L-765,314 Suppresses Melanin Synthesis by Regulating Tyrosinase Activity

Abstract

Although melanin production is a key self-defense mechanism against ultraviolet radiation (UVR)-induced skin damage, uneven or excessive deposition of melanin causes hyperpigmentary disorders. Currently available whitening agents are unsatisfactory because of issues with efficacy and safety. To develop more effective depigmenting agents, we performed high-throughput melanin content assay screening using the B16F10 melanoma cell line and identified L-765,314 as a drug that suppressed melanin production in cultured melanocytes in a dose-dependent manner as well as cAMP- or 12-O-tetradecanoylphorbol 13-acetate (TPA)-stimulated melanin production without cytotoxicity. Interestingly, melanogenic gene expression was not altered by L-765,314. Rather, diminished melanin production by L-765,314 appeared to be caused by downregulation of tyrosinase activity via inhibition of protein kinase C (PKC). Because L-765,314 did not show any adverse effect in melanocytes, altogether our data suggest that L-765,314 could be a potential therapeutic candidate for skin hyperpigmentary disorders and further discovery of selective inhibitors targeting PKC might be a promising strategy for the development of depigmenting agents to treat hyperpigmentary disorders.

Keywords: L-765,314; depigmenting agents; protein kinase C; tyrosinase activity.

Figure 1

L-765,314 reduced melanin secretion in B16F10 melanoma. (A) B16F10 melanoma cells were treated with vehicle or 0.1–10 μM L-765,314. Seventy-two hours after treatment, the melanin secreted from B16F10 cells was examined. Melanin content is given as percent change relative to vehicle-treated controls. (B) Chemical structure of L-765,314 (Benzyl (S)-4-(4-amino-6,7-dimethoxyquinazolin-2-yl)-2-(tert-butylcarbamoyl) piperazine-1-carboxylate). *** represents p < 0.001.

Figure 2

L-765,314 reduced melanin production in normal Mel-ab melanocytes. Ninety-six hours after treatment with 0.1–20 μM L-765,314, (A) microscopic images of Mel-ab cells were captured using phase-contrast microscopy and (B) melanin content was measured. Forskolin treatment was used as a positive control for melanin production. Mel-ab cells were treated with vehicle, 10 μM forskolin, 10 μM L-765,314, or 10 μM forskolin and 10 μM L-765,314 together for 96 h and (C) microscopic images were captured and (D) melanin production was compared and presented as percent changes relative to vehicle-treated controls. Statistic test for a compared to control and b compared to forskolin (FSK). Scale bar: 1000 μm, ** and *** represents p < 0.01 and p < 0.001 respectively. a** represents p < 0.01 between control and L765,314 or L765,314 + forskolin treatment, b** represents p < 0.01 between forskolin treatment and L765,314 + forskolin treatment.

Figure 3

Effect of L-765,314 on cell viability. Mel-ab cells were treated with 0.1–20 μM L-765,314 for 48 h and cell viability was examined by MTT assay.

Figure 4

Suppression of melanin production by L-765,314 did not involve the adrenoceptor signaling pathway. (A) Expression of ADRA1a, ADRA1b, and ADRA1d in Mel-ab, B16F10, and HaCat cells was analyzed by qRT-PCR. Mel-ab cells were treated with vehicle (DMSO), 10 μM forskolin (FSK), or ADRA agonists, i.e., 5 μM phenylephrine (PE5) and 5 μM cirazoline (CRZ5). Ninety-six hours after treatment, (B) microscopic images were captured and (C) melanin contents were measured. Melanin contents are given as percent changes relative to vehicle-treated controls. Scale bar: 500 μm, * represents p < 0.05.

Figure 5

L-765,314 downregulated tyrosinase activity without decreasing tyrosinase expression. (A) The expression level of microphthalmia-associated transcription factor (MITF), DCT, Tyrp1, and tyrosinase in Mel-ab cells treated with vehicle and L-765,314 was compared by immunoblotting. α-tubulin was used as an internal loading control. (B) The transcript levels of MITF, DCT, Tyrp1, and tyrosinase in L-765,314-treated Mel-Ab cells for 96 h were compared to those of control cells by qRT-PCR. L32 transcript was used as an internal control. (C) The effect of L-765,314 on MITF and tyrosinase promoter activity was assessed. Forskolin was used as a positive control for enhancing MITF promoter activity. (D) Tyrosinase activity in Mel-ab cells treated with vehicle, L-765,314, or forskolin for 96 h was examined and presented as percent change relative vehicle-treated controls. ** and *** represents p < 0.01 and p < 0.001 respectively.

Figure 6

Protein Kinase C (PKC)-stimulated tyrosinase activity was attenuated by L-765,314. (A) PKC activity was assessed in Mel-ab cells treated with L-765,314 for 96 h by immunoblotting with phospho-PKC substrate antibody. (B) The PKC activity and (C) tyrosinase activity of Mel-ab cells treated with vehicle, 12-O-Tetradecanoylphorbol 13-acetate (TPA), or L-765,314 and TPA were examined. (D) Microscopic images and (E) melanin contents of Mel-ab cells treated with vehicle, TPA, or TPA with L-765,314. *, ** and *** represents p < 0.05, p < 0.01, and p < 0.001 respectively.

Figure 7

L-765,314 suppressed melanogenesis in normal human melanocytes. (A) Melanin contents of normal human melanocyte (NHM) cells treated with 0.1–10 μM L-765,314 for 96 h. (B) PKC and (C) tyrosinase activity of NHM cells after incubation for 96 h with 10 μM L-765,314. ** and *** represents p < 0.01 and p < 0.001, respectively.