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Background: Remarkable deregulation of several microRNAs (miRNAs) is demonstrated in cutaneous melanoma. hsa-miR-193a-3p is reported to be under-expressed in tissues and in plasma of melanoma patients, but the role of both miR-193a arms in melanoma is not known yet., Methods: After observing the reduced levels of miR-193a arms in plasma exosomes of melanoma patients, the effects of hsa-miR-193a-3p and -5p transfection in cutaneous melanoma cell lines are investigated., Results: In melanoma cell lines A375, 501Mel, and MeWo, the ectopic over-expression of miR-193a arms significantly reduced cell viability as well as the expression of genes involved in proliferation (ERBB2, KRAS, PIK3R3, and MTOR) and apoptosis (MCL1 and NUSAP1). These functional features were accompanied by a significant downregulation of Akt and Erk pathways and a strong increase in the apoptotic process. Since in silico databases revealed TROY, an orphan member of the tumor necrosis receptor family, as a potential direct target of miR-193a-5p, this possibility was investigated using the luciferase assay and excluded by our results., Conclusions: Our results underline a relevant role of miR-193a, both -3p and -5p, as tumor suppressors clarifying the intracellular mechanisms involved and suggesting that their ectopic over-expression could represent a novel treatment for cutaneous melanoma patients.

Introduction : Cutaneous melanoma is the deadliest form of skin cancer, with a dramatic increase in the incidence rate worldwide over the past decade. Early detection has been shown to improve the outcome of melanoma patients. The identification of noninvasive biomarkers able to identify melanoma at an early stage remains an unmet clinical need. Circulating miRNAs (c-miRNAs), small non-coding RNAs, appear as potential ideal candidate biomarkers due to their stability in biological fluids and easy detectability. Moreover, c-miRNAs are reported to be heavily deregulated in cancer patients. Areas covered : This review examines evidence of the specific c-miRNAs or panels of c-miRNAs reported to be useful in discriminating melanoma from benign cutaneous lesions. Expert opinion : Although the interesting reported by published studies, the non-homogeneity of detection and normalization methods prevents the individuation of single c-miRNA or panel of c-miRNAs that are specific for early detection of cutaneous melanoma. In the future, prospective wide and well-designed clinical trials will be needed to validate the diagnostic potential of some of the c-miRNA candidates in clinical practice.

Cutaneous melanoma is a skin cancer with increasing incidence. Identification of novel clinical biomarkers able to detect the stage of disease and suggest prognosis could improve treatment and outcome for melanoma patients. Cell-free microRNAs (cf-miRNAs) are the circulating copies of short non-coding RNAs involved in gene expression regulation. They are released into the interstitial fluid, are detectable in blood and other body fluids and have interesting features of ideal biomarker candidates. They are stable outside the cell, tissue specific, vary along with cancer development and are sensitive to change in the disease course such as progression or therapeutic response. Moreover, they are accessible by non-invasive methods or venipuncture. Some articles have reported different cf-miRNAs with the potential of diagnostic tools for melanoma staging, recurrence and survival prediction. Although some concordance of results is already emerging, differences in analytical methods, normalization strategies and tumour staging still will require further research and standardization prior to clinical usage of cf-miRNA analysis. This article reviews this literature with the aim of contributing to a shared focusing on these new promising tools for melanoma treatment and care., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In this work, hybrid compounds 1 - 4 obtained by conjugation of the non-steroidal anti-inflammatory drug diclofenac, with natural molecules endowed with antioxidant and antiproliferative activity were prepared. The antiproliferative activity of these hybrids was evaluated on immortalized human keratinocyte (HaCaT) cells stimulated with epidermal growth factor (EGF), an actinic keratosis (AK) model, and on human squamous cell carcinoma (SCC) cells (A431). Hybrid 1 presented the best activity in both cell models. Self-assembling surfactant nanomicelles have been chosen as the carrier to drive the hybrid 1 into the skin; the in vitro permeation through and penetration into pig ear skin have been evaluated. Among the nanostructured formulations tested, Nano3Hybrid20 showed a higher tendency of the hybrid 1 to be retained in the skin rather than permeating it, with a desirable topical and non-systemic action. On these bases, hybrid 1 may represent an attractive lead scaffold for the development of new treatments for AK and SCC.

Although the anticancer properties of extra virgin olive oil (EVOO) extracts have been recognized, the role of single compounds in non-melanoma skin cancer is still unknown. The in vitro chemopreventive and anticancer action of EVOO extracts and oil-derived compounds in non-melanoma skin cancer models were evaluated on cutaneous squamous cell carcinoma cells and on immortalized human keratinocytes stimulated with epidermal growth factor. Preparation of EVOO extracts and isolation of single compounds was carried out by chromatographic methods. Antitumor activity was assessed by cell-based assays (cell viability, migration, clonogenicity, and spheroid formation) and apoptosis documented by internucleosomal DNA fragmentation. Finally, inhibition of key oncogenic signaling nodes involved in the progression from actinic keratosis to cutaneous squamous cell carcinoma was studied by western blot. EVOO extracts reduced non-melanoma skin cancer cell viability and migration, prevented colony and spheroid formation, and inhibited proliferation of atypical keratinocytes stimulated with epidermal growth factor. Such a pharmacological activity was promoted by oleocanthal and oleacein through the inhibition of Erk and Akt phosphorylation and the suppression of B-Raf expression, whereas tyrosol and hydroxytyrosol did not have effect. The current study provides in vitro evidence for new potential clinical applications of EVOO extracts and/or single oil-derived compounds in the prevention and treatment of non-melanoma skin cancers., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cutaneous melanoma is the most serious type of skin cancer, so new cytotoxic weapons against novel targets in melanoma are of great interest. Euplotin C (EC), a cytotoxic secondary metabolite of the marine ciliate Euplotes crassus , was evaluated in the present study on human cutaneous melanoma cells to explore its anti-melanoma activity and to gain more insight into its mechanism of action. EC exerted a marked cytotoxic effect against three different human melanoma cell lines (A375, 501Mel and MeWo) with a potency about 30-fold higher than that observed in non-cancer cells (HDFa cells). A pro-apoptotic activity and a decrease in melanoma cell migration by EC were also observed. At the molecular level, the inhibition of the Erk and Akt pathways, which control many aspects of melanoma aggressiveness, was shown. EC cytotoxicity was antagonized by dantrolene, a ryanodine receptor (RyR) antagonist, in a concentration-dependent manner. A role of RyR as a direct target of EC was also suggested by molecular modelling studies. In conclusion, our data provide the first evidence of the anti-melanoma activity of EC, suggesting it may be a promising new scaffold for the development of selective activators of RyR to be used for the treatment of melanoma and other cancer types.

Melanoma is a devastating disease with few therapeutic options in the advanced stage and with the urgent need of reliable biomarkers for early detection. In this context, circulating microRNAs are raising great interest as diagnostic biomarkers. We analyzed the expression profiles of 21 selected microRNAs in plasma samples from melanoma patients and healthy donors to identify potential diagnostic biomarkers. Data analysis was performed using global mean normalization and NormFinder algorithm. Linear regression followed by receiver operating characteristic analyses was carried out to evaluate whether selected plasma miRNAs were able to discriminate between cases and controls. We found five microRNAs that were differently expressed among cases and controls after Bonferroni correction for multiple testing. Specifically, miR-15b-5p, miR-149-3p, and miR-150-5p were up-regulated in plasma of melanoma patients compared with healthy controls, while miR-193a-3p and miR-524-5p were down-regulated. Receiver operating characteristic analyses of these selected microRNAs provided area under the receiver operating characteristic curve values ranging from 0.80 to 0.95. Diagnostic value of microRNAs is improved when considering the combination of miR-149-3p, miR-150-5p, and miR-193a-3p. The triple classifier had a high capacity to discriminate between melanoma patients and healthy controls, making it suitable to be used in early melanoma diagnosis.

Human cutaneous melanoma is an aggressive and chemotherapy-resistant type of cancer. AM251 is a cannabinoid type 1 (CB1) receptor antagonist/inverse agonist with off-target antitumor activity against pancreatic and colon cancer cells. The current study aimed to characterize the in-vitro antimelanoma activity of AM251. The BRAF V600E mutant melanoma cell line, A375, was used as an in-vitro model system. Characterization tools included a cell viability assay, nuclear morphology assessment, gene expression, western blot, flow cytometry with Annexin V-FITC/7-AAD double staining, cell cycle analyses, and measurements of changes in intracellular cAMP and calcium concentrations. AM251 exerted a marked cytotoxic effect against A375 human melanoma cells with potency comparable with that observed for cisplatin without significant changes in the human dermal fibroblasts viability. AM251, at a concentration that approximates the IC50, downregulated genes encoding antiapoptotic proteins (BCL2 and survivin) and increased transcription levels of proapoptotic BAX, induced alteration of Annexin V reactivity, DNA fragmentation, chromatin condensation in the cell nuclei, and G2/M phase arrest.AM251 also induced a 40% increase in the basal cAMP levels, but it did not affect intracellular calcium concentrations. The involvement of GPR55, TRPA1, and COX-2 in the AM251 mechanism of action was excluded. The combination of AM251 with celecoxib produced a synergistic antitumor activity, although the mechanism underlying this effect remains to be elucidated. This study provides the first evidence of a proapoptotic effect and G2/M cell cycle arrest of AM251 on A375 cells. This compound may be a potential prototype for the development of promising diarylpyrazole derivatives to be evaluated in human cutaneous melanoma.

Cannabinoids are implicated in the control of cell proliferation, but little is known about the role of the endocannabinoid system in human malignant melanoma. This study was aimed at characterizing the in vitro antitumor activity of anandamide (AEA) in A375 melanoma cells. The mRNA expression of genes that code for proteins involved in the metabolism and in the mechanism of AEA action was assessed by RT-PCR. Cell viability was tested using WST-1 assay and the apoptotic cell death was determined by measuring caspase 3/7 activities. A375 cells express high levels of fatty acid amide hydrolase (FAAH), cyclooxygenase (COX)-2, cannabinoid receptor 1 (CB1), transient receptor potential cation channel subfamily V member 1 (TRPV1) and G-protein-coupled receptor 55 (GPR55) genes. AEA induced a concentration-dependent cytotoxicity with an IC50 of 5.8 ± 0.7 µM and such an effect was associated to a caspase-dependent apoptotic pathway. AEA cytotoxicity was potentiated by FAAH inhibition (2-fold increase, p<0.05) and mitigated by COX-2 or lipoxygenase (LOX) inhibition (5- and 3-fold decrease, respectively; p<0.01). Blocking CB1 receptors partially decreased AEA cytotoxicity, whereas selective antagonism on the TRPV1 barely affected the mechanism of AEA action. Finally, methyl-β-cyclodextrin, a membrane cholesterol depletory, completely reversed the cytotoxicity induced by the selective GPR55 agonist, O-1602, and AEA. Overall, these findings demonstrate that AEA induces cytotoxicity against human melanoma cells in the micromolar range of concentrations through a complex mechanism, which involves COX-2 and LOX-derived product synthesis and CB1 activation. Lipid raft modulation, probably linked to GPR55 activation, might also have a role., (© 2013 Elsevier B.V. All rights reserved.)