结直肠癌（CRC）是一种常见的消化系统恶性肿瘤，尽管治疗方法取得了重大进展，但仍需要更有效的治疗方法。 Timosaponin AIII (TA-III) 是一种从知母中提取的主要甾体皂苷，是一种潜在的抗癌剂。铁死亡在癌症治疗中发挥着重要作用。研究TA-III作为新型铁死亡诱导剂通过自噬抑制结直肠癌的分子机制。铁死亡是一种自噬依赖性细胞死亡模式，与 CRC 相关。用 TA-III 处理 CRC 细胞，并通过 BODIPY493/503 染色和蛋白质印迹评估脂质自噬水平。使用 GFP-RFP-LC3B 跟踪自噬周转率。使用丙二醛试剂盒和 C11-BODIPY 流动测定对脂质过氧化进行定量。使用透射电子显微镜观察线粒体形态。 GC-MS/MS用于检测脂质代谢变化。通过蛋白质印迹和谷胱甘肽 S-转移酶下拉分析评估 ras 相关蛋白 Rab 7a (Rab7) 的作用。在体内，使用异种移植模型测试了 TA-III 的抗癌功效。RNA-seq 分析揭示了 TA-III 通过铁死亡作为抗癌剂的潜力。体内实验揭示了 TA-III 治疗如何触发 CRC 细胞中脂滴的降解，导致 FFA 积累、不饱和游离脂肪酸增加和脂质过氧化增加。这些事件最终导致线粒体收缩和铁死亡标记物（FSP1 和 GPX4）的下调。有趣的是，Rab7 蛋白成为脂噬和铁死亡之间的重要桥梁，强调了它在 TA-III 抗癌机制中的重要性。此外，异种移植肿瘤模型中的 TA-III 治疗通过铁死亡显着缩小了肿瘤体积，强调了其治疗功效。我们的研究首次证实 TA-III 通过 Rab7 基因触发 CRC 细胞中的脂肪吞噬，随后促进铁死亡。这表明其作为抗肿瘤剂的潜在用途。版权所有 © 2023。由 Elsevier GmbH 出版。

Colorectal cancer (CRC) is a common digestive system malignancy, and despite significant therapeutic advancements, more effective treatments are needed. Timosaponin AIII (TA-III), a major steroidal saponin derived from Anemarrhena asphodeloides Bge, is a potential anticancer agent. Ferroptosis plays an important role in cancer treatment.To investigate the molecular mechanism of TA-III as a novel ferroptosis inducer in suppressing CRC through lipophagy. Ferroptosis, an autophagy-dependent mode of cell death, has been implicated in CRC.CRC cells were treated with TA-III, and lipophagy levels were evaluated via BODIPY493/503 staining and western blotting. Autophagy turnover was tracked using GFP-RFP-LC3B. Lipid peroxidation was quantified using an malondialdehyde kit and C11-BODIPY flow assay. Mitochondrial morphology was observed using transmission electron microscopy. GC-MS/MS was used to detect lipid metabolism changes. The role of ras related protein Rab 7a (Rab7) was assessed by western blotting and glutathione S-transferase pull-down assays. In vivo, the anticancer efficacy of TA-III was tested using a xenograft model.RNA-seq analysis unveiled the potential of TA-III as an anticancer agent through ferroptosis. In vivo experiments revealed how TA-III treatment triggered degradation of lipid droplets in CRC cells, resulting in an accumulation of FFAs, heightened unsaturated free fatty acids, and increased lipid peroxidation. These events ultimately lead to mitochondrial shrinkage and downregulation of ferroptosis markers (FSP1 and GPX4). Intriguingly, the Rab7 protein emerged as a crucial bridge between lipophagy and ferroptosis, underlining its significance in the anticancer mechanism of TA-III. Moreover, TA-III treatment in a xenograft tumour model substantially reduced tumour volume via ferroptosis, underscoring its therapeutic efficacy.Our study is the first to establish that TA-III triggers lipophagy in CRC cells via the Rab7 gene, subsequently promoting ferroptosis. This suggests its potential use as an antitumour agent.Copyright © 2023. Published by Elsevier GmbH.