Melatonin interactions with cultured murine B16 melanoma cells

Both in vitro and in vivo observations have suggested that melatonin modulates malignant cell growth. The present studies aimed to characterize the interactions of melatonin with cultured murine B16 melanoma cells. Time- and temperature-dependent specific melatonin accumulation by B16 murine melanoma cells was observed. B16 cells possessed a high affinity binding site (KD = 1.4 nM) which exhibited structural specificity in its affinity for analogues of melatonin (melatonin > 6-hydroxymelatonin = N-acetyl-5-hydroxytryptamine > 5-methoxytryptamine >> 5-hydroxytryptamine). Evidence for a lower affinity uptake system without structural specificity was also observed. Ninety-five per cent of the specific cell-associated melatonin in B16 cells was present in the soluble subcellular fraction of lysed cells; more than 97% of the cell-associated radioactivity was authentic melatonin. When the solubilized cell extracts from the binding assay were analysed by gel filtration immediately, all of the bound counts eluted at the void volume. Continuous exposure to melatonin for 48-120 h did not affect B16 cell proliferation as determined by cell counts, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay or [3H]thymidine incorporation. After 8-h pulse exposures to melatonin daily for 3 days, a 15% stimulation of B16 cell proliferation (p < 0.02) was observed at melatonin concentrations of 0.1 and 1 nM. The anti-oestrogen, tamoxifen, inhibited B16 cell growth and increased specific melatonin accumulation by B16 cells at 1 x 10(-6) M (p < 0.02). Cultured B16 murine melanoma cells possessed a specific, high affinity uptake system for melatonin which appeared to be altered by anti-oestrogen exposure.