Tumor acidosis enhances cytotoxic effects and autophagy inhibition by salinomycin on cancer cell lines and cancer stem cells

Abstract

Sustained autophagy contributes to the metabolic adaptation of cancer cells to hypoxic and acidic microenvironments. Since cells in such environments are resistant to conventional cytotoxic drugs, inhibition of autophagy represents a promising therapeutic strategy in clinical oncology. We previously reported that the efficacy of hydroxychloroquine (HCQ), an autophagy inhibitor under clinical investigation is strongly impaired in acidic tumor environments, due to poor uptake of the drug, a phenomenon widely associated with drug resistance towards many weak bases. In this study we identified salinomycin (SAL) as a potent inhibitor of autophagy and cytotoxic agent effective on several cancer cell lines under conditions of transient and chronic acidosis. Since SAL has been reported to specifically target cancer-stem cells (CSC), we used an established model of breast CSC and CSC derived from breast cancer patients to examine whether this specificity may be associated with autophagy inhibition. We indeed found that CSC-like cells are more sensitive to autophagy inhibition compared to cells not expressing CSC markers. We also report that the ability of SAL to inhibit mammosphere formation from CSC-like cells was dramatically enhanced in acidic conditions. We propose that the development and use of clinically suitable SAL derivatives may result in improved autophagy inhibition in cancer cells and CSC in the acidic tumor microenvironment and lead to clinical benefits.

Spheroid‐based 3D cell cultures identify salinomycin as a promising drug for the treatment of chondrosarcoma

“…SAL has been reported to inhibit autophagic flux in several cancer cell lines . Since tumor cells cultured as 3D structures are characterized by an increased autophagic flux, we measured the autophagic flux in the SW1353 cells cultured as 2D or 3D.…”

“…After 24 h for 2D cultures and 1 week for 3D cultures, cells were treated with increasing doses of DXR, CIS, CQ, and SAL (0–50 µM). Doses were chosen based on previous observations and characterization of these drugs …”

“…To evaluate the autophagy basal level, SW 1353 cells were seeded and, after 24 h, the medium (IMDM) was replaced with Earle's Balanced Salt Solution (EBSS, Gibco, Monza, Italy), to induce autophagy under starvation conditions. After 2 h, the V‐ATPase inhibitor Bafilomycin A1 (BafA1) was added to the medium to monitor autophagic flux. After additional 2 h, whole‐cell extracts from SW1353 cells were obtained as described above.…”

Dissecting pharmacological effects of chloroquine in cancer treatment: interference with inflammatory signaling pathways

“…270 On the other hand, it may target the Wnt/ b-catenin signaling pathway to promote differentiation and thus elimination of CSCs, while rescuing normal fibroblasts from CQ toxicity. 274 This could allow a 'first hit' against a tumor and pave the way for a combination treatment. 272,273 This can make its synergistic antineoplastic effects with other drugs beneficial, if they decrease systemic exposure and toxicity to nontransformed cells.…”

“…To this end, under the acidic conditions that inhibit CQ activity, salinomycin reaches the acidic core of multicellular tumor spheroids and impairs the autophagic flux, thereby killing malignant cells. 274 This could allow a 'first hit' against a tumor and pave the way for a combination treatment. In line with the potential of CQ for synergy with inhibitors of DNA repair, the antineoplastic effect of salinomycin is neutralized by the induction of DNA repair via DNA-protein kinase (DNA-PK) enzymes.…”