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.