The protective role of Lactobacillus rhamnosus GG postbiotic on the alteration of autophagy and inflammation pathways induced by gliadin in intestinal models.

Celiac disease (CD) is an autoimmune enteropathy caused by an abnormal immune response to gliadin peptides in genetically predisposed individuals. For people with CD, the only available therapy thus far is the lifelong necessity for a gluten-free diet (GFD). Innovative therapies include probiotics and postbiotics as dietary supplements, both of which may benefit the host. Therefore, the present study aimed to investigate the possible beneficial effects of the postbiotic Lactobacillus rhamnosus GG (LGG) in preventing the effects induced by indigested gliadin peptides on the intestinal epithelium. In this study, these effects on the mTOR pathway, autophagic function, and inflammation have been evaluated. Furthermore, in this study, we stimulated the Caco-2 cells with the undigested gliadin peptide (P31-43) and with the crude gliadin peptic-tryptic peptides (PTG) and pretreated the samples with LGG postbiotics (ATCC 53103) (1 × 108). In this study, the effects induced by gliadin before and after pretreatment have also been investigated. The phosphorylation levels of mTOR, p70S6K, and p4EBP-1 were increased after treatment with PTG and P31-43, indicating that the intestinal epithelial cells responded to the gliadin peptides by activating the mTOR pathway. Moreover, in this study, an increase in the phosphorylation of NF-κß was observed. Pretreatment with LGG postbiotic prevented both the activation of the mTOR pathway and the NF-κß phosphorylation. In addition, P31-43 reduced LC3II staining, and the postbiotic treatment was able to prevent this reduction. Subsequently, to evaluate the inflammation in a more complex intestinal model, the intestinal organoids derived from celiac disease patient biopsies (GCD-CD) and controls (CTR) were cultured. Stimulation with peptide 31-43 in the CD intestinal organoids induced NF-κß activation, and pretreatment with LGG postbiotic could prevent it. These data showed that the LGG postbiotic can prevent the P31-43-mediated increase in inflammation in both Caco-2 cells and in intestinal organoids derived from CD patients.

Gliadin Peptide P31-43 Induces mTOR/NFkß Activation and Reduces Autophagy: The Role of Lactobacillus paracasei CBA L74 Postbiotc.

Celiac disease (CD) is an autoimmune disease characterized by an altered immune response stimulated by gliadin peptides that are not digested and cause damage to the intestinal mucosa. The aim of this study was to investigate whether the postbiotic Lactobacillus paracasei (LP) could prevent the action of gliadin peptides on mTOR, autophagy, and the inflammatory response. Most of the experiments performed were conducted on intestinal epithelial cells Caco-2 treated with a peptic-tryptic digest of gliadin (PTG) and P31-43. Furthermore, we pretreated the Caco-2 with the postbiotic LP before treatment with the previously described stimuli. In both cases, we evaluated the levels of pmTOR, p70S6k, and p4EBP-1 for the mTOR pathway, pNFkß, and pERK for inflammation and LC 3 and p62 for autophagy. For autophagy, we also used immunofluorescence analysis. Using intestinal organoids derivate from celiac (CD) patients, we analyzed the effect of gliadin after postbiotic pretreatment with LP on inflammation marker NFkß. Through these experiments, we showed that gliadin peptides are able to induce the increase of the inflammatory response in a more complex model of intestinal epithelial cells. LP postbiotic was able to induce autophagy in Caco-2 cells and prevent gliadin effects. In conclusion, postbiotic pretreatment with LP could be considered for in vivo clinical trials.

NOX4 regulates TGFß-induced proliferation and self-renewal in glioblastoma stem cells.

Glioblastoma (GBM) is the most aggressive and common glioma subtype, with a median survival of 15 months after diagnosis. Current treatments have limited therapeutic efficacy; thus, more effective approaches are needed. The glioblastoma tumoural mass is characterised by a small cellular subpopulation - glioblastoma stem cells (GSCs) - that has been held responsible for glioblastoma initiation, cell invasion, proliferation, relapse and resistance to chemo- and radiotherapy. Targeted therapies against GSCs are crucial, as is understanding the molecular mechanisms that govern the GSCs. Transforming growth factor ß (TGFß) signalling and reactive oxygen species (ROS) production are known to govern and regulate cancer stem cell biology. Among the differentially expressed genes regulated by TGFß in a transcriptomic analysis of two different patient-derived GSCs, we found NADPH oxidase 4 (NOX4) as one of the top upregulated genes. Interestingly, when patient tissues were analysed, NOX4 expression was found to be higher in GSCs versus differentiated cells. A functional analysis of the role of NOX4 downstream of TGFß in several patient-derived GSCs showed that TGFß does indeed induce NOX4 expression and increases ROS production in a NOX4-dependent manner. NOX4 downstream of TGFß regulates GSC proliferation, and NOX4 expression is necessary for TGFß-induced expression of stem cell markers and of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), which in turn controls the cell's antioxidant and metabolic responses. Interestingly, overexpression of NOX4 recapitulates the effects induced by TGFß in GSCs: enhanced proliferation, stemness and NRF2 expression. In conclusion, this work functionally establishes NOX4 as a key mediator of GSC biology.

Inflammation Is Present, Persistent and More Sensitive to Proinflammatory Triggers in Celiac Disease Enterocytes.

Celiac disease (CD) is a chronic inflammatory disease caused by a genetic predisposition to an abnormal T cell-mediated immune response to the gluten in the diet. Different environmental proinflammatory factors can influence and amplify the T cell-mediated response to gluten. The aim of this manuscript was to study the role of enterocytes in CD intestinal inflammation and their response to different proinflammatory factors, such as gliadin and viruses. Intestinal biopsies from CD patients on a gluten-containing (GCD-CD) or a gluten-free diet (GFD-CD) as well as biopsies from potential CD patients (Pot-CD) before the onset of intestinal lesions and controls (CTR) were used to investigate IL-1ß and IL-6 mRNA levels in situ. Organoids from CD patients were used to test the levels of NF-κB, ERK, IL-6, and IL-1ß by Western blot (WB), ELISA, and quantitative PCR. The Toll-like receptor ligand loxoribine (Lox) and gliadin peptide P31-43 were used as proinflammatory stimuli. In CD biopsies inflammation markers IL-1ß and IL-6 were increased in the enterocytes, and also in Pot-CD before the onset of the intestinal lesion and in GFD-CD. The inflammatory markers pNF-κB, pERK, IL-1ß, and IL-6 were increased and persistent in CD organoids; these organoids were more sensitive to P31-43 and Lox stimuli compared with CTR organoids. Taken together, these observations point to constitutive inflammation in CD enterocytes, which are more sensitive to inflammatory stimuli such as food components and viruses.

PTPRK, an EGFR Phosphatase, Is Decreased in CeD Biopsies and Intestinal Organoids.

BACKGROUND & AIMS: Celiac disease (CeD) is an immune-mediated enteropathy triggered in genetically susceptible (HLA-DQ2/8) individuals by a group of wheat proteins and related prolamins from cereals. The celiac intestine is characterized by an inversion of the differentiation/proliferation program of the enterocytes, with an increase in the proliferative compartment and crypt hyperplasia, which are the mechanisms that regulate the increased proliferation in CeD that arenot completely understood.The aim of this study is to understand the role of Protein Tyrosine Phosphatase Receptor Type K (PTPRK), a nodal phosphatase that regulates EGFR activation in the proliferation of the enterocytes from CeD biopsies and organoids. METHODS: The levels of PTPRK were evaluated by RT PCR, western blot (WB) and immunofluorescence techniques in intestinal biopsies and organoids from CeD patients and controls. Additionally, pEGFR and pERK were evaluated by WB and proliferation by BrdU incorporation. PTPRK si-RNA was silenced in CTR organoids and was overexpressed in CeD organoids. RESULTS: PTPRK was reduced in Gluten Containing Diet-Celiac Disease (GCD-CeD) and Potential-Celiac Disease(Pot-CeD) biopsies (p < 0.01-p < 0.05) whereas pEGFR (p < 0.01 p < 0.01), pERK (p < 0.01 p < 0.01) and proliferation were increased. (p < 0.05 p < 0.05) respect to the controls.The CeD organoids reproduced these same alterations. Silencing of PTPRK in CTR organoids increased pEGFR, pERK and proliferation. The overexpression of PTPRK in CeD organoids reduced pEGFR, pERK and proliferation. CONCLUSIONS: modulation of PTPRK levels can reduce or increase pEGFR, pERK and proliferation in CeD or CTR organoids, respectively. The CeD organoids can be a good model to study the mechanisms of the disease.

LXRα limits TGFß-dependent hepatocellular carcinoma associated fibroblast differentiation.

Transforming growth factor ß (TGFß) is deposited in the extracellular space of diverse tissues. Resident fibroblasts respond to TGFß and undergo myofibroblastic differentiation during tissue wound healing and cancer progression. Cancer-associated fibroblasts (CAFs) communicate with tumor cells during cancer progression, under the guidance of TGFß signaling. We report that agonist-activated liver X receptors (LXR) limit the expression of key components of myofibroblast differentiation, including the α-smooth muscle actin (αSMA) gene in liver cancer cells. CAFs derived from hepatocellular carcinoma (HCC) express high αSMA and low LXRα levels, whereas hepatocarcinoma cells exhibit an inverse expression pattern. All hepatoma cells analyzed responded to the LXRα agonist T0901317 by inducing fatty acid synthase (FASN) expression. On the other hand, T0901317 antagonized TGFß-induced fibroblastic marker responses, such as fibronectin and calponin, in a subset of hepatoma cells and all CAFs analyzed. Mechanistically, LXRα antagonized TGFß signaling at the transcriptional level. Smad3 and LXRα were recruited to adjacent DNA motifs of the ACTA2 promoter. Upon cloning the human ACTA2 promoter, we confirmed its transcriptional induction by TGFß stimulation, and LXRα overexpression repressed the promoter activity. Hepatosphere formation by HCC cells was enhanced upon co-culturing with CAFs. T0901317 suppressed the positive effects exerted on hepatosphere growth by CAFs. Taken together, the data suggest that LXRα agonists limit TGFß-dependent CAF differentiation, potentially limiting primary HCC growth.

Snail mediates crosstalk between TGFß and LXRα in hepatocellular carcinoma.

Understanding the complexity of changes in differentiation and cell survival in hepatocellular carcinoma (HCC) is essential for the design of new diagnostic tools and therapeutic modalities. In this context, we have analyzed the crosstalk between transforming growth factor ß (TGFß) and liver X receptor α (LXRα) pathways. TGFß is known to promote cytostatic and pro-apoptotic responses in HCC, and to facilitate mesenchymal differentiation. We here demonstrate that stimulation of the nuclear LXRα receptor system by physiological and clinically useful agonists controls the HCC response to TGFß. Specifically, LXRα activation antagonizes the mesenchymal, reactive oxygen species and pro-apoptotic responses to TGFß and the mesenchymal transcription factor Snail mediates this crosstalk. In contrast, LXRα activation and TGFß cooperate in enforcing cytostasis in HCC, which preserves their epithelial features. LXRα influences Snail expression transcriptionally, acting on the Snail promoter. These findings propose that clinically used LXR agonists may find further application to the treatment of aggressive, mesenchymal HCCs, whose progression is chronically dependent on autocrine or paracrine TGFß.

Somatic Ephrin Receptor Mutations Are Associated with Metastasis in Primary Colorectal Cancer.

The contribution of somatic mutations to metastasis of colorectal cancers is currently unknown. To find mutations involved in the colorectal cancer metastatic process, we performed deep mutational analysis of 676 genes in 107 stages II to IV primary colorectal cancer, of which half had metastasized. The mutation prevalence in the ephrin (EPH) family of tyrosine kinase receptors was 10-fold higher in primary tumors of metastatic colorectal than in nonmetastatic cases and preferentially occurred in stage III and IV tumors. Mutational analyses in situ confirmed expression of mutant EPH receptors. To enable functional studies of EPHB1 mutations, we demonstrated that DLD-1 colorectal cancer cells expressing EPHB1 form aggregates upon coculture with ephrin B1 expressing cells. When mutations in the fibronectin type III and kinase domains of EPHB1 were compared with wild-type EPHB1 in DLD-1 colorectal cancer cells, they decreased ephrin B1-induced compartmentalization. These observations provide a mechanistic link between EPHB receptor mutations and metastasis in colorectal cancer. Cancer Res; 77(7); 1730-40. ©2017 AACR.

Transforming growth factor ß as regulator of cancer stemness and metastasis.

Key elements of cancer progression towards metastasis are the biological actions of cancer stem cells and stromal cells in the tumour microenvironment. Cross-communication between tumour and stromal cells is mediated by secreted cytokines, one of which, the transforming growth factor ß (TGFß), regulates essentially every cell within the malignant tissue. In this article, we focus on the actions of TGFß on cancer stem cells, cancer-associated fibroblasts and immune cells that assist the overall process of metastatic dissemination. We aim at illustrating intricate connections made by various cells in the tumour tissue and which depend on the action of TGFß.

Chemical regulators of epithelial plasticity reveal a nuclear receptor pathway controlling myofibroblast differentiation.

Plasticity in epithelial tissues relates to processes of embryonic development, tissue fibrosis and cancer progression. Pharmacological modulation of epithelial transitions during disease progression may thus be clinically useful. Using human keratinocytes and a robotic high-content imaging platform, we screened for chemical compounds that reverse transforming growth factor ß (TGF-ß)-induced epithelial-mesenchymal transition. In addition to TGF-ß receptor kinase inhibitors, we identified small molecule epithelial plasticity modulators including a naturally occurring hydroxysterol agonist of the liver X receptors (LXRs), members of the nuclear receptor transcription factor family. Endogenous and synthetic LXR agonists tested in diverse cell models blocked α-smooth muscle actin expression, myofibroblast differentiation and function. Agonist-dependent LXR activity or LXR overexpression in the absence of ligand counteracted TGF-ß-mediated myofibroblast terminal differentiation and collagen contraction. The protective effect of LXR agonists against TGF-ß-induced pro-fibrotic activity raises the possibility that anti-lipidogenic therapy may be relevant in fibrotic disorders and advanced cancer.

Translating epithelial mesenchymal transition markers into the clinic: Novel insights from proteomics.

The growing understanding of the molecular mechanisms underlying epithelial-to-mesenchymal transition (EMT) may represent a potential source of clinical markers. Despite EMT drivers have not yet emerged as candidate markers in the clinical setting, their association with established clinical markers may improve their specificity and sensitivity. Mass spectrometry-based platforms allow analyzing multiple samples for the expression of EMT candidate markers, and may help to diagnose diseases or monitor treatment efficiently. This review highlights proteomic approaches applied to elucidate the differences between epithelial and mesenchymal tumors and describes how these can be used for target discovery and validation.

MOF nanoparticles coated by lipid bilayers and their uptake by cancer cells.

We report the synthesis of MOF@lipid nanoparticles as a versatile and powerful novel class of nanocarriers based on metal-organic frameworks (MOFs). We show that the MOF@lipid system can effectively store dye molecules inside the porous scaffold of the MOF while the lipid bilayer prevents their premature release. Efficient uptake of the MOF@lipid nanoparticles by cancer cells makes these nanocarriers promising for drug delivery and diagnostic purposes.

Transforming growth factor ß and bone morphogenetic protein actions in brain tumors.

Members of the transforming growth factor ß (TGF-ß) family are implicated in the biology of several cancers. Here we focus on malignancies of the brain and examine the TGFß and the bone morphogenetic protein (BMP) signaling branches of the family. These pathways exhibit context-dependent actions during tumorigenesis, acting either as tumor suppressors or as pro-tumorigenic agents. In the brain, the TGF-ßs associate with oncogenic development and progression to the more malignant state. Inversely, the BMPs suppress tumorigenic potential by acting as agents that induce tumor cell differentiation. The latter has been best demonstrated in grade IV astrocytomas, otherwise known as glioblastoma multiforme. We discuss how the actions of TGF-ßs and BMPs on cancer stem cells may explain their effects on tumor progression, and try to highlight intricate mechanisms that may link tumor cell differentiation to invasion. The focus on TGF-ß and BMP and their actions in brain malignancies provides a rich territory for mechanistic understanding of tumor heterogeneity and suggests ways for improved therapeutic intervention, currently being addressed by clinical trials.

Lapatinib/Paclitaxel polyelectrolyte nanocapsules for overcoming multidrug resistance in ovarian cancer.

The sonication-assisted layer-by-layer (SLBL) technology was developed to combine necessary factors for an efficient drug-delivery system: (i) control of nanocolloid size within 100 - 300 nm, (ii) high drug content (70% wt), (iii) shell biocompatibility and biodegradability, (iv) sustained controlled release, and (v) multidrug-loaded system. Stable nanocolloids of Paclitaxel (PTX) and lapatinib were prepared by the SLBL method. In a multidrug-resistant (MDR) ovarian cancer cell line, OVCAR-3, lapatinib/PTX nanocolloids mediated an enhanced cell growth inhibition in comparison with the PTX-only treatment. A series of in vitro cell assays were used to test the efficacy of these formulations. The small size and functional versatility of these nanoparticles, combined with their ability to incorporate various drugs, indicates that lapatinib/PTX nanocolloids may have in vivo therapeutic applications.

Drug-loaded polyelectrolyte microcapsules for sustained targeting of cancer cells.

In this review we will overview novel nanotechnological nanocarrier systems for cancer therapy focusing on recent development in polyelectrolyte capsules for targeted delivery of antineoplastic drugs against cancer cells. Biodegradable polyelectrolyte microcapsules (PMCs) are supramolecular assemblies of particular interest for therapeutic purposes, as they can be enzymatically degraded into viable cells, under physiological conditions. Incorporation of small bioactive molecules into nano-to-microscale delivery systems may increase drug's bioavailability and therapeutic efficacy at single cell level giving desirable targeted therapy. Layer-by-layer (LbL) self-assembled PMCs are efficient microcarriers that maximize drug's exposure enhancing antitumor activity of neoplastic drug in cancer cells. They can be envisaged as novel multifunctional carriers for resistant or relapsed patients or for reducing dose escalation in clinical settings.

Procedimento de desenhos de Familia com Estorias (DF-E) Avaliacoes de um Caso Clinico

O objetivo deste trabalho e investigar a eficacia do PROCEDIMENTO DE DESENHOS DE FAMILIA COM ESTORIAS (DF-E), introduzido por TRINCA como um instrumento de investigacao da personalidade. Para isto, realizou-se o confronto de varias avaliacoes independentes do extrato de um mesmo caso clinico contendo material eliciado pelo DF-E, realizadas por psicologos e um grupo de profissionais. A comparacao foi feita a partir do levantamento de topicos, procurando identificar-se os pontos de avaliacao comuns, bem como aqueles que sao especificos a determinada avaliacao. Os resultados indicam razoavel nivel de concordancia entre as avaliacoes, que se revelaram eficazes na compreensao do processo psiquico do cliente.