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.

A study of the role of cell cycle events mediating the action of coumarin derivatives 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 to the human skin cell line, SK-MEL-31. Here, we investigate the reversibility of their cytotoxicity, along with their effects on DNA synthesis and cell cycle events. Comparative studies were carried out using the main metabolite of coumarin in man, 7-hydroxycoumarin (7-OHC). 6-NO2-7-OHC and 3,6,8-NO2-7-OHC, were found to be irreversible cytotoxic agents, unlike 7-OHC. All three derivatives inhibited DNA synthesis, but 7-OHC was only nitro-derivatives which acted in an irreversible manner. Flow cytometric studies demonstrated that both nitro-derivatives caused a dose- and time-dependant S phase accumulation. 7-OHC exerted a similar effect, but appeared to be less potent. Finally, the two nitro-derivatives caused a dose-dependant inhibition of the S phase regulatory protein, cyclin A. Consequently, these and other nitro-derivatives of 7-OHC may represent novel therapeutic agents for the treatment of malignant melanoma as they are capable of selective and irreversible cytotoxicity.

Synthesis, X-ray crystal structure, anti-fungal and anti-cancer activity of [Ag2(NH3)2(salH)2] (salH2=salicylic acid).

[Ag(2)(NH(3))(2)(salH)(2)] (salH(2)=salicylic acid) was synthesised from salicylic acid and Ag(2)O in concentrated aqueous NH(3) and the dimeric Ag(I) complex was characterised using X-ray crystallography. The complex is centrosymmetric with each metal coordinated to a salicylate carboxylate oxygen and to an ammonia nitrogen atom in an almost linear fashion. The two [Ag(NH(3))(salH)] units in the complex are linked by an Ag-Ag bond. Whilst metal-free salH(2) did not prevent the growth of the fungal pathogen Candida albicans [Ag(2)(NH(3))(2)(salH)(2)], [Ag(2)(salH)(2)] and some simple Ag(I) salts greatly inhibited cell reproduction. SalH(2), [Ag(2)(NH(3))(2)(salH)(2)] [Ag(2)(salH)(2)] and AgClO(4) produced a dose-dependent cytotoxic response against the three human derived cancer cell lines, Cal-27, Hep-G2 and A-498, with the Ag(I)-containing reagents being the most effective.

Daphnetin induced differentiation of human renal carcinoma cells and its mediation by p38 mitogen-activated protein kinase.

Daphnetin has been shown to be a potent in vitro anti-proliferative agent to the human renal cell carcinoma (RCC) cell line, A-498. In the present study, we investigated its effects on mitogen-activated protein kinase (MAPK) signalling along with cell cycle events and cellular differentiation. Daphnetin-activated p38, however, higher concentrations were required to inhibit ERK1/ERK2. In addition, it did not activate SAPK or induce apoptosis, but instead inhibited S phase cell cycle transition of A-498 cells at low concentrations and time of exposure. In addition, a late G(1), early S phase inhibition was observed at higher concentrations and time of exposure, indicating that the mechanism of daphnetin-induced differentiation was concentration dependent. Increased expression of the epithelial differentiation markers cytokeratins 8 and 18, correlated with increasing concentrations of daphnetin, while pre-treatment with a specific p38-inhibitor, served to limit this effect. There was no evidence that P-glycoprotein (P-gp) mediated multi-drug resistance (MDR) played a role in the anti-proliferative activity of daphnetin. Consequently, we concluded that p38 MAP kinase is intrinsically involved in mediating the effect of daphnetin in A-498 cells, suggesting that this drug may act by promotion of cellular maturation, and consequently may represent a novel low toxic approach for the treatment of poorly differentiated RCCs.

Investigation of intracellular signalling events mediating the mechanism of action of 7-hydroxycoumarin and 6-nitro-7-hdroxycoumarin in human renal cells.

Previously, 7-hydroxycoumarin (7-OHC) and 6-nitro-7-hydroxycoumarin (6-NO2-7-OHC) have been shown to be potent and selective anti-proliferative agents to the human renal cell carcinoma (RCC) cell line, A-498. Their effect on mitogen-activated protein kinases (MAPK's) was investigated. 6-NO2-7-OHC was shown to alter the phosphorylation status of ERK1/ERK2, p38 and SAPK, while 7-OHC activated ERK1/ERK2 but had no effect on p38 and SAPK. Also, 7-OHC inhibited topoisomerase II mediated relaxation of DNA, while neither compound was a substrate for P-glycoprotein (P-gp) mediated multi-drug resistance (MDR). Therefore, 6-NO2-7-OHC, rather than 7-OHC, modulated signalling events associated with cellular differentiation and apoptosis, suggesting its mechanism of action may be the promotion of cellular maturation and/or death. Consequently, 6-NO2-7-OHC may represent a novel therapeutic agent for the treatment of RCC's.

In vitro cytotoxic potential and mechanism of action of selected coumarins, using human renal cell lines.

This study determined the selective cytotoxicity of eight coumarin compounds to human renal carcinoma cells, relative to non-carcinoma proximal tubular cells. Selectivity cytotoxicity was observed following exposure to 6-nitro-7-hydroxycoumarin (6-NO(2)-7-OHC) and 7,8-dihydroxycoumarin (7,8-OHC). 6-NO(2)-7-OHC induced cytotoxicity was irreversible in both cell lines, unlike 7,8-OHC, which was reversible in the carcinoma cells only. Mobility shift and BrdU incorporation assays showed that both compounds did not intercalate DNA but had a concentration-dependent inhibitory effect on its synthesis. All coumarins studied were found to be non-mutagenic using the standard Ames test. These results would suggest that 6-NO(2)-7-OHC and 7,8-OHC might have a therapeutic role to play in the treatment of renal cell carcinoma.