醉茄素A稳定IDH1及线粒体功能抑制皮肤肿瘤早期发生发展的机制研究

The focus of the chemoprevention has shifted toward more and more natural compounds. Although the anti-tumor activity of Withaferin A (WA, a natural compound from the traditional Ihdian medicine Ashwagandha) has been investigated in different cancer models, meanwhile, we have performed the preliminary study of WA in chemoprevention, its molecular mechanismin is unclear. The initiation and development of tumor are correlated to the switch of energy metabolism. Distingished from normal cells, tumor cells generate energy via glycolysis even under aerobic conditions. Thus, inhibition of aerobic glycolysis is an effective strategy for tumor prevention. The novel finding in our lab indicated that WA inhibited the down-regulation of expression level and activity of IDH1, and impaired both function of mitochondria in energy metabolism and carcinogenesis in the mouse JB6P+ cells treated with tumor promoter TPA. Evidences showed that Nrf2 was degraded by ubiquitioin-prosomease pathway, and Withaferin A could inhibit this pathway. Hence, we propose that Withaferin A prevents skin carcinogenesis by inhibiting ubiquition-prosomease pathway, which leads to stability of Nrf2 and up-regulation of IDH1 activity, and blocking the HIF-1α mediated pathway; and also maintains the mitochondrial function to play the essential role in energy metabolism via respiration chain, and exert its chemopreventive activity. We will utilize chemical inhibitor, immunoprecipitation, siRNA knock-down and EMSA to explore the molecular mechanism, and test our hypothesis in the two-step mouse skin tumor model in vivo. This study will not only reveal the role and machanism of Withaferin A, but also dicover the novel target molecule-IDH1 for chemoprevention of skin cancer.

醉茄素A的抗肿瘤活性已被证实，而其肿瘤预防的作用鲜有报道。我们的研究首次报道了醉茄素A的肿瘤预防，但具体的分子机制尚不清楚。肿瘤发生发展早期已涉及能量代谢方式的转换，即在氧充足的情况下，肿瘤细胞依然倾向于糖酵解方式获取能量。因此，抑制有氧糖酵解将成为肿瘤预防的有效策略。本课题前期结果揭示了醉茄素A抑制皮肤肿瘤诱变剂造成的线粒体呼吸受损及肿瘤的发生发展；抑制在异柠檬酸脱氢酶1的表达和活力的降低。有研究证实肿瘤抑制因子Nrf2受泛素-蛋白酶体途径降解，而醉茄素A抑制该途径。据此，我们推测醉茄素A是通过抑制泛素-蛋白酶体途径，稳定Nrf2，保持IDH1的活力，进而阻断HIF-1α通路，维持线粒体的正常呼吸功能来发挥肿瘤预防作用。本课题组拟在体内利用两步法小鼠皮肤肿瘤模型在整体水平上，及体外细胞水平上深入探讨其机制，以期为肿瘤化学预防提供新的作用机制及药物靶标。

作为β脱羧脱氢酶家族成员的代谢酶异柠檬酸脱氢酶1在细胞质中将异柠檬酸转化为α酮戊二酸，已经被证实可能作为一个肿瘤抑制因子。醉茄素A是从南非醉茄中分离出的一种醉茄内酯类化合物，能够抑制多种肿瘤细胞的生长与增殖。基于前期的研究结果，我们已证实醉茄素A 抑制肿瘤促进剂TPA诱导的IDH1活性减弱以及线粒体功能异常，在本项目中我们进一步研究醉茄素A是通过什么样的具体机制调节IDH1从而抑制肿瘤发生发展。IDH1作为Nrf2的靶基因，启动子区域同样具有典型的抗氧化反应元件ARE，可被直接激活。我们利用实时荧光定量PCR分别检测了在TPA和WA处理后IDH1与Nrf2的mRNA水平的变化，发现WA可显著性阻滞TPA引起的Nrf2 mRNA水平的抑制，而IDH1的mRNA水平没有显著性改变。因此我们推断WA对于IDH1活性的稳定不是发生在转录水平。通过蛋白合成抑制剂CHX预处理细胞，发现IDH1的蛋白水平在TPA和/或WA处理后没有显著性差异，我们进一步确认WA对于IDH1的作用可能发生在翻译后修饰阶段。通过检测蛋白酶体活性发现，WA抑制TPA诱导的蛋白酶体活性，运用蛋白酶体抑制剂MG132预处理细胞的细胞，发现WA通过抑制泛素蛋白酶体途径阻断TPA诱导的IDH1的下调与降解。这一结果证实WA抑制TPA诱导的IDH1泛素化降解，这与WA可作为蛋白酶体抑制剂的作用相一致。接下来利用IDH1在细胞裂解液中进行免疫沉淀，电泳后对结果进行质谱分析从而获得IDH1被泛素化的具体位点进行分析。脯氨酸羟化酶PHD作为α-酮戊二酸的下游分子，可抑制HIF-1α诱导的信号通路，后者在肿瘤发生发展中发挥重要作用。通过PHD活性的检测发现WA促进其活性，同样利用IDH1过表达也有相同的结果， 从而将WA与HIF-1α之间联系起来。我们还发现WA阻断TPA诱导的HIF-1α 的靶基因Glut1和VEGF的活化。我们的研究找到了WA发挥化学预防作用的一个机制，如何抑制肿瘤的发生发展，是通过抑制泛素蛋白酶体途径稳定IDH1，从而阻断HIF-1α途径。这些结果进一步确认肿瘤抑制基因IDH1可能作为皮肤肿瘤化学预防的靶分子，而WA作用机制的研究也为肿瘤化学预防药物的发现提供了新的思路。