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Marine compounds represent a varied source of new drugs with potential anticancer effects. Among these, sponges, including those belonging to the Irciniidae family, have been demonstrated to exert cytotoxic effects on different human cancer cells. Here, we investigated, for the first time, the therapeutic effect of an extract (referred as iSP) from the sponge, Ircinia ramosa (Porifera, Dictyoceratida, and Irciniidae), on A375 human melanoma cells. We found that iSP impaired A375 melanoma cells proliferation, induced cell death through caspase-dependent apoptosis and arrested cells in the G1 phase of the cell cycle, as demonstrated via both flow cytometry and qPCR analysis. The proapoptotic effect of iSP is associated with increased ROS production and mitochondrial modulation, as observed by using DCF-DHA and mitochondrial probes. In addition, we performed wound healing, invasion and clonogenic assays and found that iSP was able to restrain A375 migration, invasion and clonogenicity. Importantly, we observed that an iSP treatment modulated the expression of the EMT-associated epithelial markers, E-CAD and N-CAD, unveiling the mechanism underlying the effect of iSP in modulating A375 migration and invasion. Collectively, this study provides the first evidence to support the role of Ircinia ramosa sponge extracts as a potential therapeutic resource for the treatment of human melanoma.

Innate lymphoid cells (ILC) are a family of immune cells that are emerging as potent orchestrators of immune responses. In cancer, ILCs display both pro- and antitumorigenic functions depending on the nature of the tumor and the involved ILC subset. Little is known about the ILC–tumor cross-talk in human melanoma. Here, we showed that ILC1s were enriched but functionally impaired in cytokine secretion in both peripheral blood mononuclear cells and tumor-infiltrated lymph nodes of melanoma patients. These findings were confirmed in vivo in murine cutaneous melanoma. Multiple immunosuppressive mechanisms are described in the melanoma microenvironment. Among others, adenosine and kynurenines were shown to suppress antitumor immune responses. By exposing ILCs to adenosine and kynurenines, we observed a similar shift toward the ILC1 subset distribution and impairment in proinflammatory cytokine production to that of patient samples studied ex vivo. Thus, we hypothesized that the immunosuppressive microenvironment of malignant melanoma might shape ILC subpopulations. Hence, we provide a rationale for the use of drugs targeting adenosine and kynurenine pathways in melanoma patients.

Supplementary figures and legends

Organic nitrates are widely used, but their chronic efficacy is blunted due to the development of tolerance. The properties of new tolerance free organic nitrates were studied. Their lipophilicity profile and passive diffusion across polydimethylsiloxane membrane and pig ear-skin, and their efficacy in tissue regeneration using HaCaT keratinocytes were evaluated. The permeation results show that these nitrates have a suitable profile for NO topical administration on the skin. Furthermore, the derivatives with higher NO release exerted a pro-healing effect on HaCaT cells. This new class of organic nitrates might be a promising strategy for the chronic treatment of skin pathologies.

Ethnopharmacological relevance: Chamomile (M. chamomilla L.) is an herbaceous plant from family Astereaceae, that has a long history of use in traditional medicine. It has been used as herbal remedies for thousands of years to treat several diseases, including infections, neuropsychiatric, respiratory, gastrointestinal, and liver disorders. Chronic inflammation is involved in the pathogenesis of most infectious and non-infectious diseases and macrophages are considered the major cellular players that drive disease initiation and maintenance. Aim of the study: The aim of this study was to evaluate the variation in the chemical profile of the essential oil of M. chamomilla plants collected in three experimental field sites in the Molise region. Additionally, we evaluated the pharmacological mechanism behind the anti-inflammatory effect of M. chamomilla essential oils. Material and methods: Three essential oils (called GC1, GC2 and GC3) were extracted from aerial parts of M. chamomilla by hydrodistillation and chemical composition was analyzed by gas chromatography-mass spectrometry (GC-MS). The essential oils were tested for their ability to modulate pro-inflammatory murine macrophages and human peripheral blood mononuclear cells (PBMCs) functions. Results: The chemical analysis of the samples revealed the presence of a high content of the oxygenated sesquiterpenes that represented more than the half of the entire oils. GC1, GC2 and GC3 essential oils significantly attenuated LPS/IFN-γ-induced inflammation by reducing M1 polarization. In details, they showed significant anti-inflammatory property by inhibiting NO, TNF-α and IL-6 production. These effects were correlated to a suppression of LPS-mediated p65 activation, the critical transactivation subunit for NF-κB transcription factor. Oxidative stress may trigger macrophages activation and elicit strong immune responses. Our study demonstrated that GC1, GC2 and GC3 were highly effective at increasing GCL and HMOX-1 anti-oxidant enzymes expression leading to the rapid scavenging of ROS. The antioxidant activity of these oils was explained throughout the activation of NRF2 signaling pathway. Next, we demonstrated that essential oils were able to reduce CD4+ T cell activation which are also involved in inflammatory processes. Conclusions: Our data describe for the first time that chamomile essential oils exerted their anti-inflammatory and antioxidant activity by modulating macrophages and CD4+ T cells-mediate immune response.

Olive tree leaves are an abundant source of bioactive compounds with several beneficial effects for human health, including a protective role against many types of cancer. In this study, we investigated the effect of an extract, obtained from olive tree (Olea europaea L.) leaves (OLE), on proliferation, invasion, and epithelial to mesenchymal transition (EMT) on metastatic melanoma, the highly aggressive form of skin cancer and the deadliest diseases. Our results demonstrated that OLE inhibited melanoma cells proliferation through cell cycle arrest and induction of apoptotic cell death. Moreover, OLE suppressed the migration, invasion, and colonies formation of human melanoma cells. Similar to our in vitro findings, we demonstrated that the oral administration of OLE inhibited cutaneous tumor growth and lung metastasis formation in vivo by modulating the expression of EMT related factors. In addition, the anti-proliferative and anti-invasive effects of OLE against melanoma were also related to a simultaneous targeting of mitogen-activated protein kinase and PI3K pathways, both in vitro and in vivo. In conclusion, our findings suggest that OLE has the potential to inhibit the metastatic spread of melanoma cells thanks to its multifaceted mechanistic effects, and may represent a new add-on therapy for the management of metastatic melanoma.

Innate lymphoid cells (ILC) are a family of immune cells that are emerging as potent orchestrators of immune responses. In cancer, ILCs display both pro- and antitumorigenic functions depending on the nature of the tumor and the involved ILC subset. Little is known about the ILC–tumor cross-talk in human melanoma. Here, we showed that ILC1s were enriched but functionally impaired in cytokine secretion in both peripheral blood mononuclear cells and tumor-infiltrated lymph nodes of melanoma patients. These findings were confirmed in vivo in murine cutaneous melanoma. Multiple immunosuppressive mechanisms are described in the melanoma microenvironment. Among others, adenosine and kynurenines were shown to suppress antitumor immune responses. By exposing ILCs to adenosine and kynurenines, we observed a similar shift toward the ILC1 subset distribution and impairment in proinflammatory cytokine production to that of patient samples studied ex vivo. Thus, we hypothesized that the immunosuppressive microenvironment of malignant melanoma might shape ILC subpopulations. Hence, we provide a rationale for the use of drugs targeting adenosine and kynurenine pathways in melanoma patients.

Cancer immunotherapy has led to impressive advances in cancer treatment. Unfortunately, in a high percentage of patients is difficult to consistently restore immune responses to eradicate established tumors. It is well accepted that adaptive immune cells, such as B lymphocytes, CD4+helper T lymphocytes, and CD8+cytotoxic T-lymphocytes (CTLs), are the most effective cells able to eliminate tumors. However, it has been recently reported that innate immune cells, including natural killer cells (NK), dendritic cells (DC), macrophages, myeloid-derived suppressor cells (MDSCs), and innate lymphoid cells (ILCs), represent important contributors to modulating the tumor microenvironment and shaping the adaptive tumor response. In fact, their role as a bridge to adaptive immunity, make them an attractive therapeutic target for cancer treatment. Here, we provide a comprehensive overview of the pleiotropic role of tissue-resident innate immune cells in different tumor contexts. In addition, we discuss how current and future therapeutic approaches targeting innate immune cells sustain the adaptive immune system in order to improve the efficacy of current tumor immunotherapies.

Malignant melanoma is the deadliest skin cancer, due to its propensity to metastasize. MAPKs and NF-κB pathways are constitutively activated in melanoma and promote cell proliferation, cell invasion, metastasis formation, and resistance to therapeutic regimens. Thus, they represent potential targets for melanoma prevention and treatment. Phytochemicals are gaining considerable attention for the management of melanoma because of their several cellular and molecular targets. A screening of a small library of sesquiterpenes lactones selected cynaropicrin, isolated from the aerial parts of Centaurea drabifolia subsp. detonsa, for its potential anticancer effect against melanoma cells. Treatment of human melanoma cells A375 with cynaropicrin resulted in inhibition of cell proliferation and induction of caspase-3-dependent apoptosis. Furthermore, cynaropicrin reduced several cellular malignant features such migration, invasion, and colonies formation through the inhibition of ERK1/2 and NF-κB activity. Cynaropicrin was able to reduce intracellular reactive oxygen species generation, which are involved in all the stages of carcinogenesis. Indeed, cynaropicrin increased the expression of several antioxidant genes, such as glutamate-cysteine ligase and heme oxygenase-1, by promoting the activation of the transcription factor Nrf-2. In conclusion, our results individuate cynaropicrin as a potential adjuvant chemotherapeutic agent for melanoma by targeting several protumorigenic signaling pathways.

CRISPR/Cas9 has become a powerful method to engineer genomes and to activate or to repress genes expression. As such, in cancer research CRISPR/Cas9 technology represents an efficient tool to dissect mechanisms of tumorigenesis and to discover novel targets for drug development. Here, we employed the CRISPR/Cas9 technology for studying the role of prostaglandin-endoperoxide synthase 2 (PTGS2) in melanoma development and progression. Melanoma is the most aggressive form of skin cancer with a median survival of less than 1 year. Although oncogene-targeted drugs and immune checkpoint inhibitors have demonstrated a significant success in improving overall survival in patients, related toxicity and emerging resistance are ongoing challenges. Gene therapy appears to be an appealing option to enhance the efficacy of currently available melanoma therapeutics leading to better patient prognosis. Several gene therapy targets have been identified and have proven to be effective against melanoma cells. Particularly, PTGS2 is frequently expressed in malignant melanomas and its expression significantly correlates with poor survival in patients. In this study we investigated on the effect of ptgs2 knockdown in B16F10 murine melanoma cells. Our results show that reduced expression of ptgs2 in melanoma cells: i) inhibits cell proliferation, migration, and invasiveness; ii) modulates immune response by impairing myeloid derived suppressor cell differentiation; iii) reduces tumor development and metastasis in vivo. Collectively, these findings indicate that ptgs2 could represent an ideal gene to be targeted to improve success rates in the development of new and highly selective drugs for melanoma treatment.

Currently, nutraceuticals do not have a specific definition distinct from those of other food-derived categories, such as food supplements, herbal products, pre- and probiotics, functional foods, and fortified foods. Many studies have led to an understanding of the potential mechanisms of action of pharmaceutically active components contained in food that may improve health and reduce the risk of pathological conditions while enhancing overall well-being. Nevertheless, there is a lack of clear information and, often, the claimed health benefits may not be properly substantiated by safety and efficacy information or in vitro and in vivo data, which can induce false expectations and miss the target for a product to be effective, as claimed. An officially shared and accepted definition of nutraceuticals is still missing, as nutraceuticals are mostly referred to as pharma-foods, a powerful toolbox to be used beyond the diet but before the drugs to prevent and treat pathological conditions, such as in subjects who may not yet be eligible for conventional pharmaceutical therapy. Hence, it is of utmost importance to have a proper and unequivocal definition of nutraceuticals and shared regulations. It also seems wise to assess the safety, mechanism of action and efficacy of nutraceuticals with clinical data. A growing demand exists for nutraceuticals, which seem to reside in the grey area between pharmaceuticals and food. Nonetheless, given specific legislation from different countries, nutraceuticals are experiencing challenges with safety and health claim substantiation.

Background: A strong, reciprocal crosstalk between inflammation and melanoma has rigorously been demonstrated in recent years, showing how crucial is a pro-inflammatory microenvironment to drive therapy resistance and metastasis. Purpose: We investigated on the effects of Indicaxanthin, a novel, anti-inflammatory and bioavailable phytochemical from Opuntia Ficus Indica fruits, against human melanoma both in vitro and in vivo. Study Design and Methods: The effects of indicaxanthin were evaluated against the proliferation of A375 human melanoma cell line and in a mice model of cutaneous melanoma. Cell proliferation was assessed by MTT assay, apoptosis by Annexin V-Fluorescein Isothiocyanate/Propidium Iodide staining, protein expression by western blotting, melanoma lesions were subcutaneously injected in mice with B16/F10 cells, chemokine release was quantified by ELISA. Results: Data herein presented demonstrate that indicaxanthin effectively inhibits the proliferation of the highly metastatic and invasive A375 cells as shown by growth inhibition, apoptosis induction and cell invasiveness reduction. More interestingly, in vitro data were paralleled by those in vivo showing that indicaxanthin significantly reduced tumor development when orally administered to mice. The results of our study also clarify the molecular mechanisms underlying the antiproliferative effect of indicaxanthin, individuating the inhibition of NF-κB pathway as predominant. Conclusion: In conclusion, we demonstrated that indicaxanthin represents a novel phytochemical able to significantly inhibit human melanoma cell proliferation in vitro and to impair tumor progression in vivo. When considering the resistance of melanoma to the current therapeutical approach and the very limited number of phytochemicals able to partially counteract it, our findings may be of interest to explore indicaxanthin potential in further and more complex melanoma studies in combo therapy, i.e. where different check points of melanoma development are targeted.

Chemical analysis of the dichloromethane fraction obtained from aerial parts of the Northern African endemic plant Daucus virgatus led to the isolation of three previously undescribed sesquiterpenoids, namely the daucane vaginatin B, a eudesmane and the elemane elemavirgolide, along with five known metabolites. The structures of these compounds were determined by a detailed MS and NMR analysis and they were evaluated for antiproliferative activity against three human cell lines, A375 (melanoma), MCF-7 (breast adenocarcinoma), and HACAT (keratinocyte). The phytoalexin 6-methoxymellein revealed a previously unreported antiproliferative activity, while the eudesmane and the elemane derivatives exhibited a selective activity (SI = 11.1 and 3.3, respectively) against melanoma tumor cell lines.

Of the numerous gaseous substances that can act as signaling molecules, the best characterized are nitric oxide, carbon monoxide and hydrogen sulfide. Contributions of each of these low molecular weight substances, alone or in combination, to maintenance of gastrointestinal mucosal integrity have been established. There is considerable overlap in the actions of these gases in modulating mucosal defense and responses to injury, and in some instances they act in a cooperative manner. Each also play important roles in regulating inflammatory and repair processes throughout the gastrointestinal tract. In recent years, significant progress has been made in the development of novel anti-inflammatory and cytoprotective drugs that exploit the beneficial activities of one or more of these gaseous mediators.

Inflammation plays a key role in the pathogenesis of several chronic diseases. The urokinase plasminogen activator receptor (uPAR) exerts a plethora of functions in both physiological and pathological processes, including inflammation. In this study, we evaluated the anti-inflammatory effect of a novel peptide ligand of uPAR, UPARANT, in different animal models of inflammation. Rats and mice were divided in different groups (n = 5) for single or repeated administration of vehicle (9% DMSO in 0.9% NaCl), UPARANT (6, 12 and 24 mg/kg) or dexamethasone (2 mg/kg). Animals were subjected to carrageenan-induced paw oedema or zymosan-induced peritonitis. UPARANT effects were tested on: (1) the carrageenan-induced paw oedema volume, (2) the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and the nitrite/nitrate (NOx) levels in the paw exudates, (3) cells recruitment into the peritoneal cavity after zymosan injection and (4) NOx levels in the peritoneal lavage. UPARANT (12 and 24 mg/kg) reduced inflammation in both experimental paradigms. Analysis of pro-inflammatory enzymes revealed that administration of UPARANT reduced iNOS, COX2 and NO over-production. Our study provides a solid evidence that UPARANT reduces the severity of inflammation in diverse animal models, thus representing a novel anti-inflammatory drug with potential advantages with respect to the typical steroidal agents.

BACKGROUND AND PURPOSE: Myeloid‐derived suppressor cells (MDSCs) represent a major obstacle to cancer treatment, as they negatively regulate anti‐tumour immunity through the suppression of tumour‐specific T lymphocytes. Thus, the efficacy of immunotherapies may be improved by targeting MDSCs. In this study, we assessed the ability of hydrogen sulfide (H(2)S), a gasotransmitter whose anti‐cancer effects are well known, to inhibit the accumulation and immunosuppressive functions of MDSCs in melanoma. EXPERIMENTAL APPROACH: Effects of H(2)S on the host immune response to cancer were evaluated using an in vivo syngeneic model of murine melanoma. B16F10‐melanoma‐bearing mice were treated with the H(2)S donor, diallyl trisulfide (DATS) and analysed for content of MDSCs, dendritic cells (DCs) and T cells. Effects of H(2)S on expression of immunosuppressive genes in MDSCs and on T cell proliferation were evaluated. KEY RESULTS: In melanoma‐bearing mice, DATS inhibited tumour growth, and this effect was associated with a reduction in the frequency of MDSCs in the spleen, in the blood as well as in the tumour micro‐environment. In addition, we found that CD8(+) T cells and DCs were increased. Furthermore, DATS reduced the immuno‐suppressive activity of MDSCs, restoring T cell proliferation. CONCLUSIONS AND IMPLICATIONS: The H(2)S donor compound, DATS, inhibited the expansion and the suppressive functions of MDSCs, suggesting a novel role for H(2)S as a modulator of MDSCs in cancer. Therefore, H(2)S donors may provide a novel approach for enhancing the efficacy of melanoma immunotherapy. LINKED ARTICLES: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc

Olive tree (Olea europaea L.) leaves are an abundant source of bioactive compounds with several beneficial effects for human health. Recently, the effect of olive leaf extract in obesity has been studied. However, the molecular mechanism in preventing obesity-related inflammation has not been elucidated. Obesity is a state of chronic low-grade inflammation and is associated with an increase of pro-inflammatory M1 macrophages infiltration in the adipose tissue. In the current study, we explored Olea europaea L. leaf extract (OLE) anti-inflammatory activity using an in vitro model of obesity-induced inflammation obtained by stimulating murine macrophages RAW 264.7 with high dose of the free fatty acid palmitate. We found that OLE significantly suppressed the induction of pro-inflammatory mediators, tumor necrosis factor (TNF)-&alpha, interleukin (IL)-6, IL-1&beta, nitric oxide (NO), prostaglandin E2 (PGE2) and reactive oxygen species (ROS), while it enhanced the anti-inflammatory cytokine, IL-10. Moreover, we demonstrated that OLE reduced the oxidative stress induced by palmitate in macrophages by regulating the NF-E2-related factor 2 (NRF2)&minus, Kelch-like ECH-associated protein 1 (KEAP1) pathway. Finally, we showed that OLE promoted the shift of M1 macrophage toward less inflammatory M2-cells via the modulation of the associated NF-&kappa, B and proliferator-activated receptor gamma (PPAR&gamma, ) signaling pathways. Thereby, our findings shed light on the potential therapeutic feature of OLE in recovering obesity-associated inflammation via regulating M1/M2 status.

The beneficial effects of H2S-release and of COXs-inhibition have been exploited in the design of novel anti-inflammatory drugs, the H2S-releasing non-steroidal anti-inflammatory drugs (H2S-NSAIDs), showing promising potential for chemoprevention in cancers. Here, we evaluated the efficacy of a new H2S-releasing derivative of naproxen, named naproxen-4-hydroxybenzodithioate (naproxen-HBTA), in reducing metastatic melanoma features, both in vitro and in vivo. The novel H2S donor has been prepared following a synthetic scheme that provided high yields and purity. In particular, we investigated the effect of naproxen-HBTA in vitro on several metastatic features of human melanoma cells such as proliferation, migration, invasion, and colonies formation and in vivo in a model of cutaneous melanoma. Cell culture studies demonstrated that naproxen-HBTA induced caspase 3-mediated apoptosis and inhibited motility, invasiveness, and focus formation. Finally, daily oral treatment with naproxen-HBTA significantly suppressed melanoma growth and progression in mice. In conclusion, by using this dual approach we propose that the COX-2 and H2S pathways could be regarded as novel therapeutic targets/tools to generate new treatment options based on "combination therapy" for melanoma.

Malignant melanoma is one of the most leading form of skin cancer associated with a low patient survival rate. Increasing evidence revealed that microRNAs (miRNAs) play a crucial role in the occurrence and development of several form of cancer including melanoma. In this study, we aimed at investigating the expression and role of miR-143-3p in human malignant melanoma. Our results showed that the expression of miR-143-3p was lower in human melanoma cells, as well as human biopsy specimens, when compared to normal human melanocytes. Ectopic expression of miR-143-3p in human melanoma cells inhibited proliferation, migration, invasion and promoted apoptosis acting through a molecular mechanism that, at least in part, is dependent on inhibition of cyclooxygenase-2 (COX-2) gene. Collectively, these results demonstrate that miR-143-3p could represent at the same time, a new early diagnostic marker and therapeutic target acting as tumor suppressor in melanoma cancer.

Chronic inflammation contributes to tumor initiation in colitis-associated colorectal cancer (CRC). Indeed, inflammatory bowel disease (IBD) patients show an increased risk of developing CRC. Cancer immune evasion is a major issue in CRC and preclinical and clinical evidence has defined a critical role for myeloid-derived suppressor cells (MDSCs) that contribute to tumor growth and progression by suppressing T-cells and modulating innate immune responses. MDSCs comprise a heterogeneous population of immature myeloid cells that can be distinct in two subtypes: CD11b+Ly6G+Ly6Clow with granulocytic phenotype (G-MDSCs) and CD11b+Ly6G-Ly6Chigh with monocytic phenotype (M-MDSCs). Hydrogen sulfide (H2S) is an endogenous gaseous signaling molecule that regulates various physiological and pathophysiological functions. In particular, several studies support its anti-inflammatory activity in experimental colitis and ulcer. However, the role of the H2S pathway in innate immune-mediated IBD has not yet been elucidated. To better define a possible link between MDSCs and H2S pathway in colitis-associated CRC development, we used an innate immune-mediated IBD model induced by infection with the bacterium Helicobacter hepaticus (Hh), closely resembling human IBD. Here, we demonstrated an involvement of MDSCs in colitis development. A significant time-dependent increase of both G-MDSCs and M-MDSCs was observed in the colon and in the spleen of Hh-infected mice. Following, we observed that chronic oral administration of the H2S donor DATS reduced colon inflammation by limiting the recruitment of G-MDSCs in the colon of Hh-infected mice. Thus, we identify the metabolic pathway l-cysteine/H2S as a possible new player in the immunosuppressive mechanism responsible for the MDSCs-promoted colitis-associated cancer development.

Nowadays, the increasing burden of cancer has renewed the interest in plants as source of potential anticancer drugs. In fact, one of the major problems with cancer chemotherapy is the extreme toxicity of the traditional drugs. Thus, the research for new biologically active molecules against cancer is targeted on plants and their derived natural products. Plants used in traditional medicine have proved effective and safe in the treatment and management of cancers, and almost 80% of the world’s population use anticancer agents of natural origin. Thus, the aim of the present chapter is to highlight the plant-derived natural products and their analogues established as anticancer agents in the traditional and current medicinal practices, their toxicological effects, and clinical exercises along with the new technologies applied for delivering natural anticancer drugs.

Currently, nutraceuticals do not have a specific definition distinct from those of other food‐derived categories, such as food supplements, herbal products, pre‐ and probiotics, functional foods, and fortified foods. Many studies have led to an understanding of the potential mechanisms of action of pharmaceutically active components contained in food that may improve health and reduce the risk of pathological conditions while enhancing overall well‐being. Nevertheless, there is a lack of clear information and, often, the claimed health benefits may not be properly substantiated by safety and efficacy information or in vitro and in vivo data, which can induce false expectations and miss the target for a product to be effective, as claimed. An officially shared and accepted definition of nutraceuticals is still missing, as nutraceuticals are mostly referred to as pharma‐foods, a powerful toolbox to be used beyond the diet but before the drugs to prevent and treat pathological conditions, such as in subjects who may not yet be eligible for conventional pharmaceutical therapy. Hence, it is of utmost importance to have a proper and unequivocal definition of nutraceuticals and shared regulations. It also seems wise to assess the safety, mechanism of action and efficacy of nutraceuticals with clinical data. A growing demand exists for nutraceuticals, which seem to reside in the grey area between pharmaceuticals and food. Nonetheless, given specific legislation from different countries, nutraceuticals are experiencing challenges with safety and health claim substantiation.

Vitis vinifera cv Falanghina is an ancient grape variety of Southern Italy. A thorough phytochemical analysis of the Falanghina leaves was conducted to investigate its specialised metabolite content. Along with already known molecules, such as caftaric acid, quercetin-3-O-β-D-glucopyranoside, quercetin-3-O-β-D-glucuronide, kaempferol- 3-O-β-D-glucopyranoside and kaempferol-3-O-β-D-glucuronide, a previously undescribed biflavonoid was identified. For this last compound, a moderate bioactivity against metastatic melanoma cells proliferation was discovered. This datum can be of some interest to researchers studying human melanoma. The high content in antioxidant glycosylated flavonoids supports the exploitation of grape vine leaves as an inexpensive source of natural products for the food industry and for both pharmaceutical and nutraceutical companies. Additionally, this study offers important insights into the plant physiology, thus prompting possible technological researches of genetic selection based on the vine adaptation to specific pedo-climatic environments.

Hydrogen sulfide is an endogenous gasotransmitter and its mechanism of action involves activation of ATP-sensitive K+ channels and phosphodiesterase inhibition. As both mechanisms are potentially involved in malignant hyperthermia (MH), in the present study we addressed the involvement of the L-cysteine/hydrogen sulfide pathway in MH. Skeletal muscle biopsies obtained from 25 MH-susceptible (MHS) and 56 MH-negative (MHN) individuals have been used to perform the in vitro contracture test (IVCT). Quantitative real-time PCR (qPCR) and Western blotting studies have also been performed. Hydrogen sulfide levels are measured in both tissue samples and plasma. In MHS biopsies an increase in cystathionine β-synthase (CBS) occurs, as both mRNA and protein expression compared with MHN biopsies. Hydrogen sulfide biosynthesis is increased in MHS biopsies (0.128±0.12 compared with 0.943±0.13 nmol/mg of protein per min for MHN and MHS biopsies, respectively; P

H2S is a gaseous mediator, soluble in both water and lipids, sharing with nitric oxide and carbon monoxide many physiological and pathophysiological roles. H2S is a signaling molecule with a variety of functions, ranging from regulation of synaptic transmission, vascular tone, inflammation, transcription, and angiogenesis to protection of cells from oxidative stress and ischemia-reperfusion injury. The biosynthesis of H2S requires tissue-specific enzymes that generate H2S starting from L-cysteine, a metabolite of dietary methionine. Cystathionine β-synthase (CBS), and cystathionine γ-lyase (CSE) require pyridoxal-5′-phosphate as cofactor and use L-cysteine as substrate, while 3-mercaptopyruvate sulfurtransferase produces H2S using as substrate 3-mercaptopyruvate, a metabolite of L-cysteine. Recently CBS and CSE and the hydrogen sulfide pathway have been shown to be involved in cancer. In this chapter we review the role of CBS and CSE in melanoma and cancer.

Melanoma is the most common form of skin cancer. Given its high mortality, the interest in the search of preventive measures, such as dietary factors, is growing significantly. In this study we tested, in vitro and in vivo, the potential anti-cancer effect of the acetyl deacylasadisulfide (ADA), a vinyl disulfide compound, isolated and purified from asafoetida a foul-smelling oleo gum-resin of dietary and medicinal relevance. ADA markedly suppressed proliferation of human melanoma cell lines by inducing apoptosis. Moreover, treatment of melanoma cells with ADA reduced nuclear translocation and activation of NF-κB, decreased the expression of the anti-apoptotic proteins c-FLIP, XIAP and Bcl-2 and inhibited the phosphorylation and activation of both AKT and ERK proteins, two of the most frequently deregulated pathways in melanoma. Finally, the results obtained in vitro were substantiated by the findings that ADA significantly and dose-dependently reduced lung metastatic foci formation in C57BL/6 mice. In conclusion, our findings suggest that ADA significantly inhibits melanoma progression in vivo and could represent an important lead compound for the development of new anti-metastatic agents.

Background The resistance to PD-1/PD-L1 inhibitors for the treatment of melanoma have prompted investigators to implement novel clinical trials which combine immunotherapy with different treatment modalities. Moreover is also important to investigate the mechanisms which regulate the dynamic expression of PD-L1 on tumor cells and PD-1 on T cells in order to identify predictive biomarkers of response. COX-2 is currently investigated as a major player of tumor progression in several type of malignancies including melanoma. In the present study we investigated the potential relationship between COX-2 and PD-L1 expression in melanoma. Methods Tumor samples obtained from primary melanoma lesions and not matched lymph node metastases were analyzed for both PD-L1 and COX-2 expression by IHC analysis. Status of BRAF and NRAS mutations was analyzed by sequencing and PCR. Co-localization of PD-L1 and COX-2 expression was analyzed by double fluorescence staining. Lastly the BRAFV600E A375 and NRASQ61R SK-MEL-2 melanoma cell lines were used to evaluate the effect of COX-2 inhibition by celecoxib on expression of PD-L1 in vitro. Results BRAFV600E/V600K and NRASQ61R/Q61L were detected in 57.8 and 8.9% of the metastatic lesions, and in 65.9 and 6.8% of the primary tumors, respectively. PD-L1 and COX-2 expression were heterogeneously expressed in both primary melanoma lesions and not matched lymph node metastases. A significantly lower number of PD-L1 negative lesions was found in primary tumors as compared to not matched metastatic lesions (P = 0.002). COX-2 expression significantly correlated with PD-L1 expression in both primary (P = 0.001) and not matched metastatic (P = 0.048) lesions. Furthermore, in melanoma tumors, cancer cells expressing a higher levels of COX-2 also co-expressed a higher level of PD-L1. Lastly, inhibition of COX-2 activity by celecoxib down-regulated the expression of PD-L1 in both BRAFV600E A375 and NRASQ61R SK-MEL-2 melanoma cell lines. Conclusions COX-2 expression correlates with and modulates PD-L1 expression in melanoma cells. These findings have clinical relevance since they provide a rationale to implement novel clinical trials to test COX-2 inhibition as a potential treatment to prevent melanoma progression and immune evasion as well as to enhance the anti-tumor activity of PD-1/PD-L1 based immunotherapy for the treatment of melanoma patients with or without BRAF/NRAS mutations. Electronic supplementary material The online version of this article (doi:10.1186/s12967-017-1150-7) contains supplementary material, which is available to authorized users.

Background and Purpose Hydrogen sulphide (H2S) is a gaseous mediator strongly involved in cardiovascular homeostasis, where it provokes vasodilatation. Having previously shown that H2S contributes to testosterone-induced vasorelaxation, here we aim to uncover the mechanisms underlying this effect. Experimental Approach H2S biosynthesis was evaluated in rat isolated aortic rings following androgen receptor (NR3C4) stimulation. Co-immunoprecipitation and surface plasmon resonance analysis were performed to investigate mechanisms involved in NR3C4 activation. Key Results Pretreatment with NR3C4 antagonist nilutamide prevented testosterone-induced increase in H2S and reduced its vasodilator effect. Androgen agonist mesterolone also increased H2S and induced vasodilatation; effects attenuated by the selective cystathionine-γ lyase (CSE) inhibitor propargylglycine. The NR3C4-multicomplex-derived heat shock protein 90 (hsp90) was also involved in this effect; its specific inhibitor geldanamycin strongly reduced testosterone-induced H2S production. Neither progesterone nor 17-β-oestradiol induced H2S release. Furthermore, we demonstrated that CSE, the main vascular H2S-synthesizing enzyme, is physically associated with the NR3C4/hsp90 complex and the generation of such a ternary system represents a key event leading to CSE activation. Finally, H2S levels in human blood collected from male healthy volunteers were higher than those in female samples. Conclusions and Implications We demonstrated that selective activation of the NR3C4 is essential for H2S biosynthesis within vascular tissue, and this event is based on the formation of a ternary complex between cystathionine-γ lyase, NR3C4and hsp90. This novel molecular mechanism operating in the vasculature, corroborated by higher H2S levels in males, suggests that the L-cysteine/CSE/H2S pathway may be preferentially activated in males leading to gender-specific H2S biosynthesis. Linked Articles This article is part of a themed section on Pharmacology of the Gasotransmitters. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-6

Natural compounds derived from marine organisms exhibit a wide variety of biological activities. Over the last decades, a great interest has been focused on the anti-tumour role of sponges and algae that constitute the major source of these bioactive metabolites. A substantial number of chemically different structures from different species have demonstrated inhibition of tumour growth and progression by inducing apoptosis in several types of human cancer. The molecular mechanisms by which marine natural products activate apoptosis mainly include (1) a dysregulation of the mitochondrial pathway; (2) the activation of caspases; and/or (3) increase of death signals through transmembrane death receptors. This great variety of mechanisms of action may help to overcome the multitude of resistances exhibited by different tumour specimens. Therefore, products from marine organisms and their synthetic derivates might represent promising sources for new anticancer drugs, both as single agents or as co-adjuvants with other chemotherapeutics. This review will focus on some selected bioactive molecules from sponges and algae with pro-apoptotic potential in tumour cells.

Background and Purpose Proteinase-activated receptors (PARs) and toll-like receptors (TLRs) are involved in innate immune responses. The aim of this study was to evaluate the possible cross-talk between PAR2 and TLR4 in vessels in physiological condition and how it varies following stimulation of TLR4 by using in vivo and ex vivo models. Experimental Approach Thoracic aortas were harvested from both naive and endotoxaemic rats for in vitro studies. Arterial blood pressure was monitored in anaesthetized rats in vivo. LPS was used as a TLR4 agonist while PAR2 activating peptide (AP) was used as a PAR2 agonist. Aortas harvested from TLR4–/– mice were also used to characterize the PAR2 response. Key Results PAR2, but not TLR4, expression was enhanced in aortas of endotoxaemic rats. PAR2AP-induced vasorelaxation was increased in aortic rings of LPS-treated rats. TLR4 inhibitors, curcumine and resveratrol, reduced PAR2AP-induced vasorelaxation and PAR2AP-induced hypotension in both naive and endotoxaemic rats. Finally, in aortic rings from TLR4–/– mice, the expression of PAR2 was reduced and the PAR2AP-induced vasodilatation impaired compared with those from wild-type mice and both resveratrol and curcumine were ineffective. Conclusions and Implications Cross-talk between PAR2 and TLR4 contributes to vascular homeostasis.