Modulating the <scp>BI</scp> ‐1( <scp>TMBIM6</scp> )/ <scp>AKT</scp> Signaling Pathway by Licochalcone D Treatment Inhibits Human Melanoma Cell Growth

Skin cancer is a common type of cancer worldwide with increasing incidence and mortality rates. Phytochemicals, active compounds from plants, are known to exhibit diverse physiological activities with benefits of low cytotoxicity and cost. In this study, we aimed to assess the effects of Licochalcone D (LicoD) derived from Glycyrrhiza inflata on human melanoma cell proliferation and investigate its new underlying mechanisms. To assess the anticancer effects of LicoD on melanoma cells, we conducted MTT and soft agar assay, flow cytometry analysis, Western blots as well as TPA-induced inflammatory mouse model and immunohistochemical analysis. To further uncover the new mechanisms, we assessed MS/MS-based proteomic analysis and verified by knockdown and proliferation assays. LicoD (2.5, 5, and 10 μΜ) dose-dependently inhibited cell growth, induced G2/M phase arrest of cell cycle, and triggered apoptosis in SK-MEL-5 and SK-MEL-28 cells together with regulating the related markers. Importantly, we newly discovered that LicoD suppressed the expression of Bax inhibitor (BI)-1(TMBIM6), an upstream regulator of AKT signaling pathway, as confirmed by informatic analysis and Western blotting. Moreover, we demonstrated by shRNA-mediated knockdown of BI-1(TMBIM6) in melanoma cells that BI-1 plays a critical role in cell proliferation and anchorage-independent growth. Rescue assays using BIA further confirmed the functional contribution of BI-1 to the effects of LicoD. In addition, topical treatment of LicoD reduced epidermal hyperplasia and the expression of pAKT, BI-1 and COX-2 in TPA-induced inflammatory skin tissues. Taken together, our findings provide novel evidences that LicoD exerts anti-melanoma effects by down-regulation of BI-1(TMBIM6)/AKT signaling pathways in vitro and in vivo. These findings suggest essential insights for the future development of strategies in skin cancer prevention and treatment.