WIPI1, BAG1 and PEX3 autophagy-related genes are relevant melanoma markers

ROS and oxidative stress may promote autophagy; on the other hand, autophagy may help reduce oxidative damages. According to the known interplay of ROS, autophagy, and melanoma onset, we hypothesized that autophagy-related genes (ARGs) may represent useful melanoma biomarkers. We therefore analyzed the gene and protein expression of 222 ARGs in human melanoma samples, from 5 independent expression databases (overall 572 patients). Gene expression was first evaluated in the GEO database. Forty-two genes showed extremely high ability to discriminate melanoma from nevi (63 samples) according to ROC (AUC ≥ 0.85) and Mann-Whitney (p < 0.0001) analyses. The 9 genes never related to melanoma before were then in silico validated in the IST online database. BAG1, CHMP2B, PEX3, and WIPI1 confirmed a strong differential gene expression, in 355 samples. A second-round validation performed on the Human Protein Atlas database showed strong differential protein expression for BAG1, PEX3, and WIPI1 in melanoma vs control samples, according to the image analysis of 80 human histological sections. WIPI1 gene expression also showed a significant prognostic value (p < 0.0001) according to 102 melanoma patients' survival data. We finally addressed in Oncomine database whether WIPI1 overexpression is melanoma-specific. Within more than 20 cancer types, the most relevant WIPI1 expression change (p = 0.00002; fold change = 3.1) was observed in melanoma. Molecular/functional relationships of the investigated molecules with melanoma and their molecular/functional network were analyzed via Chilibot software, STRING analysis, and gene ontology enrichment analysis. We conclude that WIPI1 (AUC = 0.99), BAG1 (AUC = 1), and PEX3 (AUC = 0.93) are relevant novel melanoma markers at both gene and protein levels

Ion channel expression in human melanoma samples. in silico identification and experimental validation of molecular targets

Expression of 328 ion channel genes was investigated, by in silico analysis, in 170 human melanoma samples and controls. Ninety-one members of this gene-family (i.e., about 28%) show a significant (p 0.90 and p 90% in most cases). Such five genes (namely, SCNN1A, GJB3, KCNK7, GJB1, KCNN2) are novel potential melanoma markers or molecular targets, never previously related to melanoma. The “druggable genome” analysis was then carried out. Miconazole, an antifungal drug commonly used in clinics, is known to target KCNN2, the best candidate among the five identified genes. Miconazole was then tested in vitro in proliferation assays; it dose-dependently inhibited proliferation up to 90% and potently induced cell-death in A-375 and SKMEL-28 melanoma cells, while it showed no effect in control cells. Moreover, specific silencing of KCNN2 ion channel was achieved by siRNA transfection; under such condition miconazole strongly increases its anti-proliferative effect. In conclusion, the present study identified five ion channels that can potentially serve as sensitive and specific markers in human melanoma specimens and demonstrates that the antifungal drug miconazole, known to target one of the five identified ion channels, exerts strong and specific anti-melanoma effects in vitro

The Nutraceutical Dehydrozingerone and Its Dimer Counteract Inflammation- and Oxidative Stress-Induced Dysfunction of In Vitro

Atherosclerosis is characterized by endothelial dysfunction, mainly induced by inflammation and oxidative stress. Increased reactive oxygen species (ROS) production together with increased adhesion molecules and thrombogenic tissue factor (TF) expression on endothelial cells has a key role in proatherogenic mechanisms. Therefore downmodulation of these molecules could be useful for reducing the severity of inflammation and atherosclerosis progression. Dehydrozingerone (DHZ) is a nutraceutical compound with anti-inflammatory and antioxidant activities. In this study we evaluated the ability of DHZ and its symmetric dimer to modulate hydrogen peroxide- (H2O2-) induced ROS production in human umbilical vein endothelial cells (HUVEC). We also evaluated intercellular adhesion molecule- (ICAM-) 1, vascular cell adhesion molecule- (VCAM-) 1, and TF expression in HUVEC activated by tumor necrosis factor- (TNF-) α. HUVEC pretreatment with DHZ and DHZ dimer reduced H2O2-induced ROS production and inhibited adhesion molecule expression and secretion. Of note, only DHZ dimer was able to reduce TF expression. DHZ effects were in part mediated by the inhibition of the nuclear factor- (NF-) κB activation. Overall, our findings demonstrate that the DHZ dimer exerts a potent anti-inflammatory, antioxidant, and antithrombotic activity on endothelial cells and suggest potential usefulness of this compound to contrast the pathogenic mechanisms involved in atherosclerosis progression

Overexpression of Reelin Prevents the Manifestation of Behavioral Phenotypes Related to Schizophrenia and Bipolar Disorder

Despite the impact of schizophrenia and mood disorders, which in extreme cases can lead to death, recent decades have brought little progress in the development of new treatments. Recent studies have shown that Reelin, an extracellular protein that is critical for neuronal development, is reduced in schizophrenia and bipolar disorder patients. However, data on a causal or protective role of Reelin in psychiatric diseases is scarce. In order to study the direct influence of Reelin's levels on behavior, we subjected two mouse lines, in which Reelin levels are either reduced (Reelin heterozygous mice) or increased (Reelin overexpressing mice), to a battery of behavioral tests: open-field, black–white box, novelty-suppressed-feeding, forced-swim-test, chronic corticosterone treatment followed by forced-swim-test, cocaine sensitization and pre-pulse inhibition (PPI) deficits induced by N-methyl--aspartate (NMDA) antagonists. These tests were designed to model some aspects of psychiatric disorders such as schizophrenia, mood, and anxiety disorders. We found no differences between Reeler heterozygous mice and their wild-type littermates. However, Reelin overexpression in the mouse forebrain reduced the time spent floating in the forced-swim-test in mice subjected to chronic corticosterone treatment, reduced behavioral sensitization to cocaine, and reduced PPI deficits induced by a NMDA antagonist. In addition, we demonstrate that while stress increased NMDA NR2B-mediated synaptic transmission, known to be implicated in depression, Reelin overexpression significantly reduced it. Together, these results point to the Reelin signaling pathway as a relevant drug target for the treatment of a range of psychiatric disorders

The Role of p16INK4a Pathway in Human Epidermal Stem Cell Self-Renewal, Aging and Cancer

The epidermis is a self-renewing tissue. The balance between proliferation and differentiation processes is tightly regulated to ensure the maintenance of the stem cell (SC) population in the epidermis during life. Aging and cancer may be considered related endpoints of accumulating damages within epidermal self-renewing compartment. p16INK4a is a potent inhibitor of the G1/S-phase transition of the cell cycle. p16INK4a governs the processes of SC self-renewal in several tissues and its deregulation may result in aging or tumor development. Keratinocytes are equipped with several epigenetic enzymes and transcription factors that shape the gene expression signatures of different epidermal layers and allow dynamic and coordinated expression changes to finely balance keratinocyte self-renewal and differentiation. These factors converge their activity in the basal layer to repress p16INK4a expression, protecting cells from senescence, and preserving epidermal homeostasis and regeneration. Several stress stimuli may activate p16INK4a expression that orchestrates cell cycle exit and senescence response. In the present review, we discuss the role of p16INK4a regulators in human epidermal SC self-renewal, aging and cancer

Letter to the Editor: “Ion Channels in Brain Metastasis”—Ion Channels in Cancer Set up and Metastatic Progression

The review by Klumpp, L. et al. entitled Ion Channels in Brain Metastasis [1] discusses the role of ion channels in breast cancer, lung cancer and melanoma in metastatic tropism to the brain [...

Resveratrol Counteracts Inflammation in Human M1 and M2 Macrophages upon Challenge with 7-Oxo-Cholesterol: Potential Therapeutic Implications in Atherosclerosis

Macrophages consist of two main subsets: the proinflammatory M1 subset and the anti-inflammatory M2 one. 7-oxo-cholesterol, the most abundant cholesterol autoxidation product within atherosclerotic plaque, is able to skew the M1/M2 balance towards a proinflammatory profile. In the present study, we explored the ability of the polyphenolic compound resveratrol to counteract the 7-oxo-cholesterol-triggered proinflammatory signaling in macrophages. Resveratrol-pretreated human monocyte-derived M1 and M2 macrophages were challenged with 7-oxo-cholesterol and analyzed for phenotype and endocytic ability by flow cytometry, for metalloproteinase- (MMP-) 2 and MMP-9 by gelatin zymography, and for cytokine, chemokine, and growth factor secretome by a multiplex immunoassay. We also investigated the NF-κB signaling pathway. In the M1 subset, resveratrol prevented the downregulation of CD16 and the upregulation of MMP-2 in response to 7-oxo-cholesterol, whereas in M2 macrophages it prevented the upregulation of CD14, MMP-2, and MMP-9 and the downregulation of endocytosis. Resveratrol prevented the upregulation of several proinflammatory and proangiogenic molecules in both subsets. We identified modulation of NF-κB as a potential mechanism implicated in 7-oxo-cholesterol and resveratrol effects. Our results strengthen previous findings on the immunomodulatory ability of resveratrol and highlight its role as potential therapeutic or preventive compound, to counteract the proatherogenic oxysterol signaling within atherosclerotic plaque

Identification of Dihydrolipoamide Dehydrogenase as Potential Target of Vemurafenib-Resistant Melanoma Cells

Background: Despite recent improvements in therapy, the five-year survival rate for patients with advanced melanoma is poor, mainly due to the development of drug resistance. The aim of the present study was to investigate the mechanisms underlying this phenomenon, applying proteomics and structural approaches to models of melanoma cells. Methods: Sublines from two human (A375 and SK-MEL-28) cells with acquired vemurafenib resistance were established, and their proteomic profiles when exposed to denaturation were identified through LC-MS/MS analysis. The pathways derived from bioinformatics analyses were validated by in silico and functional studies. Results: The proteomic profiles of resistant melanoma cells were compared to parental counterparts by taking into account protein folding/unfolding behaviors. Several proteins were found to be involved, with dihydrolipoamide dehydrogenase (DLD) being the only one similarly affected by denaturation in all resistant cell sublines compared to parental ones. DLD expression was observed to be increased in resistant cells by Western blot analysis. Protein modeling analyses of DLD&rsquo;s catalytic site coupled to in vitro assays with CPI-613, a specific DLD inhibitor, highlighted the role of DLD enzymatic functions in the molecular mechanisms of BRAFi resistance. Conclusions: Our proteomic and structural investigations on resistant sublines indicate that DLD may represent a novel and potent target for overcoming vemurafenib resistance in melanoma cells