The in vitro and in vivo whitening activity of ergothioneine by autophagy induction through organic cation transporter 1 into melanocytes and keratinocytes

Abstract

Ergothioneine (EGT) has been utilized as a cosmetic ingredient, but its whitening potency has to be examined. We probed skin whitening mechanism of low concentration of EGT (0–500 nM) through autophagy induction in melanoma B16F10 and keratinocyte HaCaT cells, and suppression of ROS-intervened UVA-irradiated-α-MSH or UVB-irradiated ET-1 expression via Nrf2 pathway in HaCaT cells. EGT-incited autophagy in B16F10 and HaCaT cells was evidenced by increased LC3-II/p62 accretion, ATG4B inhibition, ATG5/ATG7 expression, Beclin-1/Bcl-2 ratio, AMPK/mTOR/ULK1 pathway, autophagosome GFP-LC3 puncta, and autolysosome AVO formation. Furthermore, EGT decreased melanosome gp100 expression and melanin accumulation by instigating autophagy in B16F10 and HaCaT cells. TEM microscopy revealed that EGT amplified melanosome-engulfing autophagosomes and autolysosomes. Further, autophagy inhibitor 3-MA/CQ or LC3 silencing attenuated EGT-mediated anti-melanogenesis in B16F10 cells and melanin degradation in melanin-feeding HaCaT cells. Interestingly, mTOR silencing triggered antimelanogenesis in B16F10 cells or enhanced melanin degradation in melanin-feeding HaCaT cells through autophagy. Notably, EGT provoked its transporter OCTN-1 expression, whereas OCTN-1 silencing prevented EGT-induced autophagy in HaCaT and B16F10 cells. In vivo zebrafish model confirmed that EGT triggered antimelanogenesis and melanin degradation by autophagy induction through OCTN-1. Additionally, EGT provoked autophagic p62-mediated Keap-1 degradation and then facilitated antioxidant Nrf2 activation in HaCaT cells. EGT triggered antimelanogenesis by inhibiting ROS-mediated UVA (3 J/cm²)-irradiated α-MSH expression and UVB (80 mJ/cm²)-irradiated ET-1 expression by upregulating Nrf2-transcribed antioxidant proteins HO-1, NQO-1, and γ-GCLC expression in HaCaT cells, whereas OCTN-1 or Nrf2 silencing reversed these effects. EGT could be utilized as a skin-whitening ingredient in the preparation of topical cosmeceutics.