Chloroquine inhibits salinomycin-induced autophagy for collaborative anticancer effect in breast cancer

INTRODUCTION: Breast cancer (BC) presents significant morbidity and mortality challenges. Autophagy plays a contradictory role in BC. The chemotherapeutic agent salinomycin exhibits anticancer effects, but its effectiveness is limited by over-activation of autophagy. This study aimed to investigate the effects and mechanisms of salinomycin and its combination with chloroquine in BC. METHODS: The MCF-7 and MCF-7 tumor spheroids (MCF-7-TS) BC models were treated separately with salinomycin and autophagy inducer/inhibitor (rapamycin/chloroquine). Cell proliferation, apoptosis, and cell cycle progression were measured using cell counting kit-8 (CCK-8), cell colony assay, and flow cytometry. The expression of apoptosis-related, autophagy-related, and phosphoinositide 3-kinases (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway-related proteins was measured via Western blot. Light chain 3 (LC3) expression was detected via immunofluorescence. RESULTS: In the MCF-7 and MCF-7-TS cells, salinomycin inhibited cell viability, p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR expression, and increased apoptosis and LC3 expression, with reduced tumor spheroid number and volume of MCF-7-TS cells. Interestingly, rapamycin enhanced LC3 expression but prevented apoptosis in salinomycin-treated cells, with elevated tumor spheroid number and volume of MCF-7-TS cells. Moreover, after screening for a suitable ratio of salinomycin and chloroquine (1:2.5), compared to salinomycin group, salinomycin+chloroquine group exhibited decreased tumor spheroid number and volume of MCF-7-TS cells; reduced B-cell lymphoma-2 (Bcl-2), LC3, LC3II/LC3I, and Beclin-1 expression; and enhanced G0/G1 phase arrest and Bcl-2-associated X protein expression in MCF-7 and MCF-7-TS cells. CONCLUSION: Chloroquine enhanced the anticancer efficacy of salinomycin by suppressing salinomycin-induced autophagy, providing a solid theoretical basis for its clinical application in BC.