Dietary Protein and Physical Exercise for the Treatment of Sarcopenia.

Sarcopenia is a multifactorial age-related disorder that causes a decrease in muscle mass, strength, and function, leading to alteration of movement, risk of falls, and hospitalization. This article aims to review recent findings on the factors underlying sarcopenia and the strategies required to delay and counteract its symptoms. We focus on molecular factors linked to ageing, on the role of low-grade chronic and acute inflammatory conditions such as cancer, which contributes to the onset of sarcopenia, and on the clinical criteria for its diagnosis. The use of drugs against sarcopenia is still subject to debate, and the suggested approaches to restore muscle health are based on adequate dietary protein intake and physical exercise. We also highlight the difference in the amount and quality of amino acids within animal- and plant-based diets, as studies have often shown varying results regarding their effect on sarcopenia in elderly people. In addition, many studies have reported that non-pharmacological approaches, such as an optimization of dietary protein intake and training programs based on resistance exercise, can be effective in preventing and delaying sarcopenia. These approaches not only improve the maintenance of skeletal muscle function, but also reduce health care costs and improve life expectancy and quality in elderly people.

Guardians and Mediators of Metastasis: Exploring T Lymphocytes, Myeloid-Derived Suppressor Cells, and Tumor-Associated Macrophages in the Breast Cancer Microenvironment.

Breast cancers (BCs) are solid tumors composed of heterogeneous tissues consisting of cancer cells and an ever-changing tumor microenvironment (TME). The TME includes, among other non-cancer cell types, immune cells influencing the immune context of cancer tissues. In particular, the cross talk of immune cells and their interactions with cancer cells dramatically influence BC dissemination, immunoediting, and the outcomes of cancer therapies. Tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs) represent prominent immune cell populations of breast TMEs, and they have important roles in cancer immunoescape and dissemination. Therefore, in this article we review the features of TILs, TAMs, and MDSCs in BCs. Moreover, we highlight the mechanisms by which these immune cells remodel the immune TME and lead to breast cancer metastasis.

Identification and Characterization of Neuroprotective Properties of Thaumatin-like Protein 1a from Annurca Apple Flesh Polyphenol Extract.

BACKGROUND: Alzheimer's disease (AD) and Parkinson's disease (PD) are multifactorial neurodegenerative disorders that are mostly treated with drugs inhibiting key enzymes of cholinergic and aminergic neurotransmission, such as acetyl and butyryl cholinesterase (AChE, BuChE) or monoamine oxidases (MAO)-A/B, and of Aß1-40 aggregation. Diet plant components with multitarget functions are promising compounds in the prevention of AD and PD. Our aim was to identify neuroprotective compounds from Annurca apple polyphenol extract (AFPE). METHODS: AFPE was fractionated by gel filtration, and the eluted peaks were subjected to chemical analyses (i.e., RP-HPLC and mass spectrometry), determination of inhibitory enzyme activity and cell effects by MTT, and morphology assays. RESULTS: In AFPE, we identified thaumatin-like protein 1a, belonging to the pathogenesis-related protein (PR) family. This protein showed the best inhibitory activity on AChE, MAO-A (IC50 = 5.53 µM and 1.71 µM, respectively), and Aß1-40 fibril aggregation (IC50 = 9.16 µM), compared to AFPE and other polyphenol-containing fractions. Among the latter, Peak 4 reverted Aß fibril formation (IC50 = 104.87 µM). Moreover, thaumatin-like protein 1a protected AGS and MKN-28 cells from serum-deprivation-induced stress conditions. CONCLUSIONS: We showed that AFPE exerted neuroprotective functions not only through its polyphenols but also through thaumatin-like protein 1a, which acted like a multitarget molecule.

Effect of Hydroxytyrosol Derivatives of Donepezil on the Activity of Enzymes Involved in Neurodegenerative Diseases and Oxidative Damage.

Monoamine oxidase and xanthine oxidase inhibitors represent useful multi-target drugs for the prevention, attenuation, and treatment of oxidative damage and neurodegenerative disorders. Chimeric molecules, constituted by naturally derived compounds linked to drugs, represent lead compounds to be explored for the discovery of new synthetic drugs acting as enzyme inhibitors. We have previously reported that seven hydroxytyrosol-donepezil hybrid compounds play a protective role in an in vitro neuronal cell model of Alzheimer's disease. In this work, we analyzed the effects exerted by the hybrid compounds on the activity of monoamine oxidase A (MAO-A) and B (MAO-B), as well as on xanthine oxidase (XO), enzymes involved in both neurodegenerative disorders and oxidative stress. The results pointed to the identification, among the compounds tested, of selective inhibitors between the two classes of enzymes. While the 4-hydroxy-3-methoxyphenethyl 1-benzylpiperidine-4-carboxylate- (HT3) and the 4-hydroxyphenethyl 1-benzylpiperidine-4-carboxylate- donepezil derivatives (HT4) represented the best inhibitors of MAO-A, with a scarce effect on MAO-B, they were almost ineffective on XO. On the other hand, the 4,5-dihydroxy-2-nitrophenethyl 1-benzylpiperidine-4-carboxylate donepezil derivative (HT2), the least efficient MAO inhibitor, acted like the best XO inhibitor. Therefore, the differential enzymatic targets identified among the hybrid compounds synthesized enhance the possible applications of these polyphenol-donepezil hybrids in neurodegenerative disorders and oxidative stress.

Inhibition of Enzymes Involved in Neurodegenerative Disorders and Aß1-40 Aggregation by Citrus limon Peel Polyphenol Extract.

Alzheimer's (AD) and Parkinson's diseases (PD) are multifactorial neurogenerative disorders of the Central Nervous System causing severe cognitive and motor deficits in elderly people. Because treatment of AD and PD by synthetic drugs alleviates the symptoms often inducing side effects, many studies have aimed to find neuroprotective properties of diet polyphenols, compounds known to act on different cell signaling pathways. In this article, we analyzed the effect of polyphenols obtained from the agro-food industry waste of Citrus limon peel (LPE) on key enzymes of cholinergic and aminergic neurotransmission, such as butyryl cholinesterase (BuChE) and monoamine oxidases (MAO)-A/B, on Aß1-40 aggregation and on superoxide dismutase (SOD) 1/2 that affect oxidative stress. In our in vitro assays, LPE acts as an enzyme inhibitor on BuChE (IC50 ~ 73 µM), MAO-A/B (IC50 ~ 80 µM), SOD 1/2 (IC50 ~ 10-20 µM) and interferes with Aß1-40 peptide aggregation (IC50 ~ 170 µM). These results demonstrate that LPE behaves as a multitargeting agent against key factors of AD and PD by inhibiting to various extents BuChE, MAOs, and SODs and reducing Aß-fibril aggregation. Therefore, LPE is a promising candidate for the prevention and management of AD and PD symptoms in combination with pharmacological therapies.

Hydroxytyrosol-Donepezil Hybrids Play a Protective Role in an In Vitro Induced Alzheimer's Disease Model and in Neuronal Differentiated Human SH-SY5Y Neuroblastoma Cells.

Alzheimer's disease (AD) is the most common neurodegenerative pathology among progressive dementias, and it is characterized by the accumulation in the brain of extracellular aggregates of beta-amyloid proteins and neurofibrillary intracellular tangles consisting of τ-hyperphosphorylated proteins. Under normal conditions, beta-amyloid peptides exert important trophic and antioxidant roles, while their massive presence leads to a cascade of events culminating in the onset of AD. The fibrils of beta-amyloid proteins are formed by the process of fibrillogenesis that, starting from individual monomers of beta-amyloid, can generate polymers of this protein, constituting the hypothesis of the "amyloid cascade". To date, due to the lack of pharmacological treatment for AD without toxic side effects, chemical research is directed towards the realization of hybrid compounds that can act as an adjuvant in the treatment of this neurodegenerative pathology. The hybrid compounds used in this work include moieties of a hydroxytyrosol, a nitrohydroxytyrosol, a tyrosol, and a homovanillyl alcohol bound to the N-benzylpiperidine moiety of donepezil, the main drug used in AD. Previous experiments have shown different properties of these hybrids, including low toxicity and antioxidant and chelating activities. The purpose of this work was to test the effects of hybrid compounds mixed with Aß1-40 to induce fibrillogenesis and mimic AD pathogenesis. This condition has been studied both in test tubes and by an in vitro model of neuronal differentiated human SH-SY5Y neuroblastoma cells. The results obtained from test tube experiments showed that some hybrids inhibit the activity of the enzymes AChE, BuChE, and BACE-1. Cell experiments suggested that hybrids could inhibit fibrillogenesis, negatively modulating caspase-3. They were also shown to exert antioxidant effects, and the acetylated hybrids were found to be more functional and efficient than nonacetylated forms.

Inhibition of Interleukin-6 Dependent Metalloproteinases-9/2 Expression in Cancer Cells by Diet Polyphenols.

Among inflammatory cytokines, Interleukin-6 (IL-6) is one of the major activators of acute phase response and is also involved in immune response and cancer progression. IL-6 is involved in the up-regulation of enzymes and growth factors acting on the extracellular matrix (ECM) remodelling components in physio-pathological processes. IL-6 enhances the expression of metalloproteases (MMP-)2/9, enzymes that play a key role in ECM degradation and therefore contribute to the process of tumor metastasis. To counteract and/or prevent cancer diseases, many efforts have been devoted to the identification of factors able to inhibit the IL-6-dependent MMP-9/2 expression. Recently, diet polyphenols have been identified as molecules manifesting anti-inflammatory and anti-cancer properties beyond their well-known capacity to promote health on the basis of their antioxidant effects. This review summarizes the recent advances in this field, focusing on the protective effects exerted by diet polyphenols on the proliferation and invasiveness of tumor cells, with specific emphasis on the ability of these molecules to inhibit the IL-6-dependent upregulation of MMP-2/9.

Celecoxib, a Non-Steroidal Anti-Inflammatory Drug, Exerts a Toxic Effect on Human Melanoma Cells Grown as 2D and 3D Cell Cultures.

Cutaneous melanoma (CM) remains one of the leading causes of tumor mortality due to its high metastatic spread. CM growth is influenced by inflammation regulated by prostaglandins (PGs) whose synthesis is catalyzed by cyclooxygenases (COXs). COX inhibitors, including non-steroidal anti-inflammatory drugs (NSAIDs), can inhibit tumor development and growth. In particular, in vitro experiments have shown that celecoxib, a NSAID, inhibits the growth of some tumor cell lines. However, two-dimensional (2D) cell cultures, used in traditional in vitro anticancer assays, often show poor efficacy due to a lack of an in vivo like cellular environment. Three-dimensional (3D) cell cultures, such as spheroids, are better models because they can mimic the common features displayed by human solid tumors. Hence, in this study, we evaluated the anti-neoplastic potential of celecoxib, in both 2D and 3D cell cultures of A2058 and SAN melanoma cell lines. In particular, celecoxib reduced the cell viability and migratory capability and triggered the apoptosis of melanoma cells grown as 2D cultures. When celecoxib was tested on 3D melanoma cell cultures, the drug exerted an inhibitory effect on cell outgrowth from spheroids and reduced the invasiveness of melanoma cell spheroids into the hydrogel matrix. This work suggests that celecoxib could represent a new potential therapeutic approach in melanoma therapy.

Novel Reversible Inhibitors of Xanthine Oxidase Targeting the Active Site of the Enzyme.

Xanthine oxidase (XO) is a flavoprotein catalysing the oxidation of hypoxanthine to xanthine and then to uric acid, while simultaneously producing reactive oxygen species. Altered functions of XO may lead to severe pathological diseases, including gout-causing hyperuricemia and oxidative damage of tissues. These findings prompted research studies aimed at targeting the activity of this crucial enzyme. During the course of a virtual screening study aimed at the discovery of novel inhibitors targeting another oxidoreductase, superoxide dismutase, we identified four compounds with non-purine-like structures, namely ALS-1, -8, -15 and -28, that were capable of causing direct inhibition of XO. The kinetic studies of their inhibition mechanism allowed a definition of these compounds as competitive inhibitors of XO. The most potent molecule was ALS-28 (Ki 2.7 ± 1.5 µM), followed by ALS-8 (Ki 4.5 ± 1.5 µM) and by the less potent ALS-15 (Ki 23 ± 9 µM) and ALS-1 (Ki 41 ± 14 µM). Docking studies shed light on the molecular basis of the inhibitory activity of ALS-28, which hinders the enzyme cavity channel for substrate entry consistently with the competitive mechanism observed in kinetic studies. Moreover, the structural features emerging from the docked poses of ALS-8, -15 and -1 may explain the lower inhibition power with respect to ALS-28. All these structurally unrelated compounds represent valuable candidates for further elaboration into promising lead compounds.

In Silico Identification of Novel Inhibitors Targeting the Homodimeric Interface of Superoxide Dismutase from the Dental Pathogen Streptococcus mutans.

The microaerophile Streptococcus mutans, the main microaerophile responsible for the development of dental plaque, has a single cambialistic superoxide dismutase (SmSOD) for its protection against reactive oxygen species. In order to discover novel inhibitors of SmSOD, possibly interfering with the biofilm formation by this pathogen, a virtual screening study was realised using the available 3D-structure of SmSOD. Among the selected molecules, compound ALS-31 was capable of inhibiting SmSOD with an IC50 value of 159 µM. Its inhibition power was affected by the Fe/Mn ratio in the active site of SmSOD. Furthermore, ALS-31 also inhibited the activity of other SODs. Gel-filtration of SmSOD in the presence of ALS-31 showed that the compound provoked the dissociation of the SmSOD homodimer in two monomers, thus compromising the catalytic activity of the enzyme. A docking model, showing the binding mode of ALS-31 at the dimer interface of SmSOD, is presented. Cell viability of the fibroblast cell line BJ5-ta was not affected up to 100 µM ALS-31. A preliminary lead optimization program allowed the identification of one derivative, ALS-31-9, endowed with a 2.5-fold improved inhibition power. Interestingly, below this concentration, planktonic growth and biofilm formation of S. mutans cultures were inhibited by ALS-31, and even more by its derivative, thus opening the perspective of future drug design studies to fight against dental caries.

Inhibition of Interleukin-6-Induced Matrix Metalloproteinase-2 Expression and Invasive Ability of Lemon Peel Polyphenol Extract in Human Primary Colon Cancer Cells.

Among matrix metalloproteinases (MMPs), MMP-9/2 are key enzymes involved in the proteolysis of extracellular matrices in the inflammatory process and in cancer. Since MMP-9/2 expression levels, activity, and secretion is up-regulated during inflammation in response to pro-inflammatory cytokines, such as interleukin-6 (IL-6), many efforts have been devoted to identifying factors that could inhibit the IL-6-induced MMP-9/2 expression. Up to now, several reports indicated that polyphenols from fruits and vegetables are among the major components of health promotion for their antioxidant properties and also for their anti-inflammatory and anti-cancer agents. Among plant derived polyphenols, lemon (Citrus limon) peel extract (LPE) shows anti-cancer properties in various cancer types. In our previous work, we demonstrated that LPE can reduce IL-6-induced migration/invasiveness and MMP-9/2 up-regulation in some gastric cancer cell lines. This study aims to exploit the anti-cancer properties of LPE using an in vitro system model of inflammation, consisting of IL-6-exposed human primary colon cancer cells. We first analyzed the effect of LPE on IL-6-induced cell migration and invasiveness by wound healing and Boyden chamber assay, respectively. The MMP-2 mRNA expression levels and gelatinolytic activity in the cell culture media were determined by q-PCR analysis and gelatin zymography, respectively, and finally, the effects of LPE on IL-6-induced JAK2/STAT3 signaling pathways have been investigated by Western blotting analysis. Our results show that LPE is able to inhibit the IL-6-dependent cell migration and invasiveness associated with the up-regulation of MMP-2 expression levels and that these effects are correlated to the STAT3 phosphorylation in human primary T88 and T93 colon cancer cells.

Tandem Mass Spectrometry as Strategy for the Selective Identification and Quantification of the Amyloid Precursor Protein Tyr682 Residue Phosphorylation Status in Human Blood Mononuclear Cells.

Background: Alzheimer's disease (AD) is a devastating neurodegenerative disease without guidelines for early diagnosis or personalized treatment. Previous studies have highlighted a crucial role of increasing phosphorylation levels of the amyloid precursor protein (APP) Tyr682 residue in predicting neuronal deficits in AD patients. However, the lack of a method for the identification and quantification of Tyr682 phosphorylation levels prevents its potential clinical applications. Methods: Here we report a method to identify and quantify APP Tyr682 phosphorylation levels in blood mononuclear cells of AD patients by tandem mass spectrometry (tMS). Results: This method showed excellent sensitivity with detection and quantification limits set respectively at 0.035 and 0.082 ng injected for the phosphorylated peptide and at 0.02 and 0.215 ng injected for the non-phosphorylated peptide. The average levels of both peptides were quantified in transfected HELA cells (2.48 and 3.53 ng/µg of protein, respectively). Preliminary data on 3 AD patients showed quantifiable levels of phosphorylated peptide (0.10-0.15 ng/µg of protein) and below the LOQ level of non-phosphorylated peptide (0.13 ng/µg of protein). Conclusion: This method could allow the identification of patients with increased APP Tyr682 phosphorylation and allow early characterization of molecular changes prior to the appearance of clinical signs.

Challenges in LC-MS-based metabolomics for Alzheimer's disease early detection: targeted approaches versus untargeted approaches.

BACKGROUND: Alzheimer's disease (AD) is one of the most common causes of dementia in old people. Neuronal deficits such as loss of memory, language and problem-solving are severely compromised in affected patients. The molecular features of AD are Aß deposits in plaques or in oligomeric structures and neurofibrillary tau tangles in brain. However, the challenge is that Aß is only one piece of the puzzle, and recent findings continue to support the hypothesis that their presence is not sufficient to predict decline along the AD outcome. In this regard, metabolomic-based techniques are acquiring a growing interest for either the early diagnosis of diseases or the therapy monitoring. Mass spectrometry is one the most common analytical platforms used for detection, quantification, and characterization of metabolic biomarkers. In the past years, both targeted and untargeted strategies have been applied to identify possible interesting compounds. AIM OF REVIEW: The overall goal of this review is to guide the reader through the most recent studies in which LC-MS-based metabolomics has been proposed as a powerful tool for the identification of new diagnostic biomarkers in AD. To this aim, herein studies spanning the period 2009-2020 have been reported. Advantages and disadvantages of targeted vs untargeted metabolomic approaches have been outlined and critically discussed.

Annurca Apple Polyphenol Extract Affects Acetyl- Cholinesterase and Mono-Amine Oxidase In Vitro Enzyme Activity.

In this study, we explored the ability of Annurca apple flesh polyphenol extract (AFPE) to affect the activity of key enzymes involved in neurodegenerative disorders-in particular, Acetyl- and Butirryl-cholinesterases, and type A and B monoamine oxidase. The effect of AFPE on enzyme activity was analyzed by in vitro enzyme assays, and the results showed concentration-dependent enzyme inhibition, with IC50 values corresponding to 859 ± 18 µM and 966 ± 72 µM for AChE and BuChE respectively, and IC50 corresponding to 145 ± 3 µM and 199 ± 7 µM for MAO-A and MAO-B, respectively, with a preference for MAO-A. Moreover, in this concentration range, AFPE did not affect the viability of human neuroblastoma SH-SY5Y and fibroblast BJ-5ta cell lines, as determined by an MTT assay. In conclusion, our results demonstrate that AFPE shows the new biological properties of inhibiting the activity of enzymes that are involved in brain functions, neurodegenerative disorders, and aging.

Quinoa as source of type 1 ribosome inactivating proteins: A novel knowledge for a revision of its consumption.

This study investigates on the presence of toxic proteins in quinoa seeds. To this aim, a plethora of biochemical approaches were adopted for the purification and characterization of quinoin, a type 1 ribosome-inactivating protein (RIP) contained in quinoa seeds. We determined its melting temperature (68.2 ± 0.6 °C) and thermostability (loss of activity after 10-min incubation at 70 °C). Considering that quinoa seeds are used as a food, we found that quinoin is cytotoxic against BJ-5ta (human fibroblasts) and HaCaT (human keratinocytes) in a dose- and time-dependent manner. Moreover, in an in vitro digestive pepsin-trypsin treatment, 30% of quinoin is resistant to enzymatic cleavage. This toxin was found in seeds (0.23 mg/g of seeds) and in sprouted seeds obtained after 24-h (0.12 mg/g of sprout) and 48-h (0.09 mg/g of sprout). We suggest a thermal treatment of quinoa seeds before consumption in order to inactivate the toxin, particularly in sprouts, generally consumed raw.

Inhibition mechanism of naphthylphenylamine derivatives acting on the CDC25B dual phosphatase and analysis of the molecular processes involved in the high cytotoxicity exerted by one selected derivative in melanoma cells.

The dual phosphatases CDC25 are involved in cell cycle regulation and overexpressed in many tumours, including melanoma. CDC25 is a promising target for discovering anticancer drugs, and several studies focussed on characterisation of quinonoid CDC25 inhibitors, frequently causing undesired side toxic effects. Previous work described an optimisation of the inhibition properties by naphthylphenylamine (NPA) derivatives of NSC28620, a nonquinonoid CDC25 inhibitor. Now, the CDC25B•inhibitor interaction was investigated through fluorescence studies, shedding light on the different inhibition mechanism exerted by NPA derivatives. Among the molecular processes, mediating the specific and high cytotoxicity of one NPA derivative in melanoma cells, we observed decrease of phosphoAkt, increase of p53, reduction of CDC25 forms, cytochrome c cytosolic translocation and increase of caspase activity, that lead to the activation of an apoptotic programme. A basic knowledge on CDC25 inhibitors is relevant for discovering potent bioactive molecules, to be used as anticancer agents against the highly aggressive melanoma.

Fyn Tyrosine Kinase as Harmonizing Factor in Neuronal Functions and Dysfunctions.

Fyn is a non-receptor or cytoplasmatic tyrosine kinase (TK) belonging to the Src family kinases (SFKs) involved in multiple transduction pathways in the central nervous system (CNS) including synaptic transmission, myelination, axon guidance, and oligodendrocyte formation. Almost one hundred years after the original description of Fyn, this protein continues to attract extreme interest because of its multiplicity of actions in the molecular signaling pathways underlying neurodevelopmental as well as neuropathologic events. This review highlights and summarizes the most relevant recent findings pertinent to the role that Fyn exerts in the brain, emphasizing aspects related to neurodevelopment and synaptic plasticity. Fyn is a common factor in healthy and diseased brains that targets different proteins and shapes different transduction signals according to the neurological conditions. We will primarily focus on Fyn-mediated signaling pathways involved in neuronal differentiation and plasticity that have been subjected to considerable attention lately, opening the fascinating scenario to target Fyn TK for the development of potential therapeutic interventions for the treatment of CNS injuries and certain neurodegenerative disorders like Alzheimer's disease.

Lemon Peel Polyphenol Extract Reduces Interleukin-6-Induced Cell Migration, Invasiveness, and Matrix Metalloproteinase-9/2 Expression in Human Gastric Adenocarcinoma MKN-28 and AGS Cell Lines.

Among plant polyphenols, lemon peels extract (LPE) from the residues of the industrial processing of lemon (Citruslimon) shows anti-proliferative properties in cancer cells and anticholinesterase activity. In this study, we analyze the anti-cancer properties of LPE on migration and invasiveness in MKN-28 and AGS human gastric cancer cell lines either in the absence or presence of the pro-inflammatory cytokine IL-6. We find that the pretreatment with non-cytotoxic concentrations (0.5-1 µg/ml of gallic acid equivalent) of LPE inhibits interleukin-6 (IL-6)-induced cell migration and invasiveness in MKN-28 and AGS cells, as analyzed by wound and matrigel assays. Pretreatment with LPE is able to prevent either IL-6-induced matrix metalloproteinases (MMP)-9/2 activity, as assessed by gel zymography, or mRNA and protein MMP-9/2 expression, as evaluated by qPCR and Western blotting analysis, respectively. These LPE effects are associated with an IL-6-dependent STAT3 signaling pathway in MKN-28 and AGS cells. Furthermore, LPE shows acetylcholinesterase inhibitory activity when assayed by the Ellman method. In conclusion, our results demonstrate that LPE reduces the invasiveness of gastric MKN-28 and AGS cancer cells through the reduction of IL-6-induced MMP-9/2 up-regulation. Therefore, these data suggest that LPE exerts a protective role against the metastatic process in gastric cancer.

Development of a Stromal Microenvironment Experimental Model Containing Proto-Myofibroblast Like Cells and Analysis of Its Crosstalk with Melanoma Cells: A New Tool to Potentiate and Stabilize Tumor Suppressor Phenotype of Dermal Myofibroblasts.

Melanoma is one of the most aggressive solid tumors and includes a stromal microenvironment that regulates cancer growth and progression. The components of stromal microenvironment such as fibroblasts, fibroblast aggregates and cancer-associated fibroblasts (CAFs) can differently influence the melanoma growth during its distinct stages. In this work, we have developed and studied a stromal microenvironment model, represented by fibroblasts, proto-myofibroblasts, myofibroblasts and aggregates of inactivated myofibroblasts, such as spheroids. In particular, we have generated proto-myofibroblasts from primary cutaneous myofibroblasts. The phenotype of proto-myofibroblasts is characterized by a dramatic reduction of α-smooth muscle actin (α-SMA) and cyclooxygenase-2 (COX-2) protein levels, as well as an enhancement of cell viability and migratory capability compared with myofibroblasts. Furthermore, proto-myofibroblasts display the mesenchymal marker vimentin and less developed stress fibers, with respect to myofibroblasts. The analysis of crosstalk between the stromal microenvironment and A375 or A2058 melanoma cells has shown that the conditioned medium of proto-myofibroblasts is cytotoxic, mainly for A2058 cells, and dramatically reduces the migratory capability of both cell lines compared with the melanoma-control conditioned medium. An array analysis of proto-myofibroblast and melanoma cell-conditioned media suggests that lower levels of some cytokines and growth factors in the conditioned medium of proto-myofibroblasts could be associated with their anti-tumor activity. Conversely, the conditioned media of melanoma cells do not influence the cell viability, outgrowth, and migration of proto-myofibroblasts from spheroids. Interestingly, the conditioned medium of proto-myofibroblasts does not alter the cell viability of both BJ-5ta fibroblast cells and myofibroblasts. Hence, proto-myofibroblasts could be useful in the study of new therapeutic strategies targeting melanoma.

Ageritin from poplar mushrooms: scale-up purification and cytotoxicity towards undifferentiated and differentiated SH-SY5Y cells.

Ageritin is the first reported ribotoxin-like protein from basidiomycetes fungi. It can induce ribosomal integrity damage and translation block, and interferes with mitochondrial redox activity of some glioma and neuroblastoma cell lines. Herein, Ageritin has been investigated as a valuable neurotoxin towards either undifferentiated or retinoic acid (RA)-differentiated SH-SY5Y neuroblastoma cells showing a selective cell toxicity against undifferentiated cells. MTT and sulforhodamine B (SRB) assays highlighted that Ageritin markedly decreases the mitochondrial redox activity and viability of undifferentiated cells, meanwhile inducing evident morphological changes eliciting neuronal-like appearance in these cells. Data from lactate dehydrogenase release assay, cytofluorimetric analysis and caspase-3 enzymatic activity measurement suggest that Ageritin promotes cell death through a caspase-dependent apoptotic pathway. The Z-VAD-FMK caspase inhibitor was able to prevent this apoptotic pathway activation. Based on the interesting behaviour of Ageritin vs. SH-SY5Y cells, the development of a scale-up procedure to obtain the purified protein in larger amounts (yield 2.5 mg per 100 g) has been optimized.