Psoralen Activity and Binding Sites in Melanotic and Amelanotic Human Melanoma Cells

Abstract

The biological activity and specific binding sites of 8-methoxypsoralen (8-MOP) are assayed using two human melanoma cell lines, melanotic SK-Mel 28 and amelanotic C32TG. Long-term (72 hr) treatment with 8-MOP at a concentration of 10(-4)M results in an increase in melanogenesis and a decrease in proliferation, similar in both cell lines. Daily exposure of these cells to ultraviolet A (UVA) irradiation (1.28 mJ/cm(2)) does not enhance the response to the compound. Daily pulse application (30 min daily) of 8-MOP does not promote any response. However, in combination with UVA, 8-MOP pulse treatment becomes as effective as the long-term treatment. A decrease in cell proliferation in the constant presence of 8-MOP is not coupled with apoptosis, since no increase in the number of apoptotic nuclei was observed after the treatment. The flow cytometry indicates that 8-MOP arrests the cells at the G0/G1 phase, irrespective of the presence or absence of UVA light. In view of the lack of epidermal growth factor (EGF) receptors in both cell lines, it is not likely that such an arrest is associated with the down-regulation of EGF receptors by 8-MOP. It is noted that this compound elicits a biphasic cell response, since cell proliferation increases after the first 24-hr treatment, whereas it decreases in the subsequent 48 hr and thereafter. Competition binding assays using 3H-8-MOP disclosed: 1) the specific binding of the compound in both cell lines occurs in the presence or absence of UVA light, and 2) a higher binding rate at low concentrations of the compound is in SK-Mel 28 (72%) rather than C32TG (58%) cells. The competition assays in the presence of UVA suggest a possible occurrence of covalent bindings between psoralen and receptor, as DNA covalent binding accounted to only 3-5% of the total binding in both cell lines.

A Melanocyte–Keratinocyte Coculture Model to Assess Regulators of Pigmentation in Vitro

“…Although 8MOP is primarily known as a photosensitizer, its ability to stimulate melanogenesis in the absence of UV radiation has been previously reported (51,52). Our experiments were performed in the absence of UV radiation.…”

The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells

“…The elevation of intracellular Ca 2+ levels by 8-MOP seems, at first, to contradict its anti-proliferative action. However, the growth curves revealed that a growth peak (≅30%) appears 24 h after the introduction of 0.1 mM 8-MOP, followed by a significant reduction of proliferation (48 and 72 h) compared to the controls (6). Spectrofluorometric calcium profiles revealed that an immediate Ca 2+ increase occurs with the addition of 8-MOP to the cell suspension.…”

“…Cells treated with 8-MOP failed to enter the G1 phase, and did not exhibit any features of apoptosis. In addition, chronic exposure to 8-MOP in the absence of UVA enhanced tyrosinase activity in both cell lines (6).…”

“…Our previous results revealed that a 24-h treatment with 8-MOP stimulated the cells to proliferate (6). In an attempt to demonstrate the need for Ca 2+ not only for this early proliferative peak, but also for the late decrease in proliferation and increase in tyrosinase activity, the cells were treated with BAPTA/AM only during the first 24 h of the assay (a total 8-MOP assay time of 72 h).…”

“…The existence of low and high affinity sites for psoralens was demonstrated in a variety of cell lines (7,8) including the two human melanoma lines (6), and in the presence of UVA, 8-MOP covalently binds to sites other than DNA (6).…”