chemotherapeutic drug. The aim of this study was to evaluate the antitumor effect of a new natural compound, veconol, on malignant prostate cells in vitro, and to further explore the mechanism of its action. Materials and methods:. The effects of veconol on apoptosis in different prostate cancer cell lines and normal prostate epithelium were evaluated by Western blotting and flow cytometry. Then the changes of ROS levels in three representative cell lines PC3DU145BPH1 were detected by reactive oxygen speciesrosis (Ros) after adding different kinds of ROS scavengers. Finally, in order to prove that the apoptosis induced by veconol is Bax protein-dependent, We evaluated the apoptosis index of PC3 cell line by RNA interference and exogenous introduction of Bax protein deletion cell line DU145. finally, we applied different kinds of Caspase protease inhibitor by transmission electron microscope and different kinds of Caspase protease inhibitor. The possible downstream pathway of Bax protein was discussed. Results:. By detecting the markers of apoptosis, For example, Caspase-3 and PARP confirmed that Vicol induced cell death was apoptosis. Ros fluorescence staining results showed that Vicol induced extensive oxidative stress and activated apoptosis pathway in prostate cancer cells, and eventually resulted in cell death. The apoptosis induced by veconol can be completely blocked by ROS scavenger N-acetyl-L-cysteine) and dihydrolipoxylic acid (DHla). We have also demonstrated that Bax protein is activated and plays an important role in the apoptosis induced by veconol. The apoptosis induced by veconol was accompanied by the activation of Bax protein. For prostate cancer cell line DU145 with Bax deletion, its resistance to veconol disappeared after exogenous transfection into Bax plasmid. It is proved that the apoptosis induced by veconol is Bax dependent pathway. The activated Bax protein eventually leads to mitochondrial damage. Finally, apoptosis of prostate cancer cells was induced by Caspase dependent and non-dependent pathway. These results suggest that Viconol may be a new chemotherapeutic agent for advanced prostate cancer in the future. Conclusion:. Vicol can induce extensive oxidative stress in prostate cancer cells and eventually lead to Bax-dependent apoptosis.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25

【参考文献】