1. Chemical Structure and Cytotoxicity of Ganodermanondiol Isolated from Ganoderma lucidum on B16F10 Melanoma Cells

Previous studies of G. lucidum have established that it contains more than 300 biologically active compounds such as triterpenoids, polysaccharides, and steroids. Initially, the ethanol (EtOH) extract of dried G. lucidum was suspended in water and partitioned successively with ethyl acetate (EtOAc). Using bioassay-guided fractionation, the EtOAc-soluble fraction was subjected to repeated column chromatography to afford ganodermanondiol. The ganodermanondiol was identified by comparing its spectroscopic nuclear magnetic resonance (NMR) data with those reported in the literature, and the results showed they were identical. Ganodermanondiol has a triterpenoid structure and is one of the major active components of G. lucidum. It has various biological effects, including inhibitory activity against human immunodeficiency virus (HIV)-1 protease, anti-complement activity, and hepatoprotective action. However, other biological activities, especially the skin-related activity of ganodermanondiol remain to be elucidated.

Therefore, in this study, we determined the cytotoxic effects of ganodermanondiol on B16F10 melanoma cells treated with indicated concentrations for 24 h. Concentrations of 2.5, 5, 7.5, and 10 μM ganodermanondiol showed no cytotoxic effects in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Therefore, the B16F10 cells were treated with ganodermanondiol at concentrations of 2.5, 5, 7.5, and 10 μM in subsequent experiments.

  1. Effects of Ganodermanondiol on Melanin Contents and Tyrosinase Activity of B16F10 Cells

Melanin plays a pivotal role in protecting human skin, especially keratinocytes, against environmental damage including ultraviolet (UV) radiation, heat, and solar radiation. Melanin is the primary determinant of skin color variation due to the melanin content of the skin of the human body. Melanin content levels correlate directly with tyrosinase’s activity, an enzyme that regulates melanin synthesis. Tyrosinase inhibitors are used as human skin-whitening agents based on their melanogenesis inhibition.

Therefore, this study demonstrated the mechanisms of melanogenesis inhibition by ganodermanondiol isolated from G. lucidum in B16F10 cells treated with various concentrations (2.5, 5, 7.5, and 10 μM) for 72 h. Treatment with ganodermanondiol significantly reduced the melanin content of B16F10 cells in a dose-dependent manner. In addition, we also examined the effects of ganodermanondiol on tyrosinase activity, which decreased B16F10 cells after treatment with ganodermanondiol. These findings indicate that ganodermanondiol significantly inhibited the production of melanin and tyrosinase activity.

  1. Effects of Ganodermanondiol on Cellular Melanogenesis-Related Proteins and MITF Protein Expression in B16F10 Cells

Tyrosinase-related enzymes, including tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2, are key factors in melanin biosynthesis. These enzymes are important for regulating melanogenesis pathways and constitute a specific family of membrane proteins. Importantly, tyrosinase protein is a catalyst for the rate-limiting reaction in the melanogenesis pathways. TRP-1 and TRP-2 are believed to determine the shape of melanosomes, which are the structures in melanocytes where melanin is produced.

In our study, to elucidate the mechanisms underlying the inhibition of melanin production and tyrosinase activity by ganodermanondiol, we examined the effects of ganodermanondiol on cellular tyrosinase protein, as well as TRP-1 and TRP-2 expression, which are all associated with melanogenesis. Western blot analysis showed that ganodermanondiol inhibited the expression of tyrosinase protein, TRP-1, and TRP-2 in B16F10 cells. The induction of these proteins by α-melanocyte-stimulating hormone (α-MSH) was markedly decreased by pretreatment with ganodermanondiol. These findings indicate that the inhibitory action of ganodermanondiol on melanogenesis is associated with the suppression of the expression of cellular melanogenesis-related proteins, including tyrosinase protein, TRP-1, and TRP-2.

Various cellular transcription factors are involved in melanogenesis, and the most notable is MITF. Generally, melanogenesis directly leads to increased MITF expression by activating tyrosinase enzymes, TRP-1 and TRP-2, and tyrosinase activity, resulting in increased melanin synthesis. Moreover, the downregulation of MITF expression through tyrosinase, TRP-1, and TRP-2 is the most significant mechanism underlying the anti-melanogenic effects of inhibitory agents.

Therefore, we investigated whether ganodermanondiol-regulated melanogenesis occurs via a MITF expression pathway. To assess the correlation of MITF regulation with a reduction in cellular melanogenesis-related protein expression by ganodermanondiol treatment, we investigated MITF protein expressions in B16F10 cells using an anti-MITF antibody. MITF protein expression was decreased by pretreatment with ganodermanondiol in α-MSH-stimulated B16F10 cells. Therefore, we suggest that the reduction in melanogenesis-related protein expression, including tyrosinase protein, TRP-1, and TRP-2 by ganodermanondiol treatment, is mediated by the MITF pathway in B16F10 cells.

  1. Effects of Ganodermanondiol on cAMP Response Element Binding Protein (CREB) Phosphorylation in B16F10 Cells

As described above, ganodermanondiol significantly inhibited tyrosinase, TRP-1, and TRP-2 protein expression by downregulating MITF. MITF primarily regulates tyrosinase, TRP-1, and TRP-2 expression. It has been reported that MITF downregulation is related to suppressing the cAMP-dependent melanogenic pathway stimulated by a-MSH in melanoma cells. The α-MSH binds to and stimulates the melanocortin-1 receptor (MC1R), which upregulates intracellular cAMP levels and induces melanogenesis through cellular signaling in melanocytes. cAMP activates protein kinase A (PKA), which subsequently phosphorylates CREB protein, thereby upregulating MITF expression. The MITF gene targets cAMP signaling induced by α-MSH, which predominantly regulates tyrosinase and TRP-1 protein expression. In the cAMP-dependent signaling pathway, the dual phosphorylation of the cAMP response element binding protein (CREB), mediated by the kinase activities of ribosomal S6 kinase (RSK) and PKA, has been found to activate MITF transcription.

Furthermore, it has been reported that suppression of the cAMP-dependent signaling pathway causes MITF degradation in α-MSH-stimulated melanoma cells. Therefore, we hypothesized that GN-induced MITF downregulation is involved in the cAMP signal transduction cascade. As expected, the phosphorylation of CREB proteins was significantly decreased by pretreatment with ganodermanondiol in the α-MSH-stimulated B16F10 cells in a dose- and time-dependent manner. Although PKA activation and cAMP levels were not determined in the present study, these results suggest that the inhibitory effect of ganodermanondiol contributed to the reduction in MITF expression and melanin production through inhibiting CREB phosphorylation.

2.5. Effects of Ganodermanondiol on Phosphorylated (p)-p38, p-c-Jun N-Terminal Kinase (JNK) and p-Extracellular Signal-Regulated Kinase (ERK) Protein Levels in B16F10 Cells

Melanogenic activation by binding stem cell factor (SCF) and endothelin-1 (EDN1) to c-kit and endothelin receptor type B (EDNRB) receptor, respectively, have been well studied in melanocytes. Binding END1 to EDNRB activates protein kinase C (PKC) by releasing diacylglycerol (DAG) from the cell membrane. The activated PKC then phosphorylates RAF proto-oncogene serine/threonine-protein kinase (Raf-1).

On the other hand, SCF triggers dimerization and autophosphorylation of c-kit receptors, followed by activation of various substrates and conversion of rat sarcoma-guanosine diphosphates (Ras-GDP) to RAS-guanosine triphosphate (GTP), leading to phosphorylation of Raf-1. The phosphorylation of Raf-1 activates (phosphorylates) a series of mitogen-activated protein kinases (MEKs), extracellular signal-regulated kinases (ERKs), RSKs, and CREB in the mitogen-activated protein kinase (MAPK) cascade.

In addition, the MAPK family proteins, including ERK, p38, and c-Jun N-terminal kinase (JNK), also play crucial roles in melanogenesis. The phosphorylation of p38 induces MITF expression, whereas the phosphorylation of ERK and JNK downregulate melanin synthesis.

Therefore, in the present study, we also evaluated whether the effects of ganodermanondiol on the phosphorylation of these proteins involved the MAPK family proteins. The phosphorylation of ERK and JNK proteins were significantly increased after 60 and 45 min, respectively, by treatment with ganodermanondiol (10 µM), whereas 15 min after treatment, phosphorylation of p38 protein was significantly decreased in B16F10 cells. These results indicate that inhibiting melanogenesis by ganodermanondiol is also associated with MAPK family proteins. In particular, ganodermanondiol induced the phosphorylation of ERK and JNK; however, phosphorylation of p38 was suppressed by ganodermanondiol treatment.