Characterization of HLA class I altered phenotypes in a panel of human melanoma cell lines

Background Altered HLA class I cell surface expression is one of the major mechanisms by which tumor cells escape from T lymphocytes. Immunohistochemistry-defined phenotypes of lost HLA class I expression have been described in human solid tumors, nut less information is available on melanoma cell lines. Objectives To describe the frequency and distribution of different types of HLA class I antigen alterations in 91 melanoma cell lines from the European Searchable Tumour Cell and Databank (ESTDAB). Methods The HLA class I expression was assessed by flow cytometry and HLA genotyping. Results We found various types of HLA class I cell surface alterations in about 67% of the melanoma cell lines. These alterations range from total to selective HLA class I loss due to loss of heterozygosity (LOH), haplotype loss, β2-microglobulin gene mutation, and/or total or selective down-regulation of HLA class I molecules. The most frequently observed phenotype is down-regulation of HLA-B locus that was reversible after treatment with IFN -γ. Conclusions In general, HLA class I alterations in the majority of the cells analyzed were of regulatory nature and could be restored by IFN-γ. Analysis of the frequency of distinct HLA class I altered phenotypes in these melanoma cell lines revealed specific differences compared to other types of tumors. Rosa Méndez and Teresa Rodríguez have equally contributed to this work and both should be considered as first authors.     

Triflavin,an Arg-Gly-Asp-containing peptide,inhibits B16-F10 mouse melanoma cell adhesion to matrix proteins via direct binding to tumor cells

Triflavin, an Arg-Gly-Asp (RGD)-containing snake venom peptide, inhibits B16-F10 mouse melanoma cell adhesion to extracellular matrices, e.g., fibronectin, vitronectin, fibrinogen, and collagen type I. In this study, GRGDS inhibits B16-F10 mouse melanoma cell adhesion to immobilized triflavin in a dose-dependent manner. In addition, flow-cytometric analysis and the fluorescence staining method in which FITC-triflavin is utilized as a binding ligand were used. GRGDS inhibits the binding of FITC-triflavin to B16-F10 cells. Additionally, the above results suggest that triflavin directly binds to its receptors expressed on B16-F10 cell surface primarily via its RGD sequence, there-by inhibiting B16-F10 cell adhesion to extracellular matrices.     

Evaluation of cytotoxic properties of organometallic ferrocifens on melanocytes, primary and metastatic melanoma cell lines

Malignant melanoma is one of the most severe forms of skin cancer, and chemotherapeutic agents currently in use are poorly effective in curing the disease. Here we describe the properties of two organometallic ferrocenyl derivatives, ferrocifen (Fc-OH-Tam) and ferrociphenol (Fc-diOH) that show a specific antiproliferative effect on melanoma cells. After a short incubation period, Fc-OH-Tam is highly cytotoxic on melanoma cells but less toxic on melanocytes. Fc-diOH is slightly toxic at a high concentration but no discrepancy is observed between malignant and normal cells. After a long incubation time the latter is highly toxic for malignant cells but not for normal cells while the former was very highly toxic for primary malignant cells and significantly less toxic for normal cells. We also found that oxidative stress is not implicated in the mechanism of cytotoxicity, since both derivatives neither induce reactive oxygen species (ROS) level in melanocytes nor in melanoma cells. Finally, investigation on hair follicle growth revealed that the two organometallic derivatives induced an irreversible ejection of the hair shaft, thus predicting a potential hair loss side effect if used as a chemotherapeutic treatment.     

Fourier-Transform InfraRed (FT-IR) spectroscopy to show alterations in molecular composition of EV subpopulations from melanoma cell lines in different malignancy

BackgroundMelanoma cells release extracellular vesicles (EVs) subpopulations which differ in size, phenotype and molecular content. Melanoma derived EVs play a role in the development and progression of cancer by delivering surface receptors and bioactive (proteins, lipids, nucleic acids) or signaling molecules to target cells.MethodsWe applied Fourier Transform Infrared Spectroscopy (FTIR) to compare infrared spectra of absorption for different subpopulations of EVs originating from normal human melanocytes, primary cutaneous melanoma (WM115) and metastatic cutaneous melanoma (WM266-4).ResultsFTIR results showed that exosome and ectosome populations differ in content of protein and lipid components. We obtained higher lipid to protein ratio for ectosomes in comparison with exosomes what confirms that exosomes are very densely packed with protein cargo. We identified the lowest value of saturated fatty acids/unsaturated fatty acids parameter in the metastatic WM266-4 cell line and ectosomes derived from WM266-4 cell line in comparison with normal melanocytes and the primary WM115 cell line. We identified the alterations in the content of secondary structures of proteins present in EV subpopulations originating from melanocytes and melanoma cells in different malignancy.ConclusionsObtained results revealed differences in the molecular composition of melanoma derived EVs subtypes, including protein secondary structure, and showed progressive structural changes during cancer development.     

Identification of proteins regulated by interferon-alpha in resistant and sensitive malignant melanoma cell lines

Treatment of patients with malignant melanoma with interferon-alpha achieves a response in a small but significant subset of patients. Currently, although much is known about interferon biology, little is known about either the particular mechanisms of interferon-alpha activity that are crucial for response or why only some patients respond to interferon-alpha therapy. Two melanoma cell lines (MeWo and MM418) that are known to differ in their response to the antiproliferative activity of interferon-alpha, have been used as a model system to investigate interferon-alpha action. Using a proteomics approach based on two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, several proteins induced in response to interferon-alpha have been identified. These include a number of gene products previously known to be type I interferon responsive (tryptophanyl tRNA synthetase, leucine aminopeptidase, ubiquitin cross-reactive protein, gelsolin, FUSE binding protein 2 and hPNPase) as well as a number of proteins not previously reported to be induced by type I interferon (cathepsin B, proteasomal activator 28alpha and alpha-SNAP). Although the proteins upregulated by interferon-alpha were common between the cell lines when examined at the level of Western blotting, the disparity in the basal level of cathepsin B was striking, raising the possibility that the higher level in MM418 may contribute to the sensitivity of this cell line to interferon-alpha treatment.     

Establishment in monolayer culture and characterization of four human melanoma cell lines

Four new human melanoma cell lines were established in monolayer culture from xenograft lines originating from different patients. Several distinct characteristics of the source xenograft lines were retained in the cell lines, e.g., number of chromosomes, DNA-index, and cell ultrastructure. Cell volume was generally larger for the cell lines than for the corresponding xenograft lines, but the differences among the lines were similar in vitro and in vivo. The cell lines showed significant differences in growth pattern, i.e., cell motility and degree of intercellular contact. Cell cycle time (T_c) during exponential growth ranged from 15 to 21 h. The differences among the lines in T_c were mainly due to differences in the duration of S. Growth fraction was close to 100% and cell loss was negligible during exponential growth. Plating efficiency was 90–100% in the presence of feeder cells. The four cell lines represent a valuable supplement to the xenograft lines for future studies of the cell biology, pathophysiology, metastatic behavior, and treatment sensitivity of malignant melanoma.     

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles’ heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.     

Radiosensitizing properties of magnetic hyperthermia mediated by superparamagnetic iron oxide nanoparticles (SPIONs) on human cutaneous melanoma cell lines

Melanoma is responsible for the majority of deaths related to skin cancer. Worryingly, prognoses show an increasing number of melanoma cases each year worldwide. Radiotherapy, which is a cornerstone of cancer treatment, has proved to be useful but insufficient in melanoma management due to exceptionally high radioresistance of melanoma cells. This problem could be overcome by superparamagnetic iron oxide nanoparticles (SPIONs) used as heat mediators in magnetic hyperthermia, which not only enhance radiosensitivity, but also enable precise targeting by exploitation of their magnetic properties.     

Durable and broad-spectrum disease protection measure against airborne phytopathogenic fungi by using the detachment action of gelatinolytic bacteria

We had previously obtained collagenolytic/gelatinolytic bacteria, which degrade the fungal extracellular matrix, to establish a novel biological control measure that inhibits germling adhesion of airborne phytopathogenic fungi on the host plant surface. By using barley-Magnaporthe oryzae pathosystem, Chryseobacterium sp. was most effective biocontrol agents as tested. The selected bacteria were evaluated for durable disease protection against M. oryzae on barley leaves by using chloramphenicol-resistant mutants. Chryseobacterium sp. from the soil was less likely to settle on leaf surfaces. Therefore, we tried to manipulate Chryseobacterium sp. to inhabit the leaf’s surface. The gelatin supplementation dramatically improved the settlement of gelatinolytic bacteria Chryseobacterium sp. from the soil, and the disease protection effect lasted for more than 2 weeks on barley. Moreover, exploitation of Chryseobacterium sp. for disease protection was extended against other airborne pathogens, Alternaria alternata Japanese pear pathotype on Japanese pear and Colletotrichum orbiculare on cucumber.     

Identification of beta1 integrin as mediator of melanoma cell adhesion to lumican

Lumican is a small leucine-rich proteoglycan (SLRP) present in the dermal extracellular matrix. Previous data from our laboratory demonstrated that lumican decreases melanoma progression in vivo. Here, we show that melanoma cell migration is decreased by lumican and that this effect is due to an enhanced cell adhesion. The adhesion of A375 human melanoma cells on lumican was dose-dependent and required Mg²⁺ and Mn²⁺ divalent cations. Using a panel of monoclonal antibodies directed against integrin subunits, we showed that A375 cells can bind to recombinant lumican through β1 type integrins. Moreover, the use of rhodocetin, an inhibitor of α2 integrin, suggested that this particular subunit might also be involved in the interaction with lumican. The increased β1 integrin-mediated adhesion of melanoma cells to lumican might explain, at least in part, the anti-invasive effect of this SLRP.     

Single cell adhesion measuring apparatus (SCAMA): application to cancer cell lines of different metastatic potential and voltage-gated Na<Superscript>+</Superscript> channel expression

We have developed a simple yet effective apparatus, based upon negative pressure directed to the tip of a micro-pipette, to measure the adhesiveness of single cells. The “single cell adhesion measuring apparatus” (SCAMA) could differentiate between the adhesion of strongly versus weakly metastatic cancer cells as well as normal cells. Adhesion was quantified as “detachment negative pressure” (DNP) or “DNP relative to cell size” (DNPR) where a noticeable difference in cell size was apparent. Thus, for rat and human prostate and human breast cancer cell lines, adhesiveness (DNPR values) decreased in line with increased metastatic potential. Using the SCAMA, we investigated the effect of tetrodotoxin (TTX), a specific blocker of voltage-gated Na⁺ channels (VGSCs), on the adhesion of rat and human prostate cancer cell lines of markedly different metastatic potential. Following pretreatment with TTX (48 h with 1 μM), the adhesion values for the Mat-LyLu cells increased significantly 4.3-fold; there was no effect on the AT-2 cells. For the strongly metastatic PC-3M cells, TTX treatment caused a significant (∼30%) increase in adhesion. The adhesion of PNT2-C2 (“normal”) cells was not affected by the TTX pretreatment. The TTX-induced increase in the adhesiveness of the strongly metastatic cells was consistent with the functional VGSC expression in these cells and the proposed role of VGSC activity in metastatic cell behaviour. In conclusion, the SCAMA, which can be constructed easily and cheaply, offers a simple and effective method to characterise single-cell adhesion and its modulation.     

Interaction between integrin alpha(5) and fibronectin is required for metastasis of B16F10 melanoma cells

In this study, we report the role of integrin alpha(5) in promoting melanoma metastasis. The alpha(5) expression was remarkably elevated in highly metastatic B16F10 melanoma cells compared to lowly metastatic B16F1 cells, whereas no significant changes were detected in those of integrin alpha(4), alpha(v), and beta(1) subunits. Neutralization of alpha(5) with anti-alpha(5) antibody significantly suppressed the potential of B16F10 cells for pulmonary metastasis in mice and inhibited cell adhesion or spreading to fibronectin in vitro. Furthermore, loss of the interaction between alpha(5) and fibronectin diminished cell survival and induced apoptosis in B16F10 cells. Above results provide clear evidence that integrin alpha(5) is positively correlated with melanoma metastasis and might be an anti-melanoma target.     

Markers of mitochondrial dysfunction during the diclofenac-induced apoptosis in melanoma cell lines

Melanoma is an aggressive cutaneous cancer, whose incidence is growing in recent years, especially in the younger population. The favorable therapy for this neoplasm consists in its early surgical excision; otherwise, in case of late diagnosis, melanoma becomes very refractory to any conventional therapy. Nevertheless, the acute inflammatory response occurring after excision of the primary melanoma can affect the activation and/or regulation of melanoma invasion and metastasis. Nonsteroidal anti-inflammatory drugs (NSAIDs), widely employed in clinical therapy as cyclooxygenase inhibitors, also display a cytotoxic effect on some cancer cell lines; therefore, their possible usage in combination with conventional chemo- and radio-therapies of tumors is being considered. In particular, diclofenac, one of the most common NSAIDs, displays its anti-proliferative effect in many tumor lines, through an alteration of the cellular redox state. In this study, the possible anti-neoplastic potential of diclofenac on the human melanoma cell lines A2058 and SAN was investigated, and a comparison was made with the results obtained from the nonmalignant fibroblast cell line BJ-5ta. Either in A2058 or SAN, the diclofenac treatment caused typical apoptotic morphological changes, as well as an increase of the number of sub-diploid nuclei; conversely, the same treatment on BJ-5ta had only a marginal effect. The observed decrease of Bcl-2/Bax ratio and a parallel increase of caspase-3 activity confirmed the pro-apoptotic role exerted by diclofenac in melanoma cells; furthermore, the drug provoked an increase of the ROS levels, a decrease of mitochondrial superoxide dismutase (SOD2), the cytosolic translocation of both SOD2 and cytochrome c, and an increase of caspase-9 activity. Finally, the cytotoxic effect of diclofenac was amplified, in melanoma cells, by the silencing of SOD2. These data improve the knowledge on the effects of diclofenac and suggest that new anti-neoplastic treatments should be based on the central role of mitochondrion in cancer development; under this concern, the possible involvement of SOD2 as a novel target could be considered.     

S100A9 mediates neutrophil adhesion to fibronectin through activation of beta2 integrins

Neutrophil migration from the blood to inflammatory sites follows a cascade of events, in which adhesion to endothelial cells and extracellular matrix proteins is essential. S100A8, S100A9, and S100A12 are small abundant proteins found in human neutrophil cytosol and presumed to be involved in leukocyte migration. Here we investigated the S100 proteins' activities in neutrophil tissue migration by evaluating their effects on neutrophil adhesion to certain extracellular matrix proteins. S100A9 induced adhesion only to fibronectin and was the only S100 protein that stimulated neutrophil adhesion to this extracellular matrix protein. Experiments with blocking antibodies revealed that neither beta1 nor beta3 integrins were strongly involved in neutrophil adhesion to fibronectin, contrary to what the literature predicted. In contrast, neutrophil adhesion to fibronectin was completely inhibited by anti-beta2 integrins, suggesting that S100A9-induced specific activation of beta2 integrin is essential to neutrophil adhesion.     

The roles of Akt and NOSs in regulation of VLA-4-mediated melanoma cell adhesion to endothelial VCAM-1 after UVB-irradiation

UVB-reduced avidity between M624 melanoma and HUVEC cells is dependent on the interaction of VLA-4 with its endothelial ligand VCAM-1. Our previous studies suggested that a spatial organization of α4 integrin, one of the two subunits of VLA-4, on the melanoma cell surface contributed to the changes in avidity for VCAM-1 upon UVB-irradiation. In this study, we demonstrate that Akt plays an important role in regulation of the expression and surface level of α4 integrin on melanoma cells upon UVB-irradiation. While the cell surface level of α4 integrin is not significantly affected by UVB-irradiation or Akt inhibitor alone, it is dynamically altered after UVB-irradiation when Akt is inhibited. Inhibition of Akt also reverses the reduction of avidity of cells after the irradiation. Our data also shows that UVB reduces the level of Akt. The inhibition of Akt activity correlates with a reduced amount of coupled cNOS and reduced amount of iNOS after UVB-irradiation. However, the effect of NOSs on melanoma cell adhesion appears due to their roles in regulation of apoptosis after UVB-irradiation. Base on these results, we propose that the UVB-induced reduction of avidity of melanoma cells is coordinatively regulated by NOSs and Akt through two differential mechanisms.     

Expression of adhesion molecules and ligands for activating and costimulatory receptors involved in cell-mediated cytotoxicity in a large panel of human melanoma cell lines

Knowledge of the interactions between MHC-unrestricted cytotoxic effector cells and solid tumour cells is essential for introducing more effective NK cell-based immunotherapy protocols into clinical practise. Here, to begin to obtain an overview of the possible universe of molecules that could be involved in the interactions between immune effector cells and melanoma, we analyse the surface expression of adhesion and costimulatory molecules and of ligands for NK-activating receptors on a large panel of cell lines from the “European Searchable Tumour Cell Line and Data Bank” (ESTDAB, http://www.ebi.ac.uk/ipd/estdab/) and discuss their potential role in the immune response against this tumour. We show that most melanoma cell lines express not only adhesion molecules that are likely to favour their interaction with cells of the immune system, but also their interaction with endothelial cells potentially increasing their invasiveness and metastatic capacity. A high percentage of melanoma cell lines also express ligands for the NK-activating receptor NKG2D; whereas, the majority express MICA/B molecules, ULBP expression, however, was rarely found. In addition to these molecules, we also found that CD155 (poliovirus receptor, PVR) is expressed by the majority of melanoma cell lines, whereas CD112 (Nectin-2) expression was rare. These molecules are DNAM-1 ligands, a costimulatory molecule involved in NK cell-mediated cytotoxicity and cytokine production that also mediates costimulatory signals for triggering naïve T cell differentiation. The phenotypical characterisation of adhesion molecules and ligands for receptors involved in cell cytotoxicity on a large series of melanoma cell lines will contribute to the identification of markers useful for the development of new immunotherapy strategies.