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Targeted therapy and immunotherapy have revolutionized the treatment of metastatic skin melanoma but around half of all patients develop resistance early or late during treatment. The situation is even worse for patients with metastatic uveal melanoma (UM). Here we hypothesized that the immunotherapy of therapy-resistant skin melanoma or UM can be enhanced by epigenetic inhibitors. Cultured B16F10 cells and human UM cells were treated with the histone deacetylase inhibitor (HDACi) entinostat or BETi JQ1. Entinostat-induced HLA expression and PD-L1, but JQ1 did not. A syngeneic mouse model carrying B16-F10 melanoma cells was treated with PD-1 and CTLA4 inhibitors, which was curative. Co-treatment with the bioavailable BETi iBET726 impaired the immunotherapy effect. Monotherapy of a B16-F10 mouse model with anti-PD-1 resulted in a moderate therapeutic effect that could be enhanced by entinostat. Mice carrying PD-L1 knockout B16-F10 cells were also sensitive to entinostat. This suggests HDAC inhibition and immunotherapy could work in concert. Indeed, co-cultures of UM with HLA-matched melanoma-specific tumor-infiltrating lymphocytes (TILs) resulted in higher TIL-mediated melanoma killing when entinostat was added. Further exploration of combined immunotherapy and epigenetic therapy in metastatic melanoma resistant to PD-1 inhibition is warranted., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

To determine whether blockade of the chemokine receptor CXCR4 might alter the tumor microenvironment and inhibit tumor growth, we tested the efficacy of the CXCR4 antagonist X4-136 as a single agent and in combination with various immune checkpoint inhibitors in the syngeneic murine melanoma model B16-OVA. We also tested its activity alone and in combination with axitinib in the renal cancer model Renca. We found that X4-136 exhibited potent single agent antitumor activity in the B16-OVA model that was additive to that of an anti-PDL1 antibody. The antitumor activities were associated with a reduction in the number of immunosuppressive regulatory T cells and myeloid-derived suppressor cells and an increase in the number of tumor-specific CD8/perforin cells in the tumor-microenvironment. Apart from these immune effects, X4-136 alone and in combination with checkpoint inhibitors inhibited the Akt/FOXO-3a cell survival pathway in vitro and in vivo, suggesting that it might have antitumor activity independent of its effects on immune cell trafficking. Similar effects on tumor growth and cytotoxic T lymphocytes infiltration were observed in the Renca model. These studies show that the effects of CXCR4 blockade on immune cell trafficking might serve as a useful adjunct to immune checkpoint inhibitors and other therapies in the treatment of cancer.

Melanoma, an aggressive skin tumor with high metastatic potential, is associated with high mortality and increasing morbidity. Multiple available chemotherapeutic and immunotherapeutic modalities failed to improve survival in advanced disease, and the search for new agents is ongoing. The aim of this study was to investigate antimelanoma effects of O,O-diethyl-(S,S)-ethylenediamine-N,N'di-2-(3-cyclohexyl) propanoate dihydrochloride (EE), a previously synthesized and characterized organic compound. Mouse melanoma B16 cell viability was assessed using acid phosphatase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, sulforhodamine B, and lactate dehydrogenase assays. Apoptosis and autophagy were investigated using flow cytometry, fluorescence and electron microscopy, and western blotting. In vivo antitumor potential was assessed in subcutaneous mouse melanoma model after 14 days of treatment with EE. Tumor mass and volume were measured, and RT-PCR was used for investigating the expression of autophagy-related, proapoptotic, and antiapoptotic molecules in tumor tissue. Investigated organic compound exerts significant cytotoxic effect against B16 cells. EE induced apoptosis, as confirmed by phosphatidyl serine externalisation, caspase activation, and ultrastructural features typical for apoptosis seen on fluorescence and electron microscopes. The apoptotic mechanism included prompt disruption of mitochondrial membrane potential and oxidative stress. No autophagy was observed. Antimelanoma action and apoptosis induction were confirmed in vivo, as EE decreased mass and volume of tumors, and increased expression of several proapoptotic genes. EE possesses significant antimelanoma action and causes caspase-dependent apoptosis mediated by mitochondrial damage and reactive oxygen species production. Decrease in tumor growth and increase in expression of proapoptotic genes in tumor tissue suggest that EE warrants further investigation as a candidate agent in treating melanoma.

Cutaneous melanoma is one of the most lethal cancers because of its increased rate of metastasis and resistance to available therapeutic options. Early studies indicate that metformin has beneficial effects on some types of cancer, including melanoma. To clarify knowledge of the mechanism of action of metformin on this disease, two treatment-based approaches are presented using metformin on melanoma progression: an in-vitro and an in-vivo model. The in-vitro assay was performed for two experimental treatment periods (24 and 48 h) at different metformin concentrations. The results showed that metformin decreased cell viability, reduced proliferation, and apoptosis was a major event 48 h after treating B16F10 cells. Oxidative stress was characterized by the decrease in total thiol antioxidants immediately following 24 h of metformin treatment and showed an increase in lipid peroxidation. The in-vivo model was performed by injecting B16F10 cells into the subcutaneous of C57/BL6 mice. Treatment with metformin began on day 3 and on day 14, the mice were killed. Treatment of mice with metformin reduced tumor growth by 54% of its original volume compared with nontreatment. The decrease in systemic vascular endothelial growth factor, restoration of antioxidants glutathione and catalase, and normal levels of lipid peroxidation indicate an improved outcome for melanoma following metformin treatment, meeting a need for new strategies in the treatment of melanoma.

The lack of an efficient agent that does not have the disadvantage of low activity (kojic acid), high cytotoxicity, and mutagenicity (hydroquinone), poor skin penetration (arbutin), or low stability in formulation (glabridin) led us to continue our research on new antipigmentation/skin-lightening agents. Therefore, research of natural products that can modulate the metabolism of pigmentation is of great interest. Otherwise, malignant melanoma is one of the most aggressive forms of skin cancer, with high metastatic potential, and currently, there is no effective chemotherapy against invasive melanoma. Therefore, it is necessary to develop new drugs with potent activity and weak side effects against melanoma. The in-vitro anticancer effect of hawthorn was analyzed against B16F10 melanoma cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effect of isolated compounds from hawthorn on melanogenesis in B16F10 melanoma cells was investigated by measuring the amounts of melanin and tyrosinase spectrophotometrically at 475 nm. Balb/c mice models inoculated with B16F10 mouse tumor cells were used to evaluate the in-vivo antitumoral potential of hawthorn by assessing its effect on the growth of transplanted tumors. The antioxidant potential of tested samples was evaluated in B16F10 and primary human keratinocyte cells using a cellular antioxidant activity assay. Hawthorn tested samples inhibited effectively the growth of melanoma cells in vitro. Furthermore, it appears that tested samples from hawthorn reduced melanogenesis by inhibiting the tyrosinase activity of B16F10 cells in a dose-dependent manner. In-vivo studies showed that hawthorn total oligomer flavonoids extract treatment at a dose of 150 mg/kg body weight for 21 days in implanted tumor mice resulted in significant inhibition of the tumor growth volume and weight. In addition, tested samples showed significant cellular antioxidant capacity against the reactive oxygen species in B16F10 and primary human keratinocyte cells. Our results indicate that hawthorn could be considered as a promising agent for the treatment of melanoma as it shows antitumor activity in vitro and in vivo. Moreover, hawthorn constituents are shown to be highly effective at inhibiting tyrosinase-mediated melanogenesis in vitro on melanoma cells by preventing oxidation in these cells and without affecting the viability of normal human keratinocyte cells. Then, hawthorn might also be used as a new candidate of natural skin depigmenting agents in skin care products.

Melanoma is a type of cancer arising from the melanocytes, which are the cells that make up the pigment melanin and are derived from the neural crest. There is no particularly effective therapy once the disease is metastatic, highlighting the need for discovery of novel potent agents. In this investigation, we adopted a zebrafish embryonic pigmentation model to identify antimelanoma agents by screening an in-house small molecule library. With this assay, we found that a small molecule compound, SKLB226, blocked zebrafish pigmentation and pigment cell migration. Mechanism of action studies showed that SKLB226 downregulated MITF mRNA level in both zebrafish embryos and mammalian melanoma cells. Further studies showed that it could efficiently suppress the viability and migration of mammalian melanoma cells. In summary, SKLB226 can be used as a chemical tool to study melanocyte development as well as an antimelanoma lead compound that should be subjected to further structural optimization.

Malignant melanoma is associated with a 5-year survival rate of less than 20% once metastasized. Malignant melanoma cells exhibit increased levels of autophagy, a process of intracellular digestion that allows cells to survive various stresses including chemotherapies, resulting in reduced patient survival. Autophagy can be inhibited by chemicals like chloroquine (CQ), which prevents fusion of autophagosomes to lysosomes, resulting in autophagosome accumulation in most systems. Here, we describe how tested CQ to see whether it could sensitize B16F10 metastatic mouse melanoma cells to the anticancer activities of the natural compounds ursolic acid (UA) and resveratrol (RES). CQ with UA or RES strongly and synergistically reduced the viability of B16F10 mouse melanoma and A375 human melanoma cells. Surprisingly, flow cytometry of acridine orange-stained cells showed that UA or RES in combination with CQ significantly reduced autophagosome levels. Western blotting analysis revealed that CQ plus UA or RES paradoxically increased LC3II, indicative of autophagosome accumulation. In addition, CQ plus RES synergistically decreased the levels of both autophagy initiator beclin-1 and autophagy supporter p62. These results indicate that CQ with UA or RES strongly and synergistically reduces the viability of B16F10 and A375 melanoma cells. However, studies on B16F10 cells have shown that the synergistic effect was not mediated by inhibition of autophagy induced by UA or RES. These compounds are well-tolerated in humans, and CQ has shown promise as an adjuvant therapy. These combinations may be valuable treatment strategies for melanoma., (Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.)

β-Elemene has been reported to be effective for the treatment of leukemia and certain solid tumors in basic and clinical studies. However, the mechanism of action of this phytochemical remains unknown. This study aimed to investigate the effect and mechanism of β-elemene in the mouse melanoma cell line B16F10. Cell viability was measured using the MTT assay. β-Elemene inhibited B16F10 melanoma cell metastasis, examined using scratch and Transwell migration/invasion assays. The mRNA and protein expression of urokinase-type plasminogen activator (uPA), the uPA receptor (uPAR), matrix metalloproteinase (MMP)-2, and MMP-9 were assayed using real-time PCR, immunocytochemistry, and western blotting methods. The results indicated that β-elemene inhibited the viability of B16F10 melanoma cells in a dose-dependent and time-dependent manner. The migratory and invasive capacities of B16F10 cells were also inhibited by β-elemene. The expression of uPA, uPAR, MMP-2, and MMP-9 was reduced by β-elemene at both the mRNA and protein level. β-Elemene inhibits the metastasis of B16F10 melanoma cells through downregulation of the expression of uPA, uPAR, MMP-2, and MMP-9. Thus, β-elemene is a natural potential anticancer drug.

Previous studies have shown that transfection of the snake venom cystatin (sv-cystatin) gene can inhibit the invasion and metastasis of tumor cells. The aim of this study was to investigate the pharmaceutical applications of sv-cystatin in melanoma gene therapy. We constructed a recombinant adenovirus carrying sv-cystatin (Ad/sv-cystatin) and a control virus (Ad/null). Matrigel assays were used to assess melanoma cell migration and invasiveness in vitro. The antimelanoma effects of Ad/sv-cystatin were assessed in a syngeneic mouse model with an experimental lung colonization assay. Ad/sv-cystatin significantly inhibited the invasion and growth of B16F10 cells in vitro compared with control and Ad/null. Ad/sv-cystatin significantly inhibited experimental lung colonization in C57BL/6 mice as compared with that in control (P<0.001) and Ad/null-treated mice (P<0.001), with an inhibition rate of 51 and 46%, respectively. Ad/sv-cystatin slowed the increase in lung weight in C57BL/6 mice as compared with that in control mice (P<0.001) and Ad/null-treated mice (P<0.001), with an inhibition rate of 40 and 35%, respectively. Our results indicate that Ad/sv-cystatin suppresses mouse melanoma invasion, metastasis, and growth in vitro and in vivo. Our findings provide support for the further examination of the pharmaceutical applications of Ad/sv-cystatin.

The HSP70 family member GRP78 is a selective tumor marker upregulated on the surface of many tumor cell types, including melanoma, where it acts as a growth factor receptor-like protein. Receptor-recognized forms of the proteinase inhibitor α2-macroglobulin (α2M*) are the best-characterized ligands for GRP78, but in melanoma and other cancer patients, autoantibodies arise against the NH2-terminal domain of GRP78 that react with tumor cell-surface GRP78. This causes the activation of signaling cascades that are proproliferative and antiapoptotic. Antibodies directed against the COOH-terminal domain of GRP78, however, upregulate p53-mediated proapoptotic signaling, leading to cell death. Here, we describe the binding characteristics, cell signaling properties, and downstream cellular effects of three novel murine monoclonal antibodies. The NH2-terminal domain-reactive antibody, N88, mimics α2M* as a ligand and drives PI 3-kinase-dependent activation of Akt and the subsequent stimulation of cellular proliferation in vitro. The COOH-terminal domain-reactive antibody, C38, acts as an antagonist of both α2M* and N88, whereas another, C107, directly induces apoptosis in vitro. In a murine B16F1 melanoma flank tumor model, we demonstrate the acceleration of tumor growth by treatment with N88, whereas C107 significantly slowed tumor growth whether administered before (P<0.005) or after (P<0.05) tumor implantation.

Resveratrol, a nontoxic natural product, exhibits multifaceted biological effects including antimutagenic and anticancer properties. We examined the effect of resveratrol on the expression and activation of Akt/protein kinase B and its impact on melanoma cell migration and invasiveness. We also explored the use of resveratrol as an antimalignant treatment option against skin melanoma in mouse models of the disease. Akt expression and activity were determined by a combination of real-time PCR and western blot analysis. Cell lines stably expressing Akt or a dominant negative variant were used to further establish the role of Akt during the response to resveratrol. Wound healing and transwell assays were used as in-vitro correlates of melanoma cell migration and invasiveness. The efficacy of resveratrol in the treatment of melanoma was assessed in two syngeneic mouse models. Resveratrol downregulated and inactivated Akt in B16F10 and B16BL6 melanoma cells. Resveratrol also inhibited the migratory and invasive properties of these highly malignant cells. The reduction of cell migration and invasion, however, was reversed in cell lines overexpressing Akt or after cotreatment with pharmacological inhibitors that blocked Akt degradation. Dominant-negative Akt cells were more sensitive to resveratrol and had diminished migratory properties. Oral treatment with resveratrol reduced primary tumor volume, Akt expression, and the propensity for metastasis in syngeneic mouse models of melanoma. These results suggest that resveratrol can reduce the malignant properties of highly invasive melanoma cells by inactivating Akt. The nontoxic targeting of Akt by resveratrol makes it an attractive treatment option for melanoma.

Biflorin, an ortho-naphthoquinone, is an active compound found in the roots of Capraria biflora L. It has been reported that biflorin presents anticancer activity, inhibiting both tumor cell line growth in culture and tumor development in mice. The aim of this study was to examine the effectiveness of biflorin treatment using both in-vitro and in-vivo melanoma models. Biflorin displayed considerable cytotoxicity against all tested cell lines, with half maximal inhibitory concentration values ranging from 0.58 μg/ml in NCI H23 (human lung adenocarcinoma) to 14.61 μg/ml in MDA-MB-231 (human breast cancer) cell lines. In a second set of experiments using B16 melanoma cells as a model, biflorin reduced cell viability but did not cause significant increase in the number of nonviable cells. In addition, the DNA synthesis was significantly inhibited. Flow cytometry analysis showed that biflorin may lead to an apoptotic death in melanoma cells, inducing DNA fragmentation and mitochondria depolarization, without affecting membrane integrity. In B16 melanoma-bearing mice, administration of biflorin (25mg/day) for 10 days inhibited tumor growth, and also increased the mean survival rate from 33.3±0.9 days (control) to 44.5±3.4 days (treated). Our findings suggest that biflorin may be considered as a promising lead compound for designing new drugs to be used in the treatment of melanoma.

Melanomas respond poorly to chemotherapy. In this study, we investigated the sensitization of B16 mouse melanoma tumors to paclitaxel by a combination of two tyrosine kinase inhibitors: vatalanib, targeting vascular endothelial growth factor receptors, and imatinib, an inhibitor targeting for example, Abl/BCR-ABL, the platelet-derived growth factor receptor, and stem cell factor receptor c-Kit. C57Bl6/J mice carrying B16/PDGF-BB mouse melanoma tumors were treated daily with vatalanib (25 mg/kg), imatinib (100 mg/kg), or a combination of these drugs. Paclitaxel was given subcutaneously twice during the study. The effects of the drugs on tumor cell proliferation in vitro were determined by counting cells. B16/PDGF-BB mouse melanoma tumors were not sensitive to paclitaxel at doses of either 5 or 20 mg/kg. However, the tumor growth was significantly reduced by 58%, in response to paclitaxel (5 mg/kg) when administered with daily doses of both vatalanib and imatinib. Paclitaxel only inhibited the in-vitro growth of B16/PDGF-BB tumor cells when given in combination with imatinib. Imatinib presumably targets c-Kit, as the cells do not express platelet-derived growth factor receptor and as another c-Abl inhibitor was without effect. This was supported by data from three c-Kit-expressing human melanoma cell lines showing varying sensitization to paclitaxel by the kinase inhibitors. In addition, small interfering RNA knockdown of c-Kit sensitized the cells to paclitaxel. These data show that combination of two tyrosine kinase inhibitors, imatinib and vatalanib, increases the effects of paclitaxel on B16/PDGF-BB tumors, thus suggesting a novel strategy for the treatment of melanomas expressing c-Kit.

There is now increasing evidence that a constitutive expression of cyclooxygenase 2 (COX-2) plays a role in the development and progression of malignant ectodermal tumours. In this study, we investigated whether the selective inhibition of COX-2 would be beneficial in melanoma treatment. Nimesulide, a selective inhibitor of COX-2 that causes the breakdown of proinflammatory 2-series prostaglandins (PG2), adversely affected the growth of B16-F10 melanoma cells through the induction of differentiation. The intracellular levels of polyamine, as a proliferation marker, were reduced by the treatment; at the same time, transglutaminase activation and increase in melanin content, as differentiation indicators, were observed. The potential antimetastatic activity of the drug was further shown by means of the Boyden invasion assay and gelatin zymography for metalloproteinase activity. Comparable results were obtained after the treatment of cells with one of the 1-series PGs (PGE1). Therefore, our hypothesis is that the antineoplastic activity observed for nimesulide may be ascribed to intracellular changes in alterations in PG levels.

Ginsenoside Rg3 is an effective chemical component extracted from the red Panix. The experiment demonstrated that it might effectively inhibit proliferation and metastasis of tumor cells. The exact molecular mechanism of Rg3 remains unclear so far. To further explore the antitumor function of Rg3, we investigated the in-vitro and in-vivo activity of Rg3 in the treatment of B16 melanoma cells, derived from C57BL/6 mouse, capable of forming tumor colonies in the lungs following intravenous injection. Cell proliferation was measured by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide assay. Morphological changes of cells were observed by staining with Giesma and Hoechst 33258. Cell cycle and apoptosis rate were analyzed by flow cytometry. The expression of caspase-3 and bcl-2 in cells was detected by immunocytochemistry and western blot analysis. We found that Rg3 could inhibit cell proliferation, regulate cell cycle, and induce cell apoptosis in vitro. B16 melanoma-bearing mice were used to evaluate in vivo the antitumor activity of Rg3. Mice that were injected with Rg3 showed significant inhibition of the tumor metastasis with lighter lung weight, lower density of microvessels, fewer metastasis nodules, and longer survival time than those in the control group (P<0.001). In conclusion, the results reveal that antitumor metastasis of Rg3 is also associated with inducing apoptosis, regulating cell cycle, and blocking angiogenesis in addition to inhibiting proliferation. This research might supply valuable data for chemotherapy with Rg3 in melanoma. Rg3 would turn out to be an anticancer drug with promising prospects.

The in-vivo antineoplastic potential of the alkylating N-(2-dialkylaminoethyl)benzamides BZA1 and BZA2, novel melanoma targeted anticancer drugs, was evaluated in a mouse melanoma model with nude mice bearing subcutaneous SkMel28, B16 or WM266-4. The maximal tolerated dose (MTD) for the intraperitoneal application of both agents was found to be 24 mg/kg. Treatment was initiated with an intraperitoneal injection of 8 mg/kg of BZA1 or BZA2 on days 0, 2 and 4 in the case of B16 melanoma on days 0, 1 and 2 after the onset of the experiment, when the mean tumor diameter ranged within 4-6 mm. The experiment was terminated when the mean tumor diameter in the control group had reached a value of 12 mm. Tumor growth delay of these agents was compared with dacarbazine (3x250 mg/kg), chlorambucil (3x5 mg/kg) and an untreated control group. Significant tumor growth delay was observed under BZA1, BZA2 and dacarbazine treatment compared with the untreated control group in all three evaluated melanomas with insignificant differences among BZA1, BZA2 and dacarbazine. The insignificant effect of chlorambucil and the strong improvement on growth delay achieved with BZA1 and BZA2 demonstrated melanoma targeting characteristics of the N-(2-dialkylaminoethyl)benzamide structure element. Dacarbazine was more effective in the in-vivo antineoplastic assay compared with the in-vitro cytotoxicity studies, probably due to hepatic bioactivation. Similar side effect intensity of BZA2 and dacarbazine was observed, whereas BZA1 was more toxic. BZA2 might represent an alternative antimelanoma drug, especially in patients not responding to dacarbazine.

N-(2-Dialkylaminoethyl)benzamides have been shown to selectively accumulate in melanoma metastases with high uptake capacity. Therefore, this class of compound has previously been evaluated as a transporter for cytostatic drugs. It has been demonstrated that this significant targeting effect improves the cytotoxicity against melanoma cells. Although these agents are not accumulated by non-melanoma cells, they have been found to be toxic. In order to identify mechanistic reasons for this effect, we investigated the DNA and melanin binding affinities of a selection of four benzamide-drug conjugates, together with their parental cytostatics. An investigation of the influence of the melanin content on the cytotoxicity of these substances in B16 melanoma and Morris hepatoma (MH3924A) cells was performed, together with their influence on melanosomal pH and tyrosinase activity. The suppression of melanin formation with phenylisothiourea and the saturation of melanin binding sites with chloroquine were also investigated. These experiments demonstrated high DNA binding and low melanin affinity, in accordance with the toxicity against tumour cells. Melanin has a concentration-dependent scavenging effect, thereby reducing cytotoxicity. These compounds lead to an increase in the acidic pH of melanosomes, resulting in an increase in tyrosinase activity. The consequence of this reaction chain is an amplification of the scavenging effect for the benzamide-drug conjugates. These effects may be considered as limiting factors for the targeting characteristics of this class of compound, necessitating further modifications to the carrier system.

Evidence indicating that hybrid steroid compounds of anti-cancer agents produce reduced toxicity, significantly lower than the cytotoxic components alone, and increased anti-cancer activity has prompted the design and development of such steroids, mostly alkylating esters. We investigated the in-vitro and in-vivo activity of a homo-aza-steroidal alkylating ester (HASE), in comparison with dacarbazine (DTIC), cisplatin (CPDD), carmustine (BCNU) and semustine (MeCCNU), in the treatment of malignant melanoma. Cytotoxicity was assessed in vitro by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using a panel of six human malignant melanoma cell lines, with or without the presence of rat liver microsome assay. B16 melanoma-bearing mice were used to evaluate in vivo the anti-tumour activity of the tested compounds. In all cases of in-vitro screening, HASE displayed a significantly higher (P<0.0001) cytostatic and cytotoxic effect than DTIC, BCNU and MeCCNU, but produced significantly lower (P<0.0001) activity than CPDD. HASE exhibited a significantly smaller range than CPDD between concentration levels that produced growth arrest and those that induced a cytotoxic effect against melanoma cells in vitro. The anti-tumour activity of HASE in B16 melanoma-bearing mice, as determined by tumour growth rate inhibition (<42%) and percentage survival prolongation (treated versus control, 167%), was significantly superior (P<0.001) to that achieved by DTIC, BCNU and MeCCNU and was equal to that of CPDD. HASE exhibited a toxicity similar to that of DTIC, BCNU and MeCCNU, but significantly lower than that of CPDD. It can be concluded that HASE displays significant in-vitro and in-vivo activity in the treatment of melanoma.

Tumour cell motility, which is dependent on the organization of the cytoskeleton, is considered to play an important role in the spread of malignant melanoma. Therefore, retinoids, which are modulators of cytoskeletal organization, may affect the motile activity of melanoma cells. In this study, the effects of the arotinoid mofarotene on single cell motility and vinculin organization of the highly metastatic melanoma cell line K-1735-M2 were determined. Melanoma cells were cultivated in a temperature- and CO2-controlled microincubator, which was located on the microscope stage. Cell movements were evaluated quantitatively from time-lapse video recordings using an IBAS image analysis system. Vinculin distribution was evaluated using confocal laser scanning microscopy and a specially developed computerized image analysing program. In addition, melanoma cell invasion was tested on the embryonic chick heart model. Although 10 microM mofarotene did not reduce the translocative movements of melanoma cells, it significantly inhibited stationary motility, including fast plasma membrane movements and changes in shape. Mofarotene also showed a pronounced effect on the organization of vinculin-containing cell-substratum adhesion plaques. In retinoid-treated cells, the numbers of vinculin plaques per cell, and particularly those in the marginal areas of the cells, were significantly increased compared with untreated controls. Furthermore, the compound reduced the invasiveness of melanoma cells in a three-dimensional tissue culture model. In conclusion, our data demonstrate that mofarotene, an already almost forgotten synthetic retinoid, shows interesting effects on melanoma cells, which may be relevant for a slowdown of tumour spread.

Radioiodinated N-(2-(diethylamino)ethyl)benzamides have recently been discovered as selective agents for melanotic melanoma and are used for scintigraphic imaging in nuclear medicine. Owing to the high binding capacity, benzamide derivatives conjugated with alkylating cytostatics were synthesized and tested for their potential for targeted drug delivery. Conjugates of chlorambucil with procainamide (1), diethylaminoethylamine (2) and 2-pyrrolidin-1-yl-ethylamine (3), as well as 4-(bis(2-chloroethyl)amino)- (6,7) and 4-(N,N-diethyltriazeno)-substituted (8-10) benzamides, were synthesized. Cell uptake studies with B16 melanoma cells revealed high uptake of radioiodinated 1 and 2, while radiolabelled chlorambucil was found to lack this characteristic. These results were confirmed by biodistribution studies in a mouse melanoma model. Viability measurements revealed that all chlorambucil-benzamide derivatives showed higher toxicity against B16 melanoma and SK-MEL-28 cells than did the parent chlorambucil itself, and that the triazene derivatives were more potent than dacarbazine, which is currently used as a standard cytostatic drug in melanoma therapy. Of all the compounds tested in this series, the triazenes 9 and 10 showed the most promising targeting effect. The toxicity of these compounds against hepatoma cells (MH3924A) and, to a lesser extent, against mouse fibroblast (NIH 3T3) and cervix carcinoma (HeLa) cells was also enhanced, but they were not as toxic as dacarbazine (HeLa). These findings support the concept of a selective, benzamide-mediated in vivo delivery of cytostatics in melanoma cells, leading to enhanced efficacy.

Melanogenesis appears to be a unique target to develop anti-tumour agents specific for malignant melanoma. Among the anti-melanoma compounds that we have examined, 4-S-cysteaminylphenol (4-S-CAP), a phenolic amine, was found to have the most promising anti-melanoma effects. To further improve the efficacy as anti-melanoma agents, we have recently synthesized enantiomers of alpha-Me-4-S-CAP and alpha-Et-4-S-CAP. The enantiomers were found to be good substrates for tyrosinase. In vitro experiments showed that the enantiomers were highly cytotoxic to B16-F1 melanoma cells, and the cytotoxic effect was proved to be tyrosinase-dependent. In the present study, in vivo cytotoxicity experiments showed that i.p. administration of R-alpha-Me-4-S-CAP and S-alpha-Et-4-S-CAP (and 4-S-CAP) strongly inhibited the subcutaneous growth of B16 melanoma in mice, while the corresponding enantiomers were much less effective. Similarly, i.p. treatment with R-alpha-Me-4-S-CAP or S-alpha-Et-4-S-CAP, but not with 4-S-CAP, caused strong depigmentation of follicular melanocytes in C57BL black mice. Among 4-S-CAP and the enantiomers, only R-alpha-Et-4-S-CAP caused a moderate decrease in blood pressure in spontaneously hypertensive rats. These results confirm that the use of enantiomers increases the efficacy of tyrosinase-dependent cytotoxic phenolic amines.

Melatonin has been reported to possess growth inhibitory action at certain physiological doses in cancer cell lines in vitro and oncostatic action under in vivo conditions. In an attempt to achieve a pharmacologically effective anticancer action of melatonin, dose-response studies with high concentrations of melatonin (10(-6) M, 10(-5) M, 10(-4) M and 10(-3) M) were conducted in the B16 murine melanoma cell line using three different numbers of exposed cells. A range of effects, including stimulatory, oncostatic and oncocidal action, were studied 3 days after exposure to melatonin. In order to standardize the results, the concentration of melatonin per cell was calculated from the amount of melatonin added to the culture, and compared with the growth patterns of the cells. Melatonin had a mild stimulatory effect on cell proliferation at the lower end of the dose spectrum and an oncostatic influence at intermediate concentrations, while the higher concentrations per cell demonstrated clear lethal (oncocidal) action. It is suggested that by using a pharmacologically appropriate dosage regimen, melatonin could be useful in the treatment of responsive cancers. Furthermore, calculation of the concentration of melatonin per cell is important in understanding the true pharmacological potential of melatonin.

Major histocompatibility complex (MHC) class I proteins are required for the formation of complexes with antigenic peptides that enable cytotoxic T lymphocytes (CTLs) to recognize and lyse target cells. The frequent loss of MHC class I expression reported in human melanomas and melanoma cell lines may therefore be an obstacle to CTL-based immunotherapy. We have investigated the expression of MHC class I proteins in the cutaneous melanomas of Tyr-SV40E (C57BL/6 strain) transgenic mice in order to evaluate their potential as experimental models for immunotherapy. The SV40 large T (LT) oncoprotein, which is expressed exclusively in the melanocytic lineages of these mice, was used as a marker for flow cytometric analysis of the parenchymal (potential target) cells of 35 freshly dissociated samples from 28 primary tumours. All the tumours were ulcerated and exceeded the Breslow thickness indicative of a poor clinical prognosis in human melanoma. Using antibodies against H-2D(b) and H-2K(b) class I proteins, the LT antigen-positive cells were found to have high levels of both these MHC class I molecules in the thinnest tumours (2 mm), whereas the levels tended to decline with increasing tumour thickness. Among the tumours > 4 mm thick, five had no detectable MHC class I expression. Unexpectedly, the apparent loss of H-2D(b) and H-2K(b) proteins was observed not only in LT-positive cells but also in LT-negative cell populations. Expression of both H-2D(b) and H-2K(b) was restored in tumours derived from a class I-low melanoma cell line by treatment of the hosts with interferon-gamma. These results implicate a regulatory defect as a principal cause of the loss of MHC class I antigens, as noted by others in some human tumours, and they demonstrate that this loss is remediable, even in advanced stages of melanomas.

Histamine is produced by many cells expressing histidine decarboxylase (HDC), the enzyme responsible for the synthesis of histamine. Since melanoma cells and tissue contain relatively large amounts of histamine, the functional significance of histamine was examined using specific antihistamines in vitro and in vivo in the human melanoma cell line HT168 and severe combined immunodeficiency (SCID) mice. It was shown that the H2 receptor antagonist cimetidine when combined with N, N-diethyl-2-[4-(phenylmethyl)phenoxy]-ethanamine-HCl (DPPE), a tamoxifen derivate, inhibits the proliferation of HT168 cells. Furthermore, it is suggested that there is a factor(s) that interferes with the exponential growth of HT168 cells xenografted to immunodeficient mice, and cimetidine and DPPE together significantly influence this factor(s). This combination of antihistamines also increases the survival of human melanoma-grafted mice. These changes are accompanied by enhanced infiltration of interferon-gamma- producing mouse macrophages into the tumour tissue. These findings suggest that two different mechanisms are probably acting concordantly: direct inhibition of tumour cell proliferation by the H2 receptor antagonists, and activation of the local immune response characterized by interferon-gamma production. These findings may help to elucidate the possibility of a rationally designed antihistamine strategy in melanoma therapy.

Although flavonoids seem to be capable of acting at all stages of the carcinogenic process, little information is available on their action in melanoma cell lines. The aim of this study was to assess the response of B16F10 and SK-MEL-1 melanoma cell lines to treatment with six different flavonoids after 24 and 72 h of exposure and to relate the response to their structure. We then compared the findings with those for melphalan treatment. When cultures were treated for 24 h, only slight inhibition at the highest concentrations (25 and 50 microM) of tangeretin and luteolin were observed, whereas melphalan caused a dose-related inhibition of growth at all concentrations. Quercetin, hesperetin, 7,3'-dimethylhesperetin and eriodictyol did not produce any effect at 24 h on B16F10 or SK-MEL-1 cells, results which point to the low toxicity of flavonoids. After 72 h of exposure culture growth was inhibited by 7,3'-dimethylhesperetin at 50 microM, but lower concentrations had no effect. Tangeretin was the most effective of the flavonoids in inhibiting B16F10 and SK-MEL-1 cell growth, showing a clear dose-response curve after 72 h. These results suggest that the absence of the C2-C3 double bond on hydroxylated flavonoids results in a loss of effect on both the cell lines, while the higher activity of tangeretin compared with 7,3'-dimethylhesperetin suggests that the presence of at least three adjacent methoxyl groups confers a more potent antiproliferative effect.

The efficacy of sequential chemoimmunotherapy involving interleukin-2 (IL2) in metastatic melanoma is limited, in part, by the severe toxicity associated with most therapeutic regimens. Glutathione (GSH), the most prevalent intracellular non-protein thiol, plays an important role in protecting against cellular injury caused by various anticancer agents. GSH is also involved in the IL2-induced proliferative activity of immune system cells and some melanoma cells expressing IL2 receptors, such as B16 melanoma cells. The present study investigated the effect of selective manipulation of GSH using the cysteine prodrug l-2-oxothiazolidine-4-carboxylate (OTZ) on the response of B16 melanoma to sequential biochemotherapy with cyclophosphamide (CY) and IL2. We found that OTZ, by depressing GSH levels, abrogates the in vitro growth-promoting effects of IL2 on B16 melanoma cells. The combination of OTZ plus IL2 in vivo also showed antitumour activity in mice bearing B16 melanoma liver metastases, significantly increasing their life span. Schedule dependency between both compounds was found; OTZ given intermittently in combination with daily IL2 administration was found to be the best therapeutic schedule. We also observed that whereas IL2 or OTZ alone added to CY resulted in a lower or non-significant improvement in the life span of the mice compared with tolerated doses of CY alone, the addition of both OTZ and IL2 to CY produced a significantly greater increase in survival than CY alone, and markedly protected mice against CY-induced toxicity, which allowed the administration of otherwise lethal doses of CY, with the CY activity/toxicity ratio being increased by four-fold.

Glutathione (GSH) is the major non-protein thiol in cells that plays a critical role against damage from electrophilic agents such as alkylating drugs. Selective therapeutic GSH elevation in normal but not in tumour cells has been suggested as a means of protecting host tissues against more intense doses of chemotherapy. The present study investigated the response of B16 melanoma to treatment with the cysteine pro-drug L-2-oxothiazolidine-4-carboxylate (OTZ), alone and in combination with cyclophosphamide (CY). We found that OTZ decreased the GSH levels and proliferation rate of B16 melanoma cells in vitro, sensitizing them to the cytotoxic action of the activated metabolite of CY, acrolein (AC). In contrast to OTZ, the cysteine deliverer N-acetylcysteine (NAC) enhanced B16 melanoma cell proliferation by increasing GSH levels, and markedly decreased the sensitivity of these tumour cells to AC. In vivo studies showed the antitumoral activity of OTZ in B16 melanoma liver metastasis-induced mice, increasing their life span. We also observed that, whereas with CY treatment the GSH levels in peripheral blood mononuclear cells (PBMCs) were reduced and a dose-dependent leukopenia was produced, OTZ significantly increased PBMC GSH content, reducing toxicity and enhancing the survival of mice bearing established melanoma liver metastases treated with lethal dose CY. These results suggest a critical role for OTZ in protecting against alkylator agent-induced immunosuppression, which may allow the dose escalation of these cytostatic drugs to improve their therapeutic benefit in the treatment of malignant melanoma.

Coumarin has antitumour effects in vivo and cytostatic effects in vitro. Its half-life in humans is short (1-1.5 h) and the monohydroxylated biotransformation products have significantly longer half-lives. One or several of these products may thus be responsible for the antitumoral effects. We have assayed the in vitro cytostatic activity of five monohydroxylated coumarins (3-, 4-, 6-, 7- and 8-monohydroxycoumarin), their acetates and methyl-ethers. Murine melanoma cells (cell line B16-F10) and murine fibroblasts (B82) were exposed to the test compounds at concentrations between 10 and 160 microg/ml. The cytostatic effects were estimated by reduction of the tetrazolium dye MTT. In the melanoma cells, some of the compounds inhibited growth after exposure for 1 day. In contrast, coumarin inhibited growth to a smaller extent, and only after exposure for 3 days. The most active compounds (3-acetoxycoumarin, 4-methoxycoumarin and 6-hydroxycoumarin), as well as coumarin, were also assayed in murine fibroblasts. The cytostatic effects of 4-methoxycoumarin and 6-hydroxycoumarin were less pronounced in fibroblasts than in melanoma cells. Our observations suggest that these compounds may have a greater therapeutic margin.

In this study we compare the effects of treatment with external sodium adenosine 5'-triphosphate (ATP) with the effects of L-buthionine-SR-sulfoximine (BSO) on B16 melanoma growth and on the modulation of the cytotoxic antimelanoma activity of cyclophosphamide (CY). We evaluated the in vitro effects of treatment with ATP or BSO on intracellular glutathione (GSH) levels, mitochondrial membrane potential (delta psi(m)) and the proliferation rate of the B16F10 melanoma cell line. Compared with untreated cells, delta psi(m) and GSH levels were already significantly decreased (25% and 57% reduction, respectively) after the first hour of incubation in culture cells exposed to 3 mM ATP. After 24 and 48 h a major reduction was observed in delta psi(m) (nearly 30%). GSH levels were also maximally depleted at 24 h (approximately 75%) and partially recovered (up to 37% of levels of control) after ATP was removed from the medium. At 24 and 48 h, the proliferation rate was decreased 1.4- and 1.7-fold, respectively, compared with control cells. Treatment with 50 microM BSO produced a time-dependent decrease in GSH levels (0.5, 21, 48 and 97.3% reduction at 1, 4, 8 and 24 h, respectively), but up to 54% of the levels of control cells was recovered after BSO was removed from the medium. In contrast to ATP, neither delta psi(m) nor proliferation rate was significantly modified in the first 24 h with BSO treatment. At 48 h, delta psi(m) was reduced by nearly 27%, and cell proliferation decreased 1.2-fold compared with controls. When the in vitro cytotoxic effect of low dose acrolein (an active metabolite of CY) in combination with BSO or ATP was analysed, a synergistic effect was found between BSO and acrolein, with a dose modification factor (DMF) of 1.98, but the antiproliferative effects of acrolein plus ATP were only approximately additive (DMF = 1.05). In addition, in in vivo studies differential effects were found between ATP and BSO. Specifically, whereas BSO alone significantly increased the survival time of mice bearing B16 melanoma liver metastases, and enhanced the cytotoxic effect of CY on this tumour model, no therapeutic benefits could be observed with ATP treatment, either alone or in combination with diethyl maleate (a GSH-depleting agent) and CY. In conclusion, our findings show that in our experimental system, both extracellular ATP and BSO have growth-inhibitory properties against B16 melanoma in vitro. In vivo, however, only BSO produces a chemosensitizing effect, whereas ATP has not proved useful as a biological modifier of chemotherapy.

Cystemustine (N'-(2-chloroethyl)-N-(2-(methylsulphonyl)ethyl)-N'-nitrosourea), a new anticancer chloroethylnitrosourea (CENU) is being tested in a phase II clinical trial of disseminated melanoma. The antitumour effect of this drug is mainly due to DNA damage in malignant melanocytes. Recently, we have shown that this damage can induce apoptosis in some melanoma cell lines. In others, apoptosis is not clearly observed, although there is a strong cytostatic effect. In this paper, we have characterized the cytological effect of cystemustine on murine malignant melanocytes (B16 cell line) which are resistant to apoptosis induced by this CENU. The results show that 3 days after cystemustine treatment, these melanocytes had accumulated in phase G2 of the cell cycle. There was then a strong morphological modification during a long cytostatic phase up to 30 days after treatment. During this cytostatic phase, there was uncontrolled DNA synthesis and marked swelling. Also, tyrosinase activity, melanin content and the number of mature melanosomes were greatly increased. These results suggest that when malignant melanocytes are not able to undergo apoptosis after treatment with CENU, they accumulate in G2 and this is followed by enhancement of melanogenesis.

In this study the response of three melanoma cell lines to single doses of radiation, to the bioreductive drug tirapazamine (SR-4233) and to the combination of radiation and tirapazamine was determined. Tirapazamine is a bioreductive drug with specific cytotoxicity in hypoxic conditions. Three melanoma cell lines (MM576, MM96L and murine B16-F10) were exposed to increasing concentrations of tirapazamine to assess cytotoxicity under aerobic and hypoxic conditions. Also, clonogenic survival after single doses of 4 MV X-rays was determined under aerobic and hypoxic conditions, with and without 20 microM tirapazamine. Tirapazamine was an effective hypoxic cytotoxin in the three cell lines. The concentrations of tirapazamine causing equal cell kill were 1000 mM for aerobic cells and 50 mM for hypoxic cells. The oxygen enhancement ratios for single X-ray doses were between 2 and 3 for all the cell lines. Addition of 20 microM tirapazamine to hypoxic cells 1 h before irradiation produced the same radiosensitivity as aerobic cells. Tirapazamine had a minimal effect on the radiosensitivity of aerobic cells. Since melanomas are known to contain hypoxic cells which may reduce their radiosensitivity, these in vitro results have demonstrated the potential of tirapazamine to overcome the radioresistance of hypoxia and give encouragement for further studies.

Taxol, an antitumour alkaloid which stabilizes microtubules, demonstrates marked activity against several tumours. However, due to its low aqueous solubility, Cremophor EL (polyoxyethylated castor oil) is used as the excipient in pharmaceutical drug preparations of taxol. This has toxic side effects, thereby limiting the clinical use of taxol in cancer therapy. The aim of this study was to design a novel taxol formulation using thermosensitive liposomes in order to eliminate the Cremophor EL vehicle and improve the antitumour activity of taxol by site-specific drug delivery. Temperature-sensitive liposomes encapsulating taxol were prepared using the natural lipids egg phosphatidylcholine and cholesterol in combination with ethanol. The liposomes have a phase transition temperature (Tm) of 43 degrees C. The in vivo efficacy of thermosensitive liposome encapsulated taxol was determined in B16F10 murine melanoma transplanted into C57BI/6 mice in combination with local hyperthermia. A significant reduction in tumour volume and an increase in survival time was observed in tumour-bearing mice treated with a combination of hyperthermia and thermosensitive liposome encapsulated taxol compared with animals treated with an equivalent dose of free taxol with or without hyperthermia. These results suggest that thermosensitive liposome encapsulated taxol in combination with hyperthermia may be useful in improving the therapeutic efficacy of taxol in the treatment of melanoma.

4-S-Cysteaminylphenol (4-S-CAP), a phenolic thioether, has been evaluated for melanocytotoxicity. We have recently shown that dihydro-1,4-benzothiazine-6,7-dione (benzothiazine BQ) is the ultimate toxic metabolite produced by tyrosinase oxidation of 4-SCAP. In this study we compared the antimelanoma effects of 4-SCAP and its two homologues, alpha-methyl-4-S-cysteaminylphenol (alpha-Me-4-SCAP) and 4-S-homocysteaminylphenol (4-S-Homo-CAP). Biochemical experiments showed that upon tyrosinase oxidation alpha-Me-S-CAP and 4-S-Homo-CAP also produced homologues of BQ which reacted rapidly with reduced glutathione (GSH) and also inhibited alcohol dehydrogenase, an SH enzyme. In vitro experiments showed that 4-S-CAP and its two homologues were taken up into B16-F1 melanoma cells at comparable rates but that 4-S-Homo-CAP was least effective in GSH deprivation, which was reflected in the low cytotoxicity of this phenol, and that the cytotoxicity of the phenols was tyrosinase dependent, as proved by the negligible effects on B16-G4F cells which have a much lower tyrosinase activity. In vivo experiments showed that direct intratumoral administration of these phenols inhibited the subcutaneous growth of B16 melanoma, with 4-S-Homo-CAP being the least effective, and that indirect Intraperitoneal administration of 4-S-CAP inhibited melanoma growth much more effectively than the two homologues. These results indicate that 4-S-CAP is the most promising antimelanoma agent among the three phenols examined.

Electrochemotherapy (ECT), chemotherapy administered in combination with electric fields, has the potential to be an effective localized treatment for cutaneous malignancies. Bleomycin's cytotoxicity was enhanced by exposing tumour cells to electrical fields following intravenous injection of the chemotherapeutic agent. Two issues associated with this procedure are the existence of a narrow but optimal time-window for effective treatment and the fact that a systemic drug dose is administered for a localized therapy. In order to address these issues, a study was initiated to examine the effectiveness of administering bleomycin by intratumoural injection. A dose-response relationship for intratumoural injection was determined. In addition, the minimal effective field strength necessary for ECT was established. Results of this study indicated drastic reductions in tumour volume for ECT-treated groups. In addition, ECT-treated groups showed increased survival over control groups. The minimum effective dose for the ECT intratumour bleomycin group was 0.025 units. The minimum effective field strength was found to be 1250 V/cm. The results demonstrate that intratumoural injection of bleomycin in combination with electric pulses is effective, and this information will be used to initiate clinical trials.

Malignant melanoma is notable for its resistance to chemotherapy, and multimodality approaches are being investigated to improve therapeutic efficacy. Melphalan and dacarbazine are commonly used for treatment of melanoma and their effect is potentiated by hyperthermia. The present study attempts to enhance the antitumour effect of melphalan by encapsulating it in temperature-sensitive liposomes and using it in combination with localized hyperthermic treatment of tumours for targeted delivery. Small unilamellar vesicles made of synthetic lipids (distearoyl phosphatidyl choline and dipalmitoyl phosphatidyl choline), showing gel to liquid crystalline phase transition at 42 degrees C, were used for encapsulation of melphalan. In vivo antitumour efficacy of the combination of liposomal melphalan and hyperthermia was determined using B16F10 murine melanoma transplanted into C57Bl/6 mice. A significant reduction in tumour volume and increased survival time was observed in tumour-bearing mice treated with a combination of hyperthermia and thermosensitive liposome-encapsulated melphalan compared with animals treated with an equivalent dose of free melphalan with or without hyperthermia. These results suggest that hyperthermia in combination with temperature-sensitive liposome-encapsulated melphalan may serve as a useful targeted drug delivery system for more effective management of melanoma.

Thymidine kinase (EC 2.7.1.21) is an enzyme supporting DNA synthesis under conditions of increased cell proliferation. Although it has proved to be a useful marker for various malignant diseases, it has not been tested in malignant melanoma. Thymidine kinase activity was measured by means of a radioenzymic assay in two classical animal models of melanoma disease--B16 and Cloudman S91 melanoma-bearing mice. Tumour cell proliferation was assessed histochemically by measuring the expression of proliferating cell nuclear antigen (PCNA). Tumour cytosolic specific thymidine kinase activity was found to be higher in less pigmented Cloudman S91 melanoma than in differentiated, ie pigmented B16 melanoma, relative to the proliferative activity of the two tumours. Serum thymidine kinase levels were increased in melanoma-bearing animals of both types compared with healthy mice; this also reflected the efficacy of the therapy: cyclophosphamide-treated B16 melanoma-bearing mice in which the tumour development was slowed down had significantly lower serum enzyme levels in comparison with the non-treated group and the same levels compared with control, healthy mice. Our results suggest that serum thymidine kinase levels might be used as a marker to follow the effect of melanoma therapy.

The relationship between the processes of melanogenesis and metastasis were investigated using metastatic variants of the B16 murine melanoma. alpha-Melanocyte stimulating hormone (MSH) enhanced lung colonization by the low metastasis variant F1 as well as inducing melanogenesis. The Ca2+ channel blocker verapamil had no effect on lung colonization by the F1 and ML8 variants but markedly enhanced melanogenesis in both these cell lines. We previously showed that the expression of the dominant metastasis associated gene mts1 was up-regulated by MSH but down-regulated by verapamil. These findings may demonstrate that the processes of melanogenesis and metastasis can be uncoupled at both genetic and phenotypic levels, and that the mechanisms involved in the regulation of metastatic behaviour and melanogenesis are different.

A subclone of Cloudman mouse melanoma cells (S91/I3) produces a resistance factor (RF) that increases the survival of a different but related subclone, S91/Amel, after exposure to either ultraviolet C (UVC) radiation or to mitomycin C (MMC). The presence of RF was deduced from experiments in which heavily irradiated S91/I3 cells were plated with the target S91/Amel cells. The effect of RF was also present in cell-free conditioned tissue culture medium (CM) from S91/I3 cultures. These results extend previous findings that both subclones produce an autocrine resistance factor (RF) that alters the radiation response of target S91/Amel cells making them less sensitive to death by low linear energy transfer (LET) ionizing radiation. S91/I3 cells are radioresistant relative to S91/Amel and produce the RF more effectively than S91/Amel. S91/I3 cells do not respond to the RF, being themselves, presumably, maximally stimulated. The significant findings are (1) the RF is effective at decreasing the killing of the target cells using cytotoxic agents that operate by different mechanisms; (2) The relative sensitivities of S91/Amel and S91/I3 to the toxic agents is not a factor in the responses of these cells to the RF: S91/Amel survivals are increased by the RF, those of S91/I3 are not; (3) the RF is elaborated by the melanoma cells whether or not they have been irradiated; it is, apparently, a normal cell product; (4) the RF is effective when added after the cytotoxic insult; its presence is not required during irradiation or drug treatment. The RF appears to act by novel mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously shown that the urinary excretion of 5-S-cysteinyldopa (5-S-CD) reflects well the progression of B16 mouse melanoma. We examined the usefulness of plasma concentration of 5-S-CD in following the progression of melanoma and evaluating the efficacy of immuno- and chemotherapeutic agents in the B16 mouse model. Murine interferon-beta (MuINF-beta; 3 x 10(5) U) or dacarbazine (DTIC; 50 mg/kg) was administered once a day for 10 or 5 consecutive days starting 8 days after subcutaneous inoculation of B16 melanoma cells in C57BL/6 mice. Blood samples were collected from the tail vein and tumour volumes were measured every other day until day 24. Levels of 5-S-CD in plasma were determined by high-performance liquid chromatography. Tumours became palpable on day 6-8 and grew exponentially thereafter in the control mice. Tumours in INF-beta-treated mice also grew exponentially, although at a reduced rate. However, the growth of tumours in the DTIC-treated mice was almost suppressed between days 12 and 18. Mean values of tumour volume on day 16 were 1.45, 1.05, and 0.83 cm3 in the control, INF-beta-treated, and DTIC-treated groups, respectively. The plasma concentration of 5-S-CD began to increase on day 8, at much slower rates in the experimental groups than those in the control group. Mean values of plasma 5-S-CD on day 16 were 14.3, 5.9, and 5.6 nmol/l in the control, INF-beta-treated, and DTIC-treated groups; 5-S-CD level on day -2 was 1.8 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Malignant melanoma is not only one of the most aggressive and lethal types of neoplasm, but its incidence in the general population is currently increasing at an alarming pace. It is interesting that most melanomas retain many of their characteristics of differentiation, including a dendritic nature and the production of melanin. This review discusses the phenotypic properties of melanoma cells, including their state of differentiation, and their tumourigenic and metastatic potentials, and attempts to provide an overview of the state of current research on the interrelationships between those parameters in murine and human systems.

Systemically administered 4-S-cysteaminylphenol (4-S-CAP) and N-acetyl-4-S-CAP inhibited the growth of xenografts of a human melanoma cell line but not of an ovarian tumour cell line. No selective cytotoxicity for melanoma cells was observed in culture, however. Further study of the in vitro mechanism of 4-S-CAP toxicity showed minimal inhibition of tyrosinase activity or DNA, RNA and protein synthesis, and there was no phase-specific arrest of the cell cycle. However, expression of an 80 kD melanosomal antigen was decreased. Cytotoxicity of 4-S-CAP in culture was decreased by simultaneous treatment with a monoamine oxidase inhibitor. An affinity column prepared from 4-S-CAP retained several proteins from a melanoma cell lysate. One protein, found also in HeLa cells, was identified by N-terminal sequencing as protein disulphide isomerase, a molecule which has multiple roles in the modification of secretory proteins. These results identify a protein target for 4-S-CAP as one possible mechanism of cytotoxicity.

2-Thiouracil and some related thioureas are receiving growing interest as selective melanoma seekers. They are incorporated into growing melanin, apparently due to covalent binding to dopaquinone, and the adduct is gradually trapped in the melanin polymer during its formation. To be clinically useful in melanoma scanning, thiouracil has been radioiodinated, and 5-iodo-2-thiouracil (ITU) was found to be localized in melanotic melanoma as selectively as thiouracil. Clinical trials with ITU, for the detection of malignant melanoma, are in progress, and the results so far are promising. Treatment with [35S]thiouracil has been performed on melanoma-bearing mice. The radiodoses needed for cure, however, were very high, which makes clinical application hazardous. Boron neutron capture therapy, on the other hand, might be a better approach. The technique is based on the irradiation of tumours with slow neutrons from an external source after the accumulation of boron in tumour tissue and clearance from normal tissues. Boron-10 undergoes instantaneous nuclear fission through the reaction 10B(n,alpha)7Li, and the emitted particles are efficient in cell killing. Boronated thioureas have been synthesized in various laboratories, and data from experiments on melanoma-bearing mice indicate that some of these compounds accumulate in the tumours in concentrations necessary for successful treatment.