新型天然铁死亡诱导剂的筛选及作用机制研究

	新型天然铁死亡诱导剂的筛选及作用机制研究
	赵越勤
导师	赵逾涵
关键词	肿瘤，天然产物，铁死亡，溶酶体，p53 Cancer, Nature products, Ferroptosis, Lysosome, p53
摘要	癌症是严重威胁人类健康的重大疾病之一。p53 作为一个经典的抑癌基因，研究表明其在肿瘤发生发展过程中参与调控铁死亡。铁死亡是依赖亚铁离子的脂质过氧化物堆积造成的一种新型细胞死亡方式。近年来的热点研究表明铁死亡与病理状态下的细胞死亡(如肿瘤、神经退行性疾病等疾病)密切相关，肿瘤细胞，尤其是间质状态细胞、部分耐药细胞、肿瘤干细胞等对铁死亡更敏感，这也使得铁死亡诱导剂有望成为新化疗药物。本研究通过构建铁死亡细胞筛选模型，从天然产物库中筛选出肿瘤细胞铁死亡诱导剂，并揭示其作用机制。首先，从筛选具有细胞毒性的天然产物出发，其中包括从云南割舌树枝叶中提取得到的柠檬苦素类化合物和从肿柄菊属植物肿柄菊中分离得到的肿丙菊内酯类化合物，筛选得到了一系列具有良好的抗肿瘤活性的小分子化合物，其中三个新的柠檬苦素类化合物 1-3 能够诱导肿瘤细胞周期阻滞及细胞凋亡、抑制肿瘤细胞的克隆形成和迁移，从而抑制肿瘤细胞的生长，通过构效关系分析发现?,?-不饱和酮结构片段是该类化合物细胞毒作用的关键基团。其次，通过实时细胞成像显微技术，对具有诱导肿瘤细胞坏死的化合物进行复筛，并联用铁死亡抑制剂(如铁离子螯合剂)发现可诱导铁死亡的化合物，对潜在活性化合物进行铁死亡标志物的检测，包括亚铁离子的累积、脂质过氧化物的累积及氧化还原系统的检测。通过复筛选发现活性天然产物 Hdj-15 可以诱导肿瘤细胞铁死亡。随后对其具体的作用机制进行探究，利用 RNAseq、基因编辑、免疫荧光等分子生物学手段，发现 Hdj-15 通过激活内质网应激反应介导的 Nrf2-HO-1 通路，催化血红素氧化成游离铁，促进铁死亡的发生；与此同时，Hdj-15可以诱导铁死亡关键蛋白谷胱甘肽过氧化物酶 4(GPX4)的降解，进一步研究发现Nrf2 的激活将进一步促进溶酶体膜蛋白 LAMP2A 的表达，介导自噬-溶酶体途径GPX4 的降解，诱导肿瘤细胞发生铁死亡。研究过程中，发现 p53 缺陷的肿瘤细胞与野生型肿瘤细胞相比对 Hdj-15 诱导的铁死亡更敏感。进一步在蛋白水平和 mRNA 水平上进行探究，发现 Hdj-15 对 p53 缺陷的肿瘤细胞毒性更大是由于 p53 缺陷的肿瘤细胞中 GPX4 的水平更低导致的。进一步研究表明在 Hdj-15 及 p53 激活剂的作用下，GPX4 在 mRNA 水平和蛋白水平上的变化趋势与 p53 相同，提示 GPX4 可能是 p53 潜在的下游靶基因。通过 ChIP 实验结果发现并验证了 GPX4 基因中的 p53 结合域。该研究结果提示 p53 转录调控 GPX4 的表达，而 Hdj-15 通过激活 p53-GPX4 途径参与调控肿瘤细胞铁死亡。进一步在体内通过裸鼠异种移植瘤模型进行Hdj-15抗肿瘤活性生物学评价，结果显示相比于对照组裸鼠，Hdj-15 给药组可以显著抑制异种移植瘤裸鼠中移植瘤的生长，并且诱导内源性脂质过氧化产物(4-羟基壬烯醛及丙二醛)在移植瘤中的积累；且 Hdj-15 在裸鼠体内未见明显毒副作用。该研究结果表明 Hdj-15 在体内也可以通过诱导铁死亡延缓肿瘤的生长。综上所述，本研究通过铁死亡筛选平台发现了诱导肿瘤细胞铁死亡的活性天然产物 Hdj-15，并揭示了 Hdj-15 促进肿瘤细胞铁死亡的分子机制，为靶向铁死亡的抗肿瘤先导化合物的发现提供了研究基础。关键词：肿瘤，天然产物，铁死亡，溶酶体，p53; Cancer is one of the major diseases that seriously threaten human health. As a classic tumor suppressor gene, p53 has been shown to be involved in the regulation of ferroptosis in the process of tumorigenesis and development. Ferroptosis is a new mode of cell death caused by iron-dependent accumulation of lipid peroxides. Researches in recent years have shown that ferroptosis is closely related to cell death in pathological conditions (such as tumors, neurodegenerative diseases, and other diseases). Tumor cells, especially cells in mesenchymal-like states, some drug-resistant cells, cancer stem cells, etc. are more sensitive to ferroptosis, which provides intriguing insights into ferroptosis inducers as novel chemotherapeutic drugs. In this study, we screened tumor cell ferroptosis-inducing agents from natural products, and explored their mechanism of action. Firstly, we screened and found a series of compounds with potent antitumor effects, including some limonoids isolated from Walsura yunnanensis and tirotundin isolated from Tithonia diversifolia. Among them, three new limonoids 1-3 can induce tumor cell cycle arrest, apoptosis, and inhibit tumor cell colony formation and migration, thereby inhibiting tumor cell growth. The structure-activity relationship analysis revealed that the α,β-unsaturated ketone moieties in rings A are essential for their cytotoxic activity. Then we further screened for potential ferroptosis inducers which exhibit ferroptotic cell death through real-time cell imaging microscopy, including the accumulation of lipid peroxides, labile iron pool and defective lipid peroxide repair capacity. What’s more, the cell death induced by the active compounds could be blocked by ferroptosis inhibitors (such as iron chelators). Here, the active natural product Hdj-15 was found to induce ferroptosis in tumor cells. Subsequently, the specific mechanism of action was explored. By using molecular biological methods such as RNAseq, gene editing, and real-time cell imaging microscopy, immunofluorescence (IF), we found that Hdj-15 catalyzed the oxidation of heme to free iron by activating the Nrf2-HO-1 pathway, which was mediated by the endoplasmic reticulum stress response, and promoted the occurrence of ferroptosis; At the same time, Hdj-15 could induce the degradation of ferroptosis key protein glutathione peroxidase 4 (GPX4), and further studies found that the activation of Nrf2 would further upregulate the expression of the lysosomal membrane protein LAMP2A, which mediated the degradation of GPX4 in the autophagy-lysosomal pathway, and induced ferroptosis in tumor cells.Interestingly, p53-deficient cells are more vulnerable to Hdj-15-induced ferroptosis. Further investigations revealed that p53 deficient cells or p53 knockdown cell lines showed decreased GPX4 at the mRNA and protein levels. In conclusion, this study identified natural products with potential anti-tumor effects.
语种	中文
	2022-05
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75149
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	赵越勤. 新型天然铁死亡诱导剂的筛选及作用机制研究[D]. 中国科学院大学,2022.

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