Synergized Anticancer Activity of Honeybee Royal Jelly and Thymoquinone Against Aggressive Human Breast Cancer

Abstract

Thymoquinone, the main constituent of Nigella sativa seeds and royal jelly (RJ), the honeybee secretion fed to queens, are effective against cancer. The anticancer activity of the combination of TQ and RJ against breast cancer is still unknown. Here, we investigated the effects of TQ alone, RJ alone, and combinations on the viability and cell cycle regulation in MDA-MB-231 human metastatic breast cancer cells and determined the cell death mechanism. Our study is the first to report prominent anticancer synergistic effects of the combination of TQ and RJ against MDA-MB-231 breast cancer cells. TQ alone inhibited cell viability in a dose-dependent manner at concentrations below and above the IC50, which was established at 19 µM. A dose of 15 µM of TQ caused a significant increase in the pre-G1 population, while a more pronounced effect was observed in response to TQ and RJ combination. Royal jelly exhibited relatively nontoxic effects against MDA-MB-231 cells and FHS 74 Int small intestinal cells at concentrations below 5 µg/ml. High doses of RJ (200 µg/ml) had greater toxicity against MDA-MB-231 cells. Interestingly, the combination of both compounds synergistically inhibited cell viability and cell death was most pronounced in response to 15 µM TQ and 5 µg/ml RJ. Immunofluorescent staining showed that TQ was the main inducer of caspase 3-dependent apoptosis when applied alone and in combination with RJ. In contrast, no significant regulation of Ki67 expression was observed, indicating that the decrease in cell viability was due to apoptosis induction rather than to the inhibition of cell proliferation. In summary, RJ synergizes with TQ to inhibit the viability of MDA-MB-231 metastatic breast cancer cells and thus could confer an advantage for cancer therapy.