Ovarian cancer (OC) is known for its high mortality rate. Although sodium citrate has anti-tumor effects in various cancers, its effect and mechanism in OC remain unclear. To analyze the inhibitory effect of sodium citrate on ovarian cancer cells and the underlying mechanism. Cell apoptosis was examined by TUNEL staining, flow cytometry, and ferroptosis was examined intracellular Fe, MDA, LPO assays, respectively. Cell metabolism was examined by OCR and ECAR measurements. Immunoblotting and immunoprecipitation were used to elucidate the mechanism. This study suggested that sodium citrate not only promoted ovarian cancer cell apoptosis but also triggeredferroptosis, manifested as elevated levels of Fe, LPO, MDA andlipid ROS production. On one hand, sodium citrate treatment led to a decrease of Ca content in the cytosol by chelatingCa, which further inhibited the Ca/CAMKK2/AKT/mTOR signaling, thereby suppressing HIF1α-dependent glycolysis pathway and inducing cell apoptosis. On the other hand, the chelation of Ca by sodium citrate resulted in inactivation of CAMKK2 and AMPK, leading to increase of NCOA4-mediated ferritinophagy, causing increased intracellular Fe levels. More importantly, the inhibition of Ca/CAMKK2/AMPK signaling pathway reduced the activity of the MCU and Ca concentration within the mitochondria, resulting in an increase in mitochondrial ROS. Additionally, metabolomic analysis indicated that sodium citrate treatment significantly increased de novo lipid synthesis. Altogether, these factors contributed to ferroptosis. As expected, Ca supplementation successfully reversed the cell death and decreased tumor growth induced by sodium citrate. Inspiringly, it was found that coadministration of sodium citrate increased the sensitivity of OC cells to chemo-drugs. These results revealed that the sodium citrate exerted its anti-cancer activity by inhibiting Ca/CAMKK2-dependent cell apoptosis and ferroptosis. Sodium citrate will hopefully serve as a prospective compound for OC treatment and for improvingthe efficacy of chemo-drugs.

中文翻译：

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柠檬酸钠靶向 Ca2+/CAMKK2 通路通过诱导卵巢癌细胞凋亡和铁死亡发挥抗肿瘤活性

卵巢癌（OC）以其高死亡率而闻名。尽管柠檬酸钠在多种癌症中具有抗肿瘤作用，但其在OC中的作用和机制仍不清楚。分析柠檬酸钠对卵巢癌细胞的抑制作用及其机制。通过TUNEL染色、流式细胞术检查细胞凋亡，并分别通过细胞内Fe、MDA、LPO测定检查铁死亡。通过 OCR 和 ECAR 测量来检查细胞代谢。使用免疫印迹和免疫沉淀来阐明其机制。该研究表明，柠檬酸钠不仅促进卵巢癌细胞凋亡，还引发铁死亡，表现为Fe、LPO、MDA和脂质ROS产生水平升高。一方面，柠檬酸钠处理通过螯合Ca导致细胞质中Ca含量降低，进一步抑制Ca/CAMKK2/AKT/mTOR信号传导，从而抑制HIF1α依赖的糖酵解途径并诱导细胞凋亡。另一方面，柠檬酸钠对Ca的螯合导致CAMKK2和AMPK失活，导致NCOA4介导的铁蛋白自噬增加，导致细胞内Fe水平增加。更重要的是，Ca/CAMKK2/AMPK信号通路的抑制降低了MCU的活性和线粒体内的Ca浓度，导致线粒体ROS增加。此外，代谢组学分析表明柠檬酸钠处理显着增加了脂质的从头合成。总而言之，这些因素导致了铁死亡。正如预期的那样，钙补充剂成功逆转了柠檬酸钠诱导的细胞死亡并减少了肿瘤生长。令人鼓舞的是，我们发现柠檬酸钠的共同给药增加了 OC 细胞对化疗药物的敏感性。这些结果表明，柠檬酸钠通过抑制 Ca/CAMKK2 依赖性细胞凋亡和铁死亡来发挥其抗癌活性。柠檬酸钠有望成为治疗 OC 和提高化疗药物疗效的有前景的化合物。