Effects of phenanthridine derivatives on melanogenic protein in B16 cells
According to the regulatory networks underlying melanogenesis, upregulation of Wnt/β -catenin signalling promotes the expression of a critical melanogenic protein, melanocyte-inducing transcription factor (MITF), which subsequently activates TYRP (tyrosinase-related protein) 1 and 2 and TYR (tyrosinase) genes and finally increases melanogenesis (Fig. 1d) (Liu et al., 2019; Manga, Sheyn, Yang, Sarangarajan & Boissy, 2006). MITF controls the production of the pigment melanin, and TYRP1 and 2 are two transmembrane proteins. TYRP-1 has been reported to influence TYR activity via forming a complex with it and/or stabilizing it. TRP-2 functions as a dopachrome tautomerase downstream of TYR in the melanogenic pathway and regulates the quantity and quality of melanin produced during melanin biosynthesis. (Wang et al., 2017). As phenanthridine derivatives could significantly increase melanin synthesis, it is necessary to further explore whether and how phenanthridine derivatives affect the expression of MITF, TYRP 1 and 2, and TYR (tyrosinase). As shown in Fig. 1e, after 48 h of treatment with HCJA121 and HCJA404 (20 μ M), the MITF gene levels were significantly enhanced in the treatment groups. Simultaneously, the levels of the TYRP-1 , TYRP-2 , and TYR genes were significantly increased in the administration groups . Particularly, HCJA121 treatment resulted in the strongest activation of MITF, TYRP-1, and TYR, the levels of which were several-fold higher than those of the control. To further elucidate the effect of HCJA121 on the expression of melanogenic proteins (MITF TYRP-1, MITF TYRP-2, and MITF TYR) at the protein level, western blotting was performed using lysates of B16 murine melanoma cells that were treated with different doses of HCJA121. As presented in Fig. 1f, compared to the control treatment, HCJA121 treatment clearly increased the expression of MITF TRP-1 and TRP-2 by in a dose-dependent manner in the B16 cells. These results indicated that HCJA121 and HCJA404 could simultaneously upregulate all key melanogenic proteins, which suggested that these compounds may be upstream regulators of the MITF-TYRP/TYR melanogenic pathway.