rhinacanthin-c and Breast-Neoplasms

Rhinacanthin-C is a natural bioactive naphthoquinone ester with potential chemotherapeutic value in cancer treatment. In this study, we investigated its apoptotic induction ability and the involved mechanisms through the mitogen-activated protein kinases (MAPK) and protein kinase B/glycogen synthase kinase-3β/nuclear factor erythroid 2-related factor 2 (Akt/GSK-3β/Nrf2) signaling pathways in doxorubicin-resistant breast cancer MCF-7 (MCF-7/DOX) cells. Our 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that rhinacanthin-C (3-28 µM) significantly decreased the viability of MCF-7/DOX cells and potentiated hydrogen peroxide cytotoxicity. This naphthoquinone was able to increase intracellular reactive oxygen species (ROS), as measured by the 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay. This compound increased the number of apoptotic cells by elevating the ratio of apoptotic checkpoint proteins Bax/Bcl-2 and by decreasing the expression of poly(ADP-ribose) polymerase (PARP) protein. Furthermore, Western blotting analyses showed that treatment with rhinacanthin-C (3-28 µM) for 24 h significantly decreased the expression levels of the phosphorylated forms of MAPK proteins (i.e., extracellular signal regulated protein kinase 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38), Akt, GSK-3β and Nrf2 proteins in MCF-7/DOX cells. Inhibition of the Akt/GSK-3β/Nrf2 pathway led to a significant reduction in heme oxygenase-1 (HO-1) and reduced nicotinamide adenine dinucleotide phosphate (NADP)(H): quinone oxidoreductase 1 (NQO1) proteins. These findings suggested that rhinacanthin-C was able to induce apoptosis in MCF-7/DOX cells through increased ROS production and suppression of the cell survival systems mediated by the MAPKs and Akt/GSK-3β/Nrf2 signaling pathways.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Cell Survival; Doxorubicin; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Female; Humans; MAP Kinase Signaling System; MCF-7 Cells; Naphthoquinones; Oxidative Stress; Reactive Oxygen Species

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 non-cytotoxic concentration (0.1µM), 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 0.1µM 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.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Breast Neoplasms; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Synergism; Humans; Intracellular Space; MCF-7 Cells; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Naphthoquinones