Cyclohexadione-aniline conjugate inhibits proliferation of melanoma cells via upregulation of Mek 1/2 kinase activity

Purpose: To investigate the antiproliferative effect of cyclohexadione-aniline conjugate (CHAC) on melanoma cells, and the mechanism of action involved. Methods: Human melanoma cell lines (B16 F1 and A375) were used in this study. The cells were cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin at 37 °C in a humidified atmosphere of 5 % CO2 and 95 % air. After attaining 70 - 80 % confluency, the cells were treated with serum-free medium and graded concentrations of CHAC (10 – 60 μM) for 24 h. Normal cell culture without CHAC served as control group. B16 F1 and A375 cells were used in logarithmic growth phase in this study. Cell viability and apoptosis were assessed using 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphe¬nyltetrazolium bromide (MTT) and flow cytometric assays, respectively. Western blotting was used to assess the levels of protein expression of X linked inhibitor of apoptosis (XIAP), survivin, p-Erk 1/2, and p-Mek 1/2. Results: Treatment of B16 F1 and A375 cells with CHAC led to significant and concentrationdependent reductions in their viability (p < 0.05). The proliferation of B16 F1 cells decreased from 93.41 to 32.87 %, while that of A375 cells was reduced from 95.23 to 36.50 %. Treatment of B16 F1 cells with CHAC significantly and concentration-dependently increased the population of cells in G0/G1 phase, and significantly reduced cell proportion in S and G2/M phases (p < 0.05). It also significantly and concentration-dependently promoted apoptosis in B16 F1 cells (p < 0.05). CHAC treatment significantly and concentration-dependently down-regulated the expressions of XIAP and survivin proteins (p < 0.05). Exposure of B16 F1 cells to CHAC significantly and concentration-dependently upregulated the expression of p-Mek 1/2, but down-regulated p-Erk 1/2 protein expression (p < 0.05). Densitometric analysis revealed that the expression of p-Mek 1/2 was increased from 12 to 91 %. Conclusion: The results of this study indicate that CHAC inhibits the proliferation of melanoma cells via upregulation of Mek 1/2 kinase activity, and therefore may find application in the management of melanoma