Synergistic effect of thymoquinone and melatonin against breast cancer implanted in mice

Aim: To test the anticancer potential of a combination of thymoquinone (TQ) and melatonin (MLT) against breast cancer implanted in mice. Materials and Methods: The antiproliferative activity of TQ, MLT, and their combination was tested against mouse epithelial breast cancer cell line (EMT6/P) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The combination index (CI) was calculated using isobolographic method. Balb/C mice were transplanted with EMT6/P cell line and in vivo antitumor activity was assessed for TQ, MLT, and their combination. Changes in tumor size were measured for each treatment. Histological examination of tumor sections was performed using standard hematoxylin/eosin staining protocol and TUNEL colorimetric assay was used to test the apoptosis induction ability for all treatments. Immunohistochemical staining was used to detect vascular endothelial growth factor (VEGF) expression in tumor section and ELISA was used to measure serum levels of interferon gamma (INF-gamma) and interleukin-4. Serum levels of the liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were used as biomarkers of hepatotoxicity of the combination therapy. Results: Synergistic anticancer effect was observed between TQ and MLT with CI value of 0.552. The combination of TQ and MLT caused a significant decrease in tumor size with a percentage cure of 60%. The combination therapy induced extensive necrosis, increased apoptosis rate, and decreased VEGF expression in tumor sections. Serum levels of INF-gamma were increased in mice treated with combination therapy and AST and ALT levels were close to their normal values. Conclusions: The combination TQ and MLT act synergistically to inhibit breast cancer implanted in mice. The anticancer effect of this combination is mediated by induction of apoptosis, angiogenesis inhibition, and activation of T helper 1 anticancer immune response.