结构新颖的中药旋覆花倍半萜二聚体多靶向治疗前列腺癌的分子机制研究

Dimeric sesquiterpenoids, as important anticancer constituents from traditional Chinese medicine (TCM) Inula plants, are found to be effective in inhibiting prostate cancer through targeting multiple oncogenes. However, the underlying molecular mechanisms for their anti-prostate cancer activities have not been systemically investigated. This proposal is based on our preliminary studies to demonstrate novel dimeric sesquiterpenoids have potent and selective effects against prostate cancer cells through a multi-targeting molecular mechanism. A lead compound, called Inulanolide A (XFH-49) selectively inhibits cancer cell growth and metastasis in vitro and in vivo, without significant cytotoxicity against normal cells and host toxicity in mice. We further discovered that XFH-49 could exert its anticancer activity by targeting the NFAT1-MDM2/MDMX network. The primary objectives of this application are to demonstrate the specificity and significance of these oncogenes for XFH-49’s anti-prostate cancer activity, as well as the importance of the entire NFAT1-MDM2/MDMX network in developing new anti-prostate cancer therapeutics. Successful completion of these specific aims will demonstrate the potential of using the novel dimeric sesquiterpenoids for the treatment of prostate cancer and provide a basis for further development of multi-targeted TCM constituents for cancer therapy, including clinical studies.

倍半萜二聚体是中药旋覆花属植物中重要的抗癌活性成分，我们前期发现该类化合物通过靶向多个癌基因有效的抑制前列腺癌。然而，旋覆花倍半萜二聚体抗前列腺癌的分子机制并未被系统性的研究。本项目拟从前期研究结果出发，证实结构新颖的倍半萜二聚体通过多靶向分子机制有效且选择性的抑制前列腺癌。前期研究中发现候选化合物双大花旋覆花内酯甲（XFH-49）在体内体外选择性抑制癌细胞的生长与转移，对正常细胞无显著毒作用，同时在小鼠体内无宿主毒性。进一步研究发现XFH-49可能通过靶向NFAT1-MDM2/MDMX网络发挥抗癌作用。在此基础上，本课题将研究XFH-49靶向各个癌基因的特异性与必要性，及整体NFAT1-MDM2/MDMX网络对发展抗前列腺癌新药的重要性。本项目对阐明倍半萜二聚体治疗前列腺癌的潜力有重要参考价值，也为开发多靶向中药活性成分作为抗癌药物及未来临床研究提供依据。

我们前期发现中药旋覆花主要药效成分倍半萜内酯具有显著的抗前列腺癌活性，然而其作用靶标与分子机制并未被系统性的研究。项目执行期间，课题组首先证实了旋覆花倍半萜内酯成分能有效抑制前列腺癌细胞生存和增殖，诱导其周期阻滞与凋亡，且能降低前列腺癌细胞的侵袭能力。分子机制研究发现旋覆花倍半萜内酯能在前列腺癌细胞中特异性抑制NFAT1、MDM2、MDMX蛋白的表达水平，诱导其泛素化与降解。利用分子对接、表面等离子共振，细胞热位移分析、免疫共沉淀等手段证实旋覆花倍半萜内酯能高亲和力高特异性地与去泛素化酶USP7结合，抑制USP7调控的NFAT1、MDM2、MDMX的去泛素化，增强这些蛋白的泛素化和降解，从而抑制NFAT1-MDM2/MDMX信号通路，并发挥抗前列腺癌作用。通过构建USP7敲减细胞模型进一步证实USP7-NFAT1-MDM2/MDMX通路对旋覆花倍半萜内酯的抗前列腺癌活性至关重要。考虑到旋覆花倍半萜内酯可能通过调控多靶标、多通路发挥作用，课题组通过转录组学分析等进一步研究了旋覆花倍半萜内酯的抗肿瘤机制，发现并证实该中药单体可通过抑制TNF-α诱导的NF-κB信号通路激活而发挥抗肿瘤作用。结合分子对接、表面等离子共振、细胞热位移分析、免疫荧光等手段发现旋覆花倍半萜内酯高亲和力高特异性地靶向泛素结合酶UbcH5c，抑制IκBα的磷酸化，稳定IκBα，降低肿瘤细胞中NF-κB p65总体和磷酸化的表达水平，抑制NF-κB p65核转入与TNF-α诱导的NF-κB信号通路激活，从而发挥抗肿瘤作用。通过构建UbcH5c敲减细胞模型证实UbcH5c-TNF-α-NF-κB通路对旋覆花倍半萜内酯的抗肿瘤活性至关重要。通过小鼠原位肿瘤模型证实旋覆花倍半萜内酯显著抑制肿瘤的生长与转移，且对小鼠无显著宿主毒性。课题组通过以上研究阐明了具有多靶标分子机制的旋覆花倍半萜内酯在治疗遗传背景复杂的恶性肿瘤中的优势，即通过同时抑制多个癌基因而发挥出协同作用，避免了因特异性靶向单个基因而最终导致的肿瘤细胞耐药性的产生。更重要的，本项目也将为后续同类型的中药活性成分研究提供思路与方法。