Rhinacanthin-C enhances doxorubicin cytotoxicity via inhibiting the functions of P-glycoprotein and MRP2 in breast cancer cells

Rhinacanthin-C is a major bioactive naphthoquinone ester found in Rhinacanthus nasutus Kurz (Acanthaceae). This compound has potential therapeutic value as an anticancer and antiviral agent. In this study, we investigated an enhancement effect of rhinacanthin-C on doxorubicin cytotoxicity in human breast cancer cell lines and the involvement of the ABC drug efflux transporters. The cytotoxicity was determined by an MTT assay. Combination between doxorubicin and rhinacanthin-C at their non-cytotoxic concentrations when giving each compound alone significantly reduced cell viability in MCF-7 and MCF-7/DOX resistant cells. At the noncytotoxic concentration (0.1 mu M), rhinacanthin-C enhanced doxorubicin cytotoxicity by 38 fold in MCF-7 cells after 48-h treatment. Moreover, intracellular doxorubicin accumulation significantly increased in both MCF-7 cells and MCF-7/DOX resistance cells in the presence of rhinacanthin-C for 6-h treatment period. The interference of rhinacanthin-C on the ABC drug transporters (P-gp, MRP1 and MRP2) was evaluated by substrate accumulation assay, using fluorescence spectroscopy technique. Our results showed that rhinacanthin-C at 0.1 mu M for 6-h treatment period could increase intracellular accumulation of transporter substrates in MCF-7 cells [i.e., CDCF by 1.65 fold (MRP2)] as well as in MCF-7/DOX resistance cells [i.e., CDCF by 1.18 fold (MRP2) and calcein by 1.38 fold (P-gp)]. In conclusion, rhinacanthin-C could enhance doxorubicin cytotoxicity through interference on MRP2 and P-gp functions. Consequently, intracellular doxorubicin accumulation in the cells increased up to its cytotoxic level. Another potential mechanism of the synergy between rhinacanthin-C and doxorubicin would be investigated further.