Synergistic breast cancer suppression efficacy of doxorubicin by combination with glycyrrhetinic acid as an angiogenesis inhibitor

Background: Therapeutic regimens of breast cancer treatment are increasingly inclined to adopt combination strategy based on the broad spectrum antitumor effect of doxorubicin (Dox). Currently, combination therapy comprises of conventional anti-cancer drugs and angiogenesis inhibitors have been corroborated as an effective approach in cancer treatment. Purpose: We explored the ability of a natural anti-angiogenic compound glycyrrhetinic acid (GA), derived from an edible-medicinal herb licorice, to enhance the breast cancer suppression effect of Dox. Study design: The drug ratio of GA and Dox with synergistic anticancer effect against MCF-7 cells was optimized by combination index (CI) value in vitro, followed by evaluation of the improved anticancer effects and reduced side-effects of this combination in vitro and in vivo. Methods: Cell viability was measured by MTT assay. Analyses of mitochondrial membrane potential and cell apoptosis on MCF-7 cells were performed by JC-1 dye and Annexin V-FITC/PI assays. The cellular accumulation of Dox when combined with GA was evaluated. Levels of apoptosis-related proteins in MCF-7 cells were measured by Western blot analysis. Synergistic anti-angiogenic effects on HUVECs were evaluated. A breast cancer mouse model was established to investigate the anti-tumor effects in vivo. Results: Based on the optimization by CI value, Dox and GA at 1:20 molar ratio was chosen as the optimal combination drug ratio that exhibited synergistic effect against MCF-7 breast cancer cells. In addition, the combination of GA and Dox exhibited significantly enhanced cytotoxicity, apoptosis, and loss of mitochondrial membrane potential via the upregulation of a mitochondrial-dependent apoptosis pathway against MCF-7 cells. Interestingly, the addition of GA increased the intracellular accumulation of Dox in MCF-7 cells. Moreover, VEGF-induced HUVECs proliferation, migration, and tube formation were strongly inhibited by Dox when used with GA via the significant down-regulation of VEGFR2-mediated pathway, indicating that the combination of Dox and GA could exhibit ideal synergistic anti-angiogenesis effect. Expectedly, the enhanced anti-tumor efficacy of Dox and reduced Dox-induced cardiotoxicity when used in combination with GA were evident in a mouse breast tumor model. Conclusions: These findings support that the combination of Dox with GA is a novel and promising therapeutic strategy for the treatment of breast cancer.