Sodium citrate targeting Ca2+/CAMKK2 pathway exhibits anti-tumor activity through inducing apoptosis and ferroptosis in ovarian cancer

Abstract

Introduction: 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.

Objectives: To analyze the inhibitory effect of sodium citrate on ovarian cancer cells and the underlying mechanism.

Methods: Cell apoptosis was examined by TUNEL staining, flow cytometry, and ferroptosis was examined intracellular Fe²⁺, MDA, LPO assays, respectively. Cell metabolism was examined by OCR and ECAR measurements. Immunoblotting and immunoprecipitation were used to elucidate the mechanism.

Results: This study suggested that sodium citrate not only promoted ovarian cancer cell apoptosis but also triggeredferroptosis, manifested as elevated levels of Fe²⁺, LPO, MDA andlipid ROS production. On one hand, sodium citrate treatment led to a decrease of Ca²⁺ content in the cytosol by chelatingCa²⁺, which further inhibited the Ca²⁺/CAMKK2/AKT/mTOR signaling, thereby suppressing HIF1α-dependent glycolysis pathway and inducing cell apoptosis. On the other hand, the chelation of Ca²⁺ by sodium citrate resulted in inactivation of CAMKK2 and AMPK, leading to increase of NCOA4-mediated ferritinophagy, causing increased intracellular Fe²⁺ levels. More importantly, the inhibition of Ca²⁺/CAMKK2/AMPK signaling pathway reduced the activity of the MCU and Ca²⁺ 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, Ca²⁺ 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.

Conclusion: These results revealed that the sodium citrate exerted its anti-cancer activity by inhibiting Ca²⁺/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.

Keywords: Apoptosis; CAMKK2; Ca(2+); Ferroptosis; Sodium citrate.