Guggulsterone and bexarotene induce secretion of exosome-associated breast cancer resistance protein and reduce doxorubicin resistance in MDA-MB-231 cells

Many breast cancer cells acquire multidrug resistance (MDR) mediated by ABC transporters such as breast cancer resistance protein (BCRP/ABCG2). Here we show that incubation of human breast cancer MDA-MB-231 cells with farnesoid X receptor antagonist guggulsterone (gug) and retinoid X receptor agonist bexarotene (bex) elevated ceramide, a sphingolipid known to induce exosome secretion. The gug+bex combination reduced cellular levels of BCRP to 20% of control cells by inducing its association and secretion with exosomes. Exogenous C6 ceramide also induced secretion of BCRP-associated exosomes, while siRNA-mediated knockdown or GW4869-mediated inhibition of neutral sphingomyelinase 2 (nSMase2), an enzyme generating ceramide, restored cellular BCRP. Immunocytochemistry showed that ceramide elevation and concurrent loss of cellular BCRP was prominent in Aldefluor-labeled breast cancer stem-like cells. These cells no longer excluded the BCRP substrate Hoechst 33342 and showed caspase activation and apoptosis induction. Consistent with reduced BCRP, ABC transporter assays showed that gug+bex increased doxorubicin retention and that the combination of gug+bex with doxorubicin enhanced cell death by more than fivefold. Taken together, our results suggest a novel mechanism by which ceramide induces BCRP secretion and reduces MDR, which may be useful as adjuvant drug treatment for sensitizing breast cancer cells and cancer stem cells to chemotherapy. What's new? Multidrug resistance (MDR) is spread from one tumor cell to the next by exosomes and microvesicles, which transfer MDR-conferring ATP-binding cassette (ABC) proteins to drug-sensitive cells. The present study explored the possibility of inducing exosome secretion as a means of depleting ABC transporters and potentially restoring drug sensitivity in tumor cells. Using breast cancer cells, the authors show that combined treatment with guggulsterone and bexarotene increased cellular levels of ceramide, a trigger for exosome budding. The drug combination also led to reductions in breast cancer resistance protein (BCRP), an ABC transporter.