Distribution of I-BZA (N-2-diethylaminoethyl-4-iodobenzamide) in grafted melanoma and normal skin: a study by secondary ion mass spectroscopy.

Iodobenzamides labelled with radioactive iodine are undergoing clinical evaluation as imaging and potential therapeutic agents in malignant melanomas. However, the uptake mechanism in melanic tissues remains controversial. Using secondary ion mass spectrometry (SIMS), we studied the microscopic distribution of N-(2 diethylaminoethyl)-4 iodobenzamide (I-BZA) in B16 murine melanoma inoculated to C57BL/6J1 Co mice as well as in normal pigmented skin. SIMS provides specific detection of iodine-127 atoms entering 127I-BZA composition. In B16 melanoma, 127I-BZA distribution was found to be heterogeneous, with focal areas of high concentration corresponding to cells rich in melanin pigments. In skin, SIMS analysis showed 127I-BZA distribution appearing as multiple small selective concentration areas within the epidermis. The number of these foci decreased from the stratum basale towards the stratum corneum. In both tissues, the intracellular location appeared specifically intracytoplasmic, with no apparent nuclear uptake. Distribution of this molecule mirrored that of melanin pigments. There was no enhancement of uptake at the membrane site. These results suggest that, in melanic tumors as well as in normal pigmented tissue, specific uptake of 127I-BZA occurs in pigment cells, with a possible link to melanin pigments.     

Endocytosis by liver cells during suppression of intralysosomal proteolysis.

The lysosomotropic agent chloroquine is widely used as a specific inhibitor of intralysosomal proteolysis in isolated hepatocytes. It was shown that in vitro chloroquine reversibly inhibited purified cathepsins H, B, L in concentrations less than those observed inside lysosomes in vivo. However, administration of high doses of chloroquine to rats (30-50 mg/kg i.p. as a single or repeated injections) was followed by increased cathepsin D and cysteine proteinase activities, as well as other lysosomal enzymes. Chloroquine administration did not induce any changes of carbon particles phagocytosis by liver cells (macrophages); modifications of fluid-phase (125I-PVP uptake) and receptor-mediated endocytosis (125I-asialo-fetuin uptake) were noted. Chloroquine administered in vivo reproduced some symptoms of lysosomal storage diseases (especially during repeated drug administration).     

Pheomelanin as a binding site for drugs and chemicals.

Certain drugs and chemicals, such as chloroquine, chlorpromazine, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), are bound to melanin and retained in pigment cells for long periods. This specific retention in pigmented tissues can cause adverse effects in the skin, eye, inner ear, and pigmented nerve cells of the substantia nigra of the brain. To date, all studies have been focused on eu- and neuromelanin. In the present study, we show that chloroquine, chlorpromazine, chlomipramine, paraquat, acridine orange, and nickel, which are bound to eumelanin, also bind to synthetic pheomelanin, but the binding to pheomelanin is lower. The binding varied with the cysteine content and pH, and the results indicate that the binding is complex and includes ionic interactions. In addition, we have shown that these substances also bind to synthetic thiourea-containing melanin, but to quite a low extent. We also present a microautoradiographic study on the binding of 14C-chloroquine to natural pheomelanin in vivo in yellow mice C57BL (Ay/a). Black (C57/BL) and albino (NMRI) mice were used as controls. The autoradiography demonstrated a pronounced uptake of chloroquine in the hair follicles and the dermal melanocytes in the ear of yellow mice, which was comparable to the corresponding accumulation of label in black mice. In the albino mouse, the uptake was lower and more homogeneously distributed in the skin. These results suggest that the toxicological risks of melanin-related adverse effects are applicable to persons with a high content of pheomelanin in the skin and hair.     

Experimental Boron Neutron Capture Therapy for Melanoma: Systemic Delivery of Boron to Melanotic and Amelanotic Melanoma

The boron-containing melanin precursor analogue p-boronophenylalanine (BPA) has previously been shown to selectively deliver boron to pigmented murine melanomas when administered in a single intragastric dose. If boron neutron capture therapy is to become a clinically useful method of radiation therapy for human malignant melanoma, the boron carrier must be capable of delivering useful amounts of boron to remote tumor sites (metastases) and to poorly pigmented melanomas. We have now determined the ability of BPA to accumulate in several nonpigmented melanoma models including human melanoma xenografts in nude mice. The absolute amount of boron in the nonpigmented melanomas was about 50% of that observed in the pigmented counterparts but was still selectively concentrated in the tumor relative to normal tissues in amounts sufficient for effective neutron capture therapy. Single intragastric doses of BPA resulted in selective localization of boron in the amelanotic Greene melanoma carried in the anterior chamber of the rabbit eye and in a pigmented murine melanoma growing in the lungs. The ratio of the boron concentration in these tumors to the boron concentration in the immediately adjacent normal tissue was in the range of 3:1 to 4:1. These distribution studies support the proposal that boron neutron capture therapy may be useful as a regional therapy for malignant melanoma.     

Differential radiosensitivity in cultured B-16 melanoma cells following interrupted melanogenesis induced by glucosamine

The relationship between cell pigmentation and radiosensitivity was investigated in a cell model in which melanogenesis was suppressed by a glycosylation inhibitor. It was found that X-irradiation of melanotic B-16 melanoma cells and their amelanotic counterparts, obtained by glucosamine treatment, showed an inverse correlation between radiosensitivity and melanin contents. Since melanogenesis interruption by glucosamine does not affect the DNA repair capacity of nonpigmented cells, it is likely that intracellular melanins play a role in the relative resistance of pigmented cells to X-irradiation.     

Distribution of 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl free radical in normal and neoplastic animal tissues.

The free radical 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (TMPO) was injected i.p. in doses of 100 mg/kg bw into Syrian hamsters: untreated, partially hepatectomized and grafted with transplantable tumours as well as into mice with B16 melanotic melanoma or with an adenocarcinoma. One hour after application of the compound its content was determined spectrometrically in livers, kidneys, lungs and all tumours. The characteristic triplet signal of TMPO was registered in the hamster and mouse melanotic melanomas but not in all other animal tissues and tumours. This may be the result of an enzyme defect in the melanotic melanoma cells or of a retention of TMPO by melanin. It was also found that TMPO is metabolized predominantly in the livers of the animals.     

Pheomelanin as a Binding Site for Drugs and Chemicals

Certain drugs and chemicals, such as chloroquine, chlorpromazine, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), are bound to melanin and retained in pigment cells for long periods. This specific retention in pigmented tissues can cause adverse effects in the skin, eye, inner ear, and pigmented nerve cells of the substantia nigra of the brain. To date, all studies have been focused on eu- and neuromelanin. In the present study, we show that chloroquine, chlorpromazine, chlomipramine, paraquat, acridine orange, and nickel, which are bound to eumelanin, also bind to synthetic pheomelanin, but the binding to pheomelanin is lower. The binding varied with the cysteine content and pH, and the results indicate that the binding is complex and includes ionic interactions. In addition, we have shown that these substances also bind to synthetic thiourea-containing melanin, but to quite a low extent. We also present a microautoradiographic study on the binding of ¹⁴C-chloroquine to natural pheomelanin in vivo in yellow mice C57BL (A^y/a). Black (C57/BL) and albino (NMRI) mice were used as controls. The autoradiography demonstrated a pronounced uptake of chloroquine in the hair follicles and the dermal melanocytes in the ear of yellow mice, which was comparable to the corresponding accumulation of label in black mice. In the albino mouse, the uptake was lower and more homogeneously distributed in the skin. These results suggest that the toxicologi-cal risks of melanin-related adverse effects are applicable to persons with a high content of pheomelanin in the skin and hair.     

Boron uptake in normal melanocytes and melanoma cells and boron biodistribution study in mice bearing B16F10 melanoma for boron neutron capture therapy

Information on (10)B distribution in normal tissues is crucial to any further development of boron neutron capture therapy (BNCT). The goal of this study was to investigate the in vitro and in vivo boron biodistribution in B16F10 murine melanoma and normal tissues as a model for human melanoma treatment by a simple and rapid colorimetric method, which was validated by HR-ICP-MS. The B16F10 melanoma cell line showed higher melanin content than human melanocytes, demonstrating a greater potential for boronophenylalanine uptake. The melanocytes showed a moderate viability decrease in the first few minutes after BNCT application, stabilizing after 75 min, whereas the B16F10 melanoma showed the greatest intracellular boron concentration at 150 min after application, indicating a different boron uptake of melanoma cells compared to normal melanocytes. Moreover, at this time, the increase in boron uptake in melanoma cells was approximately 1.6 times higher than that in normal melanocytes. The (10)B concentration in the blood of mice bearing B16F10 melanoma increased until 90 min after BNCT application and then decreased after 120 min, and remained low until the 240th minute. On the other hand, the (10)B concentration in tumors was increased from 90 min and maximal at 150 min after application, thus confirming the in vitro results. Therefore, the present in vitro and in vivo study of (10)B uptake in normal and tumor cells revealed important data that could enable BNCT to be possibly used as a treatment for melanoma, a chemoresistant cancer associated with high mortality.     

Towards in vivo melanin radicals detection in melanomas by electron paramagnetic resonance (EPR) spectroscopy: a proof-of-concept study

Melanoma is the most aggressive skin tumour type. Although complete cure can be achieved when the whole tumour is resected, prognostic dramatically drops when melanoma cells reach deeper tissues and lymph nodes. Hence, there is an urgent need to develop accurate tools allowing (i) discriminating benign naevi from malignant tumours and (ii) being able to characterise melanoma infiltration. For that purpose, we exploited the paramagnetic properties of melanin by using electron paramagnetic resonance (EPR) spectroscopy to measure the melanin content in pigmented (B16F10 cancer cells) and non-pigmented melanomas (WM2664 cancer cells) inoculated intradermally in nude mice. Specifically, we took advantage of a new clinical EPR device (1?GHz), which provides sensitive measurements of radical species in vivo. Results showed that the melanin-specific EPR signal increased with tumour growth in pigmented tumours, whereas no EPR signal could be detected in achromic melanomas. These data plead for the development of new EPR spectrometers/imagers with an improved in-depth resolution for the detection of invasive melanomas.     

Phage display library derived peptides that bind to human tumor melanin as potential vehicles for targeted radionuclide therapy of metastatic melanoma

Metastatic melanoma remains an incurable disease, and there is a great need for novel therapeutic modalities. We have recently identified melanin as a target for radionuclide therapy of melanoma and demonstrated the feasibility of this approach using a 188-rhenium ( (188)Re)-radiolabeled melanin-binding decapeptide to fungal melanin known as 4B4. Although the results indicated that radiolabeled melanin-binding decapeptide had activity against melanoma, that peptide also manifested high kidney uptake and this might become a concern during clinical trials. We hypothesized that by identifying peptides with different amino acid composition against tumor melanin we might be able to decrease their kidney uptake. Using the Heptapeptide Ph.D.-7 Phage Display Library, we identified three heptapeptides that bind to human tumor melanin. These peptides were radiolabeled with (188)Re via HYNIC ligand, and their comprehensive biodistribution in A2058 human metastatic melanoma tumor-bearing nude mice was compared to that of (188)Re-4B4 decapeptide. While tumor uptake of heptapeptides was quite similar to that of (188)Re-4B4 decapeptide, there was dramatically less uptake in the kidneys at both 3 h (6% ID/g vs 38%) and 24 h (2% ID/g vs 15%) postinjection. Administration of one of the generated heptapeptides, (188)Re-HYNIC-AsnProAsnTrpGlyProArg, to A2058 human metastatic melanoma-bearing nude mice resulted in significant retardation of the tumor growth. Immunofluorescence showed that in spite of their relatively small size heptapeptides were not able to penetrate through the membranes of viable melanoma cells and bound only to extracellular melanin, which provides assurance that they will be safe to healthy melanin-containing tissues during radionuclide therapy. Thus, these heptapeptides appear to have potentially significant advantages for targeted therapy of melanoma relative to existing melanin-binding peptides.     

Investigation of the Regulation of Pigmentation in α-Melanocyte-Stimulating Hormone Responsive and Unresponsive Cultured B16 Melanoma Cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to αMSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1°) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to αMSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro αMSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1° cells. αMSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1° cells to produce melanin in response to αMSH is not due to a lack of αMSH receptors or cAMP response to αMSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1° and F1 cells.     

Investigation of the regulation of pigmentation in alpha-melanocyte-stimulating hormone responsive and unresponsive cultured B16 melanoma cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to alpha MSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1 degree) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to alpha MSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro alpha MSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1 degree cells. alpha MSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1 degree cells to produce melanin in response to alpha MSH is not due to a lack of alpha MSH receptors or cAMP response to alpha MSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1 degree and F1 cells.     

Paraquat is not accumulated in B16 tumor cells by the polyamine transport system

We have examined the effect of difluoromethylornithine on the ability of B16 melanoma cells to take up putrescine and the 4,4'-dipyridyl herbicide paraquat. Pretreatment with difluoromethylornithine for 24 hr enhanced putrescine uptake by inducing the maximum capacity of the transport system without affecting the Km for the substrate. Paraquat uptake was minor compared with that of putrescine and was not affected by difluoromethylornithine. Neither putrescine, spermidine or spermine at concentrations up to 100 microM inhibited the accumulation of paraquat. However, paraquat competitively inhibited putrescine transport (Ki = 54 +/- 10 microM). Exposure of the B16 melanoma cells for 24 hr to increasing concentrations of paraquat produced a dose-dependent inhibition of DNA synthesis. Difluoromethylornithine pretreatment did not affect paraquat toxicity. These data show that paraquat is not taken up into B16 melanoma cells by the uptake system responsible for transporting putrescine. Moreover, it is likely that the difluoromethylornithine inducible polyamine transport system in B16 melanoma cells is characteristically different to that previously described in normal mammalian lung since the latter is reportedly capable of transporting both putrescine and paraquat.     

Cytotoxic effect of 1-methyl-4-phenylpyridinium ion on human melanoma cell lines, HMV-II and SK-MEL-44, is dependent on the melanin contents and caused by inhibition of mitochondrial electron transport

MPP+, an oxidative metabolite of a neurotoxin, MPTP, was found to be cytotoxic to human melanoma cell lines, HMV-II and SK-MEL-44. After 3 days of culture in the presence of MPP+, a larger amount of MPP+ was accumulated in HMV-II cells than in SK-MEL-44 cells, which correlated well with the melanin contents; HMV-II cells contain larger amounts of melanin than SK-MEL-44 cells. After 6 days of culture in the presence of MPP+, the cytotoxicity of MPP+ on these cell types was evaluated by counting cell numbers with the dye exclusion test and double-layer soft agar clonogenic assay. It was found that exposure to MPP+ reduced the survival of HMV-II cells more significantly than that of SK-MEL-44 cells. In HMV-II cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to elucidate the mechanism of MPP+ lethality. The formazan formation was reduced markedly by the presence of MPP+ at concentrations much lower than those required for cell death. These results suggest that cytotoxicity of MPP+ may be ascribed to its accumulation due to high affinity for melanin, and to inhibition of the enzymes utilizing ubiquinone in the mitochondrial respiratory chain.     

Distribution of the enantiomers of hydroxychloroquine and its metabolites in ocular tissues of the rabbit after oral administration of racemic-hydroxychloroquine

The disposition of the enantiomers of hydroxychloroquine (HCQ) and its major metabolites in ocular tissues of rabbits has been studied. Both albino, New Zealand White (NZW), and pigmented animals were administered daily oral doses of rac-HCQ, (S)-HCQ or (R)-HCQ (20 mg/kg) over 1, 6, or 8 day periods or for 8 days followed by a 7-day washout period. At the end of the study periods, plasma and whole blood samples were collected and the rabbits were sacrificed. The eyes were collected, the aqueous humor removed with a syringe, and the eyes separated into the cornea, lens, vitreous body, iris, choroid-retina, sclera, and conjunctiva. The concentrations of (R)-HCQ, (S)-HCQ, and their respective metabolites were determined using a validated enantioselective liquid chromatographic assay. The data from these studies indicate that HCQ accumulated in both pigmented and nonpigmented ocular tissues. In the pigmented tissues, HCQ and its metabolites were bound to melanin and the binding was not enantiospecific. In the nonpigmented tissues and in the iris and retina-choroid of the NZW rabbits, the accumulation appeared to be the result of a reversible and enantioselective binding of HCQ and its metabolites to an unidentified biopolymer present in these ocular tissues. © 1994 Wiley-liss, Inc.     

Synthesis and characterization of a (68)Ga-labeled N-(2-diethylaminoethyl)benzamide derivative as potential PET Probe for malignant melanoma

Radiolabeled benzamides have been reported to be attractive agents for targeting malignant melanoma as they bind melanin and display high accumulation in melanoma cells. Herein, we report the synthesis and bioevaluation of a novel (68)Ga-labeled benzamide as a potential PET agent for malignant melanoma. The novel radiotracer was synthesized in good radiochemical yields (80% decay corrected yield) and high specific radioactivity (10GBq/μmol). Cellular uptake of (68)Ga-SCN-NOTA-BZA was significantly higher in B16F10 cells (mouse melanoma) treated with L-tyrosine. Biodistribution and micro-PET studies of (68)Ga-SCN-NOTA-BZA in B16F10-bearing mice showed selective uptake into the tumor. The radiotracer was cleared via renal excretion without further metabolism. These results demonstrate that (68)Ga-SCN-NOTA-BZA is a potential PET probe for malignant melanoma.     

The effects of subcurative doses of chloroquine on Plasmodium vinckei petteri gametocytes and on their infectivity to mosquitoes

The effects of subcurative doses of chloroquine on rodent and human Plasmodium transmission to the mosquito have been studied by several authors who showed a short-term (12 h) enhancement of gametocyte infectivity by the drug, restricted to chloroquine-resistant strains, and a long term (4-6 days) enhancement of gametocytogenesis of chloroquine-sensitive strains of Plasmodium chabaudi. We investigated both short- and long-term effects of chloroquine on Plasmodium vinckei petteri, a chloroquine-sensitive rodent Plasmodium strain. Chloroquine treatment reduced the index of gametocytogenesis to 73% (5 mg/kg) and 55% (2.5 mg/kg) of controls, on day 6 post-infection (p.i.). The reduction was statistically significant with 5 mg/kg chloroquine. However, the reduction of gametocyte numbers did not affect the transmission capabilities of the strain. Our experiments showed that doses of 1 mg/kg chloroquine had no effect on the oocyst counts, 12 h post-administration to mice. A statistically non-significant 61% reduction of oocyst numbers was observed in mosquitoes fed on mice treated with 5 mg/kg chloroquine. The effect of 5 mg/kg chloroquine administration on the infectivity of gametocytes to mosquitoes fed 1 h post-treatment was also investigated. An overall 41% reduction of oocyst numbers was observed. This immediate effect was statistically significant in 73% of the mice. These results are consistent with the hypothesis that the short-term enhancing effect of chloroquine on transmission is restricted to the drug-resistant strains of Plasmodium.     

Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.     

Induction of supermelanin synthesis and morphological changes in interspecific reconstituted cells and its reversal by tumor promoter

Chloramphenicol-resistant (CAPr) reconstituted cells and cybrids were isolated by fusion of karyoplasts (or intact cells) of mouse amelanotic melanoma B16 cells with cytoplasts of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) -deficient, CAPr rat myoblastic cells, L6TG.CAPr, and double selection in HAT medium containing CAP. Reconstituted cells or cybrids exhibited unique cellular arrangement, and about one third of the isolated clones expressed high tyrosinase activity and marked melanin synthesis, although the parental mouse cells expressed low tyrosinase activity and the parental rat cells did not express tyrosinase activity. These phenotypic changes have been stable for more than a year. The phenotypic reversions of these clonal cells were induced by treatment with a tumor promoter. There were changes in the morphology of the treated cells to that of the mouse B16 cells and extinction of tyrosinase activity and melanin synthesis in pigmented clonal cells. These phenotypic changes and reversions induced by a promoter were repeatedly reversible.     

Cyclic amines are selective cytotoxic agents for pigmented cells

We have shown that morpholine, a cyclic amine, exerts a selective inhibition of growth on melanocytic pigmented cell lines compared to nonpigmented cells. The ID50 of morpholine for the pigmented B-16 cell line HFH was 1200 micrograms/ml, compared to values greater than 2400 micrograms/ml for baby hamster kidney, Chinese hamster ovary and NP, an unpigmented primate cell line. Two other cyclic amines piperazine and piperidine, were similarly found to be selectively toxic to melanocytes. This selective toxicity could be synergistically enhanced by pretreatment of the cells with theophylline, a stimulator of tyrosinase activity, which indicates that the selective toxicity may be associated with melanin synthesis. Low passage HFH, high passage HFH and Syrian hamster melanoma RPMI 1846 cells that were pretreated with theophylline showed between 13 and 29% greater toxicity compared to controls treated with theophylline or morpholine alone. Unpigmented NP primate cells, Chinese hamster ovary and mouse fibroblast L929 remained unaffected. These cyclic amines join a list of other amines that have also been shown to be melanocytotoxic.