Carnosic acid effectively inhibits metastasis of B16F10 melanoma cells

Abstract

Carnosic acid is a natural benzenediol abietane diterpene found in rosemary and common sage and reported to exhibit anti-inflammatory, antioxidant, and anticarcinogenic activities. The present study examined the effects of carnosic acid on the metastatic characteristics of B16F10 cells. Results from transwell migration assays revealed that carnosic acid inhibited B16F10 migration in a dose-dependent manner. Western blot analysis of cell conditioned media showed that carnosic acid significantly suppressed the secretion of MMP-9, TIMP-1, uPA, and VCAM-1. However, the secretion of TIMP-2 was significantly increased in cells treated with 10 μmol/L of carnosic acid. Moreover, carnosic acid suppressed the expression of mesenchymal markers such as snail, Slug, vimentin, and N-cadherine and increased that of E-cadherin, an epithelial marker. We previously noted that chronic consumption of a high-fat diet (HFD) increased solid tumor growth and lymph node (LN) metastasis in C57BL/6 mice injected with B16F10. To assess the effects of dietary carnosic acid on HFD-stimulated LN metastasis, male C57BL/6 mice were fed a control diet (CD, 10 kcal% fat) or a HFD (60 kcal% fat) containing 0, 0.02, or 0.04% of carnosic acid for 21 weeks. B16F10 was subcutaneously injected 16 weeks after the initiation of feeding. The body weight and solid tumor growth were significantly increased in the HFD group as compared to the CD group, and these increases were suppressed by dietary carnosic acid. Lean body mass and body fat mass were estimated by dual energy X-ray absorptiometry. The body fat mass was tremendously increased in HFD-fed mice, and this increase was substantially decreased in a carnosic acid-dose dependent manner. A small but significant increase in lean body mass was noted in HFD-fed mice, which was not altered by dietary carnosic acid. Fasting blood glucose was markedly increased in HFD-fed mice, which was suppressed by 0.04% dietary carnosic acid. The tumor was resected 3 weeks after the B16F10 injection. Immunohistochemical staining of tumor tissues revealed that the expression of Ki67, platelet endothelial cell adhesion molecule-1, lymphatic vessel endothelial hyaluronan receptor-1, phospho-tyrosine phosphatase (a leukocyte marker), and vimentin was markedly increased in the HFD group as compared to the CD group, and these increases were blocked by carnosic acid. Two weeks after the tumor resection, LN metastasis was increased in mice fed HFD, and this increase was markedly suppressed by carnosic acid. In the present study, we demonstrate that dietary carnosic acid potently inhibits HFD-induced solid tumor growth and LN metastasis of B16F10 as well as tumor angiogenesis and lymphangiogenesis. Carnosic acid effectively suppresses HFD-induced body fat gains and fasting hyperglycemia, which may be parts of the mechanisms by which carnosic acid suppresses HFD-stimulated tumor progression.