锌调控P53重建前列腺癌代谢模式提高其对放化疗敏感性的机制研究

It is reported that expression of inactivated or mutant P53 is common in human cancers and also associated with increased cancer resistance to therapy. Zinc is an essential trace element of human body and play important role in genetic stability and expression. The levels of Zn in prostate are decreased in the developing and progressive prostate cancers. The metabolic profiles from synthetic metabolism of citric acid to catabolism in patients with prostate cancer, because of low levels of systemic Zn. Our previous works also showed that zinc could inhibit metastasis and recurrence of prostate cancer, so we raised the hypothesis that zinc could reconstruct metabolic profiles through regulation of mdm2 to reduce p53 degradation reactivating p53 and induce apoptosis. The wild type p53 plasmid and co-expression of MDM2-specific siRNA and wild-type p53 plasmid (Pmp53) was tansfected to the null p53 cell line (PC-3) and mutant p53 cell line(DU145) to recover the function of wild type p53. The activity of p53 was determined by transcriptional activity and expression of downstream target gene of p53. The activity of aconitase and expression of metabolic enzyme such as TIGAR、GLUT and GLS2 was measured. The effect of Zn on MDM2 acetylization and the interaction of acetylization and ubiquitination was also measured. All above results will elucidate the mechanism of prostate cancer metabolic profiles and provide a new idea for using P53 as a target gene in the prostate cancer therapy.

P53失活或突变是肿瘤细胞对放化疗耐受的重要原因之一。锌作为人体必需的微量元素，在基因稳定性与基因表达等方面起着重要的作用。前列腺癌组织中锌浓度较正常前列腺组织下降60%-70%，其代谢模式由柠檬酸合成代谢转为分解代谢。我们在前期工作中发现锌促进凋亡抑制前列腺癌细胞转移。因此，我们提出假设：锌通过调节P53泛素化降解E3连接酶MDM2，减少对P53降解，重建代谢模式，诱导细胞凋亡。我们选取P53缺失型与变异型前列腺癌细胞，转染野生型P53与抑制MDM2的共表达质粒Pmp53，重建其野生型P53功能。观察上述细胞对放化疗敏感性的基础上，从P53转录活性与P53靶基因的表达等评价P53的活性，通过检测顺乌头酸酶、TIGAR、GLUT及GLS2等代谢酶类的变化；通过检测锌对MDM2乙酰化的影响，结合乙酰化与泛素化之间的交互调控，阐明代谢模式改变的机制，为以P53为靶点的前列腺癌治疗提供新的思路。

前列腺癌是危害中老年男性健康的常见恶性肿瘤。由于前列腺癌好发于60岁以上的老年人，转移率高而且发现就已是晚期，所以化疗治疗显得尤为重要。本课题组前期对前列腺癌标本进行检测中发现大约53%的前列腺癌患者中存在p53的突变和失活，严重影响治疗和预后，更为重要的p53失活或突变是肿瘤细胞对化疗耐受的重要原因之一。引起 p53 突变和失活的原因很多，比如锌的缺失和MDM2 对其的降解。其中锌的缺失与前列腺癌的发生和发展密切相关。锌作为人体必需的微量元素，在基因稳定性与基因表达等方面起着重要的作用。前列腺癌组织中锌浓度较正常前列腺组织下降60%-80%，其代谢模式由柠檬酸合成代谢转为分解代谢。我们在工作中发现锌可以增强前列腺癌化疗敏感性，其机制与诱导细胞发生线粒体凋亡途径有关，进一步我们同时恢复野生型p53的表达干涉MDM2的表达并补充锌，发现可以增强前列腺癌的化疗敏感性，其机制为影响了ACO2的活性，抑制TCA循环，以及改变了VDAC1和HK2的共定位，降低了线粒体膜通透性，线粒体的动态平衡和结构受损，最终启动凋亡。