Tyrosinase Expression In Melanoma Cells Research Articles

The Inhibitory Effect of Polyphenon 60 from Green Tea on Melanin and Tyrosinase in Zebrafish and A375 Human Melanoma Cells.

Polyphenon 60 (PP60) from green tea has long been used as an antioxidant, anticancer, antimicrobial, and antimutagenic. Aim of the Study. To investigate tyrosinase inhibition-related kinetic mechanism and antimelanogenesis potential of PP60. Materials and Methods. The effect of PP60 on melanin and tyrosinase was evaluated in A375 melanoma cells and zebrafish embryos. The melanoma cells were treated with 20, 40, and 60 µg/mL of PP60, and tyrosinase expression was induced by using L-DOPA. The western blot method was used for the evaluation of tyrosinase expression. Cell lysates were prepared from treated and untreated cells for cellular tyrosinase and melanin quantification. Furthermore, zebrafish embryos were treated with 20, 40, and 60 µg/mL of PP60 and reference drug kojic acid for determination of depigmentation and melanin quantification. In vitro assays were also performed to examine the impact of PP60 on mushroom tyrosinase activity. To determine cytotoxicity, MTT was used against melanoma cell line A375. Results. PP60 showed good tyrosinase inhibitory activity with an IC50 value of 0.697 ± 0.021 µg/mL as compared to kojic acid a reference drug with an IC50 value of 2.486 ± 0.085 µg/mL. Kinetic analysis revealed its mixed type of inhibition against mushroom tyrosinase. In addition, western blot analysis showed that at 60 µg/mL dose of PP60 significantly reduced L-DOPA-induced tyrosinase expression in melanoma cells. PP60 significantly inhibits the cellular tyrosinase (p < 0.05) and reduces the melanin (p < 0.05) contents of melanoma cells. Furthermore, PP60 was found to be very potent in significantly reducing the zebrafish embryos' pigmentation (p < 0.05) and melanin (p < 0.05) content at the dose of 60 µg/mL. Conclusions. Our results demonstrate that PP60 has a strong potency to reduce pigmentation. It may be useful for the cosmetic industries to develop skin whitening agents with minimal toxic effects.

MicroRNA-203 Regulates Melanosome Transport and Tyrosinase Expression in Melanoma Cells by Targeting Kinesin Superfamily Protein 5b

MicroRNA (miR)-203 is known to be downregulated and to act as an anti-oncomir in melanoma cells. At present, we found that exogenous miR-203 increased pigmentation and protein expression levels of the melanoma antigen recognized by T cells (Melan-As/MART1s) and/or tyrosinase (TYR) in the human melanoma cells tested. Inversely, treatment with an inhibitor of miR-203 downregulated the expression level of TYR. The target gene of miR-203 involved in the mechanism was kinesin superfamily protein 5b (kif5b), which was revealed by gene silencing using short interfering RNA and luciferase activity assay. Furthermore, immunocytochemistry showed obvious accumulation of melanosomes around nuclei of human melanoma Mewo cells transfected with miR-203 or siR-kif5b. Importantly, treatment with the miR-203 inhibitor, but not miR-203, exhibited effects on human epidermal melanocytes isolated from lightly pigmented adult skin similar to those on melanoma cells. In addition, the data indicated that exogenous miR-203 also negatively regulated the cAMP response element-binding protein 1 (CREB1)/microphthalmia-associated transcription factor (MITF)/Rab27a pathway, which is one of the main pathways active in melanoma cells. In conclusion, our data indicated that anti-oncogenic miR-203 had a pivotal role in melanoma through reducing melanosome transport and promoting melanogenesis by targeting kif5b and through negative regulation of the CREB1/MITF/Rab27a pathway.

Selective down-regulation of tyrosinase family gene TYRP1 by inhibition of the activity of melanocyte transcription factor, MITF.

Tyrosinase (TYR), tyrosinase-related protein-1 (TYRP1/gp75) and dopachrome tautomerase (DCT/TYRP2) belong to a family of melanocyte-specific gene products involved in melanin pigmentation. During melanocyte development expression of tyrosinase family genes is thought to be orchestrated in part by the binding of a shared basic helix-loop-helix transcription factor MITF to the M box, a regulatory element conserved among these genes. In transformed melanocytes, expression of tyrosinase and TYRPs is highly variable. Whereas TYR expression in melanoma cells is regulated by both transcriptional and post-translational mechanisms, TYRP1/gp75 transcription is often completely extinguished during melanoma tumor progression. In this study, we investigated the mechanisms of selective repression of TYRP1 transcription. Interestingly, in early stage melanoma cells TYRP1 mRNA could be induced by inhibition of protein synthesis. Transient transfection experiments with a minimal TYRP1 promoter showed that the promoter activity correlates with expression of the endogenous TYRP1 gene. Nucleotide deletion analysis revealed novel regulatory sequences that attenuate the M box-dependent MITF activity, but which are not involved in the repression of TYRP1. Gel mobility shift analysis showed that binding of the transcription factor MITF to the TYRP1 M box is selectively inhibited in TYRP1(-) cells. These data suggest that protein factors that modulate the activity of MITF in melanoma cells repress TYRP1 and presumably other MITF target genes.