The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells.
8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 micro M). The intracellular Ca2+ chelator BAPTA/AM (1 micro M) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 micro M protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92% increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.
Isoldi
M C
MC
Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, Sao Paulo, SP, Brazil.
Pereira
E A
EA
Visconti
M A
MA
Castrucci
A M L
AM
D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008545:Melanoma / Q000378:metabolism* / Q000473:pathology; D008730:Methoxsalen / Q000494:pharmacology*; D017319:Photosensitizing Agents / Q000494:pharmacology*; D024681:Potassium Channels, Calcium-Activated / Q000187:drug effects* / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000187:drug effects
D007211:Indoles; D017319:Photosensitizing Agents; D024681:Potassium Channels, Calcium-Activated; D011505:Protein-Tyrosine Kinases; D008730:Methoxsalen; C064758:Ro 31-8220