Activation of mitogen activated protein kinase pathways and melanogenesis by novel nitro-derivatives of 7-hydroxycomarin in human malignant melanoma cells.
6-Nitro-7-hydroxycoumarin (6-NO2-7-OHC) and 3,6,8-trinitro-7-hydroxycoumarin (3,6,8-NO2-7-OHC) have previously been shown to be potent and selective anti-proliferative agents in a human melanoma cell line. These agents functioned by decreasing DNA synthesis, through an inhibition of the S phase regulatory protein, cyclin A. However, the key molecular target(s) for these drugs remained undefined. Here, we attempted to elucidate the exact nature of the relationship between drug exposure and signal transduction, particularly their effects on the mitogen activated protein kinase (MAPK) cascades, and the consequent effect on cell growth, death and differentiation. Comparative studies were carried out using 7-hydroxycoumarin (7-OHC). Both nitro-derivatives were found to alter the phosphorylation status of ERK1/ERK2 and p38. However, 7-OHC exerted this effect only at higher concentrations and longer incubation times. Also, none of the three drugs had any effect on SAPK phosphorylation. Tyrosinase activity assays and morphological studies were used to show drug-induced effects on cellular differentiation. Unlike 7-OHC, both 6-NO2-7-OHC and 3,6,8-NO2-7-OHC caused a dramatic increase in tyrosinase activity in a manner similar to the cAMP elevating agent, forskolin. Also, the MEK inhibitor (PD98059) in combination with nitro-derivatives stimulated an even greater increase in tyrosinase activity when compared to either drug. In addition, the p38 inhibitor (SB203580) reduced the activity of both drugs. Morphological examination of treated cells showed nitro-derivatives caused changes consistent with altered cellular differentiation. Taken together, we have established that exposure of human malignant melanoma cells to these drugs leads to a modulation of p38 MAP kinase phosphorylation. This implies that these drugs may function by altering both melanogenesis and cellular differentiation. However, their effect on the levels of these proteins rather than their phosphorylation status remains to be determined. Therefore, additional studies are underway in order to identify the exact binding partners for these drugs.
Cancer Research Program, Beth Israel Deaconess Medical Centre, Harvard Medical School, Boston, MA, USA.
Gregory J
GJ
Finn
Bernadette S
BS
Creaven
Denise A
DA
Egan
D000970:Antineoplastic Agents / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008543:Melanins / Q000096:biosynthesis*; D008545:Melanoma; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014442:Monophenol Monooxygenase / Q000378:metabolism; D009574:Nitro Compounds / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology; D014468:Umbelliferones / Q000494:pharmacology*
C492875:3,6,8-trinitro-7-hydroxycoumarin; C492874:6-nitro-7-hydroxycoumarin; D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D008543:Melanins; D009574:Nitro Compounds; D011725:Pyridines; D014468:Umbelliferones; D005576:Colforsin; D014442:Monophenol Monooxygenase; D020928:Mitogen-Activated Protein Kinases; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one