Drug Transporter-Mediated Protection of Cancer Stem Cells From Ionophore Antibiotics

Abstract

Ionophore antibiotics were reported to selectively kill cancer stem cells and to overcome multidrug resistance, but mechanistic studies of the significance of drug transporters for treatment with these compounds are lacking. We applied chemosensitivity testing of well-characterized human cancer cell lines to elaborate on whether drug transporters are involved in protection from the cytotoxic effects of the ionophore antibiotics salinomycin and nigericin. Our experiments demonstrated that ionophore antibiotics were ineffective against both stem-like ovarian cancer side population cells (expressing either ABCB1 or ABCG2) and K562/Dox-H1 cells, which constitute a genetically defined model system for ABCB1 expression. Considering that cancer stem cells often express high levels of drug transporters, we deduced from our results that ionophore antibiotics are less suited to cancer stem cell-targeted treatment than previously thought.

Significance: Ionophore antibiotics such as salinomycin have repeatedly been shown to target cancer stem and progenitor cells from various tumor entities. Meanwhile, cancer stem cell (CSC)-selective toxicity of ionophore antibiotics seems to be a commonly accepted concept that is about to encourage their clinical testing. This study provides data that challenge the concept of targeted elimination of CSC by ionophore antibiotics. Stem-like ovarian cancer side population (SP) cells expressing high levels of ABC drug transporters are shown to largely resist the cytotoxic effects of salinomycin and nigericin. Furthermore, using a small interfering RNA-based knockdown model specific for ABCB1, this study demonstrates that ABC drug transporters are indeed causally involved in mediating protection from ionophore antibiotics. Considering that it is a hallmark of CSCs to exhibit drug resistance conferred by ABC drug transporters, it must be deduced from these results that CSCs may also be protected from ionophore antibiotics by means of drug-transporter mediated efflux.

Keywords: Cancer stem cell; Drug transporter; Ionophore antibiotic; Nigericin; Salinomycin.

Figure 1.

Drug transporter status of cell lines used within this study. (A): Histogram overlay of fluorescence-activated cell sorting-purified SP and NSP fractions of the ovarian cancer cell lines A2780V, IGROV1 (both ABCB1 positive), and B17/92 (ABCG2 positive). (B): Dot plot overlay of ABCB1-positive K562/Dox-H1 (empty vector control; gray) and ABCB1-negative K562/Dox-MM (MDR1-targeting small interfering RNA; black) chronic myelogenous leukemia cells. (C): Dot plot showing the ABCG2-expressing subpopulation of A2780 ovarian cancer cells (rectangular gate). Plots are representative examples of at least three independent stainings. Abbreviations: H1, ABCB1-positive K562/Dox-H1 leukemia cells; MM, ABCB1-negative K562/Dox-MM leukemia cells; NSP, non-side population; SP, side population.

Figure 2.

Resistance of ABCB1-positive SP cells to paclitaxel. Fluorescence-activated cell sorting-purified SP and NSP fractions of the ovarian cancer cell lines A2780V and IGROV1 were treated with increasing concentrations of paclitaxel and subsequently subjected to the metabolic colorimetric MTT assay. Nontreatment controls were included and used for calculation of the degree of specific inhibition of cell viability. ∗, p < .05; ∗∗, p < .01. Abbreviations: NSP, non-side population; SP, side population.

Figure 3.

Resistance of ABCB1- and ABCG2-expressing SP cells to ionophore antibiotics. Fluorescence-activated cell sorting-purified SP and NSP fractions of the ovarian cancer cell lines A2780V (ABCB1-positive SP) and B17/92 (ABCG2-positive SP) were treated with increasing concentrations of salinomycin (A) or nigericin (B) and subsequently subjected to the metabolic colorimetric MTT assay. Nontreatment controls were included and used for calculation of the degree of specific inhibition of cell viability. ∗, p < .05; ∗∗, p < .01. Abbreviations: NSP, non-side population; SP, side population.

Figure 4.

Drug transporter expression protects from the cytotoxic effects of ionophore antibiotics. A mixture of 20% ABCB1-positive K562/Dox-H1 cells (empty vector control) and 80% ABCB1-negative K562/Dox-MM cells (MDR1-targeting small interfering RNA) was generated and exposed to increasing concentrations of salinomycin (A) or nigericin (B) in a competitive assay. The same assay was also applied to A2780 ovarian cancer cells, which naturally harbor an ABCG2-positive side population (C, D). After 7 days of treatment, the proportion of drug transporter-positive cells was determined using staining with respective monoclonal antibodies and flow cytometry. Representative fluorescence-activated cell sorting plots are shown. ∗, p < .05; ∗∗, p < .01.