Melanotic Melanoma Research Articles

Synthesis, characterization and biological evaluation of tricarbonyl M(i) (M = Re, 99mTc) complexes functionalized with melanin-binding pharmacophores

Aiming to evaluate their potential as radioactive probes for in vivo targeting of melanotic melanoma and its metastases, we have synthesized 99mTc(I) tricarbonyl complexes (Tc1–Tc8) anchored by pyrazolyl-containing chelators with (N3) or (N2O) donor atom sets and functionalized with 2-aminoethyldiethylamine and 4-amino-N-(2-diethylaminoethyl)benzamide groups as melanin-binding pharmacophores. The chemical identification of the several 99mTc complexes has been accomplished by HPLC comparison with the Re congeners (Re1–Re8), which were synthesized at the macroscopic level and fully characterized by common analytical techniques. The biological evaluation of the 99mTc(I) complexes comprised the determination of their in vitro binding to synthetic melanin, measurement of cellular uptake in B16F1 murine melanoma cells, as well as biodistribution studies in B16F1 melanoma-bearing mice. All the tested complexes have shown a moderate to high in vitro affinity to melanin, with percentages of binding spanning between 60 and 94%. In agreement with the poor cellular uptake measured in vitro, the in vivo tumor uptake of the complexes was in general relatively low, ranging between 0.12 and 1.69% ID g−1 at 4 h p.i. However, some complexes have shown favorable tumor-to-organ ratios with values as high as 28 and 5.3 for tumor–muscle and tumor–blood ratios, respectively. This seems to indicate that some selectivity towards melanoma tissue was conserved, and encourages further optimization of the in vitro/in vivo biological properties of this type of complexes aimed at finding novel radioactive probes for non-invasive imaging of melanoma.

Melanin-Targeted Preclinical PET Imaging of Melanoma Metastasis

Dialkylamino-alkyl-benzamides possess an affinity for melanin, suggesting that labeling of such benzamides with (18)F could potentially produce melanin-targeted PET probes able to identify melanotic melanoma metastases in vivo with high sensitivity and specificity. In this study, N-[2-(diethylamino)ethyl]-4-(18)F-fluorobenzamide ((18)F-FBZA) was synthesized via a 1-step conjugation reaction. The sigma-receptor binding affinity of (19)F-FBZA was determined along with the in vitro cellular uptake of radiofluorinated (18)F-FBZA in B16F10 cells. In vivo distribution and small-animal PET studies were conducted on mice bearing B16F10 melanoma, A375M amelanotic melanoma, and U87MG tumors, and comparative studies were performed with (18)F-FDG PET in the melanoma models. In vitro, uptake of (18)F-FBZA was significantly higher in B16F10 cells treated with l-tyrosine (P < 0.001). In vivo, (18)F-FBZA displayed significant tumor uptake; at 2 h, 5.94 +/- 1.83 percentage injected dose (%ID) per gram was observed in B16F10 tumors and only 0.75 +/- 0.09 %ID/g and 0.56 +/- 0.13 %ID/g was observed in amelanotic A375M and U87MG tumors, respectively. Lung uptake was significantly higher in murine lungs bearing melanotic B16F10 pulmonary metastases than in normal murine lungs (P < 0.01). Small-animal PET clearly identified melanotic lesions in both primary and pulmonary metastasis B16F10 tumor models. Coregistered micro-CT with small-animal PET along with biopsies further confirmed the presence of tumor lesions in the mouse lungs. (18)F-FBZA specifically targets primary and metastatic melanotic melanoma lesions with high tumor uptake and may have translational potential.

8721 Evaluation of objective response as a predictor of survival in bevacizumab-treated patients with glioblastoma at first or second relapse in the BRAIN Study

The nitrosoureas BCNU, CCNU, ACNU, and Fotemustine covalently deactivate thioredoxin reductase, glutathione reductase and ribonucleotide reductase by alkylating their thiolate active sites. Since thioredoxin reductase and glutathione reductase function as alternative electron donors in the biosynthesis of deoxyribonucleotides, catalyzed by ribonucleotide reductase, the inhibition of these electron transfer systems by the nitrosoureas could determine the cytostatic property of this homologous series of drugs. A detailed study of the kinetics and mechanism for the inhibition of purified thioredoxin reductases from human metastatic melanotic and amelanotic melanomas by the nitrosoureas showed significantly different inhibitor constants. This difference is due to the regulation of these proteins by calcium. Calcium protects thioredoxin reductase from deactivation by the nitrosoureas. In addition, it has been shown that reduced thioredoxin displaces the nitrosourea-inhibitor complex from the active site of thioredoxin reductase to fully reactivate enzyme purified from human metastatic amelanotic melanoma. It has been possible to label the active sites of thioredoxin reductase and glutathione reductase by using chloro[14C]ethyl Fotemustine, resulting in the alkylation of the thiolate active sites to produce chloro[14C]ethyl ether-enzyme inhibitor complexes. These complexes can be reactivated via reduced thioredoxin and reduced glutathione, respectively, by a β-elimination reaction yielding [14C]ethylene and chloride ions as reaction products.

8718 A review of the use of anti-epileptic drugs (AEDs) in high grade gliomas

The nitrosoureas BCNU, CCNU, ACNU, and Fotemustine covalently deactivate thioredoxin reductase, glutathione reductase and ribonucleotide reductase by alkylating their thiolate active sites. Since thioredoxin reductase and glutathione reductase function as alternative electron donors in the biosynthesis of deoxyribonucleotides, catalyzed by ribonucleotide reductase, the inhibition of these electron transfer systems by the nitrosoureas could determine the cytostatic property of this homologous series of drugs. A detailed study of the kinetics and mechanism for the inhibition of purified thioredoxin reductases from human metastatic melanotic and amelanotic melanomas by the nitrosoureas showed significantly different inhibitor constants. This difference is due to the regulation of these proteins by calcium. Calcium protects thioredoxin reductase from deactivation by the nitrosoureas. In addition, it has been shown that reduced thioredoxin displaces the nitrosourea-inhibitor complex from the active site of thioredoxin reductase to fully reactivate enzyme purified from human metastatic amelanotic melanoma. It has been possible to label the active sites of thioredoxin reductase and glutathione reductase by using chloro[14C]ethyl Fotemustine, resulting in the alkylation of the thiolate active sites to produce chloro[14C]ethyl ether-enzyme inhibitor complexes. These complexes can be reactivated via reduced thioredoxin and reduced glutathione, respectively, by a β-elimination reaction yielding [14C]ethylene and chloride ions as reaction products.

Glycosylated DOTA−α-Melanocyte-Stimulating Hormone Analogues for Melanoma Targeting: Influence of the Site of Glycosylation on in Vivo Biodistribution

alpha-Melanocyte-stimulating hormone (alpha-MSH) is known to bind to the melanocortin receptor 1 (MC1R) which is overexpressed on melanotic and amelanotic melanoma cells. alpha-MSH analogues are potential candidates for specific targeting of melanoma metastases. Several linear and cyclic radiolabeled MSH peptides have been designed and tested in the past, showing both high affinity for the MC1R in vitro and good incorporation in tumor xenografts in vivo. However, considerable kidney reabsorption of the radiopeptides could not be avoided. With the aim to increase the tumor-to-kidney ratio, we synthesized six glycosylated derivatives of NAPamide, an alpha-MSH octapeptide analogue with high tumor selectivity and coupled them to the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The peptides were evaluated in vitro for MC1R binding and bioactivity and, after labeling with (111)In, for in vitro cellular uptake and in vivo tissue distribution in mice carrying B16F1 melanoma tumors. The glycopeptides showed excellent binding affinities in the low nanomolar to subnanomolar range using both murine and human melanoma cell lines. However, five glycopeptides displayed lower selectivity in vivo than the parent DOTA-NAPamide, because of either a lower tumor uptake or a higher kidney uptake. In particular C-terminal extension of the amide group by a galactosyl moiety increased the kidney retention dramatically. By contrast, an N-terminally positioned galactose residue in DOTA-Gal-NAPamide improved the tumor-to-kidney ratio (4-48 h AUC of 1.34) by a factor of about 1.2 as compared to the parent DOTA-NAPamide (4-48 h AUC of 1.11), thus serving as new lead compound for MC1R-targeting molecules.

A novel boronated-porphyrin as a radio-sensitizing agent for boron neutron capture therapy of tumours: In vitro and in vivo studies

A water-soluble [meso-tetra(4-nido-carboranylphenyl)porphyrin] (H 2TCP) bearing 36 boron atoms was studied for its accumulation and its radio/photo-sensitization efficiency towards murine melanotic melanoma cells. The amount of H 2TCP in the cells increased with the porphyrin dose in the incubation medium up to 100 μM with no significant dark toxicity. Fluorescence microscopy observations showed that the porphyrin was largely localized intracellularly. Based on these “in vitro” results our investigations were pursued using the B16F1 melanotic melanoma subcutaneously transplanted in C57BL6 mice as “in vivo” model. Phormacokinetic studies were performed by injection of H 2TCP intratumorally (1 mg/kg) and intravenously (10 mg/kg). At 0.5 h after i.t. administration or at 24 h after i.v. injection, the amountsof 10B in the tumour were about 60 ppm and about 6 ppm, respectively. The distribution of H 2TCP in the tumour after intravenous or intratumoural injection was also assessed by fluorescence microscopy analyses. Under these conditions, preliminary BNCT studies were carried out using a new thermal column called HYTOR (HYbrid Thermal spectrum sHifter TapirO Reactor) inserted in the fast nuclear reactor Tapiro at Enea Casaccia, Italy. The mice were exposed to HYTHOR radiation field for 20 min at a reactor power of 5 kW. In spite of different amounts of 10B in the tumour at the irradiation time, a similar significant delay in tumour growth (5–6 days) was induced by neutron irradiation in intratoumorally and intravenously injected mice. The response of the melanotic melanoma to H 2TCP-BNCT was compared with that obtained by irradiation after intraperitoneal injection of boron-phenylalanine.

Synthesis, inhibition of NO production and antiproliferative activities of some indole derivatives

The synthesis and the biological evaluation of pyrano[3,2-e]indoles and their reaction intermediates are described. The compounds prepared were evaluated for their inhibition of NO production, antioxidant activity and also for their ability to inhibit in vitro the growth of four human tumor cell lines: large lung carcinoma (COR-L23), alveolar basal epithelial carcinoma (A549), amelanotic melanoma (C32) and melanoma (A375). The two reaction intermediates, 5a and 5b, showed the highest inhibition of NO production in murine monocytic macrophage (IC50 = 1.1 μM and IC50 = 2.3 μM respectively). Compound 5a was the most active against melanotic melanoma (IC50 = 11.8 μM) while the other compounds exhibited weak cytotoxicity with IC50 values >50 μM on all cell lines.

Effusion cytomorphology and immunocytochemistry of malignant melanoma: Five cases of melanotic melanoma and one case of amelanotic melanoma

Effusion cytological analyses of amelanotic malignant melanoma (AMM) are very rare and no concise description of AMM related cytomorphologic features using effusion have been reported. Here, we report the cytomorphological, immunohistochemical, and immunocytochemical findings in the effusion cytology of six cases of malignant melanoma (MM), one case of AMM, and five cases of melanotic malignant melanoma. Papanicolaou-stained smears exhibited conspicuous nucleoli, multinucleation, and cytoplasmic vacuolization in all of the MM cases. In addition, the AMM case displayed numerous mitotic figures and intranuclear cytoplasmic inclusions. With regard to the immunohistochemistry findings, all six cases of melanoma were positive for Melan-A/MART-1, HBME-1, and S-100. In the immunohistochemistry analyses, five of six cases of melanoma were positive for WT-1, as was the AMM specimen. Furthermore, because the effusion analysis of malignant mesothelioma proved positive for WT-1, it should be noted that WT-1 effusion analysis is not an appropriate means to distinguish between MM and malignant mesothelioma. We suggest that it is important to recognize cytomorphologic characteristics, such as melanin pigment, conspicuous nucleoli, multinucleation, and cytoplasmic vacuolization, and to choose appropriate antibodies for the correct diagnosis of MM in effusion.

Early diagnosis of melanotic melanoma based on laser-induced melanin fluorescence

Because of the increasing incidence of skin cancer, interest in using the autofluorescence of skin tissue as a noninvasive tool for early diagnosis is enforced. Focus is especially on malignant melanotic melanoma. On the basis of a newly developed method to selectively excite melanin fluorescence of skin tissue by stepwise two-photon excitation with nanosecond laser pulses at 810 nm, we have investigated information from this melanin fluorescence with respect to the differentiation of pigmented lesions. A distinct difference in the melanin fluorescence spectrum of malignant melanoma (including melanoma in situ) when compared to that of benign melanocytic lesions (i.e., common nevi) has been found for freshly excised samples as well as for histopathological samples. There is also specific fluorescence from dysplastic nevi. In this way, early detection of malignant melanoma is possible.

High-resolution magic angle spinning nuclear magnetic resonance analysis of metabolic changes in melanoma cells after induction of melanogenesis

Melanin synthesis affects melanoma behavior and tumor responsiveness to therapy; therefore, we investigated metabolic changes in melanoma cells after induction of melanogenesis. Amelanotic and melanotic melanoma cells were labeled with 13C precursors and changes in their metabolism was analyzed by high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR). HRMAS NMR demonstrated clear differences in the pattern of metabolic intermediates between amelanotic and melanotic cells. Although the exact nature of the metabolites requires further investigations, our comparative studies clearly show that induction of melanogenesis is associated with changes of glucose and sodium acetate metabolism, demonstrating HRMAS NMR as a powerful and noninvasive technique to investigate such process.

Inhibitors of melanogenesis increase toxicity of cyclophosphamide and lymphocytes against melanoma cells

High mortality rate for metastatic melanoma is related to its resistant to the current methods of therapy. Melanogenesis is a metabolic pathway characteristic for normal and malignant melanocytes that can affect the behavior of melanoma cells or its surrounding environment. Human melanoma cells in which production of melanin pigment is dependent on tyrosine levels in medium were used for experiments. Peripheral blood mononuclear cells were derived from the buffy coats purchased from Lifeblood Biological Services. Cell pigmentation was evaluated macroscopically, and tyrosinase activity was measured spectrophotometrically. Cell proliferation and viability were measured using lactate dehydrogenase release MTT, [(3)H]-thymidine incorporation and DNA content analyses, and gene expression was measured by real time RT-PCR. Pigmented melanoma cells were significantly less sensitive to cyclophosphamide and to killing action of IL-2-activated peripheral blood lymphocytes. The inhibition of melanogenesis by either blocking tyrosinase catalytic site or chelating copper ions sensitized melanoma cells towards cytotoxic action of cyclophosphamide, and amplified immunotoxic activities of IL-2 activated lymphocytes. Exogenous L-DOPA inhibited lymphocyte proliferation producing the cell cycle arrest in G1/0 and dramatically inhibited the production of IL-1beta, TNF-alpha, IL-6 and IL-10. Thus, the active melanogenesis could not only impair the cytotoxic action of cyclophosphamid but also has potent immunosuppressive properties. This resistance to a chemotherapeutic agent or immunotoxic activity of lymphocytes could be reverted by the action of tyrosinase inhibitors. Thus, the inhibition of melanogenesis might represent a valid therapeutic target for the management of advanced melanotic melanomas.

Fas and FasL expression on cells of two transplantable melanoma lines according to their different biological properties.

Fas and FasL interaction induces apoptotic cell death. In immunocompetent cells it plays a crucial role in the effector functions of the cells and in the regulation of host immune response. In tumours (e.g. melanoma), FasL expression possibly counteracts the Fas-positive effector T cells that infiltrate into tumours, and consequently the Fas/FasL interaction can contribute to the escape of tumour cells from the systemic immune response. In this study we examined differences in Fas and FasL expression on cells from the hamster melanotic melanoma line (Ma) and a more aggressive amelanotic melanoma line (Ab). We also tried to find out whether the Fas/FasL expression induces an ability to undergo spontaneous apoptosis in these two transplantable melanoma lines. Our previous studies have shown that cells of the Ma line have a higher ability to undergo spontaneous apoptosis than cells of the Ab line. Isolated transplantable melanoma cells were incubated for 4 and 24 hours and after that time the expression of Fas and FasL was estimated by flow cytometry. The results show that there was no Fas expression, although FasL was detected on both melanoma cell lines. Therefore the data reported by other authors indicate that a lack or a low level of Fas expression and an ectopic expression of FasL on melanoma cells can be an escape mechanism of the tumour, to avoid host immune responses. The content of FasL-positive melanotic melanoma cells was higher than in amelanotic melanoma cells and increased with the prolongation of the incubation time. FasL expression on amelanotic melanoma cells was detected after 24 hours at a level similar to that on melanotic melanoma cells after 4 hours incubation time. FasL expression on melanoma cells can induce apoptosis in cytotoxic T lymphocytes and NK cells which are responsible for tumour cells elimination. The results obtained suggest that the Fas/FasL system does not play any significant role in spontaneous apoptosis of two melanoma cell lines. But these results may indicate the presence of immune privilege of tumour cells with FasL expression.

Synthesis and evaluation of novel radioiodinated nicotinamides for malignant melanoma

A series of iodonicotinamides based on the melanin-binding iodobenzamide compound N-2-diethylaminoethyl-4-iodobenzamide was prepared and evaluated for the potential imaging and staging of disseminated metastatic melanoma. [(123)I]Iodonicotinamides were prepared by iododestannylation reactions using no-carrier-added iodine-123 and evaluated in vivo by biodistribution and competition studies and by single photon emission computed tomography (SPECT) imaging in black and albino nude mice bearing B16F0 murine melanotic and A375 human amelanotic melanoma tumours, respectively. The iodonicotinamides displayed low-affinity binding for sigma(1)-sigma(2) receptors (K(i)>300 nM). In biodistribution studies in mice, N-(2-(diethylamino)ethyl)-5-[(123)I]iodonicotinamide ([(123)I]1) exhibited the fastest and highest uptake of the nicotinamide series in the B16F0 tumour at 1 h ( approximately 8% ID/g), decreasing slowly over time. No uptake was observed in the A375 tumour. Clearance from the animals by urinary excretion was more rapid for N-alkyl-nicotinamides than for piperazinyl derivatives. At 1 h postinjection, the urinary excretion was 66% ID for [(123)I]1, while the gastrointestinal tract amounted to 17% ID. Haloperidol was unable to reduce the uptake of [(123)I]1 in pigmented mice, indicating that this uptake was likely due to an interaction with melanin. SPECT imaging of [(123)I]1 in black mice bearing the B16F0 melanoma indicated that the radioactivity was predominately located in the tumour and eyes. No specific localisation was observed in nude mice bearing A375 amelanotic tumours. These findings suggest that [(123)I]1, which displays high tumour uptake with rapid clearance from the body, could be a promising imaging agent for the detection of melanotic tumours.

A method for quantifying melanosome transfer efficacy from melanocytes to keratinocytes in vitro

Several different in vivo and in vitro bioassays are used to evaluate melanosome transfer efficacy from melanocytes to keratinocytes. However, these methods are complicated and time consuming. Here, we report on a simple, rapid, direct, and reliable in vitro method for observing the process of melanosome transfer from melanocytes to keratinocytes. First, we selected and tested a melanoma cell line RPMI-7951 that can normally synthesize melanin and transfer from mature melanosomes to keratinocytes in vitro. We cocultured these cells with a human ovarian teratoma transformed epidermal carcinoma cell line, which is also capable of accepting melanosomes transferred from melanocytes, as in normal keratinocytes. The cells were cocultured for 24-72 h and double labeled with FITC-conjugated antibody against the melanosome-associated protein TRP-1, and with Cy5-conjugated antibody against the keratinocyte-specific marker keratin 14. The cells were examined by fluorescence microscope and flow cytometry. Melanosome transfer from melanocytes to keratinocytes increased in a time-dependent manner. To verify the accessibility of this method, the melanosome transfer inhibitor, a serine protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and a melanosome transfer stimulator, alpha-melanocyte-stimulating hormone, were added. The serine protease inhibitor decreased melanosome transfer, and alpha-melanocyte-stimulating hormone increased melanosome transfer, in a dose-dependent manner. In conclusion, this is a simple, rapid, and effective model system to quantify the melanosome transfer efficacy from melanocytes to keratinocytes in vitro.

Tumor-localizing and radiosensitizing properties of meso-tetra(4-nido-carboranylphenyl)porphyrin (H2TCP)

Background and Purpose: Boron Neutron Capture Therapy (BNCT) is a binary cancer treatment that exploits the short range particles released from a nuclear fission reaction involving the non-radioactive 10 B nucleus and low-energy neutrons for the destruction of tumor cells. In this perspective, porphyrins and phthalocyanines can represent a vehicle for the transport of significant amounts of boron to the neoplastic lesion. Material and Methods: B16F1 melanotic melanoma subcutaneously transplanted in C57/BL6 mice has been used as an in vivo model. Pharmacokinetic studies were performed by intratumoral and intravenous injection of a meso-substituted porphyrin containing 36 B atoms per molecule ( H 2 TCP ) and the distribution of H 2 TCP in the tumor was assessed by fluorescence microscopy analysis. The tumor-bearing mice were exposed to the radiation field for 20 min at a reactor power of 5 kW. Results: At 0.5 h after intratumoral administration or at 24 h after intravenous injection, the amount of 10 B in the tumor was found to be about 60 ppm and about 6 ppm, respectively. In spite of the different amounts of 10 B in the tumor at the time of irradiation, a very similar delay in tumor growth (5-6 days) was induced by neutron irradiation in the two groups of injected mice with respect to control mice. Conclusions: Our results demonstrate that a suitable boron-loaded porphyrin displays a significant affinity for subcutaneous tumors, and upon activation by thermal neutrons, can promote an important response even in a fairly aggressive and generally radioresistant tumor such as melanotic melanoma.

Synthesis and biological investigations of tetrakis(p-carboranylthio-tetrafluorophenyl)chlorin (TPFC)

We describe the total synthesis and biological properties of a new carboranyl-containing chlorin (TPFC) that might find application as a dual sensitizer in the PDT and BNCT treatment of cancer. TPFC was found to be non-toxic in the dark but showed extensive photosensitizing ability both in vitro and in vivo despite its relatively low singlet oxygen quantum yield. In particular, TPFC exhibited significant photosensitizing activity against highly pigmented melanotic melanoma tumors in mice.

Tumour-localizing and -photosensitising properties of meso-tetra(4-nido-carboranylphenyl)porphyrin (H 2TCP)

A water-soluble meso-substituted porphyrin (H 2TCP) bearing 36 boron atoms, which appeared to be an efficient photodynamic sensitiser (singlet oxygen quantum yield = 0.44), was studied for its accumulation by murine melanotic melanoma cells (B16F1). The amount of H 2TCP in the cells increased with the porphyrin dose in the incubation medium up to, and at least, 100 μM concentrations with no significant cytotoxic effect in the dark. Moreover, the H 2TCP uptake increased with the incubation time reaching a plateau value corresponding with the recovery of 0.4 nmol of H 2TCP per mg of cell proteins after 24 h incubation. Fluorescence microscopy observations showed that the porphyrin was largely localized intracellularly, exhibiting a discrete distribution in the cytoplasm with a pattern which was closely similar to that observed for the endosomal probe Lucifer yellow. The photosensitising efficiency of the H 2TCP toward B16F1 cells was studied for different irradiation (1–15 min) and incubation (1–24 h) times. Nearly complete (>95%) cell mortality was obtained upon incubation with 20 μM H 2TCP and 10 min irradiation with red light (600–700 nm, 20 mW/cm 2). The porphyrin was also accumulated in appreciable amounts by the tumour tissue after intravenous injection to C57BL/6 mice bearing a subcutaneously transplanted melanotic melanoma. Maximum accumulation in the tumour was achieved by administration of H 2TCP dissolved in the ternary mixture 20% dimethylsulfoxide (DMSO)–30% polyethyleneglycol (PEG 400)–50% water. Thus, this porphyrin could act as both a photodynamic therapy agent and a radiosensitising agent for boron neutron capture therapy.

Antivascular Treatment of Solid Melanoma Tumors with Bacteriochlorophyll-serine-based Photodynamic Therapy¶

We describe here a strategy for photodynamic eradication of solid melanoma tumors that is based on photoinduced vascular destruction. The suggested protocol relies on synchronizing illumination with maximal circulating drug concentration in the tumor vasculature attained within the first minute after administrating the sensitizer. This differs from conventional photodynamic therapy (PDT) of tumors where illumination coincides with a maximal concentration differential of sensitizer in favor of the tumor, relative to the normal surrounding tissue. This time window is often achieved after a delay (3–48 h) following sensitizer administration. We used a novel photosensitizer, bacteriochlorophyll–serine (Bchl–Ser), which is water soluble, highly toxic upon illumination in the near-infrared (λmax 765–780 nm) and clears from the circulation in less than 24 h. Nude CD1 mice bearing malignant M2R melanotic melanoma xenografts (76–212 mm3) received a single complete treatment session. Massive vascular damage was already apparent 1 h after treatment. Changes in vascular permeability were observed in vivo using contrast-enhanced magnetic resonance imaging (MRI), with the contrast reagent Gd-DTPA, by shortening spin–spin relaxation time because of hemorrhage formation and by determination of vascular macromolecular leakage. Twenty-four hours after treatment a complete arrest of vascular perfusion was observed by Gd-DTPA–enhanced MRI. Histopathology performed at the same time confirmed primary vascular damage with occlusive thrombi, hemorrhage and tumor necrosis. The success rate of cure of over 80% with Bchl–Ser indicates the benefits of the short and effective treatment protocol. Combining the sensitizer administration and illumination steps into one treatment session (30 min) suggests a clear advantage for future PDT of solid tumors.

The activity of caspases in spontaneous and camptothecin-induced death of melanotic and amelanotic melanoma cell

Loss of pigment in hamster amelanotic melanoma line is accompanied by a faster growth rate, higher tumorigenicity and shorter animal survival time. Thus, the malignancy of melanoma increases during the alteration of melanotic (Ma) into amelanotic (Ab) line. As changes in the ability to undergo a spontaneous or induced apoptosis, and the role of caspases in this process during melanoma progression are not well defined, they were investigated in this work. Our results show that the proportion of spontaneously early apoptotic (caspase+/PI-) cells in the Ab line decreased in comparison to the Ma line. Cytochrome c release into cytosol, and the activation of effector caspases, estimated by PARP degradation clearly showed that during the spontaneous death in the cells from both melanoma lines intrinsic way of apoptosis was activated. Confocal and cytometric flow analyses indicate that camptothecin (CPT) induced apoptosis with caspase activation by the intrinsic way only in the amelanotic melanoma cells, even though cells of the Ma line also underwent CPT-induced apoptosis (the content of TUNEL- positive cells increased). Thus, our results suggest that melanoma progression, associated with a decreased ability to undergo spontaneous apoptosis but an increased susceptibility to CPT-induced apoptosis, relates to different levels of caspase activation; they also show that intrinsic way of apoptotis depends on the phenotype of melanoma cells, being more pronounced in the melanotic melanoma cells. On the other hand, melanotic melanoma cells resistance to camptothecin-induced apoptosis suggests that the melanogenic apparatus or melanin itself may have the protective effect on the ability of the melanoma cells to undergo apoptosis.

A New Method for Photodynamic Therapy of Melanotic Melanoma—Effects of Depigmentation with Violet Light Photodynamic Therapy

Melanotic melanomas have a poor response to photodynamic therapy (PDT). The reason for this is that melanin absorbs light over the entire wavelength region used for PDT (400-750 nm). Photobleaching of melanin is an approach to overcome this obstacle. In the present work, reflectance spectroscopy was applied to study depigmentation of human and murine skin with different melanin contents, and effects induced by PDT with topical application of methyl 5-aminolevulinate (MAL) on B16F10 melanotic melanomas transplanted to nude mice. Depigmentation and inhibition of tumor growth after violet light (420 nm) exposure, red light (634 nm) exposure, and combinations of both were studied. Reflectance spectroscopy was suitable for evaluation of the pigmentation of both human and murine skin. Skin depigmentation leads to increase in reflectance. PDT with violet light bleached some of the melanin in the skin above the B16F10 melanomas, and possibly also in the upper part of the melanomas. This resulted in a larger growth inhibition of tumors first given PDT with violet light and then with red light compared to treatments using the reverse order of illumination, namely, red light before violet light. It is concluded that violet light PDT can bleach melanin in melanotic tumors and therefore increase their sensitivity to red light PDT. This finding indicates a new PDT modality that can be further developed for treatment of superficial melanotic melanomas and possibly other diseases where pigmentation is a problem.