Abstract

Natural and synthetic (−)-kusunokinin inhibited breast cancer, colon cancer and cholangiocarcinoma cells at the G2/M phase and induced apoptosis. However, there is no report on the action and adverse effects of (−)-kusunokinin in animal models. In this study, we investigated the cytotoxic effect of (−)-kusunokinin from Piper nigrum on cancer cells. NMU-induced rat mammary tumors, an ER positive breast cancer model, were treated with (−)-kusunokinin. Proteins of interest related to cell cycle, angiogenesis, migration and signaling proteins were detected in tumor tissues. Results showed that (−)-kusunokinin exhibited strong cytotoxicity against breast, colon and lung cancer cells and caused low toxicity against normal fibroblast cells. For in vivo study, 7.0 mg/kg and 14.0 mg/kg of (−)-kusunokinin reduced tumor growth without side effects on body weight, internal organs and bone marrow. Combination of (−)-kusunokinin with a low effective dose of doxorubicin significantly inhibited tumor growth and provoked cell death in cancer tissues. Mechanistically, 14.0 mg/kg of (−)-kusunokinin decreased cell proliferation (c-Src, PI3K, Akt, p-Erk1/2 and c-Myc), cell cycle (E2f-1, cyclin B1 and CDK1), and metastasis (E-cadherin, MMP-2 and MMP-9) proteins in tumor tissues, which supports its anticancer effect. We further confirmed the antimigration effect of (−)-kusunokinin; the results show that this compound inhibited breast cancer cell (MCF-7) migration in a dose-dependent manner. In conclusion, the results suggest that 14 mg/kg of (−)-kusunokinin inhibited tumors through the reduction of signaling proteins and their downstream molecules. Therefore, (−)-kusunokinin becomes an intriguing candidate for cancer treatment as it provides a strong potency in cancer inhibition.