Specific Activity In Presence Research Articles

In vitro modulation of proliferation and melanization of melanoma cells by citrate.

B16/F10 murine melanoma cells were grown for 24 and 36 h in Dulbecco's modified Eagle medium in presence of 10-20 mM trisodium citrate. The intracellular melanin concentration and the melanin secreted in the extracellular medium was estimated. It is observed that 20 mM citrate stimulates extracellular melanin secretion in B16/F10 melanoma cells by 200% at 36 h treatment. The intracellular melanin content increased by 90%. This stimulatory effect of citrate was totally abolished when these cells were grown in presence of 1 mM phenyl thiourea, a specific inhibitor of tyrosinase activity. Citrate (0.1-5 mM) had no effect on dopa oxidase activity either at pH 5.0 or at pH 6.8. There was no increase in the tyrosinase specific activity in presence of citrate. The increased melanin synthesis was shown to be due to stimulation of cellular tyrosine hydroxylase activity by citrate. It has been suggested that enhanced melanin synthesis results in an increased production of metabolites that are toxic to the growth of melanoma cells. We have studied the effect of citrate on cellular proliferation. Following 24 and 36 h treatment with citrate, the cells exhibited a dose-dependent decrease in proliferation. In presence of 20 mM citrate the cell number was only up to 50% of the control cultures after 36 h of incubation. The growth retardation was not due to cytotoxicity. Citrate, a natural metabolite, is a unique molecule which may be involved in the regulation of melanin biosynthetic pathway, since it enhances melanogenesis by increasing the hydroxylase activity of tyrosinase which is the regulatory enzyme of this pathway. These observations add further support to the critical role of intramelanosomal pH in regulation of melanogenesis.