基于SREBP-2调控的甲羟戊酸途径研究荷叶生物碱抗胰腺癌及化疗增敏作用机制

As the mevalonate pathway can promote the malignant transformation of tumors, sterol regulatory element binding protein-2 (SREBP-2) attracts attention in tumor research. Our previous studies have found that SREBP-2 is a potential target for pancreatic cancer treatment. The results of drug screening by targeting SREBP-2 show that the natural medicine alkaloids of lotus leaf can significantly inhibit the activity of SREBP-2, accompanied by the decrease of cholesterin, reduced cell viability and increased apoptosis. Moreover, overexpression of SREBP-2 can significantly impair the anti-pancreatic cancer effect of alkaloids of lotus leaf. Therefore, we hypothesized that alkaloids of lotus leaf could exhibit anti-pancreatic cancer effects by inhibiting SREBP-2. To elucidate the molecular mechanism of alkaloids of lotus leaf anti-pancreatic cancer and chemotherapeutic sensitization, this project plants to reveal the exact mechanism by which alkaloids of lotus leaf inhibits the transcriptional activity of SREBP-2 at the cellular level, and clarify the changes of mevalonate pathway-related enzymes, products and the downstream signaling pathway of SREBP-2 after SREBP-2 inhibition. Furthermore, we will verify the impact of SREBP-2 on the anti-tumor activity of alkaloids of lotus leaf and combined with gemcitabine to investigate the chemosensitivity of alkaloids of lotus leaf in vivo. This research project approach to clarify the molecular anti- pancreatic cancer mechanisms of alkaloids of lotus leaf and provides theoretical basis for its application in the clinical treatment of pancreatic cancer.

随着甲羟戊酸途径促进肿瘤恶性转化的发现，甾醇调控元件结合蛋白-2（SREBP-2）在肿瘤研究中引起关注。我们前期研究证实SREBP-2是胰腺癌治疗潜在靶点，并发现荷叶生物碱能抑制SREBP-2活性、降低胰腺癌细胞中胆固醇水平并伴随细胞活力下降和细胞凋亡，过表达SREBP-2能显著削弱其对胰腺癌细胞活力的影响。据此，我们推测荷叶生物碱可通过抑制SREBP-2发挥抗胰腺癌及化疗增敏作用。本项目拟在细胞层面揭示荷叶生物碱抑制SREBP-2转录活性的确切机制，明确荷叶生物碱抑制SREBP-2后胰腺癌细胞中甲羟戊酸途径相关酶、中间体及SREBP-2下游信号通路的变化，进而阐明荷叶生物碱抗胰腺癌及化疗增敏的分子机制；动物层面验证荷叶生物碱通过抑制SREBP-2发挥抗胰腺癌作用并联合吉西他滨考察荷叶生物碱体内化疗增敏作用。本课题将阐明荷叶生物碱抗胰腺癌分子机制，为其应用于胰腺癌临床治疗提供理论依据。

荷叶生物碱是荷叶降脂减肥的主要活性成分，临床研究表明高血脂和肥胖是胰腺癌发生发展的独立危险因素，但荷叶生物碱是否具有抗胰腺癌作用尚不明确，关于荷叶生物碱抗胰腺癌有效成分也不清楚。课题组前期研究发现固醇调节元件结合蛋白 2 （sterol regulatory element⁃binding protein 2, SREBP2）是胰腺癌治疗的潜在靶点，并发现荷叶生物碱能抑制SREBP-2活性来发挥抗胰腺癌作用。本项目在前期研究基础上首先揭示荷叶碱是荷叶生物碱中通过SREBP-2抑制甲羟戊酸途径来发挥抗胰腺癌作用的主要药效成分。机制研究发现荷叶碱下调SREBP-2 前体蛋白（pre-SREBP-2）和核内形式（nSREBP-2）表达，进一步研究发现荷叶碱不是通过作用于SCAP与Insig-1来影响SREBP-2在高尔基体的蛋白酶切来发挥作用，而是通过激活AMPK来影响SREBP-2蛋白裂解成熟和核内转位来抑制SREBP-2活性。荷叶碱对SREBP-2的抑制下调了甲羟戊酸途径合成酶HMGCS、 HMGCR、FPPS 和 SQS的表达。靶向脂质组学数据也显示荷叶碱降低了甲羟戊酸途径中间产物及胆固醇水平。另一方面，体内外研究发现荷叶碱能够增强胰腺癌细胞对吉西他滨敏感性，并揭示荷叶碱对甲羟戊酸途径的抑制上调了YAP Ser127磷酸化水平从而抑制YAP核转位及靶基因的表达是荷叶碱化疗增敏作用的分子机制。该研究为荷叶生物碱防治胰腺癌提供实验和理论基础，同时为靶向抑制肿瘤细胞过度活跃的甲羟戊酸途径来治疗胰腺癌提供依据。本项目共培养硕士研究生6名，第一或通讯作者在Pyotomedicne (IF=6.656, 医学1区)、Biochemical Pharmacology （IF=6.1, 医学2区Top）和European journal of pharmaceutical sciences（IF=4.432, 医学2区）发表SCI论文3篇。基于此项目研究发现，考虑到莲子和荷叶都是收入中国药典的中药，其所含生物碱是各组织中均存在的最重要的活性成分，我们还扩展研究了荷叶碱抗肝癌及莲子心生物碱抗胰腺癌作用及机制，发现荷叶碱通过改变脂质代谢重编程发挥抗肝癌作用，莲子心生物碱促进胆固醇在溶酶体蓄积而抑制自噬流来发挥抗胰腺癌作用。