背景:结直肠癌(CRC)是一种常见的消化系统恶性肿瘤,尽管治疗上取得了显著进步,需要更有效的治疗。知母皂苷AIII(TA-III),一种来自知母的主要甾体皂苷,是一种潜在的抗癌剂。Ferroptosis在癌症治疗中起着重要作用。
目的:探讨TA-III作为一种新型的铁凋亡诱导剂通过吞噬抑制CRC的分子机制。Ferroptosis,细胞死亡的自噬依赖性模式,与CRC有牵连。
方法:用TA-III处理CRC细胞,通过BODIPY493/503染色和蛋白质印迹评估脂质吞噬水平。使用GFP-RFP-LC3B追踪自噬周转。使用丙二醛试剂盒和C11-BODIPY流动测定定量脂质过氧化。使用透射电子显微镜观察线粒体形态。GC-MS/MS检测脂质代谢变化。通过蛋白质印迹和谷胱甘肽S-转移酶下拉测定来评估ras相关蛋白Rab7a(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。
结果:RNA-seq分析揭示了TA-III通过铁凋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的积累,增加的不饱和游离脂肪酸,和增加脂质过氧化。这些事件最终导致线粒体收缩和铁凋亡标志物(FSP1和GPX4)的下调。有趣的是,Rab7蛋白是吸脂性和铁性凋亡之间的重要桥梁,强调其在TA-III抗癌机制中的意义。此外,异种移植肿瘤模型中的TA-III治疗通过铁性凋亡大大减少了肿瘤体积,强调其治疗功效。
结论:我们的研究是首次确定TA-III通过Rab7基因在CRC细胞中触发吸脂性,随后促进铁性凋亡。这表明其作为抗肿瘤剂的潜在用途。
BACKGROUND: 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.
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.