Nanomechanical analysis of pigmented human melanoma cells

Based on hitherto measurements of elasticity of various cells in vitro and ex vivo, cancer cells are generally believed to be much softer than their normal counterparts. In spite of significant research efforts on the elasticity of cancer cells, only few studies were undertaken with melanoma cells. However, there are no reports concerning pigmented melanoma cells. Here, we report for the first time on the elasticity of pigmented human melanoma cells. The obtained data show that melanin significantly increases the stiffness of pigmented melanoma cells and that the effect depends on the amount of melanin inside the cells. The dramatic impact of melanin on the nanomechanical properties of cells puts into question widely accepted paradigm about all cancer cells being softer than their normal counterparts. Our findings reveal significant limitations of the nanodiagnosis approach for melanoma and contribute to better understanding of cell elasticity.     

An Endogenous Electron Spin Resonance (ESR) Signal Discriminates Nevi from Melanomas in Human Specimens: A Step Forward in Its Diagnostic Application

Given the specific melanin-associated paramagnetic features, the Electron Spin Resonance (ESR, called also Electron Paramagnetic Resonance, EPR) analysis has been proposed as a potential tool for non-invasive melanoma diagnosis. However, studies comparing human melanoma tissues to the most appropriate physiological counterpart (nevi) have not been performed, and ESR direct correlation with melanoma clinical features has never been investigated. ESR spectrum was obtained from melanoma and non-melanoma cell-cultures as well as mouse melanoma and non-melanoma tissues and an endogenous ESR signal (g = 2.005) was found in human melanoma cells and in primary melanoma tissues explanted from mice, while it was always absent in non-melanoma samples. These characteristics of the measured ESR signal strongly suggested its connection with melanin. Quantitative analyses were then performed on paraffin-embedded human melanoma and nevus sections, and validated on an independent larger validation set, for a total of 112 sections (52 melanomas, 60 nevi). The ESR signal was significantly higher in melanomas (p = 0.0002) and was significantly different between “Low Breslow’s and “High Breslow’s” depth melanomas (p<0.0001). A direct correlation between ESR signal and Breslow’s depth, expressed in millimetres, was found (R = 0.57; p<0.0001). The eu/pheomelanin ratio was found to be significantly different in melanomas “Low Breslow’s” vs melanomas “High Breslow’s” depth and in nevi vs melanomas “High Breslow’s depth”. Finally, ROC analysis using ESR data discriminated melanomas sections from nevi sections with up to 90% accuracy and p<0.0002. In the present study we report for the first time that ESR signal in human paraffin-embedded nevi is significantly lower than signal in human melanomas suggesting that spectrum variations may be related to qualitative melanin differences specifically occurring in melanoma cells. We therefore conclude that this ESR signal may represent a reliable marker for melanoma diagnosis in human histological sections.     

Anti-melanoma efficacy of internal radionuclide therapy in relation to melanin target distribution

Targeted internal radionuclide therapy (TRT) could be an efficient, specific way to treat disseminated melanoma. Based on a previous pharmacomodulation study, we selected a quinoxaline-derived molecule (ICF01012) for its melanin specificity and kinetic properties suitable for TRT. Here, we determined the efficacy of [¹³¹I]ICF01012 radiotherapy in vitro and in vivo in relation to melanogenesis using human melanoma models. [¹²⁵I]ICF01012 uptake was first assessed in relation to melanin content. We found that melanin distribution in different models was representative of pathology seen in human tumours: melanin content was high in the extracellular space of SKMel3 tumours, and accumulated primarily in melanophages in M4Beu tumours. Targeted [¹³¹I]ICF01012 radiotherapy had a strong anti-tumoural efficacy in pigmented versus unpigmented tumours, regardless of target distribution and content. This study supports the use of melanin targeting with ¹³¹I-labelled iodoquinoxaline for effective treatment of melanoma.     

Co‐localization of β1,6‐branched Oligosaccharides and Coarse Melanin in Macrophage–Melanoma Fusion Hybrids and Human Melanoma Cells In Vitro

Fusion hybrids between normal macrophages and Cloudman S91 melanoma cells were shown earlier to have increased metastatic potential, along with high expression of β1,6‐N‐acetylglucosaminyltransferase  V and β1,6‐branched oligosaccharides. Curiously, hybrids, but not parental melanoma cells, also produced ‘coarse melanin’– autophagic vesicles with multiple melanosomes. As β1,6‐branched oligosaccharides were known to be associated with metastasis, and coarse melanin had been described in invasive human melanomas, we looked for potential relationships between the two. Using lectin‐ and immunohistochemistry, we analyzed cell lines producing coarse melanin for β1,6‐branched oligosaccharides: gp100/pmel‐17 (a melanosomal structural component) and CD63 (a late endosome/lysosome component associated with melanoma and certain other human cancers). Cell lines used in this study were (i) hybrid 94‐H48, a highly metastatic, macrophage–melanoma experimental fusion hybrid; (ii) 6^neo mouse melanoma cells, the weakly metastatic, parental fusion partner; and (iii) SKmel‐23, a human melanoma cell line derived from a metastasis. Coarse melanin granules were prominent both in hybrids and in SKmel‐23 cells, and co‐localized with stains for β1,6‐branched oligosaccharides, gp100/pmel 17, and CD63. This is the first report of this phenotype being expressed in vitro, although co‐expression of β1,6‐branched oligosaccharides and coarse melanin was recently shown to be a common and pervasive characteristic in archival specimens of human melanomas, and was most prominent in metastases. The results suggest that pathways of melanogenesis in melanoma may differ significantly from those in normal melanocytes. In vitro expression of this phenotype provides new biological systems for more detailed analyses of its genesis and regulation at the molecular genetic level.     

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.     

Histopathologic abundance of pigmentation correlates with disease-specific survival in malignant melanoma but is not independent of current AJCC pT stage

The increasing number of melanoma patients makes it necessary to develop best possible strategies for prognosis assessment in order to recommend appropriate therapy and follow-up. The prognostic significance of tumor cell pigmentation has not been fully elucidated. Hematoxylin and eosin (H&E)-stained sections of 775 melanomas diagnosed between 2012 and 2015 were independently assessed for melanin pigment abundance by two investigators, and the impact on melanoma-specific survival was calculated. Unpigmented melanomas (n = 99) had a melanoma-specific survival of 67.7%, melanomas with moderate pigmentation (n = 384) had a melanoma-specific survival of 85.9%, and strongly pigmented melanomas (n = 292) had a melanoma-specific survival of 91.4% (p < .001). In an analysis of melanoma-specific survival adjusted for pT stage and pigmentation, we found a nonsignificant impact of pigmentation abundance with a hazard ratio of 1.277 (p = .74). The study presented here provides evidence in a German cohort that patients with pigmented melanomas have a more favorable prognosis than those diagnosed with nonpigmented melanomas. Moreover, the abundance of pigmentation already seems to provide a first prognostic estimate. However, it does not appear to provide significant additional value for prognostic assessment according to the AJCC 2017 pT classification.     

Pitfalls in the diagnosis of malignant melanoma

The histological appearance of benign melanocytic naevi and malignant melanomas can be variable, causing in a significant number of cases severe differential diagnostic problems. The early, thin (less than 1 mm) melanomas have to be differentiated from naevi containing dominant junctional or lentiginous component or pagetoid melanocytosis and from some epithelial tumours, while in cases of thick lesion the diagnosis of thick melanoma, Spitz naevus, deep penetrating naevus or cellular blue naevus should be considered for example. The morphology of the so-called atypical Spitz naevus and atypical pigmented spindle cell naevus show overlapping with malignant melanoma and sometimes in these cases the biological behaviour cannot be assessed. The variable appearance of malignant melanoma is illustrated by the fact that different superficial soft tissue tumours with epithelioid and/or spindle cells or with pigment can mimic it. The rare balloon cell and signet ring cell melanoma is a mimicker of primary or metastatic carcinoma and the desmoplastic variant is often misdiagnosed as benign mesenchymal lesion. Lymph node metastasis of melanoma, when the primary tumour is not known, may raise the possibility of interdigitating reticulum cell tumour or anaplastic large cell lymphoma.     

Enhanced Melanogenesis Induced by Tyrosinase Gene-Transfer Increases Boron-Uptake and Killing Effect of Boron Neutron Capture Therapy for Amelanotic Melanoma

Specific and powerful cancer killing effect for melanoma by boron neutron capture therapy (BNCT) using DOPA analogue, ¹⁰B-p-boronophenylalanine (¹⁰B-BPA), has been established, but amelanotic melanoma is insufficiently responsive to ¹⁰B-BPA BNCT in comparison with actively melanin-producing melanoma. Although the accumulation mechanism of ¹⁰B-BPA within melanoma was not established, we have recently obtained findings suggesting that melanin monomers, key intermediates for melanin polymer formation, play a critical role in ¹⁰B-BPA accumulation. In addition, there are some kinds of human amelanotic melanomas, such as MEL2A, in which expression of tyrosinase is repressed or lacking though tyrosinase-related protein (TRP)-l and TRP-2 are well expressed. Thus, by using a similarly tyrosinase-lacking mouse amelanotic melanoma cell line, A1059, we constructed TA1059 cells by transfecting human tyrosinase-cDNA into these cells. TA1059 cells acquired higher DOPA-oxidase and DOPAchrome tautomerase activity as well as eumelanin content at even higher levels than those of B16F10 cells. TA1059 cells showed about 2.5 times higher ^p-boronophenylalanine (BPA) uptake than A1059 cells in culture. In animal experiments, by using these cell lines, tumor growth of TA1059 was significantly suppressed by ¹⁰B-BPA BNCT as compared with A1059. These findings indicate that the induction of active melanin biosynthesis by melanogenic gene-transfer effectively improves the treatment of amelanotic melanoma by BNCT.     

Photodynamic therapy-induced killing is enhanced in depigmented metastatic melanoma cells

The resistance of pigmented human melanomas over their unpigmented counterparts to a number of therapies has suggested that the presence of intracellular melanin plays a role in rendering these cells less susceptible to cell death, probably through the ability of this pigment to act as an intracellular antioxidant, thus neutralizing chemotherapeutic-induced ROS (reactive oxygen species). PDT (photodynamic therapy) was recently suggested as an attractive, adjunctive therapy owing to its cellular specificity and limited side effects. In the present study, we propose that first depigmenting melanomas with a reversible TYR (tyrosinase) inhibitor such as PTU (phenylthiourea) increases their susceptibility to HYP-PDT (hypericin-mediated PDT). Pigmented [UCT Mel-1 (University of Cape Town melanoma cell line 1)] and unpigmented (A375) melanomas were first characterized with respect to their TYR activities and melanin quantities and then treated with a TYR inhibitor for 48 h. Cell viability assays after treatment with 3 μM HYP-PDT showed a significant increase in cell death in depigmented melanomas compared with untreated melanomas that returned to the level of untreated melanoma cells on removing the TYR inhibitor. The present study supports the hypothesis that combining the inhibition of melanogenesis with PDT should be explored as a valid therapeutic target for the management of advanced melanoma.     

Cytomorphology of Metastatic Melanoma—Use of S-100 Protein In the Diagnosis of Amelanotic Melanoma

The cytomorphological features of cells from 52 cases of metastatic melanoma obtained by fine needle aspiration cytodiagnosis were studied. Morphologically, 11, 19 and 22 cases were classified as spindle, epithelial, and mixed cell types of metastatic melanoma respectively. There were 34 melanotic and 18 amelanotic melanomas. Besides melanin, the presence of intranuclear cytoplasmic inclusions, eosinophilic macronucleoli and giant cells were helpful in the diagnosis of a melanoma. Where attempted, staining for S-100 protein was positive in all the 19 cases (eight amelanotic and 11 sparsely pigmented melanomas). In addition eight cases of metastatic tumour where a differential diagnosis of poorly differentiated carcinoma or large cell lymphoma was entertained, were also studied for localization of S-100 protein and all were found to be negative. Electron microscopy was performed in five cases and showed the presence of melanosomes and/or premelanosomes.     

Biomarkers of tumour redox status in response to modulations of glutathione and thioredoxin antioxidant pathways

The ability of certain cancer cells to maintain a highly reduced intracellular environment is correlated with aggressiveness and drug resistance. Since the glutathione (GSH) and thioredoxin (TRX) systems cooperate to a tight regulation of ROS in cell physiology, and to a stimulation of tumour initiation and progression, modulation of the GSH and TRX pathways are emerging as new potential targets in cancer. In vivo methods to assess changes in tumour redox status are critically needed to assess the relevance of redox-targeted agents. The current study assesses in vitro and in vivo biomarkers of tumour redox status in response to treatments targeting the GSH and TRX pathways, by comparing cytosolic and mitochondrial redox nitroxide electron paramagnetic resonance (EPR) probes, and cross-validation with redox dynamic fluorescent measurement. For that purpose, the effect of the GSH modulator buthionine sulfoximine (BSO) and of the TRX reductase inhibitor auranofin were measured in vitro using both cytosolic and mitochondrial EPR and roGFP probes in breast and cervical cancer cells. In vivo, mice bearing breast or cervical cancer xenografts were treated with the GSH or TRX modulators and monitored using the mito-TEMPO spin probe. Our data highlight the importance of using mitochondria-targeted spin probes to assess changes in tumour redox status induced by redox modulators. Further in vivo validation of the mito-tempo spin probe with alternative in vivo methods should be considered, yet the spin probe used in vivo in xenografts demonstrated sensitivity to the redox status modulators.     

Investigation of scavenging activities and distribution of paramagnetic species in Zanthoxylum limonella seeds

We investigated the scavenging activities of methanol-extracted oil and the distribution of paramagnetic species in Zanthoxylum limonella (ZL) seeds using noninvasive 9?GHz electron paramagnetic resonance (EPR) imaging and continuous wave EPR. EPR detected three different stable paramagnetic species that were assigned to stable organic radicals, Mn²⁺, and other paramagnetic metal complexes. Two-dimensional EPR imaging showed that the stable paramagnetic species were located in the pigmented seed region with a strong intensity. Gas chromatography–mass spectrophotometric (GC–MS) analyses were then performed to identify the compound possibly related to the scavenging activity. The DPPH scavenging activities of ZL were slightly higher than those of Piper nigrum and Coriandrum sativum. Based on the results of EPR, GC–MS, and other methods, limonene in ZL is one of the major compounds that can be related to the scavenging activities.     

The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes

Malignant transformation of melanocytes is associated with changes in melanogenesis. Therefore, fluorescence of melanin may be an informative indicator of this process. But the conventionally excited autofluorescence of melanin in skin tissue is ultra-weak and its main part in the visible spectral region is hidden by the much stronger fluorescence from other endogenous fluorophores. Here, using a new mode of stepwise two-photon excitation, melanin-dominated fluorescence spectra of pigmented skin lesions are reported. From these, pure melanin fluorescence spectra of normal pigmented skin, melanocytic nevi and malignant pigmented melanoma were analyzed. They show distinctly different spectral shapes: melanoma gave a characteristic fingerprint with a fluorescence band peaking at 640 nm, independent of the melanoma subtype. The melanin fluorescence spectra peaked at 590 nm for all types of common melanocytic nevi. These differences in the fluorescence spectra are probably based on different contents of eumelanin and pheomelanin. In a series of 167 cases with melanocytic nevi and melanomas, the sensitivity of this new method to diagnose melanoma was 93.5%, the specificity 80.0% and the diagnostic accuracy 82.6%. The two-photon excitation fluorescence method is a new diagnostic tool which may in future supplement conventional dermatohistopathology.     

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.     

Melanin quantification by in vitro and in vivo analysis of near‐infrared fluorescence

Objective measurements of melanin can provide important information for differentiating melanoma from benign pigmented lesions and in assessing pigmentary diseases. Herein, we evaluate near‐infrared (NIR) fluorescence as a possible tool to quantify melanin. Various concentrations of in vitro Sepia melanin in tissue phantoms were measured with NIR fluorescence and diffuse reflectance spectroscopy. Similar optic measurements were conducted in vivo on 161 normal human skin sites. Diffuse reflectance spectroscopy was used to quantify the melanin content via Stamatas–Kollias algorithm. At physiologic concentrations, increasing in vitro melanin concentrations demonstrated higher fluorescence that was linearly correlated (R² = 0.99, p < .001). At higher concentrations, the fluorescence signal plateaued. A linear relationship was also observed with melanin content in human skin (R² = 0.59, p < .001). Comparing the fluorescence and reflectance signals with in vitro and in vivo samples, the estimated melanin concentration in human skin ranged between 0 and 1.25 mg/ml, consistent with previous quantitative studies involving invasive methods.     

Chloroform extract from Moricandia arvensis inhibits growth of B16-F0 melanoma cells and promotes differentiation in vitro

Objectives: Poor therapeutic results have been reported for treatment of malignant melanoma; therefore in this study we have investigated inhibitory capacity of ethyl acetate, chloroform (Chl) and methanol extracts from Moricandia arvensis on mouse melanoma (B16‐F0) and human keratinocyte (HaCaT) cell proliferation. Influence of Chl extract on percentage distribution in cell cycle phases and melanogenesis was also studied. Material and methods: Cell viability was determined at various periods using the MTT assay, and flow cytometry was used to analyse effects of Chl extract on progression through the cell cycle and apoptosis. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475 nm. Results: Chl extract exhibited significant anti‐proliferative activity after incubation with the two types of tumour skin cells. Morphological changes in B16‐F0 cells, accompanied by increase of tyrosinase activity, and of melanin synthesis were observed, which are markers of differentiation of malignant melanoma cells. Furthermore, cell cycle analysis revealed that B16‐F0 cells treated with Chl extract were arrested predominantly in G₁ phase. Conclusion: Chl extract had the ability to reverse malignant melanoma cells from proliferative to differentiated state, thus providing a new perspective in developing novel strategies for prevention and treatment of malignant melanoma, possibly through consumption of the extract in an appropriate cancer prevention diet. Moreover, there is scope for the extract being introduced into cosmetic products as a natural tanning agent.     

A cancer‐causing gene is positively correlated with male aggression in Xiphophorus cortezi

The persistence of seemingly maladaptive genes in organisms challenges evolutionary biological thought. In Xiphophorus fishes, certain melanin patterns form malignant melanomas because of a cancer‐causing gene (Xiphophorus melanoma receptor kinase; Xmrk), which arose several millions years ago from unequal meiotic recombination. Xiphophorus melanomas are male biased and induced by androgens however male behaviour and Xmrk genotype has not been investigated. This study found that male X. cortezi with the spotted caudal (Sc) pattern, from which melanomas originate, displayed increased aggression in mirror image trials. Furthermore, Xmrk males (regardless of Sc phenotype) bit and performed more agonistic displays than Xmrk deficient males. Male aggressive response decreased when males viewed their Sc image as compared with their non‐Sc image. Collectively, these results indicate that Xmrk males experience a competitive advantage over wild‐type males and that intrasexual selection could be an important component in the evolutionary maintenance of this oncogene within Xiphophorus.     

Multiple roles of NF1 in the melanocyte lineage

NF1 is a tumour suppressor gene, germline mutations of which lead to neurofibromatosis type 1 syndrome. Patients develop benign tumours from several types of cells including neural crest‐derived cells. NF1 somatic mutations also occur in 15% of sporadic melanoma, a cancer originating from melanocytes. Evidence now suggests the involvement of NF1 mutations in melanoma resistance to targeted therapies. Although NF1 is ubiquitously expressed, genetic links between NF1 and genes involved in melanocyte biology have been described, implying the lineage‐specific mechanisms. In this review, we summarize and discuss the latest advances related to the roles of NF1 in melanocyte biology and in cutaneous melanoma.     

EPR imaging and HPLC characterization of the pigment-based organic free radical in black soybean seeds

We investigated the location and distribution of paramagnetic species in dry black, brown, and yellow (normal) soybean seeds using electron paramagnetic resonance (EPR), X-band (9?GHz) EPR imaging (EPRI), and HPLC. EPR primarily detected two paramagnetic species in black soybean. These two different radical species were assigned as stable organic radical and Mn^2+?species based on the g values and hyperfine structures. The signal from the stable radical was noted at g?≈?2.00 and was relatively strong and stable. Subsequent noninvasive two-dimensional (2D) EPRI of the radical present in black soybean revealed that the stable radical was primarily located in the pigmented region of the soybean coat, with very few radicals observed in the soybean cotyledon (interior). Pigments extracted from black soybean were analyzed using HPLC. The major compound was found to be cyanidin-3-glucoside. Multi-EPR and HPLC results indicate that the stable radical was only found within the pigmented region of the soybean coat, and it could be cyanidin-3-glucoside or an oxidative decomposition product.     

Ribonucleotide reductase in ascites tumour cells detected by electron paramagnetic resonance spectroscopy

Tyrosine radicals localized in the M2 subunits of ribonucleotide reductase have been detected by electron paramagnetic resonance (EPR) in ordinary ascites tumour cells. The intensity of its doublet EPR spectrum is higher in rapidly proliferating cells. Hydroxyurea, a specific inhibitor of this enzyme, decreases the concentration of the tyrosine radical. Whereas in different ascites tumours the doublet EPR spectrum dominates at g = 2.004, in solid tumours another more intense EPR spectrum from nitrosyl-hemoproteins appears. In conclusion, EPR spectroscopy can be used to monitor the content and variations of active M2 subunits of ribonucleotide reductase in intact ascites tumour cells.