phenylthiourea and Melanoma

Human tyrosinase (

Topics: Agaricales; Amino Acid Sequence; Biological Factors; Drug Delivery Systems; Drug Design; Enzyme Inhibitors; Humans; Melanins; Melanocytes; Melanoma; Monophenol Monooxygenase; Pigmentation; Protein Structure, Secondary; Skin Lightening Preparations

Melanosomes have been considered crucial targets in melanoma treatments. In this study we explored the role of melanosomes in photodynamic therapy (PDT), employing the synthetic Zn(II) phthalocyanine Pc13, a potent photosensitizer that promotes melanoma cell death after irradiation. Phototoxic action is mediated by reactive oxygen species increase. The internalization mechanism of Pc13 and its consequent subcellular localization were evaluated in melanotic B16-F0 cells. Pharmacological inhibitors of dynamin or caveolae, but not of clathrin, decreased Pc13 cellular uptake and phototoxicity. Similar results were obtained when cells over-expressed dominant negative mutants of dynamin-2 and caveolin-1, indicating that Pc13 is internalized by caveolae-mediated endocytosis. Confocal microscopy analysis revealed that Pc13 targets melanosomes and damage of these structures after irradiation was demonstrated by transmission electron microscopy. Treatment of pigmented B16-F0 and WM35 melanoma cells with the melanin synthesis inhibitor phenylthiourea for 48 h led to cell depigmentation and enhanced cell death after irradiation, whereas a 3-h period of inhibition did not modify melanin content but produced a marked reduction of Pc13 phototoxicity, together with a decrease of oxidative melanin synthesis intermediates. In contrast, the effect of Pc13 in amelanotic A375 cells was not altered by phenylthiourea treatment. These results provide evidence that melanosomes have a dual role in the efficacy of PDT. While melanin antagonizes the phototoxic action of Pc13, the release of cytotoxic synthetic intermediates to cytosol after irradiation and melanosome damage is conducive to the phototoxic response. Based on these findings, we demonstrate that melanosome-targeted PDT could be an effective approach for melanoma treatment.

Topics: Caveolin 1; Dermatitis, Phototoxic; Endocytosis; Humans; Indoles; Isoindoles; Melanins; Melanoma; Melanosomes; Phenylthiourea

Melanoma is the most threatening form of metastatic skin cancer that develops from melanocytes and causes a large majority of deaths due to poor therapeutic prognosis. It has significant limitations in treatment because it shows great resistance to chemotherapy, radiotherapy, and other therapeutic methods. A noninvasive and clinically accepted therapeutic modality, photodynamic therapy (PDT), is a promising treatment option, but it is limitedly applied for melanoma skin cancer treatment. This is because most of the photosensitizers are unlikely to be expected to have a remarkable effect on melanoma due to drug efflux by melanin pigmentation and intrinsic antioxidant defense mechanisms. Moreover, melanin is a dominant absorber in the spectral region of 500-600 nm that can cause the decreased photoreaction efficiency of photosensitizers. Herein, to overcome these drawbacks, we have developed a phenylthiourea-conjugated BODIPY photosensitizer (

Topics: Antineoplastic Agents; Biocompatible Materials; Boron Compounds; Cell Line, Tumor; Cell Survival; Drug Screening Assays, Antitumor; Humans; Materials Testing; Melanoma; Melanoma, Cutaneous Malignant; Molecular Structure; Monophenol Monooxygenase; Particle Size; Phenylthiourea; Photochemotherapy; Photosensitizing Agents; Skin Neoplasms

The resistance of pigmented human melanomas over their unpigmented counterparts to a number of therapies has suggested that the presence of intracellular melanin plays a role in rendering these cells less susceptible to cell death, probably through the ability of this pigment to act as an intracellular antioxidant, thus neutralizing chemotherapeutic-induced ROS (reactive oxygen species). PDT (photodynamic therapy) was recently suggested as an attractive, adjunctive therapy owing to its cellular specificity and limited side effects. In the present study, we propose that first depigmenting melanomas with a reversible TYR (tyrosinase) inhibitor such as PTU (phenylthiourea) increases their susceptibility to HYP-PDT (hypericin-mediated PDT). Pigmented [UCT Mel-1 (University of Cape Town melanoma cell line 1)] and unpigmented (A375) melanomas were first characterized with respect to their TYR activities and melanin quantities and then treated with a TYR inhibitor for 48 h. Cell viability assays after treatment with 3 μM HYP-PDT showed a significant increase in cell death in depigmented melanomas compared with untreated melanomas that returned to the level of untreated melanoma cells on removing the TYR inhibitor. The present study supports the hypothesis that combining the inhibition of melanogenesis with PDT should be explored as a valid therapeutic target for the management of advanced melanoma.

Topics: Anthracenes; Cell Line, Tumor; Cell Survival; Humans; Melanoma; Monophenol Monooxygenase; Perylene; Phenylthiourea; Photochemotherapy; Photosensitizing Agents; Pigmentation; Reactive Oxygen Species

A series of N-hydroxy-N'-phenylthiourea and N-hydroxy-N'-phenylurea analogues were prepared and evaluated as inhibitors of tyrosinase and melanin formation. The most active analogue 1 inhibited mushroom tyrosinase with an IC(50) of around 0.29 microM and also retained a substantial potency in cell culture by reducing pigment synthesis by 78%. Therefore, compound 1 could be considered as a promising candidate for preclinical drug development for skin hyperpigmentation application.

Topics: Agaricales; Animals; Cell Line, Tumor; Dose-Response Relationship, Drug; Inhibitory Concentration 50; Melanins; Melanocytes; Melanoma; Mice; Molecular Structure; Monophenol Monooxygenase; Peptides; Phenylthiourea; Stereoisomerism; Structure-Activity Relationship; Toxicity Tests

Melanin pigment displays strong photo- and radioprotective properties, suggesting that inhibition melanogenesis could increase sensitivity of melanoma to ionizing radiation. We tested this concept in human melanoma cells cultured in either Ham's F10 medium to maintain amelanotic phenotype or DMEM to induce/stimulate melanin production, respectively; N-phenylthiourea (PTU) and D-penicillamine were used as an inhibitor of melanogenesis. Melanogenic activity was evaluated by visual inspection (color of cell pellets) or by measurement of tyrosinase (dopa oxidase) activity assay. Amelanotic cells or cells with various melanin content were exposed to gamma radiation and tested for viability and colony forming capability. Gamma radiation at doses of 2-15 Gy inhibited cell viability and colony forming efficiency in dose- and time-dependent manner, but pigmented melanoma cells were significantly more resistant to gamma radiation than nonpigmented cells (p < 0.05-0.001). Both PTU and D-penicillamine inhibited strongly tyrosinase activity and melanin production in melanoma cells (p < 0.05-0.001). Furthermore, inhibition of melanogenesis by PTU or D-penicillamine resulted in enhancement of melanoma cells sensitivity to killing by gamma rays. In conclusion, the results of these cell culture experiments give support to a clinical trial of pharmacologically induced decrease in melanin synthesis to enhance the efficacy of radiotherapy in advanced melanomas.

Topics: Cell Line, Tumor; Cell Survival; Humans; Melanins; Melanoma; Melanoma, Amelanotic; Penicillamine; Phenylthiourea; Radiation Tolerance; Radiation-Sensitizing Agents; Radiation, Ionizing

It was previously found that L-tyrosine oxidation product(s) are cytotoxic, genotoxic and increase the sister chromatid exchange (SCE) levels in human melanoma cells. In this work, the micronucleus assay has been performed on human melanotic and amelanotic melanoma cell lines (Carl-1 MEL and AMEL) in the presence of 1.0, 0.5 and 0.1 mM L-tyrosine concentrations to investigate if melanin synthesis intermediate(s) increase micronuclei production. L-Tyrosine oxidation product(s) increased the frequency of micronuclei in melanoma cells; 0.1 mM phenylthiourea (PTU), an inhibitor of L-tyrosine oxidation by tyrosinase, lowered the micronucleus production to the control levels. The culture of melanoma cells with high L-tyrosine in the culture medium resulted in a positive response to an ELISA-based apoptotic test. For comparison the effect of L-tyrosine on micronuclei production in human amelanotic melanoma cells was also investigated; the micronucleus production in the presence of 1 mM L-tyrosine in the culture medium was lower than that found with melanotic melanoma cells of the same cell line. The data suggest that melanin synthesis intermediates arising from L-tyrosine oxidation may cause micronuclei production in Carl-1 human melanoma cells; the addition of PTU in the presence of L-tyrosine decreased the frequency of micronuclei to about the control values thus the inhibition of melanogenesis may have some clinical implication in melanotic melanoma.

Topics: Apoptosis; Cell Nucleus; Cytochalasin B; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Humans; Melanoma; Melanoma, Amelanotic; Micronucleus Tests; Monophenol Monooxygenase; Phenylthiourea; Skin Neoplasms; Tumor Cells, Cultured; Tyrosine

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

Our previous studies have shown that 4-S-cysteaminylphenol (4-S-CAP) causes a significant inhibition of in vivo melanoma growth and a marked depigmentation of black skin and hair follicles. These studies have suggested a role of tyrosinase in the manifestation of these in vivo effects. In this study 4-S-CAP and its analogues were examined for their effects on the growth of human melanoma cells in vitro. 4-S-CAP and 4-S-HomoCAP exhibited strong cytotoxicity with effects much greater than those of alpha-methyl-4-S-CAP and N,N-dimethyl-4-S-CAP. The cytotoxicity of the former two amines was completely prevented by semicarbazide, an inhibitor of plasma monoamine oxidase, while that of the latter two was not prevented by semicarbazide, catalase, and phenylthiourea, a tyrosinase inhibitor. In culture medium 4-S-CAP was rapidly converted by the action of monoamine oxidase present in fetal bovine serum to the aldehyde which was then metabolized to the alcohol and the carboxylic acid when cells were present. alpha-Methyl-4-S-CAP was found to exert higher cytotoxicity to cells with higher tyrosinase activity and melanin content. These results suggest that the in vitro cytotoxicity of 4-S-CAP and 4-S-HomoCAP is mediated through conversion to the aldehydes while that of alpha-methyl-4-S-CAP appears to be dependent on tyrosinase activity to some extent.

Topics: Antineoplastic Agents; Cell Division; Chromatography, High Pressure Liquid; Cysteamine; Humans; Melanins; Melanoma; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Monophenol Monooxygenase; Phenylthiourea; Semicarbazides; Tumor Cells, Cultured

The cytotoxic activities of three new synthetic catechol analogues, beta-[(p-hydroxyphenyl)amino]alanine (Compound 1), N delta-(p-hydroxyphenyl)ornithine (Compound 2), and N delta-(m-hydroxyphenyl)ornithine (Compound 3), were determined against 10 human melanoma and 5 nonmelanoma cell lines. Activities of L-DOPA and 3,4-dihydroxybenzylamine were also measured. Dose-response curves were obtained and concentrations in micrograms/ml required to give 90% inhibition of colony formation (IC90) were calculated. Using a cutoff IC90 of less than 2.5 as a definition of activity. Compound 2 was active in 6 of 10 melanoma and 0 of 5 nonmelanoma cell lines while both Compound 1 and L-DOPA methyl ester were active in 1 of 10 melanomas and 0 of 5 nonmelanomas. Compound 3 was inactive in all cell lines and all IC90 values exceeded 100. 3,4-Dihydroxybenzylamine was active in 3 of 8 melanomas and 1 of 5 nonmelanomas. Regression analysis of IC90 values for Compound 2 and tyrosinase levels in the 15 cell lines yielded a correlation coefficient of 0.93 (P less than 0.001). By contrast, a similar analysis for 3,4-dihydroxybenzylamine gave a correlation coefficient of 0.17 (P greater than 0.05). Spectrophotometric data indicated that Compounds 1 and 2 were oxidized by tyrosinase to quinones. Cytotoxic activity was blocked by the tyrosinase inhibitor phenylthiocarbamide. Since the rates of activation of Compounds 1 and 2 were identical, the higher activity of Compound 2 was probably due to its higher lipophilicity and greater intracellular accumulation. Compounds 1 and 2 exhibited greater potency and selectivity against malignant melanoma than did the natural product L-DOPA methyl ester.

Topics: Antineoplastic Agents; Catechols; Cell Survival; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Melanoma; Monophenol Monooxygenase; Phenylthiourea; Structure-Activity Relationship; Tumor Stem Cell Assay

The uptake (total radioactivity in intact cells) and incorporation (radioactivity bound to acid-precipitable material) of 14C-chlorpromazine (CPZ) and 14C-thiouracil (TU) were studied using a library of 3 human fibroblast strains and 13 tumour cell lines. In contrast to previous studies using rodent melanomas in vivo, the melanoma lines, including lines with high tyrosinase and melanin contents, did not take up more CPZ and TU than non-melanoma cells (fibroblasts, HeLa cells). Incorporation of CPZ was also broadly similar in all cell types studied. TU was selectively incorporated into the melanoma line having a high tyrosinase and melanin content but not into lines with high tyrosinase activity and low melanin content. While supporting the possibility of selective therapy for heavily-pigmented melanomas using labelled TU derivatives, these results suggest that the action of potentially melanoma-affined compounds should be further evaluated in human cells. Unlabelled CPZ or TU was not selectively toxic to melanoma cells. Unexpectedly, methylation-sensitive tumour cells (Mer-phenotype) were highly resistant to TU, thus providing a new experimental tool for understanding the genesis of this phenotype in vivo.

Topics: Cell Line; Chlorpromazine; Humans; Melanins; Melanoma; Monophenol Monooxygenase; Phenylthiourea; Thiouracil

A spontaneous cyclic phenomenon characterized by successive waves of either high proliferative rate or intense melanogenesis is described in non-confluent B16 melanoma cells subcultivated during 2 months (or more). Dopaoxidase activity is quantified in individual cells after L-dopa reaction, by an original method of visible light absorption cytophotometry. A 24-hr treatment with alpha-MSH increases dopa-oxidase activity. This increase is also noted during the following 14 hr, in a fresh medium devoid of alpha-MSH, in which cell proliferation resumes after 24 hr. Phenylthiourea, cycloheximide or actinomycin D inhibit dopaoxidase activity, but also cell proliferation in alpha-MSH pre-stimulated cells. The effects of the two latter agents suggest that de novo synthesis of the enzyme takes place following alpha-MSH treatment.

Topics: Animals; Cell Division; Cells, Cultured; Cycloheximide; Dactinomycin; Densitometry; Melanins; Melanocyte-Stimulating Hormones; Melanoma; Mice; Mice, Inbred C57BL; Monophenol Monooxygenase; Phenylthiourea

PTU markedly enhanced the cytotoxic effects of CuCl2 on chick embryonic PECs cultured in vitro. We investigated this newly discovered effect of PTU and its analogues in relation to the toxic effects of Cu ion. Most PECs maintained in medium containing 0.5 mM PTU were lysed within 4 h by the addition of 0.1 mM CuCl2, which addition killed no PECs in the absence of PTU. The effect of PTU was not specific to PECs. All the cell lines tested, KB, N-18, N-115 and B-16, reacted against exogenous Cu in the presence of PTU as did the PECs. Analogues of PTU had effects on PECs similar to those on PTU in the presence of Cu ion. ANTU had a greater effect than PTU. MTU and TU had less effect than PTU. PTU did not affect the cytolysis induced by the addition of the divalent cations Mn, Co and Zn. About 6-fold the 64Cu-uptake by PECs was scored in the presence of PTU. The relation between this cytotoxic-enhancing effect and other biological activities of PTU are discussed.

Topics: Animals; Carcinoma, Squamous Cell; Cations, Divalent; Cell Division; Cell Line; Cell Survival; Chick Embryo; Copper; Dose-Response Relationship, Drug; Drug Interactions; Epithelium; Humans; Kinetics; Melanoma; Mice; Neuroblastoma; Phenylthiourea; Pigmentation; Thiourea

The incorporation of [2-14C]-2-thiouracil and a series of [125I]-5-iodo-2-thiouracils ([125I]ISUra(s)) into cultured Greene hamster melanoma cells was determined in order to establish their properties as false precursors in the melanin-biosynthetic pathway. The cold trichloroacetic acid-precipitable incorporation of [2-14C]-2-thiouracil as well as [125I]ISUra into melanoma cells after a 24- to 48-hr labeling period proved to be completely tyrosinase dependent (more than 99.5% inhibition could be achieved by 0.5 mM phenylthiourea). [125I]ISUra incorporation was 3-fold higher than was [2-14C]-2-thiouracil incorporation and was enhanced by 1 mM theophylline treatment. [125I]ISUra incorporation into hamster, rabbit, and human melanoma cells showed a linear relationship with cell melanin content. Methylation of the sulfur completely prevented the incorporation, while propylation but not methylation at position 6 resulted in lower incorporation. [125I]ISUra proved to be a marker for melanogenesis and may be useful in studies on the differentiation of cultured melanoma cells.

Topics: Animals; Cell Division; Cells, Cultured; Cricetinae; Humans; Melanins; Melanoma; Phenylthiourea; Rabbits; Thiouracil

Topics: Animals; Carbon Isotopes; Culture Techniques; Depression, Chemical; Dihydroxyphenylalanine; Leucine; Melanins; Melanoma; Mice; Phenylthiourea; Thymidine; Tyrosine

Topics: Animals; Culture Techniques; Depression, Chemical; Dihydroxyphenylalanine; Melanins; Melanoma; Mice; Neoplasms, Experimental; Phenylthiourea