Melanocortin receptor 4 as a new target in melanoma therapy: Anticancer activity of the inhibitor ML00253764 alone and in association with B-raf inhibitor vemurafenib.

The aim of our study is to investigate in vitro and in vivo MC4R as a novel target in melanoma using the selective antagonist ML00253764 (ML) alone and in combination with vemurafenib, a B-rafV600E inhibitor. The human melanoma B-raf mutated A-2058 and WM 266-4 cell lines were used. An MC4R null A-2058 cell line was generated using a CRISPR/Cas9 system. MC4R protein expression was analysed by western blotting, immunohistochemistry, and immunofluorescence. Proliferation and apoptotic assays were performed with ML00253764, whereas the synergism with vemurafenib was evaluated by the combination index (CI) and Loewe methods. ERK1/2 phosphorylation and BCL-XL expression were quantified by western blot. In vivo experiments were performed in Athymic Nude-Foxn1nu male mice, injecting subcutaneously melanoma cells, and treating animals with ML, vemurafenib and their concomitant combination. Comet and cytome assays were performed. Our results show that human melanoma cell lines A-2058 and WM 266-4, and melanoma human tissue, express functional MC4R receptors on their surface. MC4R receptors on melanoma cells can be inhibited by the selective antagonist ML, causing antiproliferative and proapoptotic activity through the inhibition of phosphorylation of ERK1/2 and a reduction of BCL-XL. The concomitant combination of vemurafenib and ML caused a synergistic effect on melanoma cells in vitro and inhibited in vivo tumor growth in a preclinical model, without causing mouse weight loss or genotoxicity. Our original research contributes to the landscape of pharmacological treatments for melanoma, providing MC4R antagonists as drugs that can be added to established therapies.

Inhibiting immunoregulatory amidase NAAA blocks ZIKV maturation in Human Neural Stem Cells.

Recent evidence suggests that lipids play a crucial role in viral infections beyond their traditional functions of supplying envelope and energy, and creating protected niches for viral replication. In the case of Zika virus (ZIKV), it alters host lipids by enhancing lipogenesis and suppressing ß-oxidation to generate viral factories at the endoplasmic reticulum (ER) interface. This discovery prompted us to hypothesize that interference with lipogenesis could serve as a dual antiviral and anti-inflammatory strategy to combat the replication of positive sense single-stranded RNA (ssRNA+) viruses. To test this hypothesis, we examined the impact of inhibiting N-Acylethanolamine acid amidase (NAAA) on ZIKV-infected human Neural Stem Cells. NAAA is responsible for the hydrolysis of palmitoylethanolamide (PEA) in lysosomes and endolysosomes. Inhibition of NAAA results in PEA accumulation, which activates peroxisome proliferator-activated receptor-α (PPAR-α), directing ß-oxidation and preventing inflammation. Our findings indicate that inhibiting NAAA through gene-editing or drugs moderately reduces ZIKV replication by approximately one log10 in Human Neural Stem Cells, while also releasing immature virions that have lost their infectivity. This inhibition impairs furin-mediated prM cleavage, ultimately blocking ZIKV maturation. In summary, our study highlights NAAA as a host target for ZIKV infection.

Targeting SIRT1 Rescues Age- and Obesity-Induced Microvascular Dysfunction in Ex Vivo Human Vessels.

BACKGROUND: Experimental evidence suggests a key role of SIRT1 (silent information regulator 1) in age- and metabolic-related vascular dysfunction. Whether these effects hold true in the human microvasculature is unknown. We aimed to investigate the SIRT1 role in very early stages of age- and obesity-related microvascular dysfunction in humans. METHODS: Ninety-five subjects undergoing elective laparoscopic surgery were recruited and stratified based on their body mass index status (above or below 30 kg/m2) and age (above or below 40 years) in 4 groups: Young Nonobese, Young Obese, Old Nonobese, and Old Obese. We measured small resistance arteries' endothelial function by pressurized micromyography before and after incubation with a SIRT1 agonist (SRT1720) and a mitochondria reactive oxygen species (mtROS) scavenger (MitoTEMPO). We assessed vascular levels of mtROS and nitric oxide availability by confocal microscopy and vascular gene expression of SIRT1 and mitochondrial proteins by qPCR. Chromatin immunoprecipitation assay was employed to investigate SIRT1-dependent epigenetic regulation of mitochondrial proteins. RESULTS: Compared with Young Nonobese, obese and older patients showed lower vascular expression of SIRT1 and antioxidant proteins (FOXO3 [forkhead box protein O3] and SOD2) and higher expression of pro-oxidant and aging mitochondria proteins p66Shc and Arginase II. Old Obese, Young Obese and Old Nonobese groups endothelial dysfunction was rescued by SRT1720. The restoration was comparable to the one obtained with mitoTEMPO. These effects were explained by SIRT1-dependent chromatin changes leading to reduced p66Shc expression and upregulation of proteins involved in mitochondria respiratory chain. CONCLUSIONS: SIRT1 is a novel central modulator of the earliest microvascular damage induced by age and obesity. Through a complex epigenetic control mainly involving p66Shc and Arginase II, it influences mtROS levels, NO availability, and the expression of proteins of the mitochondria respiratory chain. Therapeutic modulation of SIRT1 restores obesity- and age-related endothelial dysfunction. Early targeting of SIRT1 might represent a crucial strategy to prevent age- and obesity-related microvascular dysfunction.

Lipid balance remodelling by human positive-strand RNA viruses and the contribution of lysosomes.

A marked reorganization of internal membranes occurs in the cytoplasm of cells infected by single stranded positive-sense RNA viruses. Most cell compartments change their asset to provide lipids for membrane rearrangement into replication organelles, where to concentrate viral proteins and enzymes while hiding from pathogen pattern recognition molecules. Because the endoplasmic reticulum is a central hub for lipid metabolism, when viruses hijack the organelle to form their replication organelles, a cascade of events change the intracellular environment. This results in a marked increase in lipid consumption, both by lipolysis and lipophagy of lipid droplets. In addition, lipids are used to produce energy for viral replication. At the same time, inflammation is started by signalling lipids, where lysosomal processing plays a relevant role. This review is aimed at providing an overview on what takes place after human class IV viruses have released their genome into the host cell and the consequences on lipid metabolism, including lysosomes.

Palmitoylethanolamide (PEA) Inhibits SARS-CoV-2 Entry by Interacting with S Protein and ACE-2 Receptor.

Lipids play a crucial role in the entry and egress of viruses, regardless of whether they are naked or enveloped. Recent evidence shows that lipid involvement in viral infection goes much further. During replication, many viruses rearrange internal lipid membranes to create niches where they replicate and assemble. Because of the close connection between lipids and inflammation, the derangement of lipid metabolism also results in the production of inflammatory stimuli. Due to its pivotal function in the viral life cycle, lipid metabolism has become an area of intense research to understand how viruses seize lipids and to design antiviral drugs targeting lipid pathways. Palmitoylethanolamide (PEA) is a lipid-derived peroxisome proliferator-activated receptor-α (PPAR-α) agonist that also counteracts SARS-CoV-2 entry and its replication. Our work highlights for the first time the antiviral potency of PEA against SARS-CoV-2, exerting its activity by two different mechanisms. First, its binding to the SARS-CoV-2 S protein causes a drop in viral infection of ~70%. We show that this activity is specific for SARS-CoV-2, as it does not prevent infection by VSV or HSV-2, other enveloped viruses that use different glycoproteins and entry receptors to mediate their entry. Second, we show that in infected Huh-7 cells, treatment with PEA dismantles lipid droplets, preventing the usage of these vesicular bodies by SARS-CoV-2 as a source of energy and protection against innate cellular defenses. This is not surprising since PEA activates PPAR-α, a transcription factor that, once activated, generates a cascade of events that leads to the disruption of fatty acid droplets, thereby bringing about lipid droplet degradation through ß-oxidation. In conclusion, the present work demonstrates a novel mechanism of action for PEA as a direct and indirect antiviral agent against SARS-CoV-2. This evidence reinforces the notion that treatment with this compound might significantly impact the course of COVID-19. Indeed, considering that the protective effects of PEA in COVID-19 are the current objectives of two clinical trials (NCT04619706 and NCT04568876) and given the relative lack of toxicity of PEA in humans, further preclinical and clinical tests will be needed to fully consider PEA as a promising adjuvant therapy in the current COVID-19 pandemic or against emerging RNA viruses that share the same route of replication as coronaviruses.

A low-cost simple test for weekly detection of Mycoplasma hyorhinis and arginini contaminations in cell cultures and viral preparations.

Mollicutes (Mycoplasma and Acholeplasma) are parasitic bacteria that adhere to cellular surfaces, naturally resistant to many antibiotics and extremely small. They are often found as contaminants in cultured cells, where they go unnoticed. They may be present in viral stocks because they are present in supernatants of cells where cultured viruses are released. The best way to keep laboratories free of Mycoplasma is to discard infected cultures, but, as judged by the very common finding of Mycoplasma-contaminated cultures in many laboratories, this is not done as often as it should be. A possible reason is that most procedures recommended take as long as performing a simple experiment and many laboratories delay testing to save money and time. Indeed, many methods exist to detect Mycoplasma infection of cell lines, but they take at least a couple of hours of hands-on work, if not more. Here we describe a procedure to screen viral stocks and tissue cultures for Mycoplasma presence. It relies on isolation of Mycoplasma on ordinary horse blood agar directly from exhausted tissue culture supernatants and does not require experienced personnel or expensive equipment. It only requires minutes of hands-on work, and, for this, it may be useful for weekly screening of cultures. It yields semiquantitative results in roughly 5 days, which is the time that usually passes between one subculture passage of cells in vitro to another. Because of its simplicity, it may be useful for detecting Mycoplasma in viral stocks and for frequent screening of cultures in research laboratories.

Acid ceramidase controls apoptosis and increases autophagy in human melanoma cells treated with doxorubicin.

Acid ceramidase (AC) is a lysosomal hydrolase encoded by the ASAH1 gene, which cleaves ceramides into sphingosine and fatty acid. AC is expressed at high levels in most human melanoma cell lines and may confer resistance against chemotherapeutic agents. One such agent, doxorubicin, was shown to increase ceramide levels in melanoma cells. Ceramides contribute to the regulation of autophagy and apoptosis. Here we investigated the impact of AC ablation via CRISPR-Cas9 gene editing on the response of A375 melanoma cells to doxorubicin. We found that doxorubicin activates the autophagic response in wild-type A375 cells, which effectively resist apoptotic cell death. In striking contrast, doxorubicin fails to stimulate autophagy in A375 AC-null cells, which rapidly undergo apoptosis when exposed to the drug. The present work highlights changes that affect melanoma cells during incubation with doxorubicin, in A375 melanoma cells lacking AC. We found that the remarkable reduction in recovery rate after doxorubicin treatment is strictly associated with the impairment of autophagy, that forces the AC-inhibited cells into apoptotic path.

Ablation of Acid Ceramidase Impairs Autophagy and Mitochondria Activity in Melanoma Cells.

Cutaneous melanoma is often resistant to therapy due to its high plasticity, as well as its ability to metabolise chemotherapeutic drugs. Sphingolipid signalling plays a pivotal role in its progression and metastasis. One of the ways melanoma alters sphingolipid rheostat is via over-expression of lysosomal acid ceramidase (AC), which catalyses the hydrolysis of pro-apoptotic long-chain ceramides into sphingosine and fatty acid. In this report, we examine the role of acid ceramidase in maintaining cellular homeostasis through the regulation of autophagy and mitochondrial activity in melanoma cell lines. We show that under baseline conditions, wild-type melanoma cells had 3-fold higher levels of the autophagy marker, microtubule-associated proteins 1A/1B light chain 3B (LC3 II), compared to AC-null cells. This difference was further magnified after cell starvation. Moreover, we noticed autophagy impairment in A375 AC-null cells, possibly due to local accumulation of non-metabolized ceramides. Nonetheless, we observed that AC-null cells exhibited a significant increase in mitochondrial membrane potential compared to control cells. Consistent with this observation, we found that, after total starvation, ~30% of AC-null cells undergo apoptosis compared to ~6% of wild-type cells. As expected, AC transfection restored viability in A375 AC-null cells. Together, these findings suggest that AC-null melanoma cells change and adapt their metabolism to survive in the absence of AC, although in a way that does not allow them to cope with the stress of nutrient deprivation.

Sphingolipid/Ceramide Pathways and Autophagy in the Onset and Progression of Melanoma: Novel Therapeutic Targets and Opportunities.

Melanoma is a malignant tumor deriving from neoplastic transformation of melanocytes. The incidence of melanoma has increased dramatically over the last 50 years. It accounts for most cases of skin cancer deaths. Early diagnosis leads to remission in 90% of cases of melanoma; conversely, for melanoma at more advanced stages, prognosis becomes more unfavorable also because dvanced melanoma is often resistant to pharmacological and radiological therapies due to genetic plasticity, presence of cancer stem cells that regenerate the tumor, and efficient elimination of drugs. This review illustrates the role of autophagy in tumor progression and resistance to therapy, focusing on molecular targets for future drugs.