Biochemical basis of 4-hydroxyanisole induced cell toxicity towards B16-F0 melanoma cells

In the current work we investigated for the first time the biochemical basis of 4-hydroxyanisole (4-HA) induced toxicity in B16-F0 melanoma cells. It was found that dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased 4-HA induced toxicity towards B16-F0 cells whereas dithiothreitol, a thiol containing agent, and ascorbic acid (AA), a reducing agent, largely prevented 4-HA toxicity. TEMPOL and pyrogallol, free radical scavengers, did not significantly prevent 4-HA toxicity towards B16-F0 cells. GSH>AA>NADH prevented the o-quinone formation when 4-HA was metabolized by tyrosinase/O(2). 4-HA metabolism by horseradish peroxidase/H(2)O(2) was prevented more effectively by AA than NADH>GSH. We therefore concluded that quinone formation was the major pathway for 4-HA induced toxicity in B16-F0 melanoma cells whereas free radical formation played a negligible role in the 4-HA induced toxicity.     

The fatty acid synthase inhibitor orlistat reduces experimental metastases and angiogenesis in B16-F10 melanomas

Background:Fatty acid synthase (FASN) is overexpressed and associated with poor prognosis in several human cancers. Here, we investigate the effect of FASN inhibitors on the metastatic spread and angiogenesis in experimental melanomas and cultured melanoma cells.Methods:The lung colonisation assay and cutaneous melanomas were performed by the inoculation of mouse melanoma B16-F10 cells in C57BL6 mice. Blood vessel endothelial cells (RAEC and HUVEC) were applied to determine cell proliferation, apoptosis, and the formation of capillary-like structures. Vascular endothelial growth factor A (VEGFA) expression was evaluated by quantitative RT-PCR and ELISA in B16-F10, human melanoma (SK-MEL-25), and human oral squamous carcinoma (SCC-9) cells. Conditioned media from these cancer cell lines were used to study the effects of FASN inhibitors on endothelial cells.Results:B16-F10 melanoma-induced metastases and angiogenesis were significantly reduced in orlistat-treated mice. Fatty acid synthase inhibitors reduced the viability, proliferation, and the formation of capillary-like structures by RAEC cells, as well as the tumour cell-mediated formation of HUVEC capillary-like structures. Cerulenin and orlistat stimulated the production of total VEGFA in B16-F10, SK-MEL-25, and SCC-9 cells. Both drugs also enhanced VEGFA(121), (165), (189,) and (165b) in SK-MEL-25 and SCC-9 cells.Conclusion:FASN inhibitors reduce metastasis and tumour-induced angiogenesis in experimental melanomas, and differentially modulate VEGFA expression in B16-F10 cells.     

Synthesis and selective in vitro anti-melanoma effect of enantiomeric alpha-methyl- and alpha-ethyl-4-S-cysteaminylphenol

Melanogenesis provides a unique target for the development of antitumour agents specific for malignant melanoma. Among the anti-melanoma compounds 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 its efficacy as an anti-melanoma agent, we synthesized the R- and S-enantiomers (99% enantiomer excess) of alpha-methyl- 4-S-cysteaminylphenol (alpha-Me-4-S-CAP) and alpha-ethyl- 4-S-cysteaminylphenol (alpha-Et-4-S-CAP) by coupling 4-hydroxythiophenol with the oxazolines obtained from the (R)- and (S)-enantiomers of 2-amino-1-propanol and 2-amino-1-butanol, respectively. The enantiomers of alpha-Me-4-S-CAP and alpha-Et-4-S-CAP were found to be better substrates for tyrosinase than the natural substrate, L-tyrosine. In vitro experiments showed that all four enantiomers were highly cytotoxic to pigmented B16-F1 melanoma cells, the effect being 70-fold and 160-fold greater than that on non-pigmented B16-G4F melanoma cells and 3T3 fibroblasts, respectively. The cytotoxic effect against B16-F1 cells was completely inhibited by phenylthiourea, a tyrosinase inhibitor, or by N-acetyl-L-cysteine, which increases the intracellular reduced glutathione (GSH) level. 4-S-CAP and the enantiomers were taken up into B16-F1 cells at comparable rates, but showed varying rates of GSH depletion that were inversely correlated to the cytotoxicity. These results suggest that the use of enantiomers would increase the efficacy of tyrosinase-dependent cytotoxic phenols.     

Efficacy of acetylsalicylic acid (aspirin) in skin B16-F0 melanoma tumor-bearing C57BL/6 mice

Several epidemiological studies show that aspirin can act as a chemopreventive agent and decrease the incidences of various cancers including melanoma. In this work, we investigated the in vitro and in vivo efficacy of acetylsalicylic acid (ASA) as an antimelanoma agent in B16-F0 cells and skin B16-F0 melanoma tumor mouse model. Our findings indicate that the IC₅₀ (48 h) for ASA in B16-F0 melanoma cells was 100 μM and that ASA caused a dose- and time-dependent GSH depletion and increase in reactive oxygen species (ROS) formation in B16-F0 melanoma cells. Male C57BL/6 mice were inoculated s.c. with 1?×?10⁶ B16-F0 melanoma cells. ASA (80, 100, and 150 mg/kg) was initiated on day 1 or day 7, or day 9 after cell inoculation and continued daily for 13, 7, and 5 days, respectively. Animals were weighed daily and sacrificed on day 13. The tumors were excised and weighed. The animals receiving 13 days of ASA therapy at 80, 100, and 150 mg/kg demonstrated tumor growth inhibition by 1?±?12 %, 19?±?22 %, and 50?±?29 %, respectively. Animals receiving 7 days of therapy at 80, 100, and 150 mg/kg demonstrated tumor growth inhibition by 12?±?14 %, 27?±?14 %, and 40?±?14 %, respectively. No significant tumor growth inhibition was observed with 5 days of therapy. ASA at 100 and 150 mg/kg caused significant tumor growth inhibition in C57BL/6 mice when administered for 13 and 7 days, respectively. The results obtained in this study are consistent with the recent epidemiologically based report that aspirin is associated with lower melanoma risk in humans.     

The killing effect of 4-S-cysteaminylphenol, a newly synthesised melanin precursor, on B16 melanoma cell lines

We have examined the killing effect of 4-S-cysteaminylphenol (4-S-CAP), a newly synthesised melanin precursor, on B16 melanoma cell lines possessing different melanin-producing activities and found it to be particularly effective in heavily melanised melanoma cells, but less so in moderately melanised melanoma cells, and having no effect on amelanotic melanoma cells and nonmelanoma cells. Thus, it was found that the killing effect of 4-S-CAP is highly dependent upon the synthesis of melanin and tyrosinase in melanoma cells, suggesting that 4-S-CAP may become toxic to melanoma cells only after oxidation by tyrosinase. The killing activity of 4-S-CAP also was found to be associated with a profound inhibition of the thymidine incorporation in pigmented melanoma cells, as compared to the uridine and leucine incorporation. Further, the inhibition of DNA synthesis was most pronounced in heavily melanised melanoma cells, less so in moderately melanised melanoma cells, and not seen in amelanotic melanoma cells. As a possible mechanism that might account for this action, it may be that 4-S-CAP is oxidised by tyrosinase to the o-quinone form via the catechol derivative and that some of the quinones then conjugate with sulfhydryl enzymes including DNA polymerase, thus exerting a killing activity for pigmented melanoma cells. Thus, 4-S-CAP appears to provide a new, effective cytotoxic agent for rational chemotherapy of malignant melanomas.     

Effect of glutathione depletion on the cytotoxicity of cisplatin and iproplatin in a human melanoma cell line

 Previous studies from our laboratory have indicated that glutathione (GSH) may affect the cytotoxicity of iproplatin to a greater extent than four other platinum agents tested including cisplatin. Therefore we studied the effect of GSH depletion by buthionine sulfoximine (BSO) on the cytotoxicity of iproplatin and cisplatin in a human melanoma cell line SK-MEL-2. Depletion of GSH was dependent on the concentration and time of incubation with BSO. BSO (100 μM) depleted GSH by 85% at 24 h and by 91% at 48 h. BSO (10 to 100 μM) by itself was not cytotoxic to SK-MEL-2 cells. At 85% depletion of GSH, cytotoxicity of iproplatin was increased by a factor of >7 and that of cisplatin by <2. These results confirm the previous finding that GSH interferes with the cytotoxicity of iproplatin to a significantly greater extent than that of cisplatin. Equitoxic IC₆₅ and IC₉₀ values of cisplatin (2 μM and 5 μM) or iproplatin (25 μM and 50 μM) had no effect on the intracellular GSH levels in SK-MEL-2 cells. Also, depletion of GSH by BSO had no effect on the accumulation of platinum from either cisplatin or iproplatin in this cell line. Our results suggest that the effect of GSH on the cytotoxicity of cisplatin and iproplatin in this cell line was not a consequence either of differences in GSH–Pt conjugate formation, or of differences in platinum accumulation induced by GSH depletion. GSH may have modulated the cytotoxicity of these platinum complexes by other means such as effects on DNA repair, apoptosis, free radical scavenging or through other yet unidentified mechanisms. Received: 1 August 1995 / Accepted: 12 September 1996     

Specification and use of a mouse monoclonal antibody raised against melanosomes for the histopathologic diagnosis of amelanotic malignant melanoma

The positive reactivity and specificity of a mouse monoclonal antibody (MoAb) human melanosome-specific antigen (HMSA) 2 raised against the melanosomal protein with amelanotic malignant melanoma on routine paraffin sections is reported. MoAb HMSA-2 identified cytoplasmic antigen with the following specifications: (1) a sharp heterogeneous expression in melanoma cells (acral lentiginous melanoma [ALM], 11 of 14; superficial spreading melanoma [SSM], 13 of 14; nodular melanoma [NM], one of three; and lentigo maligna melanoma [LMM], zero of two), whereas a diffuse homogeneous expression in cells of benign pigmented melanocytic nevi; and (2) an intense expression on amelanotic melanoma cells as opposed to a weak or negative expression on highly melanotic cells. The positive reactivity of MoAb HMSA-2 with amelanotic melanomas was exemplified by two shave-biopsy specimens of amelanotic subungual and plantar lesions, and by two cases of axillary and cervical amelanotic nodes that were left undiagnosed on routine hematoxylin and eosin (H & E) sections because of lack of melanin pigments. These were found, after diagnosis with MoAb HMSA-2, to possess the regressed primary lesions (both ALM).     

pEGFC1-IGFBP7诱导恶性黑素瘤SK-MEL-28 细胞的凋亡

目的：构建胰岛素样生长因子结合蛋白7（insulinlike growth factor binding protein 7,IGFBP7）表达质粒(pEGFC1-IGFBP7),研究IGFBP7对恶性黑素瘤细胞SK-MEL-28凋亡的影响。方法：构建pEGFC1-IGFBP7质粒,并将pEGFC1-IGFBP7质粒及空质粒分别转染入SK-MEL-28细胞,荧光显微镜观测细胞转染效率,Annexin-FITC/PI检测转染后SK-MEL-28细胞的凋亡。结果：成功构建pEGFC1IGFBP7质粒,用Effectene试剂能将pEGFC1IGFBP7质粒有效转染入SK-MEL-28细胞,转染效率达61%。流式细胞仪结果显示pEGFC1IGFBP7可明显促进SKMEL28细胞凋亡,转染24 h后的凋亡率达（28.4±257）%,转染空质粒及未转染组细胞凋亡率分别为（5.8±0.44）%和（6.4±0.71）%（F=406.138,P<0.05）。结论：pEGFC1-IGFBP7能有效诱导黑素瘤SK-MEL-28细胞凋亡,为IGFBP7为基础的黑素瘤基因治疗提供了实验依据。     

The effect of functional groups on reduction and activation of quinone bioreductive agents by DT-diaphorase

PURPOSE: Bioreductive antitumor agents are an important class of anticancer drugs that include the clinically used drug, mitomycin C, and new agents such as EO9 and tirapazamine that have recently been tested in clinical trials. These agents require activation by reductive enzymes such as DT-diaphorase or NADPH:cytochrome P450 reductase. A major focus for improving cancer chemotherapy has been to increase the selectivity and targeting of antitumor drugs to tumor cells. Bioreductive antitumor agents are ideally suited to improving tumor selectivity by an enzyme-directed approach to tumor targeting. However, none of the bioreductive agents developed to date has been specific for activation by a single reductive enzyme. This is in part due to a lack of knowledge about structural factors that confer selectivity for activation by reductive enzymes. The purpose of this study was to investigate the ability of specific functional groups to modify reduction and activation of quinone bioreductive agents by DT-diaphorase. METHODS: We used a series of model benzoquinone mustard (BM) bioreductive agents and compared the parent compound BM to MBM, which has a strong electron-donating methoxy group, MeBM, which has a weaker electron-donating methyl group, CBM, which has an electron-withdrawing chloro group, and PBM and its structural isomer, meta-PBM (m-PBM), which both have sterically bulky benzene rings attached to the quinone moiety. We determined the rate of reduction of these agents by purified human DT-diaphorase under hypoxic and aerobic conditions. We also measured the cytotoxic activity of these agents in human tumor cell lines with and without the DT-diaphorase inhibitor, dicoumarol. RESULTS: Under hypoxic conditions in vitro, the t(1/2) values for reduction of the analogs by purified DT-diaphorase were 4, 6, 8, 9, 10 and 21 min for BM, MeBM, CBM, MBM, PBM and m-PBM, respectively. Under aerobic conditions the rank order of redox cycling after two-electron reduction by DT-diaphorase was MBM > MeBM > BM approximately CBM approximately PBM approximately m-PBM. The rate of reduction by DT-diaphorase of HBM, a non-alkylating analog of BM, was similar to that of BM under hypoxic conditions, and the rate of redox cycling under aerobic conditions was comparable to that of BM, suggesting that structural changes to the cytotoxic group of these BMs do not affect DT-diaphorase-mediated reduction and redox cycling potential. MBM, MeBM and PBM were more toxic than BM in the NCI-H661 human non-small-cell lung cancer cells and SK-MEL-28 human melanoma cells, while CBM displayed significantly increased cytotoxic activity compared to BM only in the NCI H661 cells. m-PBM had similar cytotoxic activity compared with BM in both cell lines. These cell lines have moderate to high levels of DT-diaphorase activity. When cells were pretreated with the DT-diaphorase inhibitor, dicoumarol, the cytotoxic activity of BM increased while that of MBM decreased in both cell lines, suggesting that BM was inactivated by DT-diaphorase while MBM was activated by this enzyme. Pretreatment of the SK-MEL-28 melanoma cells with dicoumarol resulted in an increased cytotoxic activity of MeBM, but pretreatment of the NCI-H661 cells did not affect the cytotoxicity of MeBM. This suggests, that similar to the results with BM, DT-diaphorase is an inactivating enzyme for MeBM in the SK-MEL-28 cell line. Dicoumarol had no significant effect on the cytotoxicity of CBM, PBM or m-PBM in both cell lines. CONCLUSIONS: These studies demonstrated that functional groups can significantly affect the reduction and activation of bioreductive agents by DT-diaphorase. All the functional groups decreased the rate of reduction of the quinone group by DT-diaphorase. Since MeBM and MBM, with electron-donating functional groups, and CBM with an electron-withdrawing functional group had similar half-lives of reduction by DT-diaphorase, steric rather than electronic effects of the functional groups appear to be more important for modifying the rate of reduction by DT-diaphorase. Steric effects on reduction by DT-diaphorase were also influenced by the position of the functional group on the quinone ring moiety, as the reduction of m-PBM was much slower than the reduction of PBM. The electron-donating methoxy and methyl functional groups increased the ability of the reduced products of MBM and MeBM to undergo redox cycling. DT-diaphorase appeared to be an activating enzyme for MBM. This may have resulted in part from increased formation of reactive oxygen species resulting from the increased redox cycling by MBM. In contrast, DT-diaphorase was an inactivating enzyme for BM, and for MeBM in the SK-MEL-28 melanoma cells, possibly because the hydroquinone product of BM and MeBM may be less cytotoxic than the semiquinone produced by one-electron reduction by NADPH:cytochrome P450 reductase.     

gamma-L-Glutaminyl-4-hydroxybenzene, an inducer of cryptobiosis in Agaricus bisporus and a source of specific metabolic inhibitors for melanogenic cells.

A stable phenol, gamma-L-glutaminyl-4-hydroxybenzene (GHB), is oxidized by tyrosinase in the gill tissues of the mushroom Agaricus bisporus to a quinone and a second oxidation product which together suppress mitochondrial energy production and the synthesis of proteins and nucleic acids in the zygote, thus establishing dormancy in the spores. Brief incubation of cultured murine L1210 leukemia and B-16 melanoma cells with muM concentrations of the purified quinone notably prolonged survival times or blocked tumor growth in histocompatible mice inoculated i.p. with high concentrations of the exposed cells. The instability of the quinone precluded in vivo administration. The short incubation of cultured B-16 melanoma cells with mM concentrations of GHB markedly prolonged survival times or abolished tumor growth in histocompatible C57BL/6J mice inoculated i.p. with 5 X 10(6) exposed cells. This response did not occur with L1210 leukemia cells, which lack the enzyme tyrosinase. The survival times of mice bearing B-16 melanoma, but not of those with L1210 leukemia, were slightly prolonged by a single injection and were significantly extended by daily i.p. injections of GHB. Normal C57BL/6J mice, given GHB i.p. as single or multiple 400-mg/kg doses, manifested no systemic toxicity but showed depigmentation of the hair after 2 to 3 weeks. These studies provide evidence that GHB exerts cytotoxicity specifically for cells that by their content of tyrosinase convert the phenol to the quinone. This targeted response minimizes systemic toxocity and underscores the potential therapeutic application of this agent to melanocarcinoma.     

Correlation of overexpression of the low-affinity p75 neurotrophin receptor with augmented invasion and heparanase production in human malignant melanoma cells.

The role of growth factor receptors in regulating the progression of human melanocytes toward tumorigenicity and ultimately a malignant phenotype is poorly understood. In particular, the autocrine and paracrine influences that modulate cellular invasion and extracellular matrix (ECM)-degradative enzymes in melanoma cells remain undefined at the molecular level. The low-affinity p75 neurotrophin receptor (p75NTR), a cysteine-rich transmembrane glycoprotein, is frequently expressed in advanced stages of human melanoma, but the biological consequences of this expression are unknown. p75NTR can enhance the invasive potential of brain-metastatic melanoma cells in vitro. We have extended here these results and related the level of p75NTR in human metastatic melanoma cells to their invasive potential to target organs other than brain. Fluorescence activated cell sorting (FACS) analysis showed that 3 melanoma cell lines (SK-MEL-146, SK-MEL-119, 70W) had differential p75NTR contents, whereas SK-MEL-147 cells had elevated amounts of p75NTR. Two other melanoma cell lines (SK-MEL-94, SK-MEL-110) with point mutations in the p75NTR transmembrane domain had reduced (SK-MEL-94) or absent (SK-MEL-110) p75NTR. We also examined these cell lines for presence of TrkA receptor, the high-affinity receptor for nerve growth factor (NGF), the prototypic neurotrophin. No TrkA receptor expression was detected in any of the cell lines. The extent of p75NTR expression correlated with the capability of NGF to promote cellular invasion and with production of heparanase, an important ECM-degradative enzyme. Melanoma cells sorted for high p75NTR expression (p75NTR-H cells) had markedly greater (9- to 13-fold increase) invasive capabilities in response to NGF exposure than those sorted for low p75NTR expression (p75NTR-L cells). Additionally, NGF induced a 8- to 10-fold increase of heparanase activity in p75NTR-H cells. Thus, we propose that p75NTR-mediated trophic support profoundly affects melanoma cell invasion to neurotrophin-rich organs.     

The role of mitogen-activated protein kinase activation in determining cellular outcomes in polyamine analogue-treated human melanoma cells

The clinically relevant polyamine analogue N(1),N(11)-diethylnorspermine (DENSPM) inhibits cell growth by down-regulating polyamine biosynthesis, up-regulating polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase (SSAT), and depleting intracellular polyamine pools. Among human melanoma cell lines, the analogue causes rapid apoptosis in SK-MEL-28 cells and a sharp G(1) arrest in MALME-3M cells. This study reveals that DENSPM potently activates the mitogen-activated protein kinase (MAPK) pathways in melanoma cells and investigates the role of this response in determining cellular outcomes. Onset of apoptosis was preceded by an intense phosphorylation of the MAPKs, including extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase, and p38 in both SK-MEL-28 and MALME-3M cells. A panel of DENSPM analogues differing only in their ability to induce SSAT was used to show that MAPK activation was causally linked to induction of SSAT activity and related oxidative events. The latter was confirmed with the polyamine oxidase inhibitor MDL-75275 and the antioxidant N-acetyl-L-cysteine, which when used in combination with DENSPM, decreased MAPK activation and as previously shown, reduced apoptosis. The MAP/extracellular signal-regulated kinase-1 inhibitor PD 98059 reduced activation of all three kinases but failed to alter apoptosis in DENSPM-treated SK-MEL-28 cells. By contrast, the inhibitor prevented p21(waf1/cip1) induction and enhanced apoptosis in MALME-3M cells as indicated by accelerated caspase-3 activation and positive annexin V staining. The generality of this effect was demonstrated in DENSPM-treated A375 and LOX human melanoma cells. Taken together, the importance of the MAPK pathways in determining the biological response to DENSPM treatment is dependent on the genetic environment of the cell.     

Depletion of glutathione in vivo as a method of improving the therapeutic ratio of misonidazole and SR 2508

Depletion of intracellular glutathione (GSH) can enhance misonidazole (MISO) radiosensitizing efficacy both in vivo and in vitro. However, such treatments may also enhance the systemic toxicity in animals. The purpose of the present study was to test various ways of depleting GSH levels in a variety of experimental mouse tumors, to measure the improvement in the efficacy of MISO and its less toxic analog SR 2508 by this depletion, and to determine the effect of daily GSH depletion on the toxicity of MISO and SR 2508. GSH levels were measured daily for 5 days in tumors, livers and brains of mice injected daily with buthionine sulfoximine (BSO), with or without diethylmaleate (DEM). To investigate tumor variability we studied 5 different tumors: EMT-6, RIF-1, KHT, SCC VII, and B16 melanoma. The efficacy of MISO and SR 2508 was evaluated using the KHT and SCC VII tumors either by the regrowth delay assay or by the in vivo/in vitro clonogenic assay. The drug toxicity was evaluated by weight loss and by death. Daily doses of 3 mmole/kg BSO depleted tumor levels of GSH to 20 to 40% of controls by 6 hr after each injection. Injection of DEM (300 mg/kg) 6 hr after BSO further enhanced the depletion. Administration of MISO or SR 2508 at the time of maximum GSH depletion enhanced the MISO efficacy by factors of 2.5 to 8 for depletion to 8% of controls by BSO + DEM, but no enhancement of SR 2508 was seen with tumors at 20% GSH levels achieved with BSO alone in the preliminary experiment. The chronic toxicity of MISO was enhanced not at all or by a factor of up to 2 for BSO and BSO + DEM respectively. Further studies are needed before it can be concluded that GSH depletion by BSO alone may be a useful adjunct to the clinical use of radiosensitizers.     

Improvement of the tumor-suppressive effect of boron neutron capture therapy for amelanotic melanoma by intratumoral injection of the tyrosinase gene

Boron neutron capture therapy (BNCT) is successful when there is a sufficient (10)B concentration in tumor cells. In melanoma, (10)B-para-boronophenylalanine (BPA) accumulation is proportional to melanin-producing activity. This study was done to confirm enhancement of the tumor-suppressive effect of BNCT on amelanotic melanoma by intratumoral injection of the tyrosinase gene. D178 or FF amelanotic melanomas were implanted s.c. in Syrian hamsters. One group of D178- or FF-bearing hamsters (TD178 or TFF group) received intratumoral injections of pcDNA-Tyrs constructed as a tyrosinase expression plasmid. The other hamsters (pD178 and pFF groups) were injected with pUC119, and control hamsters (D178 and FF groups) only with transfection reagents. All the groups underwent immunofluorescence analysis of tyrosinase expression and BPA biodistribution studies. BNCT experiments were done at the Kyoto University Research Reactor. Tyrosinase expression increased in the tumors of the TD178 and TFF groups but remained the same in the pD178 and pFF groups. Tumor boron concentrations in the TD178 and TFF groups increased significantly (TD178: 49.7 +/- 12.6 versus D178: 27.2 +/- 4.9 microg/g, P < 0.0001; TFF: 30.7 +/- 6.6 versus FF: 13.0 +/- 4.7 microg/g, P < 0.0001). The BNCT tumor-suppressive effect was marked in the TD178 and TFF groups. In vivo transfection with the tyrosinase gene increased BPA accumulation in the tumors, the BNCT tumor-suppressive effect on amelanotic melanoma being significantly enhanced. These findings suggest a potential new clinical strategy for the treatment of amelanotic melanoma with BNCT.     

4-S-Cysteaminylphenol-loaded magnetite cationic liposomes for combination therapy of hyperthermia with chemotherapy against malignant melanoma

Tyrosine analogs are good candidates for developing melanoma chemotherapies because melanogenesis is inherently toxic and expressed uniquely in melanocytic cells. The sulfur homolog of tyrosine, 4-S-cysteaminylphenol (4-S-CAP), was shown to be a substrate of melanoma tyrosinase and can cause selective cytotoxicity of melanocytes and melanoma cells. Previously, in order to improve the adsorption of magnetite nanoparticles to target cell surfaces, and generate heat in an alternating magnetic field (AMF) for cancer hyperthermia, we produced hyperthermia using magnetite cationic liposomes (MCL) that have a positive charge at the liposomal surface. In the present study, we constructed 4-S-CAP-loaded MCL (4-S-CAP/MCL), which act as a novel modality, combining melanoma-specific chemotherapy by 4-S-CAP with intracellular hyperthermia mediated by MCL. The 4-S-CAP/MCL exerted 4-S-CAP-mediated anticancer effects on B16 melanoma cells in vitro and in vivo. Moreover, after intratumoral injection of 4-S-CAP/MCL in vivo, the melanoma nodules were heated to 45 degrees C under an AMF. Significantly higher therapeutic effects were observed in mice treated with the combination therapy mediated by 4-S-CAP/MCL plus AMF irradiation compared with mice treated with 4-S-CAP/MCL alone (without AMF) or mice treated with hyperthermia alone (MCL + AMF irradiation). These results suggest that this novel therapeutic tool is applicable to the treatment of malignant melanoma.     

Photothermal sensitisation as a novel therapeutic approach for tumours: studies at the cellular and animal level

Irradiation of B78H1 murine amelanotic melanoma cells with 850 nm light emitted from a Ti:sapphire laser, operated in a pulsed mode at high fluence rates and in the presence of Ni(II)-octabutoxy-naphthalocyanine (NiNc), promoted a photothermally sensitised process leading to fast and irreversible cell death. This resulted in the ejection of a consistent mass of cytoplasmic material from the irradiated cells that was detected by scanning electron microscopy. The extensive chemical and mechanical damage was probably caused by the photoinduced generation of an acoustic shock wave. The efficiency of the photoprocess was modulated by intracellular concentration of NiNc and maximally by the formation of aggregated naphthalocyanine clusters in specific subcellular areas. Very similar results were obtained upon irradiation of NiNc-loaded C32 human amelanotic melanoma cells and transformed murine HT-1080 and HaCaT fibroblasts. From these results, photothermal sensitisation appears to be a general phenomenon and preliminary studies with mice bearing subcutaneously transplanted amelanotic melanomas, irradiated with 850 nm light 24 h after intravenous injection of NiNc, suggest that this approach has potential for the therapy of some types of skin tumours.