Protocatechuic Aldehyde Inhibits α-MSH-Induced Melanogenesis in B16F10 Melanoma Cells via PKA/CREB-Associated MITF Downregulation

Abstract

Protocatechuic aldehyde (PA) is a naturally occurring phenolic compound that is a potent inhibitor of mushroom tyrosinase. However, the molecular mechanisms of the anti-melanogenesis activity of PA have not yet been reported. The aim of the current study was to clarify the melanogenesis inhibitory effects of PA and its molecular mechanisms in murine melanoma cells (B16F10). We first predicted the 3D structure of tyrosinase and used a molecular docking algorithm to simulate binding between tyrosinase and PA. These molecular modeling studies calculated a binding energy of -527.42 kcal/mol and indicated that PA interacts with Cu400 and 401, Val283, and His263. Furthermore, PA significantly decreased α-MSH-induced intracellular tyrosinase activity and melanin content in a dose-dependent manner. PA also inhibited key melanogenic proteins such as tyrosinase, tyrosinase-related protein 1 (TRP-1), and TRP-2 in α-MSH-stimulated B16F10 cells. In addition, PA decreased MITF expression levels by inhibiting phosphorylation of cAMP response element-binding protein (CREB) and cAMP-dependent protein kinase A (PKA). These results demonstrate that PA can effectively suppress melanin synthesis in melanoma cells. Taken together, our results show that PA could serve as a potential inhibitor of melanogenesis, and hence could be explored as a possible skin-lightening agent.

Keywords: anti-melanogenesis activity; melanoma cells; molecular mechanisms; protocatechuic aldehyde.

Figure 1

Chemical structure of protocatechuic aldehyde (PA) (A) and the specific interactions between PA and tyrosinase after automated docking of PA to the tyrosinase enzyme binding site. Predicted 3D structure of the tyrosinase (Protein Data Bank; PDB code: 2Y9X)–arbutin complex and 2D diagram (B). Predicted 3D structure of the tyrosinase (PDB 2Y9X)–PA complex and 2D diagram (C). Binding energy values were obtained from the Discovery Studio (DS) 3.0 binding energy calculation program.

Figure 2

Effects of PA on cellular melanin synthesis and intracellular tyrosinase activity in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. Cells were treated with the indicated concentrations of PA for 72 h and then cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (A). The relative cellular melanin content (B) and intracellular tyrosinase activity (C) were measured at 72 h after treatment. Cells were exposed to 100 nM α-MSH in the presence of PA at the indicated concentrations or 50 µg/mL arbutin. Values are expressed as means ± SDs of triplicate experiments (n = 3). Note: ^## p < 0.01 compared to the untreated control group; * p < 0.05 and ** p < 0.01 compared to the α-MSH only group.

Figure 3

Inhibitory effects of PA on the expression levels of proteins related to melanogenesis in α-MSH-stimulated B16F10 cells. Cells were exposed to 100 nM α-MSH in the presence of PA at the indicated concentrations or 50 µg/mL arbutin. The expression levels of MITF, tyrosinase, TRP-1, and TRP-2 were measured using Western blot analysis and quantified using Image J. Values are expressed as means ± SDs of triplicate experiments (n = 3). Note: ^## p < 0.01 compared to the untreated control group; * p < 0.05 and ** p < 0.01 compared to the α-MSH group.

Figure 4

PA suppresses the PKA and CREB signaling pathways in α-MSH-stimulated B16F10 cells. Cells were exposed to 100 nM α-MSH in the presence of PA at the indicated concentrations or 50 µg/mL arbutin. The protein expression levels of p-PKA/PKA and p-CREB/CREB were determined using Western blot analysis and quantified using Image J. Values are expressed as means ± SDs of triplicate experiments (n = 3). Note: ^## p < 0.01 compared to the untreated control group; ** p < 0.01 compared to α-MSH group.