Vemurafenib and concomitant stereotactic radiation for the treatment of melanoma with spinal metastases: A case report

A 56-year-old man with BRAFV600E melanoma and spinal metastases treated with vemurafenib and stereotactic radiation showed a partial response without neurological, skin or mucosal toxicity, 8 months after completion of this combination. This case suggests that stereotactic radiation spares normal tissues and might be safer than conventional fractionated radiation with vemurafenib.     

Biologic,Immunocytochemical, and Cytogenetic Characterization of Two New Human Melanoma Cell Lines: IIB-MEL-LES and IIB-MEL-IAN

Two human melanoma cell lines, derived from metastases of two patients with epithelioid malignant amelanotic melanomas, and designated IIB-MEL-LES and IIB-MEL-IAN, have been established. Both cell lines have been in continuous culture over 2 years and were propagated continuously for 85 and 75 serial passages, respectively. Morphologically, IIB-MEL-LES is composed predominantly of spindle shaped cells, whereas IIB-MEL-IAN grows as a monolayer of cuboid and stellate shaped cells with many rounded cells in suspension. Immunocytochemical studies revealed that both cell lines express S-100 protein, vimentin, and GD₃ and GD₂ gangliosides but are negative for keratin and collagen. Both cell lines express HLA class I and HLA-DR antigens in variable proportions. The MAGE-1 gene is expressed only by the IIB-MEL-IAN cell line, as revealed by PCR analysis. Cytogenetic analysis of both cell lines revealed abnormal karyotypes; the modal chromosome numbers of IIB-MEL-LES and IIB-MEL-IAN were 48 and 81, respectively. IIB-MEL-LES cells presented rearrangements in chromosomes 1, 14 and X, gains in chromosomes 10,20, and 21 losses in chromosomes 15 and Y. The most frequent markers observed in IIB-MEL-IAN cells were 7q+, 10p+, 2p+, i(6p), 2q+, and 10q-. Clonal gains were observed in chromosomes 12 and 21, whereas losses were seen in chromosomes 1, 2, 3, 4, 6, 7, 11, and 17. Both cell lines were capable of forming colonies in soft agar and developed tumors when transplanted into nude mice, reproducing and maintaining the characteristics of the original tumors. These cell lines and their xenografts appear to provide useful systems for studying the biology, genetics and histogenesis of human malignant melanoma and could be utilized for the development of melanoma vaccines.     

Ganglioside Expression in Melanomas From Japanese Individuals: Unusual Pattern in Two Patients With Metastatic Lesions of Acral Lentiginous Melanomas

Melanoma among Japanese is rare, and differs in its clinical and histological characteristics from that found in Caucasians. In this study, the ganglioside expression of melanoma specimens obtained from Japanese was determined and compared with previously published data on Caucasians. The ganglioside composition of 25 biopsy melanoma specimens, including 13 primary and 12 metastatic lesions, was studied using thin layer chromatography. Four gangliosides (G_M3, G_D3, G_M2, G_D2) were most commonly expressed in melanomas in Japanese. The expression of gangliosides was quite variable in both primary and metastatic melanomas as seen in previous reports. No significant differences were observed between gangliosides from primary and metastatic sites. A new type of ganglioside expression, in which G_M3 was nearly the only ganglioside (>95%), was found in metastatic tumors from two Japanese patients with acral lentiginous melanoma (ALM), which is the most common clinical and histopathological type of melanoma among Japanese but is very unusual among Caucasians. The patterns of expression were similar to those in Caucasians except for the detection of a “new” pattern.     

Preparation of Purified Tyrosinase Devoid of Dopachrome Tautomerase From Mammalian Malignant Melanocytes

Although tyrosinase has been considered for a long time the only enzyme involved in mammalian melanosynthesis, it has been shown that mouse melanoma melanosomes contain high levels of dopachrome tautomerase (DCT²), an enzyme catalyzing DC tautomerization to DHICA. At least in B16 mouse melanoma, DCT is present in higher catalytic amounts than tyrosinase. Moreover, it can be anticipated that tyrosinase and DCT should be very difficult to resolve by most conventional biochemical techniques because of the structural similarity between these enzymes, as predicted from the sequence of their corresponding cDNAs. It is shown that the presence of DCT can cause serious artifacts when tyrosinase activity is determined by most of the currently available methods, such as the Dopa oxidase and melanin formation assays. We describe a simple and convenient method for the preparation of tyrosinase devoid of DCT. The method takes advantage of the different thermal stability of both enzymes. Heating of crude melanosomal extracts at 60°C for 1 hr results in a complete denaturation of DCT, while tyrosinase activity is recovered almost quantitatively. The resulting tyrosinase preparation is considerably purified and the electrophoretic, immunologic and kinetic characteristics of the enzyme appear unaltered. Because if its high yield and simplicity, the method can be used for the microscale partial purification of DCT-free tyrosinase from mammalian malignant melanocytes grown in culture.     

Autocrine/paracrine actions of growth hormone in human melanoma cell lines

Melanoma is the most aggressive skin cancer. Its aggressiveness is most commonly attributed to ERK pathway mutations leading to constitutive signaling. Though initial tumor regression results from targeting this pathway, resistance often emerges. Interestingly, interrogation of the NCI-60 database indicates high growth hormone receptor (GHR) expression in melanoma cell lines. To further characterize melanoma, we tested responsiveness to human growth hormone (GH). GH treatment resulted in GHR signaling and increased invasion and migration, which was inhibited by a GHR monoclonal antibody (mAb) antagonist in WM35, SK-MEL 5, SK-MEL 28 and SK-MEL 119 cell lines. We also detected GH in the conditioned medium (CM) of human melanoma cell lines. GHR, JAK2 and STAT5 were basally phosphorylated in these cell lines, consistent with autocrine/paracrine GH production. Together, our results suggest that melanomas are enriched in GHR and produce GH that acts in an autocrine/paracrine manner. We suggest that GHR may constitute a therapeutic target in melanoma.     

Apatinib-loaded nanoparticles inhibit tumor growth and angiogenesis in a model of melanoma

Anti-angiogenic drugs are an effective therapeutic method for the treatment of melanomas. Apatinib is a small-molecule tyrosine kinase inhibitor, which has potent inhibitory activity on tumor angiogenesis. Due to the low water solubility and stability of Apatinib, we aimed to design and develop poly (lactic-co-glycolic acid) (PLGA) and Poloxamer 407 nanoparticles to encapsulate Apatinib (Apa/p NPs) to improve the efficacy of application in melanoma treatment. The size and morphology of the nanoparticles were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). In vitro proliferation assays were used to assess the capacity of Apa/p NPs to suppress the growth of B16 cells. Furthermore, we constructed melanoma models using C57BL/6 mice, and preliminary evaluation of the effect and mechanism of Apa/p NPs on tumor inhibition was performed in vivo. The results showed that the size of Apa/p NPs averaged 136 ± 0.27 nm and the nanoparticles were evenly dispersed. Moreover, Apa/p NPs significantly inhibited the growth of B16 cells and melanoma tumors, compared with the naked drug treatment and control groups. The protein levels of VEGFR-2, phosphorylated (p)-VEGFR-2 and p-ERK1/2 in tumor tissues were inhibited by Apa/p NP treatment, as detected by Western blot. The results of this study suggested that Apa/p NPs could inhibit the growth of melanoma tumors by inhibiting the phosphorylation and expression of VEGFR-2 and downstream ERK1/2, providing a theoretical basis for the clinical application of Apatinib in the treatment of melanoma.     

The DHICA Oxidase Activity of the Melanosomal Tyrosinases LEMT and HEMT

Although melanins can be formed in vitro by the unique action of tyrosinase on L-tyrosine, it is now well accepted that other enzymes termed tyrosinase-related proteins are involved in mammalian melanogenesis. However, some aspects of their roles in the regulation of the pathway are still unknown. The action of dopachrome tautomerase on L-dopachrome yields DHICA, a stable dihydroxyindole with a low rate of spontaneous oxidation. However, DHICA is efficiently incorporated to the pigment, as judged by the high content of carboxylated indole units in natural melanins. Therefore, the fate of this melanogenic intermediate and the mechanisms of its incorporation to the melanin polymer are major issues in the study of melanogenesis. We have recently shown that mouse melanosomes contain two electrophoretically distinguishable tyrosinase isoenzymes, LEMT and HEMT, that can be purified and completely resolved (Jiménez-Cervantes et al., 1993a). Herein, we have compared the ability of these tyrosinases to catalyze DHICA oxidation. Although highly purified LEMT shows a very low specific activity for dopa oxidation in comparison to HEMT, it is able to catalyze DHICA oxidation. However, the DHICA oxidase activity of HEMT was very low, if significant. The ability of purified LEMT to catalyze DHICA oxidation was abolished by heat, trypsin, or phenylthiourea treatments. LEMT acting on DHICA caused the formation of a brownish soluble color similar to DHICA-melanin. Immunoprecipitation of the DHICA oxidase activity of LEMT by specific antibodies suggests that this activity corresponds to TRP1. These results indicate that LEMT, most probably identical to the product of the b locus, is a tyrosinase having a specific DHICA oxidase activity. Opposite to HEMT, the true tyrosinase encoded by the albino locus, its role in melanogenesis would be related to the incorporation of DHICA into eumelanin rather than to the first steps of the pathway.     

Characterization of Vitiligo Antigens

Patients with vitiligo have circulating antibodies directed in part to pigment cell antigens with MWs of approximately 90, 75, and 40-45 kDs. These antigens are denominated VIT 90, VIT 75, and VIT 40, respectively. To further characterize these “vitiligo” antigens, we examined their relation to antigens defined by a panel of 25 monoclonal antibodies (moab) to pigment cell antigens. We found by immunoprecipitation and SDS-PAGE analysis of ¹²⁵I labelled, detergent soluble, human melanocyte macromolecules, that 24 (83%) of 29 patients with vitiligo had antibodies to one or more vitiligo antigens vs. 2 (7%) of 28 control individuals. Seventeen of the 25 moabs did not react with any labelled antigen in the same lysate. Of the remaining eight moabs, only four precipitated an antigen that co-migrated with one of the vitiligo antigens. Moab TA99, HMSA-5, and TMH-1 (all directed to the 75 kD tyrosinase-related protein [TRP1]) co-migrated with VIT 75. Moab W6/32 (directed to class I HLA antigen) co-migrated with VIT 40. Immunodepletion studies with vitiligo antibodies selectively depleted the antigen defined by W6/32 but not the antigen defined by TA99 and HMSA-5, indicating that VIT 75 was not the 75 kD tyrosinase-related protein. The vitiligo antigens were easily labelled by the lactoperoxidase technique but poorly labelled with ³⁵S-methionine, suggesting they are expressed on the cell surface. These studies indicate that VIT 90 and VIT 75 differ from antigens defined by currently available moabs to pigment cell antigens. VIT 40 appears to share a cross-reactive epitope, or be tightly bound to, class I HLA antigen.     

Selective and Synergistic Activity of L-S,R-Buthionine Sulfoximine on Malignant Melanoma Is Accompanied by Decreased Expression of Glutathione-S-Transferase

L-buthionine-S,R-sulfoximine (BSO) selectively inhibits glutathione (GSH) synthesis. Malignant melanoma may be uniquely dependent on GSH and its linked enzymes, glutathione S-transferase (GST) and GSH-peroxidase, for metabolism of reactive orthoquinones and peroxides produced during melanin synthesis. We compared the in vitro effects of BSO on melanoma cell lines and fresh melanoma specimens (n = 118) with breast and ovarian cell lines and solid tumors (n = 244). IC₅₀ values (μM) for BSO on melanoma, breast and ovarian tumor specimens were 1.9, 8.6, and 29, respectively. The IC₉₀ for melanoma was 25.5 μM, a level 20-fold lower than steady state levels achieved clinically. The sensitivity of individual specimens of melanoma correlated with their melanin content (r = 0.63). BSO synergistically enhanced BCNU activity against melanoma cell lines and human tumors. We followed GSH levels, GST enzyme activity, GST isoenzyme profiles and mRNA levels after BSO. BSO (50 μM) treatment for 48 hr resulted in a 95% decrease in ZAZ and M14 melanoma cell line GSH levels, and a 60% decrease in GST enzyme activity. GST-μ. protein and mRNA levels were significantly reduced in both cell lines. GST expression was unaffected. These data suggest that BSO action on melanoma may be related to GSH depletion, diminishing the capacity to scavenge toxic metabolites produced during melanin synthesis. We report here for the first time that BSO enhancement of alkylator action may be related in part to down regulation of GST. BSO may be a clinically useful adjunct in the treatment of malignant melanoma.     

Dysplastic Melanocytic Nevi Contain High Levels of Pheomelanin: Quantitative Comparison of Pheomelanin/Eumelanin Levels Between Normal Skin,Common Nevi,and Dysplastic Nevi

The degree and type of melanogenesis, i.e., either eumelanin of pheomelanin, has been shown to be a reliable marker for the differentiation of the melanocyte. If exposed to UV light, these two melanins were reported to behave differently; eumelanin was photoprotective whereas pheomelanin was phototoxic to cultured tumor cells. Our previous study indicated that dysplastic melanocytic nevus (DMN) undergoes altered melanogenesis, forming pheomelanosome-like granules. The present study examined chemically the type and degree of melanin synthesized in 31 melanocytic nevi excised from 27 patients as compared with that occurring in the surrounding normal skin. The tissue content of eumelanin and pheomelanin was expressed by the amounts of pyrrole-2,3,5-tricarboxylic acid (PTCA) and aminohydroxyphenylalanine (AHP), respectively. We found that DMN lesions contain significantly higher amounts of pheomelanin than either common melanocytic nevus (CMN) or normal skin. Differences in pheomelanin content between DMN and CMN could not be accounted for by inherently higher levels of pheomelanin within the skin in general from DMN patients. Our present finding substantiates our previous claim that epidermal melanocytes in DMN undergo deranged melanogenesis.