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Role of Epigenetic Bromodomain Protein in Metastatic Melanoma Open Access

Metastatic melanoma is the most aggressive skin cancer and is the sixth most common cancer, of all age groups in the USA. Introduction of BRAF inhibitors and cancer immunotherapy has greatly improved treatment of melanoma, however, the problem of tumour relapse and therapy resistance persists. A better understanding of the mechanism of development of the diseases is urgently needed to help devise an improved therapeutic intervention. Bromodomain and extra-terminal domain BET family is represented by three members in humans BRD2, BRD3, BRD4 and play key mediators of transcriptional activation. Specific BET inhibitors have been developed and are already in dose escalation clinical trials to investigate safety, tolerability and pharmacokinetics. Goal of this study is to identify the role of BET proteins in melanoma cells. We, therefore examined the effect of BET pharmacological inhibitor JQ1 on the proliferation and survival of the three melanoma cell lines (A375, A2058 and WM164). Melanoma cells were grown and maintained in the RPMI media supplemented with 15% fetal bovine serum and 1% antibiotic mixture. 70-80% confluent melanoma cells were plated overnight, then treated with JQ1 inhibitor. MTT: Melanoma cells were treated with various concentrations of JQ1 (1, 5.0 and 10.0 uM) for 72 hours in the humidified 37 C CO2 chamber. MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium was added, followed by incubation at 37 C for 4 hours. After addition of isopropanol containing 0.04N HCL, absorbance readings were taken at a wavelength of 570nm using spectrophotometer. MTT results showed a dose-dependent decrease in the cell proliferation in all the three melanoma cell lines tested, suggesting anti-proliferative role of BET inhibitor in the melanoma cells. Invasion and migration: In order to understand the role of BET inhibitors in the metastasis, we performed Invasion and migration assays in the melanoma cells. We used uncoated (for migration) and matrigel coated (for invasion) trans-well assays to detect the migratory potential of A375, and A2058 WM164 cells, with or without JQ1 in concentration of 5.0 and 10. uM. Interestingly, both migration and invasion were found to be reduced in JQ treated cells relative to the control cells in all the cell lines examined. In summary, our results suggest that BET proteins play an important role in the growth and progression of melanoma cells. Further studies are ongoing to delineate the mechanistic role of BRD proteins in the metastatic melanoma.

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