葫芦素E靶向花生四烯酸P450酶代谢通路防治乳腺癌以及对P450酶作用机制的研究

Arachidonic acid (AA) can significantly increase the incidence of breast cancer. Among the AA metabolites, the prostaglandins and leukotrienes are the best known. They are generated by two pathways controlled by the enzyme systems cyclooxygenase and lipoxygenase, respectively. However, AA is also substrate for a third enzymatic pathway, the cytochrome P450 (CYP) system. This third eicosanoid pathway consists of two main branches: ω-hydroxylases convert arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) and epoxygenases convert it to epoxyeicosatrienoic acids (EETs). These lipid autacoids may be involved in cancer in a few different ways: either in cell-autonomous tumor survival and growth or in modulating stromal processes, such as angiogenesis and inflammation that can support tumor progression. The EETs and HETEs pathways should be evaluated as potential targets in cancer therapy, directed both against tumor cells and their surrounding stroma. Cucurbitacin E (CuE) has significant anti-tumor activity, and is a potential candidate as new anti-tumor herbal medicines. Previous our studies found CuE inhibits tumor angiogenesis through VEGFR2 mediated Jak2/STAT3 signaling pathway and inhibits breast tumor metastasis by suppressing cell migration and invasion. Further studies in this project will investigate the effects of CuE on the pathway of CYP-derived AA in the breast cancer and the CYP isoforms activities. Moreover, the project will reveal the relationship of AA, CYP, HETEs and EETs in breast cancer, and provide novel mechanisms for the pathogenesis of breast cancer and evidence for development new strategy for breast cancer treatment. Furthermore, CYP-associated CuE metabolic studies not only are one of the major attritions in drug development, but also are considered cost-effective for predicting the potential drug-drug interaction in the clinic.

花生四烯酸（AA）显著提高乳腺癌的发生率。除了环氧化酶和脂氧合酶调控的两条代谢通路外，AA还有第三条代谢通路经细胞色素P450（CYP）酶生成羟基花生四烯酸（HETEs）和环氧花生四烯酸（EETs）。靶向CYP通路既能杀伤肿瘤细胞，同时也抑制肿瘤周围的间质微环境，因此在乳腺癌的防治中具有重要的潜在应用前景。我们先前的研究发现葫芦素E(CuE)是潜在的治疗乳腺癌及其转移的新药物。然而，CuE是否靶向AA的CYP代谢通路以及对CYP酶的作用机理未见报道。本项目拟通过细胞和动物模型，结合临床样本，系统研究CuE对AA的CYP代谢通路以及CYP酶的作用机制。本项目的实施不仅确定CuE通过AA代谢通路防治乳腺癌及对CYP酶的作用机理；还揭示AA、CYP、EETs和HETEs与乳腺癌之间的联系，为乳腺癌的发病机制提供新的内容，并对其治疗提供新的策略。

花生四烯酸（AA）显著提高乳腺癌的发生率。除了环氧化酶和脂氧合酶调控的两条代谢通路外，AA 还有第三条代谢通路经细胞色素P450（CYP）酶生成羟基花生四烯酸（HETEs）和环氧花生四烯酸（EETs）。靶向CYP通路既能杀伤肿瘤细胞，同时也抑制肿瘤周围的间质微环境，因此在乳腺癌的防治中具有重要的潜在应用前景。本项目研究了葫芦素E对AA的代谢通路尤其CYP通路的作用机理，以期建立AA、CYP、EETs和HETEs与乳腺癌之间的联系，为乳腺癌的发病机制提供新的内容，并对其治疗提供新的策略。本项目首先系统研究了葫芦素E对CYP酶的作用机制：通过体内体外实验，检测CYP酶的表达及活性；确定了葫芦素E与CYP酶的具体亚型的表达以及活性的关系，对其进一步开发成为新药物提供了帮助，并且为临床合理用药例如是否可以和其他主流治疗乳腺癌药物合用提供指导。本项目还建立了LC-MS/MS 分析AA及其主要代谢产物EETs和HETEs的分析方法, 并在乳腺癌自发小鼠模型中发现AA、12-HETE、19-HETE及8,9-EET在血浆和肿瘤组织中存在显著性差异, 为乳腺癌的快速诊断及治疗提供了帮助。更为重要的是在项目实施过程中，项目组创造性的取得了一些科学技术上的突破，包括建立了一种重组人源CYP3A4/CPR/cyt b5 蛋白共转染共表达的方法，成功构建体外研究代谢转运蛋白P-糖蛋白的人小肠3D类器官模型，在国际上率先报道利用CRISPR/Cas系统进行特定Cyp基因敲除大鼠的构建。Cyp基因敲除大鼠作为一种新颖且可用的啮齿类动物模型，将是一个强有力的研究工具，用来探究CYP酶在化学物代谢、毒性、致癌性，以及药物相互作用中的重要作用。目前，本项目已发表标注受本项目资助SCI科研论文13篇，学术专著论文1篇，申请国内发明专利1项。总体而言，本项目顺利开展实施，取得了较好的研究成果。