A mitochondria-targeting and G-quadruplex structure-binding ligand inducing calcium overload and ferroptosis in human cancer cells

Background and Purpose: Regulation of mitochondrial calcium overload and ferroptosis with mitochondria-targeting ligands is an attractive anticancer strategy but it remains a challenge. The aim of the present study was to demonstrate that a mitochondria-targeting and mtDNA G-quadruplex-binding ligand, BYB, induced mitochondrial calcium overload and ferroptosis in HeLa cells and showed potent in vitro and in vivo anticancer activity. Experimental Approach: Cellular functions and molecular mechanism were studied using cell viability assay, live-cell imaging, western blotting, immunofluorescence, cell uptake, cell cycle arrest and apoptosis analysis, mitochondrial metabolism analysis, Comet assay, and wound-healing analysis. Pharmacokinetic studies were conducted in rat. In vivo antitumor activity was studied in a cervical cancer HeLa cell xenograft mouse model. Key Results: Cellular results showed that BYB induced mitochondrial calcium overload, attributed to ligand-induced mitochondrial dysfunction via the mechanism of inhibiting mitochondrial DNA replication and transcription. The expression of respiratory chain complexes was markedly downregulated in BYB-treated HeLa cells. The respiratory chain function was also dysregulated. Mitophagy and mitochondrial calcium overload were induced in BYB-treated HeLa cells. Mitochondrial calcium overload markedly induced mtROS production. The induced mtDNA stress activated cGAS-STING pathway, leading to autophagy-dependent ferroptosis. The antitumour efficacy of BYB, evaluated in a HeLa tumour xenograft mouse model, achieved over 60% tumour weight reduction. Conclusion and Implications: BYB, via targeting mitochondria and mtDNA G-quadruplexes, induced mitochondrial calcium overload and ferroptosis, exhibited high in vivo antitumour efficacy and low toxicity. It shows high potential to be a mitochondria-targeting lead compound for chemical biology and drug discovery.