allopurinol and Melanoma

We recently demonstrated that two new prenylflavanones, propolin A and propolin B, isolated and characterized from Taiwanese propolis, induced cytotoxicity effect in human melanoma A2058 cells and shows a strong capability to scavenge free radicals. In this study, propolin A effectively induced a cytotoxic effect on five different cancer cell lines. Similar results were obtained for propolin B. DNA flow cytometric analysis and DNA fragmentation ladder indicated that propolin A and propolin B actively induced apoptosis in A2058 cells. To address the mechanism of the apoptosis effect of propolin A and propolin B, we evaluated the apoptosis-related proteins in A2058 cells. The levels of procaspase-8, Bid, procaspase-3, DFF45, and PARP were decreased in dose- and time course-dependent manners. Furthermore, also found propolin A and propolin B was capable of releasing cytochrome c from mitochondria to cytosol. The findings suggest that propolin A and propolin B may activate a mitochondria-mediated apoptosis pathway. On the other hand, our data show that propolin B inhibitied xanthine oxidase activity more efficiently than propolin A or CAPE. However, CAPE suppressed ROS-induced DNA strand breakage more efficiently than propolin A or propolin B. All these results indicated that propolin A and propolin B may trigger apoptosis of A2058 cells through mitochondria-dependent pathways and also shown that propolin A and propolin B were strong antioxidants.

Topics: Apoptosis; Blotting, Western; Caffeic Acids; Caspase 3; Caspase 8; Cell Cycle; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Breaks; DNA Fragmentation; DNA, Circular; Dose-Response Relationship, Drug; Flavonoids; Flow Cytometry; HL-60 Cells; Humans; Melanoma; Mitochondria; Molecular Structure; Phenylethyl Alcohol; Propolis; Reactive Oxygen Species; Signal Transduction; Xanthine Oxidase

We had demonstrated that two prenylflavanones, propolin A and propolin B, isolated and characterized from Taiwanese propolis, induced apoptosis in human melanoma cells and significantly inhibited xanthine oxidase activity. Here, we have isolated a third compound called propolin C. The chemical structure of propolin C has been characterized by NMR and HRMS spectra, and was identical to nymphaeol-A. However, no biological activities of this compound have ever been reported. In the present study, propolin C effectively induced a cytotoxic effect on human melanoma cells, with an IC(50) of about 8.5 microM. DNA flow cytometric analysis indicated that propolin C actively induced apoptosis in human melanoma cells and there is a marked loss of cells from the G2/M phase of the cell cycle. To address the mechanism of the apoptosis effect of propolin C, we evaluated the effect of propolin C on induction of apoptosis-related proteins in human melanoma cells. The levels of procaspase-8, Bid, procaspase-3, and poly(ADP-ribose) polymerase were decreased in dose- or time course-dependent manners. Moreover, propolin C was capable of releasing cytochrome c from mitochondria to cytosol. The findings suggest that propolin C may activate a mitochondria-mediated apoptosis pathway. On other hand, propolin C is a potential antioxidant agent and shows a strong capability to scavenge free radicals and inhibit on xanthine oxidase activity with IC(50) of about 17.0microM. In conclusion, the isolation and characterization of propolin C from bee propolis are described for the first time, and this compound is a powerful inducer of apoptosis in human melanoma cells.

Topics: Antineoplastic Agents; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Caffeic Acids; Carrier Proteins; Caspases; Cell Cycle; Cell Division; Cell Survival; Cytochromes c; DNA Fragmentation; Enzyme Activation; Flavonoids; Free Radicals; Humans; Melanoma; Mitochondria; Phenylethyl Alcohol; Propolis; Xanthine Oxidase

Cytotoxicity indicated by increased release of prelabeled 51chromium (51Cr) and lactate dehydrogenase (LDH) was studied in human prostate cancer and melanoma cells in cell culture following irradiation or exposure to several injurious substances. These changes were compared to those observed in bovine aortic endothelial cells (BAEC) subjected to identical treatments. Further, the effect of irradiation on plasminogen activator (PA) secretion from prostate cancer cells, and the effect of glycine on radiation-induced cytotoxicity in BAEC were also investigated. Radiation, lipopolysaccharide and xanthine/xanthine oxidase stimulated no release of 51Cr or LDH from tumor cells, while these treatments induced a dose- and time-related loss of those cytotoxic indicators from BAEC. Protease, elastase and Triton X-100 incited loss of 51Cr and LDH from all three cell types. Radiation, lipopolysaccharide and xanthine/xanthine oxidase have been shown to cause cell injury via a common pathogenic pathway of oxidant generation. Tumor cells appear quite resistant to oxidant stress. Cell damage precipitated by protease, elastase and Triton probably involves hydrolysis of proteins and phospholipids in the cell membrane, leading to an increased leakage of intracellular proteins such as LDH and those bound with 51Cr. Radiation caused a dose- and time-related reduction in the secretion of PA from prostate cancer cells. PA is alleged to play a role in tumor metastasis; the reduced secretion could be another beneficial effect of radiation, in addition to interruption of cell proliferation, in the impediment of tumor growth and spread. Glycine diminished cytotoxic injury of BAEC inflicted by radiation. This amino acid may prove useful in offering a degree of protection of normal tissue against radiation associated side-effects.

Topics: Animals; Cattle; Cells, Cultured; Chromium Radioisotopes; Endopeptidases; Endothelium, Vascular; Glycine; Hot Temperature; Humans; L-Lactate Dehydrogenase; Lipopolysaccharides; Male; Melanoma; Pancreatic Elastase; Plasminogen Activators; Polyethylene Glycols; Prostatic Neoplasms; Radiation Injuries; Radiation-Protective Agents; Time Factors; Tumor Cells, Cultured; Xanthine Oxidase; Xanthines

Tyrosinase may protect against oxidative stress by using the superoxide anion (O2-1.) in the production of melanin. We have examined this by comparing its cytotoxic effects in B16/F10 and B16/F10-differential deficient (-DD) mouse melanoma cells that express high and low levels of tyrosinase activity respectively. Xanthine oxidase (XO) was used to generate O2.1 and cytotoxicity assessed by measuring cell survival. XO increased O2.- concentrations and 3 h later dose related decreases in cell survival were seen. F10 cells were more resistant to these cytotoxic effects than the F10-DD cells. [Nle4, DPhe7]MSH increased tyrosinase activity and melanin content, reduced O2.- concentration and increased the resistance of F10 cells to the cytotoxic effects of O2.-. No such effects were seen in F10-DD cells. The effect of [Nle4, DPhe7]MSH on the resistance of the F10 cells was time-dependent and noticeable when tyrosinase activity but not melanin was increased. This suggests that it was the activation of tyrosinase rather than the increase in the melanin that provided the protection against O2.-. In support of this, inhibition of tyrosinase with phenylthiocarbamide reduced the increased resistance induced by [Nle4, DPhe7]MSH. Moreover, although melanin was capable of scavenging O2.- it had little effect at concentrations comparable to those in the activated F10 cells. XO also increased the melanin content of F10 but not F10-DD cells. We conclude that tyrosinase is able to utilise O2.- to produce melanin and this provides pigment cells with a unique anti-oxidant mechanism.

Topics: Animals; Enzyme Activation; Enzyme Inhibitors; Free Radical Scavengers; Hydrogen Peroxide; Melanins; Melanoma; Mice; Monophenol Monooxygenase; Oxidative Stress; Phenylthiourea; Superoxides; Tumor Cells, Cultured; Xanthine Oxidase

Tyrosinase isolated from cultured human melanoma cells was studied for tyrosine oxygenation activity. L-Tyrosine and D-tyrosine were used as substrates and dopa was measured with HPLC and electrochemical detection as the product of oxygenation. Incubations were performed in the presence or absence of dopamine as co-substrate. Oxygenation of L-tyrosine occurred only in the presence of dopamine as co-substrate. No oxygenation of D-tyrosine was found, and we conclude that human tyrosinase is characterised by exclusive specificity for the L-isomer of tyrosine in its oxygenase function. It has recently been suggested that superoxide anion is a preferential oxygen substrate for human tyrosinase. Incubations were therefore performed with L- and D-tyrosine, human tyrosine, and xanthine/xanthine oxidase in the system, generating superoxide anion and hydrogen peroxide. Considerable formation of dopa was observed, but the quantity was the same irrespective of whether D-tyrosine or L-tyrosine was used as the substrate. Furthermore, formation of dopa occurred in a xanthine/xanthine oxidase system when bovine serum albumin (BSA) was substituted for tyrosinase. Our results provide no evidence that superoxide anion is an oxygen substrate for human tyrosinase. In the incubate containing xanthine/xanthine oxidase, catalase completely inhibited dopa formation, and superoxide dismutase and mannitol each strongly inhibited dopa formation. The results are compatible with hydroxyl radicals being responsible for the formation of dopa, since such radicals may be secondarily formed in the presence of superoxide anion and hydrogen peroxide.

Topics: Animals; Catalase; Cattle; Chromatography, High Pressure Liquid; Dihydroxyphenylalanine; Dopamine; Humans; Hydrogen Peroxide; Melanoma; Monophenol Monooxygenase; Oxygen; Substrate Specificity; Superoxide Dismutase; Superoxides; Tumor Cells, Cultured; Tyrosine; Xanthine; Xanthine Oxidase; Xanthines

The effects of systems generating active oxygen species (superoxide anion, hydrogen peroxide, hydroxyl radical) on tyrosinase have been studied in cultured human melanoma cells. Tyrosinase activity was determined by measuring the quantity of 5-S-L-cysteinyl-L-dopa (5-S-CD) formed in the presence of D,L-dopa and L-cysteine. In some experiments, the enzyme protein was determined by radio immunoassay [RIA]. Exposure of cells to xanthine/xanthine oxidase or glucose/glucose oxidase resulted in a dose-related elevation of tyrosinase. Catalase, but not superoxide dismutase, prevented this increase indicating that hydrogen peroxide may be the agent responsible for the action, whereas superoxide anion is not involved. Hydroxyl radicals formed by the Haber-Weiss or Fenton type reactions were not found to produce elevation of tyrosinase. Catalase determinations showed no enzyme in the medium but a high concentration in the cells. Inhibition of intracellular catalase by 3-amino-1,2,4-triazole caused an increase in the tyrosinase level. The effects of dopac, xanthine/xanthine oxidase, and glucose/glucose oxidase all producing hydrogen peroxide, and increasing tyrosinase, were enhanced by the inhibition of catalase. It is concluded that hydrogen peroxide, formed by the systems, accounts for the elevation of tyrosinase level. When tyrosinase activities determined by 5-S-CD formation were compared to enzyme amounts found by RIA, the ratios of these values were always constant. This fact indicates that the increase in the tyrosinase activities was not due to an activation of the enzyme, but mirrored the quantities of enzyme protein present in the samples. On the basis of our findings, it is assumed that hydrogen peroxide is a regulator of tyrosinase in normal melanocytes and melanoma cells.

Topics: 3,4-Dihydroxyphenylacetic Acid; Amitrole; Catalase; Glucose Oxidase; Humans; Hydrogen Peroxide; Melanoma; Monophenol Monooxygenase; Radioimmunoassay; Tumor Cells, Cultured; Xanthine Oxidase