Selective incorporation and specific cytocidal effect as the cellular basis for the antimelanoma action of sulphur containing tyrosine analogs. (1/130)

Tyrosine analogs are good candidates for developing melanoma chemotherapy because melanogenesis is inherently toxic and uniquely expressed in melanocytic cells. Sulphur containing substrate (tyrosine) analogs, N-acetyl-4-S-cysteaminylphenol (NAcCAP) and N-propionyl-4-S-cysteaminylphenol (NPrCAP), have been shown to have potent antimelanoma activity in mice bearing melanoma. Both NAcCAP and NPrCAP show selective cytotoxicity towards melanoma cell lines. But the mechanism leading to selectivity is not clear as these drugs are also toxic to other cell lines to a lesser extent. Here we show that these drugs have both cytostatic and cytocidal effects, which could account for this. Cytostatic effect is suggested by DNA flow cytometry. The drug causes cell cycle changes in four human cell lines (normal skin fibroblasts, HeLa cells, and melanoma cell lines, C32 and SK-MEL-23) in a dose-dependent manner blocking cells in S phase with concomitant decrease in the number of cells in G1 phase. There is also a gradual decrease in cells in G2 + M phases. The dose-concentration curves give IC50 values in the range of 50-400 microM and the melanotic melanoma cell line SK-MEL-23 has the lowest IC50 value consistent with our hypothesis that these drugs are selective towards melanoma cells. The concentration-dependent accumulation of cells in S phase suggest a cytostatic effect as a consequence of inhibition of DNA synthesis in agreement with [3H] thymidine incorporation assay. There is a highly specific uptake of [14C]NAcCAP and irreversible damage to DNA synthesis machinery in SK-MEL-23 cells, indicating a melanotic-specific cytocidal effect as well. Trypan blue exclusion study and competitive inhibition assay indicated that visible cytocidal effect occurs slowly and oxidative stress resulting from tyrosinase mediated oxidation of the drug appears to be the underlying mechanism. The primary antimelanoma effect of cysteaminylphenols derives from a selective cytostatic effect, but is followed by a specific cytocidal action rendering the drugs useful for targeted melanoma chemotherapy.  (+info)

Inhibition of melanin synthesis by cystamine in human melanoma cells. (2/130)

In studies to determine whether pigmentation can be regulated physiologically by thiols, human melanoma cells (MM418c5) and melanocytes were found to become depigmented when cultured continuously in 50 microM cystamine. Cystamine was depleted from the culture medium and the treatment was nontoxic and reversible. Cysteamine, dithiothreitol, and phenylthiourea were less effective, and glutathione, cysteine, and cystine were inactive. Tyrosinase (dopa oxidase) activity was not greatly affected except for induction of a lag period. In contrast, tyrosinase activity in an amelanotic melanoma cell line (MM96L) was rapidly inhibited without consumption of cystamine/cysteamine, in association with the generation of free thiol in the culture medium, and could be enhanced by the cystine transport inhibitor, glutamate. Tyrosinase expressed by a recombinant vaccinia virus was inhibited by cystamine treatment of MM96L and HeLa cells. Cystamine treatment lowered the degree of cross-linking of the pigmentation antigen gp75/TRP-1 in MM418c5 cells. Tyrosinase protein and mRNA levels in MM418c5 cells were not affected by cystamine. The results show that cystamine at a concentration close to physiologic levels has multiple effects on the melanogenic pathway. In amelanotic cells, tyrosinase has a short half-life and is readily inhibited by cystamine/cysteamine whereas tyrosinase in the more mature melanosomes of the pigmented cell appears to be less accessible to proteolytic and thiol attack. Inhibition of melanin synthesis in the latter cell type may arise to a significant degree from reduction of cystamine to cysteamine, which sequesters quinones.  (+info)

Phentolamine inhibits exocytosis of glucagon by Gi2 protein-dependent activation of calcineurin in rat pancreatic alpha -cells. (3/130)

Capacitance measurements were used to investigate the molecular mechanisms by which imidazoline compounds inhibit glucagon release in rat pancreatic alpha-cells. The imidazoline compound phentolamine reversibly decreased depolarization-evoked exocytosis >80% without affecting the whole-cell Ca(2+) current. During intracellular application through the recording pipette, phentolamine produced a concentration-dependent decrease in the rate of exocytosis (IC(50) = 9.7 microm). Another imidazoline compound, RX871024, exhibited similar effects on exocytosis (IC(50) = 13 microm). These actions were dependent on activation of pertussis toxin-sensitive G(i2) proteins but were not associated with stimulation of ATP-sensitive K(+) channels or adenylate cyclase activity. The inhibitory effect of phentolamine on exocytosis resulted from activation of the protein phosphatase calcineurin and was abolished by cyclosporin A and deltamethrin. Exocytosis was not affected by intracellular application of specific alpha(2), I(1), and I(2) ligands. Phentolamine reduced glucagon release (IC(50) = 1.2 microm) from intact islets by 40%, an effect abolished by pertussis toxin, cyclosporin A, and deltamethrin. These data suggest that imidazoline compounds inhibit glucagon secretion via G(i2)-dependent activation of calcineurin in the pancreatic alpha-cell. The imidazoline binding site is likely to be localized intracellularly and probably closely associated with the secretory granules.  (+info)

Retinoic acid-induced tissue transglutaminase and apoptosis in vascular smooth muscle cells. (4/130)

Retinoids exert antiproliferative and prodifferentiating effects in vascular smooth muscle cells (SMCs) and reduce neointimal mass in balloon-injured blood vessels. The mechanisms through which retinoids carry out these effects are unknown but likely involve retinoid receptor-mediated changes in gene expression. Here we report the cloning, chromosomal mapping, and biological activity of the retinoid-response gene rat tissue transglutaminase (tTG). Northern blotting studies showed that tTG is rapidly and dose-dependently induced in a protein synthesis-independent manner after stimulation with the natural retinoid all-trans retinoic acid (atRA). The induction of tTG was selective for atRA and its stereoisomers 9-cis and 13-cis RA, because little or no elevation in mRNA expression was observed with a panel of growth factors. Western blotting and immunofluorescence confocal microscopy showed an accumulation of cytosolic tTG protein after atRA stimulation. Radiolabeled cross-linking studies revealed a corresponding elevation in in vitro tTG activity. The increase in tTG activity was reduced in the presence of 2 distinct inhibitors of tTG (monodansylcadaverine and cystamine). atRA-induced tTG mRNA and protein expression were followed by a significant elevation in SMC apoptosis. Such retinoid-induced programmed cell death could be partially inhibited with each tTG inhibitor and was completely blocked when both inhibitors were used simultaneously. These results establish a role for atRA in the sequential stimulation of tTG and apoptosis in cultured SMCs. atRA-mediated apoptosis in SMCs seems to require the participation of active tTG, suggesting a potential mechanistic link between this retinoid-inducible gene and programmed cell death.  (+info)

Catalytic selenols couple the redox cycles of metallothionein and glutathione. (5/130)

Co-ordination of zinc to the thiol group of cysteine allows mobilization of zinc through oxidation of its ligand. This molecular property links the binding and release of zinc in metallothionein (MT) to the cellular redox state [Maret W. & Vallee B.L. (1998) Proc. Natl Acad. Sci. USA 95, 3483-3488]. Biological disulfides such as glutathione disulfide (GSSG) oxidize MT with concomitant release of zinc, while glutathione (GSH) reduces the oxidized protein to thionein, which then binds to available zinc. Neither of these two redox processes is very efficient, even at high concentrations of GSSG or GSH. However, the GSH/GSSG redox pair can efficiently couple with the MT/thionein system in the presence of a selenium compound that has the capacity to form a catalytic selenol(ate). This coupling provides a very effective means of modulating oxidation and reduction. Remarkably, selenium compounds catalyze the oxidation of MT even under overall reducing conditions such as those prevailing in the cytosol. In this manner, the binding and release of zinc from zinc-thiolate co-ordination sites is linked to redox catalysis by selenium compounds, changes in the glutathione redox state, and the availability of either a zinc donor or a zinc acceptor. The results also suggest that the pharmacological actions of selenium compounds in cancer prevention and other antiviral and anti-inflammatory therapeutic applications, as well as unknown functions of selenium-containing proteins, may relate to coupling between the thiol redox state and the zinc state.  (+info)

Drosophila melanogaster glutamate-cysteine ligase activity is regulated by a modifier subunit with a mechanism of action similar to that of the mammalian form. (6/130)

Glutamate-cysteine ligase (GCL) plays an important role in regulating glutathione homeostasis. In mammals, it comprises a catalytic (GCLC) and modifier (GCLM) subunit. The existence of a modifier subunit in invertebrates has not been described to date. We now demonstrate that GCL from Drosophila melanogaster has a functional modifier subunit (DmGCLM). A putative DmGCLM was obtained as an expressed sequence tag with 27% identity to human GCLM at the amino acid level. D. melanogaster GCLC (DmGCLC) and the candidate DmGCLM were expressed separately in Escherichia coli, purified, mixed, and then subjected to gel filtration, where they eluted as an approximately 140-kDa complex. DmGCLC co-immunoprecipitated with DmGCLM from S2 cell extracts, suggesting that they also associate in vivo. Enzyme kinetic analyses showed that DmGCLC has a K(m) for glutamate of 2.88 mm, but when complexed with DmGCLM, the K(m) for glutamate is 0.45 mm. Inhibition of DmGCLC activity by glutathione was found to be competitive with respect to glutamate (K(i) = 0.03 mm), whereas inhibition of the GCL complex was mixed (K(i) = 0.67 mm), suggesting allosteric effects. In accordance with this, DmGCLC and DmGCLM have the ability to form reversible intermolecular disulfide bridges. A further mechanism for control of D. melanogaster GCL was found to be induction of DmGCLC by tert-butylhydroquinone in S2 cells. DmGCLM levels were, however, unaffected by tert-butylhydroquinone.  (+info)

Dimethylnitrosamine-induced inhibition of hepatic protein synthesis in vitro and the effect of pretreatment with cystamine or pregnenolone-16alpha-carbonitrile. (7/130)

Hepatic protein synthesis was investigated using a postmitochondrial supernatant system derived from the livers of rats that were given injections of a single dose of dimethylnitrosamine (DMN), 30 mg/kg. The time course and extent of DMN-induced inhibition in vitro were identical to those reported for the incorporation of amino acids into liver proteins in vivo, maximum inhibition being about 70% at 5 hr. Addition of specific inhibitors of chain initiation (polyinosinic acid and aurin tricarboxylic acid) to the postmitochrondrial supernatant system from DMN-treated rats caused only a slight additional inhibition, indicating that DMN predominantly affects translation by a block of initiation. Treatment with cystamine prior to DMN administration completely abolished the depression of protein synthesis and reduced by more than 90% the methylation by [14C]DMN of purine bases in liver DNA. Pretreatment with pregnenolone-16alpha-carbonitrile stimulated protein synthesis in controls but had no preventive effect in DMN-treated rats and did not reduce the extent of DNA alkylation in vivo.  (+info)

Discrimination by SZL49 between contractions evoked by noradrenaline in longitudinal and circular muscle of human vas deferens. (8/130)

The effects of irreversible alpha1-adrenoceptor antagonists, SZL-49 (an alkylating analogue of prazosin), dibenamine and benextramine on contractions to noradrenaline (NA) in longitudinal and circular muscle of human epididymal vas deferens were investigated. Competitive alpha1-adrenoceptor antagonists were also used to further characterize the alpha1-adrenoceptor subtype stimulated by NA in longitudinal and circular muscle. NA evoked concentration-dependent contractions of both muscle types (pD2; 5.4 and 5.2 respectively). The contraction of circular muscle was comparatively more sensitive than that of longitudinal muscle to pretreatment with SZL-49. In contrast, dibenamine or benextramine produced comparable effects in both muscle types. The relationship between receptor occupancy and contraction in either longitudinal or circular muscle was nonlinear, with half-maximal response requiring similar receptor occupancy (longitudinal muscle 14%, circular muscle 16%). Maximal response in both muscle types occurred with little or no receptor reserve (<10%). The competitive alpha1-adrenoceptor antagonists produced dextral shifts of the dose-response curves to NA in longitudinal and circular muscle. The inhibitory potencies, estimated from the apparent pKB values were significantly different in longitudinal and circular muscle respectively for either WB 4101 (pKB, 8.6 and 9.5) or RS-17053 (pKB, 7.1 and 9.0) but not for Rec 15/2739 (pKB, 9.2 and 9.8) or HV 723 (pKB, 8.3 and 8.4). In conclusion, the potency profile of the competitive alpha1-adrenoceptor antagonists and the lack of different receptor reserves for NA in the muscle types suggest that the discriminatory effects of SZL-49 is primarily due to a predominance of the alpha1L-adrenoceptor subtype in longitudinal muscle and alpha1A-subtype in circular muscle.  (+info)

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