Albendazole negatively regulates keratinocyte proliferation

Abstract Background: Increased keratinocyte proliferation occurs in the skin of psoriatic patients and is supposed to play a role in the pathogenesis of this disorder. Compounds interfering with keratinocyte proliferation could be useful in the management of psoriatic patients. Aim: To investigate whether albendazole, an anti-helmintic drug that regulates epithelial cell function in various systems, inhibits keratinocyte proliferation in models of psoriasis. Methods: Aldara-treated mice received daily topical application of albendazole. Keratinocyte proliferation and keratin (K) 6 and K16 expression were evaluated by immunohistochemistry and Western blotting and inflammatory cells/mediators were analysed by immunohistochemistry and real-time PCR. In human keratinocytes (HEKa and HaCaT) treated with albendazole, cell cycle and proliferation, keratins and cell cycle-associated factors were evaluated by flow cytometry, colorimetric assay and Western blotting respectively. Results: Aldara-treated mice given albendazole exhibited reduced epidermal thickness, decreased number of proliferating keratinocytes and K6/K16 expression. Reduction of CD3- and Ly6G-positive cells in the skin of albendazole-treated mice associated with inhibition of IL-6, TNF-α, IL-1β, IL-17A, IL-36, CCL17, CXCL1, CXCL2 and CXCL5 expression. Treatment of keratinocytes with albendazole reduced K6/K16 expression and reversibly inhibited cell growth by promoting accumulation of cells in S-phase. This phenomenon was accompanied by down-regulation of CDC25A, a phosphatase regulating progression of cell cycle through S-phase, and PKR-dependent hyper-phosphorylation of eIF2α, an inhibitor of CDC25 translation. In Aldara-treated mice, albendazole activated PKR, enhanced eIF2α phosphorylation and reduced CDC25A expression. Conclusions: Data show that albendazole inhibits keratinocyte proliferation and exerts therapeutic effect in a murine model of psoriasis

Antisense Oligonucleotide: Basic Concepts and Therapeutic Application in Inflammatory Bowel Disease

Several molecular technologies aimed at regulating gene expression that have been recently developed as a strategy to combat inflammatory and neoplastic diseases. Among these, antisense technology is a specific, rapid, and potentially high-throughput approach for inhibiting gene expression through recognition of cellular RNAs. Advances in the understanding of the molecular mechanisms that drive tissue damage in different inflammatory diseases, including Crohn’s disease (CD) and ulcerative colitis (UC), the two major inflammatory bowel diseases (IBDs) in humans, have facilitated the identification of novel druggable targets and offered interesting therapeutic perspectives for the treatment of patients. This short review provides a comprehensive understanding of the basic concepts underlying the mechanism of action of the oligonucleotide therapeutics, and summarizes the available pre-clinical and clinical data for oligonucleotide-based therapy in IBD

NPD-0414-2 and NPD-0414-24, Two Chemical Entities Designed as Aryl Hydrocarbon Receptor (AhR) Ligands, Inhibit Gut Inflammatory Signals

Defects in counterregulatory mechanisms contribute to amplify the detrimental inflammatory response leading to the pathologic process occurring in the gut of patients with Crohn’s disease (CD) and ulcerative colitis (UC), the major inflammatory bowel diseases (IBDs), in human beings. One such mechanism involves aryl hydrocarbon receptor (AhR), a transcription factor activated by natural and synthetic ligands, which induces the production of interleukin (IL)-22 and down-regulates inflammatory signals. In IBD, AhR expression is down-regulated and its activation by natural ligands promotes clinical and endoscopic benefit. Since the use of AhR natural ligands can associate with serious adverse events, we developed new chemical ligands of AhR and assessed their regulatory effects. Among these derivatives, we selected the compounds NPD-0414-2 and NPD-0414-24, as they displayed the more pronounced capacity to induce IL-22. Peripheral blood mononuclear cells and lamina propria mononuclear cells (LPMC) were isolated from CD and UC patients. Cells were treated in vitro with Ficz, AhR ligands, and AhR antagonist and then cytokines’ expression was evaluated by real-time PCR and flow cytometry. After the induction of TNBS colitis, Ficz and AhR ligands were injected intra-peritoneally to wild type and AhR knock-out mice. After 4 days, mice were sacrificed and colonic tissues were collected for histologic examination and real-time PCR analysis. Treatment of IBD LPMC with NPD-0414-2 and NPD-0414-24 reduced IFN-γ and increased IL-22 transcripts, and these effects were abrogated by CH223191, a specific inhibitor of AhR interaction with its ligands. Mice given NPD-0414-2 and NPD-0414-24 developed a significantly less severe form of TNBS colitis and exhibited reduced expression of IFN-γ and increased expression of IL-22. The therapeutic effect of NPD-0414-2 and NPD-0414-24 on the ongoing colitis was abrogated in AhR-deficient mice. Collectively, these data show that NPD-0414-2 and NPD-0414-24 exert Ahr-dependent regulatory effects in the gut

The Fragile X Mental Retardation Protein Regulates RIPK1 and Colorectal Cancer Resistance to Necroptosis

Background & aims: The fragile X mental retardation protein (FMRP) affects multiple steps of the mRNA metabolism during brain development and in different neoplastic processes. However, the contribution of FMRP in colon carcinogenesis has not been investigated. Methods: FMR1 mRNA transcript and FMRP protein expression were analyzed in human colon samples derived from patients with sporadic colorectal cancer (CRC) and healthy subjects. We used a well-established mouse model of sporadic CRC induced by azoxymethane to determine the possible role of FMRP in CRC. To address whether FMRP controls cancer cell survival, we analyzed cell death pathway in CRC human epithelial cell lines and in patient-derived colon cancer organoids in presence or absence of a specific FMR1 antisense oligonucleotide or siRNA. Results: We document a significant increase of FMRP in human CRC relative to non-tumor tissues. Next, using an inducible mouse model of CRC, we observed a reduction of colonic tumor incidence and size in the Fmr1 knockout mice. The abrogation of FMRP induced spontaneous cell death in human CRC cell lines activating the necroptotic pathway. Indeed, specific immunoprecipitation experiments on human cell lines and CRC samples indicated that FMRP binds receptor-interacting protein kinase 1 (RIPK1) mRNA, suggesting that FMRP acts as a regulator of necroptosis pathway through the surveillance of RIPK1 mRNA metabolism. Treatment of human CRC cell lines and patient-derived colon cancer organoids with the FMR1 antisense resulted in up-regulation of RIPK1. Conclusions: Altogether, these data support a role for FMRP in controlling RIPK1 expression and necroptotic activation in CRC

Protective Effects of Aryl Hydrocarbon Receptor Signaling in Celiac Disease Mucosa and in Poly I:C-Induced Small Intestinal Atrophy Mouse Model

Aryl hydrocarbon receptor (AhR), a transcription factor activated by a large number of natural and synthetic agents, modulates the activity of immune cells in the gut and represents an important link between the environment and immune-mediated pathologies. In this study, we investigated the role of AhR in celiac disease (CD), a gluten-driven enteropathy. AhR expression was evaluated in intestinal biopsies taken from patients with CD and controls by real-time polymerase chain reaction (PCR), immunohistochemistry and flow cytometry. AhR was also analyzed in ex vivo organ cultures of duodenal biopsies taken from inactive CD patients incubated in presence or absence of peptic-tryptic digest of gliadin. IFN-γ, TNF-α, granzyme B, and perforin expression was evaluated in anti-CD3/CD28-activated intestinal lamina propria mononuclear cells (LPMC) and intestinal intra-epithelial cells (IEL) of active CD patients cultured in the presence or absence of the AhR agonist 6-formylindolo(3, 2-b)carbazole (Ficz). Finally, the protective role of AhR was evaluated in a mouse model of poly I:C-driven small intestine damage. AhR RNA transcripts were reduced in active CD samples as compared to inactive CD and normal controls. Flow cytometry confirmed such results and showed a reduction of AhR in both IEL and LPMC of active CD patients. The addition of a peptic-tryptic digest of gliadin to ex vivo organ cultures of duodenal biopsies taken from inactive CD patients reduced AhR expression. Treatment of CD IEL and LPMC with Ficz reduced the levels of inflammatory cytokines, granzyme B and perforin. Mice injected with Ficz were protected against poly I:C-induced intestinal lesions. Our findings suggest that defective AhR-driven signals could contribute to amplify pathogenic responses in the gut of CD patients

A Novel Smad7 Genetic Variant Mapping on the Genomic Region Targeted by Mongersen Is Associated with Crohn's Disease

Down-regulation of Smad7 with a specific Smad7 antisense (AS) oligonucleotide-containing oral drug (Mongersen) was effective in pre-clinical studies and initial clinical trials in Crohn's disease (CD) patients. A recent phase 3 trial was discontinued due to an apparent inefficacy of the drug, but factors contributing to the failure of this study remain unknown. Here, we analysed the frequency in CD of rs144204026 C/T single nucleotide polymorphism (SNP), which maps on the corresponding region targeted by the Smad7 AS contained in the Mongersen formulation and examined whether such a variant allele affects the ability of Smad7 AS to knockdown Smad7

Inflammatory bowel disease: new therapies from antisense oligonucleotides

Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of the gastrointestinal tract encompassing two main clinical entities: Crohn's disease (CD) and ulcerative colitis (UC). These disorders are characterized by various grades of tissue damage and development of local complications and extra-intestinal manifestations. The cause of IBD remains unknown but accumulating evidence indicates that both CD and UC arise in genetically predisposed individuals as a result of the action of multiple environmental factors, which ultimately trigger excessive and poorly controlled immune response against antigens of the luminal flora. Despite this realization, a full understanding of IBD pathogenesis is still out of reach and, consequently, treatment is far from optimal. However, in recent years, several pathways of intestinal damage have been delineated and the improved knowledge has contributed to the development of new therapies. Various approaches have been used to either inhibit the expression and/or function of inflammatory molecules or enhance counter-regulatory mechanisms. This review summarizes the available pre-clinical and clinical data for antisense oligonucleotides and oligonucleotide-based therapy to provide a comprehensive understanding of the rationale and mechanism of action of these compounds in IBD. Key messages Preclinical studies and clinical trials show that antisense oligonucleotide (ASO)-based therapy could be of benefit in inflammatory bowel diseases. ASOs have an excellent safety profile. Technical issues emerged from clinical trials suggest that changes in drug formulation and/or route of administration could improve ASO efficacy

Transforming Growth Factor-β1/Smad7 in Intestinal Immunity, Inflammation, and Cancer

In physiological conditions, the activity of the intestinal immune system is tightly regulated to prevent tissue-damaging reactions directed against components of the luminal flora. Various factors contribute to maintain immune homeostasis and diminished production and/or function of such molecules trigger and/or propagate detrimental signals, which can eventually lead to chronic colitis and colon cancer. One such a molecule is transforming growth factor-β1 (TGF-β1), a cytokine produced by many inflammatory and non-inflammatory cells and targeting virtually all the intestinal mucosal cell types, with the down-stream effect of activating intracellular Smad2/3 proteins and suppressing immune reactions. In patients with inflammatory bowel diseases (IBD), there is defective TGF-β1/Smad signaling due to high Smad7, an inhibitor of TGF-β1 activity. Indeed, knockdown of Smad7 with a specific antisense oligonucleotide restores endogenous TGF-β1 activity, thereby inhibiting inflammatory pathways in patients with IBD and colitic mice. Consistently, mice over-expressing Smad7 in T cells develop severe intestinal inflammation in various experimental models. Smad7 expression is also upregulated in colon cancer cells, in which such a protein controls positively intracellular pathways that sustain neoplastic cell growth and survival. We here review the role of TGF-β1 and Smad7 in intestinal immunity, inflammation, and cancer

Role of TGF-Beta and Smad7 in Gut Inflammation, Fibrosis and Cancer

The human gastrointestinal tract contains the largest population of immune cells in the body and this is a reflection of the fact that it is continuously exposed to a myriad of dietary and bacterial antigens. Although these cells produce a variety of inflammatory cytokines that could potentially promote tissue damage, in normal conditions the mucosal immune response is tightly controlled by counter-regulatory factors, which help induce and maintain gut homeostasis and tolerance. One such factor is transforming growth factor (TGF)-β1, a cytokine produced by multiple lineages of leukocytes, stromal cells and epithelial cells, and virtually targets all the gut mucosal cell types. Indeed, studies in animals and humans have shown that defects in TGF-β1 production and/or signaling can lead to the development of immune-inflammatory pathologies, fibrosis and cancer in the gut. Here, we review and discuss the available evidence about the role of TGF-β1 and Smad7, an inhibitor of TGF-β1 activity, in gut inflammation, fibrosis and cancer with particular regard to the contribution of these two molecules in the pathogenesis of inflammatory bowel diseases and colon cancer