Targeted suppression on MITF-related proteins in human melanin-producing cells strengthens the potential development of cajanin as an effective treatment for human hyperpigmented disorders

Targeted suppression on MITF-related proteins in human melanin-producing cells strengthens the potential development of cajanin as an effective treatment for human hyperpigmented disorders. Roxb. tyrosinase, TRP-1 and Dct (TRP-2) were observed in MNT1 cells treated with 50 M cajanin for 24C72 h. Correspondingly, treatment with cajanin modulated the signaling pathway of CREB and ERK which both regulate MITF expression level. Targeted suppression on MITF-related proteins in human melanin-producing cells strengthens the potential development of cajanin as an effective treatment for human hyperpigmented disorders. Roxb. (Leguminosae) Tacrine HCl Hydrate has been previously reported to have potent inhibitory effect against enzymatic activity of tyrosinase isolated from mushroom [17]. However, the effect of cajanin on melanin production in human melanocytes has not been thoroughly examined. This study investigated anti-melanogenic activity and related mechanism of cajanin in human melanin-producing cells. The findings may facilitate the further development of cajanin as an effective depigmenting agent for the treatment of hyperpigmentation disorders. 2. Results 2.1. Antiproliferative Effect of Cajanin in Human Melanoma MNT1 Cells Before investigation on anti-melanogenic effect, cytotoxic profile of cajanin (Figure 1) in human melanin-producing cells was primarily investigated. Cell viability was evaluated through MTT assay in human melanoma MNT1 cells (5 103 cells/well in 96-well plates) treated with cajanin (0C100 M) for 72 h. The results showed that treatment with cajanin at 1C50 M did not cause significant alteration of viability in MNT1 cells compared with non-treated control (Figure 2a). Open in a separate window Figure 1 Schematic chemical structure of cajanin. Open in a separate window Figure 2 Cytotoxic profile of cajanin in human melanin-producing cells. (a) The significant reduction of %cell viability NOTCH1 determined by MTT assay was noted in human melanoma MNT1 cells treated with 100 M cajanin for 72 h. Although there was no alteration of viability observed with the lower concentrations (1C50 M), (b) the incubation with 50 M cajanin for 48C72 h obviously suppressed proliferation in MNT1 cells. Experiments were performed in triplicate and data are expressed as mean SEM. * 0.05, ** 0.01, *** 0.005 versus non-treated cells. The cytotoxic effect of 1C50 M cajanin which did not alter %cell viability after 72-h treatment was further evaluated by a cell proliferation assay. Although 72 h had been reported as a doubling time of human melanocytes [18], the MNT1 cells cultured under the present condition was found to have the doubling time of 13.75 h (Supplementary Data; Figure S1). Thus, the effect of cajanin on the proliferation of human melanoma MNT1 cells was observed after the incubation period of 24C72 h that covers the logarithmic growth phase of the cells [19]. Figure 2b demonstrates that there was a clear increase in the %proliferation after culturing MNT1 cells Tacrine HCl Hydrate at low density (2 103 cells/well in 96-well plates) which was not repressed Tacrine HCl Hydrate by 0C10 M of cajanin for 48C72 h. At 50 M cajanin, on the other hand, the proliferative activity was significantly suppressed compared with the untreated control cells. 2.2. Cajanin Tacrine HCl Hydrate Diminishes Melanin Content in Human Melanin-Producing Cells Based on cell viability results, the cajanin range of 1C50 M which caused no alteration of %cell viabilty after 72-h treatment was selected for further investigation on anti-melanogenic activity. Due to melanin-generating activity, forskolin was used as a positive control [20], while 4-butylresorcinol, a tyrosinase inhibitor, was selected for a negative control [21]. The capability to generate melanin pigment was noted in human melanoma MNT1 cells cultured in DMEM supplemented with 20% FBS, 10% AIM-V medium for 72 h (Figure 3a) and for 24C72 h (Figure 3b). It should be noted that the gradually augmented melanin content in human MNT1 cells was early observed at 12 h of the incubation Tacrine HCl Hydrate time (Supplementary Data; Figure S2). As forskolin is an activator of adenylyl cyclase which consequently stimulates CREB and melanin synthesis, the accumulation of melanin content was obviously observed in MNT1 cells treated with 10 M forskolin (Figure 3a). The ratio between the cellular melanin and total protein content extracted from MNT1 cells was represented as a relative value to untreated control cells to minimize the interference from antiproliferative effect of cajanin. Figure 3c.