Inhibitory effect of capsaicin on B16-F10 melanoma cell migration via the phosphatidylinositol 3-kinase/Akt/Rac1 signal pathway Dong-Hoon Shin,1 Ok-Hee Kim,2 Hye-Seung Jun,2 and Mi-Kyung Kang3
1Department of General Surgery, Kosin University College of Medicine, Busan 602-739, Korea.3Institute for Medical Science, Kosin University College of Medicine, Busan 602-739, Korea .2National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul 122-704, Korea.
Abstract Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the major pungent ingredient of red pepper, has been reported to possess anti-carcinogenic and anti-mutagenic activities. In this study, the anti-migration activity of capsaicin on highly metastatic B16-F10 melanoma cells was investigated. Capsaicin significantly inhibited the migration of melanoma cells without showing obvious cellular cytotoxicity at low doses. This effect correlated with the down-regulation of phosphatidylinositol 3-kinase (PI3-K) and its downstream target, Akt. Although B16-F10 cell migration was increased by the PI3-K activator through the activation of Akt, these PI3-K activator-induced phenomena were attenuated by capsaicin. Moreover, capsaicin was found to significantly inhibit Rac1 activity in a pull-down assay. These results demonstrate that capsaicin inhibits the migration of B16-F10 cells through the inhibition of the PI3-K/Akt/Rac1 signal pathway. The present investigation suggests that capsaicin targets PI3-K/Akt/Rac1-mediated cellular events in B16-F10 melanoma cells. Consequently, capsaicin administration should be considered an effective approach for the suppression of invasion and metastasis in malignant melanoma chemotherapy.
Discussion Capsaicin is an active component of red peppers of the genus Capsicum (Cordell and Araujo, 1993). It has been reported to possess inhibitory effects on colon and gastric carcinogenesis (Jung et al., 2001; Kim et al., 2004), and was also found to inhibit carcinogeneisis in mouse skin (Park and Surh, 1997; Park et al., 1998). Recently, Mori et al. (2006) reported that capsaicin could be effective against prostate cancer by inhibiting androgen-independent growth. Furthermore, capsaicin had strong apoptotic activity in B16-F10 cells via the down-regulation of Bcl-2 (Jun et al., 2007). In the present study, the anti-migratory properties of capsaicin in highly metastatic B16-F10 melanoma cells were investigated. Capsaicin efficiently suppressed the migration of B16-F10 cells without obvious cellular cytotoxicity (Figure 1). These results suggest that capsaicin could have an effective role in the management of melanoma cancer patients.PI3-K is considered to be one of the main intracellular factors responsible for the transmission of cell migration signals (Krasilnikov, 2000). In addition, Akt, one of the major downstream targets of PI3-K, promotes cell motility and migration in tumor cells (Morales-Ruiz et al., 2000). It has been indicated that the PI3-K/Akt signaling pathway is required for the invasion and metastasis of cancer cells. Thus, we hypothesized that the inhibition of the PI3-K/Akt pathway was the underlying mechanism responsible for the inhibitory effect of capsaicin on the migration of B16-F10 cells. This hypothesis is supported by the observation that capsaicin suppresses the phosphorylation of Akt (Figure 2). In addition, capsaicin inhibited migration and Akt phosphorylation in B16-F10 cells; these processes are activated by a PI3-K activator (Figure 3). These results demonstrate that capsaicin is an inhibitor of the PI3-K/Akt signaling pathway in B16-F10 melanoma cells. Our findings are basically consistent with previous reports on the inhibition of the PI3-K pathway and the suppression of cell migration by other chemopreventive agents such as red wine polyphenols (Iijima et al., 2002), sulindac sulfide, and caffeic acid phenethyl ester (Shigeoka et al., 2003).Rac1 was reported to act as a downstream effector of PI3-K in several growth factor-stimulated pathways (Higuchi et al., 2001), and to induce invasion and metastasis in cancer cells (Price and Collard, 2001). Conversely, several studies suggest that Rac1 acts upstream of PI3-K to increase cellular motility and invasiveness in T47D mammary carcinoma cells and H-Ras-activated MCF-10A breast epithelial cells (Keely et al., 1997; Shin et al., 2005). Rac1 activation also played a critical role in the migration of cancer cells (Stem et al., 1998). Although we did not address the signaling pathways between PI3-K and Rac1, our results showing inhibition of Rac1 activation following capsaicin treatment in B16-F10 cells are in line with these observations (Figure 4). This inhibition might lead to the inactivation of Akt and may, in turn, affect the migration of B16-F10 cells. These results suggest that, in addition to its inhibitory effect on the PI3-K/Akt signaling pathway, capsaicin might also inhibit cell migration by down-regulating Rac1. However, a previous study reported that activation of PI3-K/Rac1 signaling inhibits cell migration in human melanoma cells (Kallergi et al., 2007). Therefore, the functions of Rac1 on migration are at least in part cell type- and cell substrate-dependent. However, further studies are needed to investigate whether capsaicin-induced Rac1 activation is PI3-K-dependent in B16-F10 melanoma cells.It is well known that capsaicin is a specific agonist of the transient receptor potential vanilloid 1 (TRPV1) family of ion channels (Caterina et al., 1997). Several reports have indicated that the anti-cancer effects of capsaicin are not to activate TRPV1 (Raisinghani et al., 2005), but to inhibit plasma membrane NADH oxidase (Morre et al., 1995). Moreover, alternative anti-cancer mechanisms of capsaicin have been associated with the production of reactive oxygen species (Qiao et al., 2005) and the reduction of TNF-α (Park et al., 2004). In a review, Surh (2002) indicated that capsaicin could mediate suicide in human skin cancer cells via the excessive generation of reactive oxygen species (ROS), according to the inhibition of mitochondrial and plasma membrane electron transport systems. Therefore, further study is required to determine whether the inhibition of migration by capsaicin is due to the function of capsaicin-sensitive TRPV1 channels and the availability and efficiency of antioxidant capacity.Collectively, we found that non-toxic levels of capsaicin could efficiently suppress the migration of B16-F10 melanoma cells; this suppression is achieved through the inhibition of the PI3-K/Akt signal pathway and Rac1 activity. Thus, the present study provides novel insights into the molecular mechanisms of capsaicin. Furthermore, anti-migration of highly metastatic B16-F10 melanoma cells by capsaicin can be considered an effective approach for the suppression of cancer invasion and metastasis.