Figure 1 . Identification of new phenanthridine
Wnt/β -catenin signalling agonists, HCJA121 and HCJA404, and their
effects on melanogenesis in B16-F10 cells. (a) Structures of HCJA121 and
HCJA404. (b) The levels of melanin in phenanthridine-treated B16-F10
cells. B16 cells were seeded in a 6-well plate and incubated with Wnt3a
before being treated with 50 µ M of HCJA121, HCJA404, and
compounds 1 –6 for 48 h; 8-MOP served as a positive
control and DMSO as blank control. The optical density was detected at
405 nm. The melanin content was calculated relative to that in the
control group and was corrected on the basis of the protein
concentrations, considering the control group as representing 100%
melanin content. (c) HCJA121 and HCJA404 (20 μ M) significantly
increased tyrosinase activity in B16-F10 cells. The cellular tyrosinase
activity was estimated by measuring the rate of
ʟ-3,4-dihydroxyphenylalanine (ʟ-DOPA) oxidase activity. The tyrosinase
activity of each sample was calculated and corrected for the
concentrations of proteins, considering the control group as
representing 100% activity. (d) The MITF regulatory network
underlying melanogenesis. (e) HCJA121 and HCJA404 increased the
expression of the MITF , TYR , and TYRP 1 and2 genes. B16 cells pre-treated with HCJA121, HCJA404, or 8-MOP
for 2 h were incubated with Wnt3a-conditioned medium containing the same
dose of compounds for an additional 48 h. MITF , TYR , andTYRP 1 and 2 were analysed by qPCR. The results were
normalized to β -actin expression. (f) Compound HCJA121 increases
the levels of the MITF, TYR TYRP 1, and TYR TYRP 2 proteins. B16 cells
treated with HCJA121 for 48 h were fractionated as described in the
Methods section and subjected to western blot analysis. β -actin
served as the loading control. The immunoblots were quantified by
densitometry, and the intensity values were normalized toβ -actin. The values are indicated beneath each band. The data
shown represent means ± standard deviations (S.E.M) from five
independent experiments (n=5). Significantly different as indicated
(*P < 0.05, **P < 0.01, ***P< 0.001, and ****P < 0.0001).