Bile acids serve as endogenous antagonists of the Leukemia inhibitory factor (LIF) receptor in oncogenesis.

The leukemia inhibitory factor (LIF) is member of interleukin (IL)-6 family of cytokines involved immune regulation, morphogenesis and oncogenesis. In cancer tissues, LIF binds a heterodimeric receptor (LIFR), formed by a LIFRß subunit and glycoprotein(gp)130, promoting epithelial mesenchymal transition and cell growth. Bile acids are cholesterol metabolites generated at the interface of host metabolism and the intestinal microbiota. Here we demonstrated that bile acids serve as endogenous antagonist to LIFR in oncogenesis. The tissue characterization of bile acids content in non-cancer and cancer biopsy pairs from gastric adenocarcinomas (GC) demonstrated that bile acids accumulate within cancer tissues, with glyco-deoxycholic acid (GDCA) functioning as negative regulator of LIFR expression. In patient-derived organoids (hPDOs) from GC patients, GDCA reverses LIF-induced stemness and proliferation. In summary, we have identified the secondary bile acids as the first endogenous antagonist to LIFR supporting a development of bile acid-based therapies in LIF-mediated oncogenesis.

Identification of Mortalin as the Main Interactor of Mycalin A, a Poly-Brominated C-15 Acetogenin Sponge Metabolite, by MS-Based Proteomics.

Mycalin A (MA) is a polybrominated C-15 acetogenin isolated from the marine sponge Mycale rotalis. Since this substance displays a strong antiproliferative bioactivity towards some tumour cells, we have now directed our studies towards the elucidation of the MA interactome through functional proteomic approaches, (DARTS and t-LIP-MS). DARTS experiments were performed on Hela cell lysates with the purpose of identifying MA main target protein(s); t-LiP-MS was then applied for an in-depth investigation of the MA-target protein interaction. Both these techniques exploit limited proteolysis coupled with MS analysis. To corroborate LiP data, molecular docking studies were performed on the complexes. Finally, biological and SPR analysis were conducted to explore the effect of the binding. Mortalin (GRP75) was identified as the MA's main interactor. This protein belongs to the Hsp70 family and has garnered significant attention due to its involvement in certain forms of cancer. Specifically, its overexpression in cancer cells appears to hinder the pro-apoptotic function of p53, one of its client proteins, because it becomes sequestered in the cytoplasm. Our research, therefore, has been focused on the possibility that MA might prevent this sequestration, promoting the re-localization of p53 to the nucleus and facilitating the apoptosis of tumor cells.

Defective Bile Acid Signaling Promotes Vascular Dysfunction, Supporting a Role for G-Protein Bile Acid Receptor 1/Farnesoid X Receptor Agonism and Statins in the Treatment of Nonalcoholic Fatty Liver Disease.

BACKGROUND: Patients with nonalcoholic fatty liver disease are at increased risk to develop atherosclerotic cardiovascular diseases. FXR and GPBAR1 are 2 bile acid-activated receptors exploited in the treatment of nonalcoholic fatty liver disease: whether dual GPBAR1/FXR agonists synergize with statins in the treatment of the liver and cardiovascular components of nonalcoholic fatty liver disease is unknown. METHODS AND RESULTS: Investigations of human aortic samples obtained from patients who underwent surgery for aortic aneurysms and Gpbar1-/-, Fxr-/-, and dual Gpbar1-/-Fxr-/- mice demonstrated that GPBAR1 and FXR are expressed in the aortic wall and regulate endothelial cell/macrophage interactions. The expression of GPBAR1 in the human endothelium correlated with the expression of inflammatory biomarkers. Mice lacking Fxr and Gpbar1-/-/Fxr-/- display hypotension and aortic inflammation, along with altered intestinal permeability that deteriorates with age, and severe dysbiosis, along with dysregulated bile acid synthesis. Vasomotor activities of aortic rings were altered by Gpbar1 and Fxr gene ablation. In apolipoprotein E-/- and wild-type mice, BAR502, a dual GPBAR1/FXR agonist, alone or in combination with atorvastatin, reduced cholesterol and low-density lipoprotein plasma levels, mitigated the development of liver steatosis and aortic plaque formation, and shifted the polarization of circulating leukocytes toward an anti-inflammatory phenotype. BAR502/atorvastatin reversed intestinal dysbiosis and dysregulated bile acid synthesis, promoting a shift of bile acid pool composition toward FXR antagonists and GPBAR1 agonists. CONCLUSIONS: FXR and GPBAR1 maintain intestinal, liver, and cardiovascular homeostasis, and their therapeutic targeting with a dual GPBAR1/FXR ligand and atorvastatin holds potential in the treatment of liver and cardiovascular components of nonalcoholic fatty liver disease.

The leukemia inhibitory factor regulates fibroblast growth factor receptor 4 transcription in gastric cancer.

PURPOSE: The gastric adenocarcinoma (GC) represents the third cause of cancer-related mortality worldwide, and available therapeutic options remain sub-optimal. The Fibroblast growth factor receptors (FGFRs) are oncogenic transmembrane tyrosine kinase receptors. FGFR inhibitors have been approved for the treatment of various cancers and a STAT3-dependent regulation of FGFR4 has been documented in the H.pylori infected intestinal GC. Therefore, the modulation of FGFR4 might be useful for the treatment of GC. METHODS: To investigate wich factors could modulate FGFR4 signalling in GC, we employed RNA-seq analysis on GC patients biopsies, human patients derived organoids (PDOs) and cancer cell lines. RESULTS: We report that FGFR4 expression/function is regulated by the leukemia inhibitory factor (LIF) an IL-6 related oncogenic cytokine, in JAK1/STAT3 dependent manner. The transcriptomic analysis revealed a direct correlation between the expression of LIFR and FGFR4 in the tissue of an exploratory cohort of 31 GC and confirmed these findings by two external validation cohorts of GC. A LIFR inhibitor (LIR-201) abrogates STAT3 phosphorylation induced by LIF as well as recruitment of pSTAT3 to the promoter of FGFR4. Furthermore, inhibition of FGFR4 by roblitinib or siRNA abrogates STAT3 phosphorylation and oncogentic effects of LIF in GC cells, indicating that FGFR4 is a downstream target of LIF/LIFR complex. Treating cells with LIR-201 abrogates oncogenic potential of FGF19, the physiological ligand of FGFR4. CONCLUSIONS: Together these data unreveal a previously unregnized regulatory mechanism of FGFR4 by LIF/LIFR and demonstrate that LIF and FGF19 converge on the regulation of oncogenic STAT3 in GC cells.

Activation of GPBAR1 attenuates vascular inflammation and atherosclerosis in a mouse model of NAFLD-related cardiovascular disease.

While patients with nonalcoholic fatty liver disease (NAFLD) are at increased risk to develop clinically meaningful cardiovascular diseases (CVD), there are no approved drug designed to target the liver and CVD component of NAFLD. GPBAR1, also known as TGR5, is a G protein coupled receptor for secondary bile acids. In this study we have investigated the effect of GPBAR1 activation by BAR501, a selective GPBAR1 agonist, in Apolipoprotein E deficient (ApoE-/-) mice fed a high fat diet and fructose (Western diet), a validated model of NAFLD-associated atherosclerosis. Using aortic samples from patients who underwent surgery for abdominal aneurism, and ex vivo experiments with endothelial cells and human macrophages, we were able to co-localize the expression of GPBAR1 in CD14+ and PECAM1+ cells. Similar findings were observed in the aortic plaques from ApoE-/- mice. Treating ApoE-/- mice with BAR501, 30 mg/kg for 14 weeks, attenuated the body weight gain while ameliorated the insulin sensitivity by increasing the plasma concentrations of GLP-1 and FGF15. Activation of GPBAR1 reduced the aorta thickness and severity of atherosclerotic lesions and decreased the amount of plaques macrophages. Treating ApoE-/- mice reshaped the aortic transcriptome promoting the expression of anti-inflammatory genes, including IL-10, as also confirmed by tSNE analysis of spleen-derived macrophages. Feeding ApoE-/- mice with BAR501 redirected the bile acid synthesis and the composition of the intestinal microbiota. In conclusion, GPBAR1 agonism attenuates systemic inflammation and improve metabolic profile in a genetic/dietetic model of atherosclerosis. BAR501 might be of utility in the treatment for NAFLD-related CVD.

Identification of a Protein Arginine Methyltransferase 7 (PRMT7)/Protein Arginine Methyltransferase 9 (PRMT9) Inhibitor.

Less studied than the other protein arginine methyltransferase isoforms, PRMT7 and PRMT9 have recently been identified as important therapeutic targets. Yet, most of their biological roles and functions are still to be defined, as well as the structural requirements that could drive the identification of selective modulators of their activity. We recently described the structural requirements that led to the identification of potent and selective PRMT4 inhibitors spanning both the substrate and the cosubstrate pockets. The reanalysis of the data suggested a PRMT7 preferential binding for shorter derivatives and prompted us to extend these structural studies to PRMT9. Here, we report the identification of the first potent PRMT7/9 inhibitor and its binding mode to the two PRMT enzymes. Label-free quantification mass spectrometry confirmed significant inhibition of PRMT activity in cells. We also report the setup of an effective AlphaLISA assay to screen small molecule inhibitors of PRMT9.

Targeting USP-7 by a Novel Fluorinated 5-Pyrazolyl-Urea Derivative.

The impact of innovative technologies on the target discovery has been employed here to characterize the interactome of STIRUR 41, a promising 3-fluoro-phenyl-5-pyrazolyl-urea derivative endowed with anti-cancer activity, on neuroblastoma-related cells. A drug affinity responsive target stability-based proteomic platform has been optimized to elucidate the molecular mechanism at the basis of STIRUR 41 action, together with immunoblotting analysis and in silico molecular docking. Ubiquitin Specific Protease 7 (USP-7), one of the deubiquitinating enzymes which protect substrate proteins from proteasomal degradation, has been identified as the most affine STIRUR 41 target. As further demonstrated by in vitro and in-cell assays, STIRUR 41 was able to inhibit both the enzymatic activity of USP-7 and its expression levels in neuroblastoma-related cells, thus laying an encouraging base for the blockade of USP-7 downstream signaling.

A multidisciplinary functional proteomics-aided strategy as a tool for the profiling of a novel cytotoxic thiadiazolopyrimidone.

In recent years, thiadiazolopyrimidine derivatives have been acknowledged for their striking poly-pharmacological framework, thus representing an interesting scaffold for the development of new therapeutic candidates. This paper examines the synthesis and the interactome characterization of a novel bioactive thiadiazolopyrimidone (compound 1), endowed with cytotoxic activity on HeLa cancer cells. In detail, starting from a small set of synthesized thiadiazolopyrimidones, a multi-disciplinary strategy has been carried out on the most bioactive one to disclose its potential biological targets by functional proteomics, using a label-free mass spectrometry based platform coupling Drug Affinity Responsive Target Stability and targeted Limited Proteolysis-Multiple Reaction Monitoring. The identification of Annexin A6 (ANXA6) as compound 1 most reliable cellular partner paved the way to deepen the protein-ligand interaction through bio-orthogonal approaches and to prove compound 1 action on migration and invasion processes governed by ANXA6 modulation. The identification of compund 1 as the first ANXA6 protein modulator represents a relevant tool to further explore the biological role of ANXA6 in cancer, as well as to develop novel anticancer candidates.

Discovery of BAR502, as potent steroidal antagonist of leukemia inhibitory factor receptor for the treatment of pancreatic adenocarcinoma.

Introduction: The leukemia inhibitory factor (LIF), is a cytokine belonging to IL-6 family, whose overexpression correlate with poor prognosis in cancer patients, including pancreatic ductal adenocarcinoma (PDAC). LIF signaling is mediate by its binding to the heterodimeric LIF receptor (LIFR) complex formed by the LIFR receptor and Gp130, leading to JAK1/STAT3 activation. Bile acids are steroid that modulates the expression/activity of membrane and nuclear receptors, including the Farnesoid-X-Receptor (FXR) and G Protein Bile Acid Activated Receptor (GPBAR1). Methods: Herein we have investigated whether ligands to FXR and GPBAR1 modulate LIF/LIFR pathway in PDAC cells and whether these receptors are expressed in human neoplastic tissues. Results: The transcriptome analysis of a cohort of PDCA patients revealed that expression of LIF and LIFR is increased in the neoplastic tissue in comparison to paired non-neoplastic tissues. By in vitro assay we found that both primary and secondary bile acids exert a weak antagonistic effect on LIF/LIFR signaling. In contrast, BAR502 a non-bile acid steroidal dual FXR and GPBAR1 ligand, potently inhibits binding of LIF to LIFR with an IC50 of 3.8 µM. Discussion: BAR502 reverses the pattern LIF-induced in a FXR and GPBAR1 independent manner, suggesting a potential role for BAR502 in the treatment of LIFR overexpressing-PDAC.

Combinatorial therapy with BAR502 and UDCA resets FXR and GPBAR1 signaling and reverses liver histopathology in a model of NASH.

Non-alcoholic steatosis (NAFLD) and steatohepatitis (NASH) are two highly prevalent human disorders for which therapy remains suboptimal. Bile acids are signaling molecules acting on two main receptors the Farnesoid-x-receptor (FXR) and G protein coupled receptor GPB AR1. Clinical trials have shown that FXR agonism might result in side effects along with lack of efficacy in restoring liver histopathology. For these reasons a multi-targets therapy combined FXR agonists with agent targeting additional molecular mechanisms might have improved efficacy over selective FXR agonists. In the present study we have compared the effects of BAR502, a dual FXR/GPBAR1 ligand) alone or in combination with ursodeoxycholic acid (UDCA) in a model of NAFLD/NASH induced by feeding mice with a Western diet for 10 weeks. The results demonstrated that while BAR502 and UDCA partially protected against liver damage caused by Western diet, the combination of the two, reversed the pro-atherogenic lipid profile and completely reversed the histopathology damage, attenuating liver steatosis, ballooning, inflammation and fibrosis. Additionally, while both agents increased insulin sensitivity and bile acid signaling, the combination of the two, modulated up top 85 genes in comparison of mice feed a Western diet, strongly reducing expression of inflammatory markers such as chemokines and cytokines. Additionally, the combination of the two agents redirected the bile acid metabolism toward bile acid species that are GPBAR1 agonist while reduced liver bile acid content and increased fecal excretion. Together, these data, highlight the potential role for a combinatorial therapy based on BAR502 and UDCA in treating of NAFLD.

Curcumin Has Beneficial Effects on Lysosomal Alpha-Galactosidase: Potential Implications for the Cure of Fabry Disease.

Fabry disease is a lysosomal storage disease caused by mutations in the GLA gene that encodes alpha-galactosidase (AGAL). The disease causes abnormal globotriaosylceramide (Gb3) storage in the lysosomes. Variants responsible for the genotypic spectrum of Fabry disease include mutations that abolish enzymatic activity and those that cause protein instability. The latter can be successfully treated with small molecules that either bind and stabilize AGAL or indirectly improve its cellular activity. This paper describes the first attempt to reposition curcumin, a nutraceutical, to treat Fabry disease. We tested the efficacy of curcumin in a cell model and found an improvement in AGAL activity for 80% of the tested mutant genotypes (four out of five tested). The fold-increase was dependent on the mutant and ranged from 1.4 to 2.2. We produced evidence that supports a co-chaperone role for curcumin when administered with AGAL pharmacological chaperones (1-deoxygalactonojirimycin and galactose). The combined treatment with curcumin and either pharmacological chaperone was beneficial for four out of five tested mutants and showed fold-increases ranging from 1.1 to 2.3 for DGJ and from 1.1 to 2.8 for galactose. Finally, we tested a long-term treatment on one mutant (L300F) and detected an improvement in Gb3 clearance and lysosomal markers (LAMP-1 and GAA). Altogether, our findings confirmed the necessity of personalized therapies for Fabry patients and paved the way to further studies and trials of treatments for Fabry disease.

Repositioning Mifepristone as a Leukaemia Inhibitory Factor Receptor Antagonist for the Treatment of Pancreatic Adenocarcinoma.

Pancreatic cancer is a leading cause of cancer mortality and is projected to become the second-most common cause of cancer mortality in the next decade. While gene-wide association studies and next generation sequencing analyses have identified molecular patterns and transcriptome profiles with prognostic relevance, therapeutic opportunities remain limited. Among the genes that are upregulated in pancreatic ductal adenocarcinomas (PDAC), the leukaemia inhibitory factor (LIF), a cytokine belonging to IL-6 family, has emerged as potential therapeutic candidate. LIF is aberrantly secreted by tumour cells and promotes tumour progression in pancreatic and other solid tumours through aberrant activation of the LIF receptor (LIFR) and downstream signalling that involves the JAK1/STAT3 pathway. Since there are no LIFR antagonists available for clinical use, we developed an in silico strategy to identify potential LIFR antagonists and drug repositioning with regard to LIFR antagonists. The results of these studies allowed the identification of mifepristone, a progesterone/glucocorticoid antagonist, clinically used in medical abortion, as a potent LIFR antagonist. Computational studies revealed that mifepristone binding partially overlapped the LIFR binding site. LIF and LIFR are expressed by human PDAC tissues and PDAC cell lines, including MIA-PaCa-2 and PANC-1 cells. Exposure of these cell lines to mifepristone reverses cell proliferation, migration and epithelial mesenchymal transition induced by LIF in a concentration-dependent manner. Mifepristone inhibits LIFR signalling and reverses STAT3 phosphorylation induced by LIF. Together, these data support the repositioning of mifepristone as a potential therapeutic agent in the treatment of PDAC.

Role of Intracellular and Extracellular Annexin A1 in MIA PaCa-2 Spheroids Formation and Drug Sensitivity.

Among solid tumors, pancreatic cancer (PC) remains a leading cause of death. In PC, the protein ANXA1 has been identified as an oncogenic factor acting in an autocrine/paracrine way, and also as a component of tumor-deriving extracellular vesicles. Here, we proposed the experimental protocol to obtain spheroids from the two cell lines, wild-type (WT) and Annexin A1 (ANXA1) knock-out (KO) MIA PaCa-2, this last previously obtained through CRISPR/Cas9 genome editing system. The use of three-dimensional (3D) models, like spheroids, can be useful to mimic tumor characteristics and for preclinical chemo-sensitivity studies. By using PC spheroids, we have assessed the activity of intracellular and extracellular ANXA1. Indeed, we have proved that the intracellular protein influences in vitro tumor development and growth by spheroids analysis, in addition to defining the modification about cell protein pattern in ANXA1 KO model compared to the WT one. Moreover, we have tested the response to FOLFIRINOX chemotherapy regimen whose cytostatic effect appeared notably increased in ANXA1 KO spheroids. Additionally, this study has highlighted that the extracellular ANXA1 action is strengthened through the EVs supporting spheroids growth and resistance to drug treatment, mainly affecting tumor progression. Thus, our data interestingly suggest the relevance of ANXA1 as a potential therapeutic PC marker.

Drug affinity-responsive target stability unveils filamins as biological targets for artemetin, an anti-cancer flavonoid.

Artemetin is a valuable 5-hydroxy-3,6,7,3',4'-pentamethoxyflavone present in many different medicinal plants with very good oral bioavailability and drug-likeness values, owing to numerous bioactivities, such as anti-inflammatory and anti-cancer ones. Here, a multi-disciplinary plan has been settled and applied for identifying the artemetin target(s) to inspect its mechanism of action, based on drug affinity-responsive target stability and targeted limited proteolysis. Both approaches point to the disclosure of filamins A and B as direct artemetin targets in HeLa cell lysates, also giving detailed insights into the ligand/protein-binding sites. Interestingly, also 8-prenyl-artemetin, which is an artemetin more permeable semisynthetic analog, directly interacts with filamins A and B. Both compounds alter filamin conformation in living HeLa cells with an effect on cytoskeleton disassembly and on the disorganization of the F-actin filaments. Both the natural compound and its derivative are able to block cell migration, expectantly acting on tumor metastasis occurrence and development.

Beneficial effects of UDCA and norUDCA in a rodent model of steatosis are linked to modulation of GPBAR1/FXR signaling.

Non-alcoholic steatosis (NAFLD) and steatohepatitis (NASH) are two highly prevalent human disorders for which therapy remains suboptimal. Bile acids play an essential role in regulating liver metabolism, and several bile acids-based therapy are currently investigated for their potential therapeutic efficacy in NAFLD/NASH. Bile acids exert their functions, at least in part, by modulating two main receptors the Farnesoid-x-receptor (FXR) and the G protein-coupled receptor, GPBAR1. In the present study we have compared the pharmacological effects of two bile acids, the ursodeoxycholic acid (UDCA) and its derivative norUDCA, in a model of NAFLD/NASH induced by feeding mice with a Western diet for 12 weeks. The results of these studies demonstrated that both UDCA and norUDCA protected against development of steatosis and fibrosis, but did not reduce the hepatocytes ballooning nor the development of a pro-atherogenic lipid profile. Both agents reduced liver lipogenesis and ameliorated insulin sensitivity and adipocytes signaling as shown by increased expression of adiponectin. Mechanistically, UDCA acts as weak GPBAR1 agonist, while norUDCA exerted no effect on both GPBAR1 and FXR. In vivo administration of UDCA resets bile acid synthesis and promotes a shift toward bile acids species that are GPBAR1 agonists, UDCA, TUDCA and hyodeoxycholic acid, and increases GLP1 expression in the ileum. In contrast norUDCA is poorly metabolized exerting a minimal impact on GPBAR1 signaling. Together, these data, highlight the potential role of UDCA and norUDCA in treating of NAFLD, though these beneficial effects are supported by different mechanisms.

Drug Repositioning for Fabry Disease: Acetylsalicylic Acid Potentiates the Stabilization of Lysosomal Alpha-Galactosidase by Pharmacological Chaperones.

Fabry disease is caused by a deficiency of lysosomal alpha galactosidase and has a very large genotypic and phenotypic spectrum. Some patients who carry hypomorphic mutations can benefit from oral therapy with a pharmacological chaperone. The drug requires a very precise regimen because it is a reversible inhibitor of alpha-galactosidase. We looked for molecules that can potentiate this pharmacological chaperone, among drugs that have already been approved for other diseases. We tested candidate molecules in fibroblasts derived from a patient carrying a large deletion in the gene GLA, which were stably transfected with a plasmid expressing hypomorphic mutants. In our cell model, three drugs were able to potentiate the action of the pharmacological chaperone. We focused our attention on one of them, acetylsalicylic acid. We expect that acetylsalicylic acid can be used in synergy with the Fabry disease pharmacological chaperone and prolong its stabilizing effect on alpha-galactosidase.

Exosomal CD73 from serum of patients with melanoma suppresses lymphocyte functions and is associated with therapy resistance to anti-PD-1 agents.

BACKGROUND: CD73 is an ectonucleotidase producing the immunosuppressor mediator adenosine. Elevated levels of circulating CD73 in patients with cancer have been associated with disease progression and poor response to immunotherapy. Immunosuppressive pathways associated with exosomes can affect T-cell function and the therapeutic efficacy of anti-programmed cell-death protein 1 (anti-PD-1) therapy. Here, we conducted a retrospective pilot study to evaluate levels of exosomal CD73 before and early during treatment with anti-PD-1 agents in patients with melanoma and its potential contribution to affect T-cell functions and to influence the clinical outcomes of anti-PD-1 monotherapy. METHODS: Exosomes were isolated by mini size exclusion chromatography from serum of patients with melanoma (n=41) receiving nivolumab or pembrolizumab monotherapy. Expression of CD73 and programmed death-ligand 1 (PD-L1) were evaluated on exosomes enriched for CD63 by on-bead flow cytometry. The CD73 AMPase activity was evaluated by mass spectrometry, also in the presence of selective inhibitors of CD73. Interferon (IFN)-γ production and granzyme B expression were measured in CD3/28 activated T cells incubated with exosomes in presence of the CD73 substrate AMP. Levels of CD73 and PD-L1 on exosomes were correlated with therapy response. Exosomes isolated from healthy subjects were used as control. RESULTS: Isolated exosomes carried CD73 on their surface, which is enzymatically active in producing adenosine. Incubation of exosomes with CD3/28 activated T cells in the presence of AMP resulted in a significant reduction of IFN-γ release, which was reversed by the CD73 inhibitor APCP or by the selective A2A adenosine receptor antagonist ZM241385. Expression levels of exosomal CD73 from serum of patients with melanoma were not significantly different from those in healthy subjects. Early on-treatment, expression levels of both CD73 and PD-L1 on exosomes isolated from patients receiving pembrolizumab or nivolumab monotherapy were significantly increased compared with baseline. Early during therapy exosomal PD-L1 increased in responders, while exosomal CD73 resulted significantly increased in non-responders. CONCLUSIONS: CD73 expressed on exosomes from serum of patients with melanoma produces adenosine and contributes to suppress T-cell functions. Early on-treatment, elevated expression levels of exosomal CD73 might affect the response to anti-PD-1 agents in patients with melanoma who failed to respond to therapy.

Label-Free Quantitative Proteomics to Explore the Action Mechanism of the Pharmaceutical-Grade Triticum vulgare Extract in Speeding Up Keratinocyte Healing.

Plant extracts have shown beneficial properties in terms of skin repair, promoting wound healing through a plethora of mechanisms. In particular, the poly-/oligosaccharidic aqueous extract of Triticum vulgare (TVE), as well as TVE-based products, shows interesting biological assets, hastening wound repair. Indeed, TVE acts in the treatment of tissue regeneration mainly on decubitus and venous leg ulcers. Moreover, on scratched monolayers, TVE prompts HaCat cell migration, correctly modulating the expression of metalloproteases toward a physiological matrix remodeling. Here, using the same HaCat-based in vitro scratch model, the TVE effect has been investigated thanks to an LFQ proteomic analysis of HaCat secretomes and immunoblotting. Indeed, the unbiased TVE effect on secreted proteins has not yet been fully understood, and it could be helpful to obtain a comprehensive picture of its bio-pharmacological profile. It has emerged that TVE treatment induces significant up-regulation of several proteins in the secretome (153 to be exact) whereas only a few were down-regulated (72 to be exact). Interestingly, many of the up-regulated proteins are implicated in promoting wound-healing-related processes, such as modulating cell-cell interaction and communication, cell proliferation and differentiation, and prompting cell adhesion and migration.

Atorvastatin protects against liver and vascular damage in a model of diet induced steatohepatitis by resetting FXR and GPBAR1 signaling.

Farnesoid-x-receptor (FXR) agonists, currently trialed in patients with non-alcoholic steatosis (NAFLD), worsen the pro-atherogenic lipid profile and might require a comedication with statin. Here we report that mice feed a high fat/high cholesterol diet (HFD) are protected from developing a pro-atherogenic lipid profile because their ability to dispose cholesterol through bile acids. This protective mechanism is mediated by suppression of FXR signaling in the liver by muricholic acids (MCAs) generated in mice from chenodeoxycholic acid (CDCA). In contrast to CDCA, MCAs are FXR antagonists and promote a CYP7A1-dependent increase of bile acids synthesis. In mice feed a HFD, the treatment with obeticholic acid, a clinical stage FXR agonist, failed to improve the liver histopathology while reduced Cyp7a1 and Cyp8b1 genes expression and bile acids synthesis and excretion. In contrast, treating mice with atorvastatin mitigated liver and vascular injury caused by the HFD while increased the bile acids synthesis and excretion. Atorvastatin increased the percentage of 7α-dehydroxylase expressing bacteria in the intestine promoting the formation of deoxycholic acid and litocholic acid, two GPBAR1 agonists, along with the expression of GPBAR1-regulated genes in the white adipose tissue and colon. In conclusion, present results highlight the central role of bile acids in regulating lipid and cholesterol metabolism in response to atorvastatin and provide explanations for limited efficacy of FXR agonists in the treatment of NAFLD.

The Pyrazolyl-Urea Gege3 Inhibits the Activity of ANXA1 in the Angiogenesis Induced by the Pancreatic Cancer Derived EVs.

The pyrazolyl-urea Gege3 molecule has shown interesting antiangiogenic effects in the tumor contest. Here, we have studied the role of this compound as interfering with endothelial cells activation in response to the paracrine effects of annexin A1 (ANXA1), known to be involved in promoting tumor progression. ANXA1 has been analyzed in the extracellular environment once secreted through microvesicles (EVs) by pancreatic cancer (PC) cells. Particularly, Gege3 has been able to notably prevent the effects of Ac2-26, the ANXA1 mimetic peptide, and of PC-derived EVs on endothelial cells motility, angiogenesis, and calcium release. Furthermore, this compound also inhibited the translocation of ANXA1 to the plasma membrane, otherwise induced by the same ANXA1-dependent extracellular stimuli. Moreover, these effects have been mediated by the indirect inhibition of protein kinase Cα (PKCα), which generally promotes the phosphorylation of ANXA1 on serine 27. Indeed, by the subtraction of intracellular calcium levels, the pathway triggered by PKCα underwent a strong inhibition leading to the following impediment to the ANXA1 localization at the plasma membrane, as revealed by confocal and cytofluorimetry analysis. Thus, Gege3 appeared an attractive molecule able to prevent the paracrine effects of PC cells deriving ANXA1 in the tumor microenvironment.