Biological activity and binding of estradiol to SK-Mel 23 human melanoma cells

Abstract

Patients expressing estradiol receptors in melanoma cells have been reported to have a better prognosis. We therefore decided to investigate the in vitro effects of β-estradiol and tamoxifen on the growth and tyrosinase activity of SK-Mel 23 human melanoma cells. Twenty-four-hour treatment with 0.4 nM β-estradiol inhibited cell proliferation in 30% (0.70 ± 0.03 × 105 cells) and increased tyrosinase activity in 50% (7130.5 ± 376.5 cpm/105 cells), as compared to untreated cells 1.0± 0.05 × 105 cells and 4769 ± 25.5 CpM/105 cells, respectively). Both responses were completely (100%) blocked by 1 μM tamoxifen. Higher concentrations (up to 1.6 nM or longer treatments (up to 72 h) did not result in a larger effect of the hormone on proliferation or tyrosinase activity. Competition binding assays demonstrated the presence of binding sites to [2,4,6,7-3H]-β-estradiol, and that the tritiated analogue was displaced by the unlabeled hormone (1 nM to 100 μM, Kd = 0.14 μM, maximal displacement of 93%) or by 10 μM tamoxifen (displacement of 60%). β-estradiol also increased the phosphorylated state of two proteins of 16 and 46 kDa, after 4-h treatment, as determined by Western blot. The absorbance of each band was 1.9- and 4-fold the controls, respectively, as determined with Image-Pro Plus software. Shorter incubation periods with β -estradiol did not enhance phosporylation; after 6-h treatment with the hormone, the two proteins returned to the control phosphorylation levels. The growth inhibition promoted by estradiol may explain the better prognosis of melanoma-bearing women as compared to men, and open new perspectives for drug therapy.