Correlation between melanogenesis and Wnt activation
According to the results of the aforementioned bioassays, we concluded that phenanthridine derivatives could truly promote melanogenesis and significantly upregulate the expression of melanogenic proteins. Because these compounds have been demonstrated to be potent Wnt agonists and because the Wnt signalling pathway plays an important role in melanogenesis regulation, the correlation between melanogenesis and Wnt activation were further investigated. To investigate whether Wnt was upregulated while melanogenesis was promoted, the effect of HCJA121 and HCJA404 on endogenous Wnt target genes was initially examined in B16 cells. The results indicated that the compounds induced a dose-dependent increase in the expression of AXIN2 and DKK1 (Hoverter & Waterman, 2008) in B16 cells and that there was a positive correlation between Wnt activation and melanogenesis promotion. In addition, since these compounds are Wnt ligand-dependent agonists, we hypothesized that they would be more efficient in B16 cells treated with Wnt3a-conditioned media than in B16 cells that were not been treated with Wnt3a-conditioned media. The data showed that HCJA121 and HCJA404 increased the levels of the melanogenesis gene to a lesser extent in the cells without Wnt3a-conditioned media than in those treated with Wnt3a-conditioned media (Fig. 2a), which suggested that these derivatives increase melanogenesis by activating Wnt.
To further confirm that phenanthridine derivatives could promote melanin synthesis through specific activation of Wnt, Xav939, a reported Wnt inhibitor 30 (Huang et al., 2009), was added to drug-treated B16 cells. XAV939 treatment decreased the expression of melanogenesis markers (MITF , TYR , TRP1 , andTRP2 ) (Fig. 2b), antagonizing phenanthridine-derivative-mediated promotion of melanogenesis and tyrosinase activity (Fig. 2c). These preliminary results indicated that HCJA121 and HCJA404 truly promote melanogenesis by activating the Wnt signalling pathway.