Effects of histone deacetylase inhibitors Tricostatin A and Quisinostat on tight junction proteins of human lung adenocarcinoma A549 cells and normal lung epithelial cells.

Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for non-small cell lung cancer (NSCLC). However, more preclinical studies of HDAC inhibitors in NSCLC and normal lung epithelial cells are required to evaluate their antitumor activities and mechanisms. The bicellular tight junction molecule claudin-2 (CLDN-2) is highly expressed in lung adenocarcinoma tissues and increase the proliferation of adenocarcinoma cells. Downregulation of the tricellular tight junction molecule angulin-1/LSR induces malignancy via EGF-dependent CLDN-2 and TGF-ß-dependent cellular metabolism in human lung adenocarcinoma cells. In the present study, to investigate the detailed mechanisms of the antitumor activities of HDAC inhibitors in lung adenocarcinoma, human lung adenocarcinoma A549 cells and normal lung epithelial cells were treated with the HDAC inibitors Trichostatin A (TSA) and Quisinostat (JNJ-2648158) with or without TGF-ß. Both HDAC inhibitors increased anguin-1/LSR, decrease CLDN-2, promoted G1 arrest and prevented the migration of A549 cells. Furthermore, TSA but not Quisinostat with or without TGF-ß induced cellular metabolism indicated as the mitochondrial respiration measured using the oxygen consumption rate. In normal human lung epithelial cells, treatment with TSA and Quisinostat increased expression of LSR and CLDN-2 and decreased that of CLDN-1 with or without TGF-ß in 2D culture. Quisinostat but not TSA with TGF-ß increased CLDN-7 expression in 2D culture. Both HDAC inhibitors prevented disruption of the epithelial barrier measured as the permeability of FD-4 induced by TGF-ß in 2.5D culture. TSA and Quisinostat have potential for use in therapy for lung adenocarcinoma via changes in the expression of angulin-1/LSR and CLDN-2.

Δ9-Tetrahydrocannabinol reverses TNFα-induced increase in airway epithelial cell permeability through CB2 receptors.

Despite pharmacological treatment, bronchial hyperresponsiveness continues to deteriorate as airway remodelling persists in airway inflammation. Previous studies have demonstrated that the phytocannabinoid Δ9-tetrahydrocannabinol (THC) reverses bronchoconstriction with an anti-inflammatory action. The aim of this study was to investigate the effects of THC on bronchial epithelial cell permeability after exposure to the pro-inflammatory cytokine, TNFα. Calu-3 bronchial epithelial cells were cultured at air-liquid interface. Changes in epithelial permeability were measured using Transepithelial Electrical Resistance (TEER), then confirmed with a paracellular permeability assay and expression of tight junction proteins by Western blotting. Treatment with THC prevented the TNFα-induced decrease in TEER and increase in paracellular permeability. Cannabinoid CB1 and CB2 receptor-like immunoreactivity was found in Calu-3 cells. Subsequent experiments revealed that pharmacological blockade of CB2, but not CB1 receptor inhibited the THC effect. Selective stimulation of CB2 receptors displayed a similar effect to that of THC. TNFα decreased expression of the tight junction proteins occludin and ZO-1, which was prevented by pre-incubation with THC. These data indicate that THC prevents cytokine-induced increase in airway epithelial permeability through CB2 receptor activation. This highlights that THC, or other cannabinoid receptor ligands, could be beneficial in the prevention of inflammation-induced changes in airway epithelial cell permeability, an important feature of airways diseases.

Gold Nanoparticles Increase Endothelial Paracellular Permeability by Altering Components of Endothelial Tight Junctions, and Increase Blood-Brain Barrier Permeability in Mice.

Gold nanoparticles (Au-NPs) are being increasingly used as constituents in cosmetics, biosensors, bioimaging, photothermal therapy, and targeted drug delivery. This elevated exposure to Au-NPs poses systemic risks in humans, particularly risks associated with the biodistribution of Au-NPs and their potent interaction with biological barriers. We treated human umbilical vein endothelial cells with Au-NPs and comprehensively examined the expression levels of tight junction (TJ) proteins such as occludin, claudin-5, junctional adhesion molecules, and zonula occludens-1 (ZO-1), as well as endothelial paracellular permeability and the intracellular signaling required for TJ organization. Moreover, we validated the effects of Au-NPs on the integrity of TJs in mouse brain microvascular endothelial cells in vitro and obtained direct evidence of their influence on blood-brain barrier (BBB) permeability in vivo. Treatment with Au-NPs caused a pronounced reduction of PKCζ-dependent threonine phosphorylation of occludin and ZO-1, which resulted in the instability of endothelial TJs and led to proteasome-mediated degradation of TJ components. This impairment in the assembly of TJs between endothelial cells increased the permeability of the transendothelial paracellular passage and the BBB. Au-NPs increased endothelial paracellular permeability in vitro and elevated BBB permeability in vivo. Future studies must investigate the direct and indirect toxicity caused by Au-NP-induced endothelial TJ opening and thereby address the double-edged-sword effect of Au-NPs.

Targeting tight junctions during epithelial to mesenchymal transition in human pancreatic cancer.

Pancreatic cancer continues to be a leading cause of cancer-related death worldwide and there is an urgent need to develop novel diagnostic and therapeutic strategies to reduce the mortality of patients with this disease. In pancreatic cancer, some tight junction proteins, including claudins, are abnormally regulated and therefore are promising molecular targets for diagnosis, prognosis and therapy. Claudin-4 and -18 are overexpressed in human pancreatic cancer and its precursor lesions. Claudin-4 is a high affinity receptor of Clostridium perfringens enterotoxin (CPE). The cytotoxic effects of CPE and monoclonal antibodies against claudin-4 are useful as novel therapeutic tools for pancreatic cancer. Claudin-18 could be a putative marker and therapeutic target with prognostic implications for patients with pancreatic cancer. Claudin-1, -7, tricellulin and marvelD3 are involved in epithelial to mesenchymal transition (EMT) of pancreatic cancer cells and thus might be useful as biomarkers during disease. Protein kinase C is closely related to EMT of pancreatic cancer and regulates tight junctions of normal human pancreatic duct epithelial cells and the cancer cells. This review focuses on the regulation of tight junctions via protein kinase C during EMT in human pancreatic cancer for the purpose of developing new diagnostic and therapeutic modalities for pancreatic cancer.

Progressive proximal-to-distal reduction in expression of the tight junction complex in colonic epithelium of virally-suppressed HIV+ individuals.

Effective antiretroviral therapy (ART) dramatically reduces AIDS-related complications, yet the life expectancy of long-term ART-treated HIV-infected patients remains shortened compared to that of uninfected controls, due to increased risk of non-AIDS related morbidities. Many propose that these complications result from translocated microbial products from the gut that stimulate systemic inflammation--a consequence of increased intestinal paracellular permeability that persists in this population. Concurrent intestinal immunodeficiency and structural barrier deterioration are postulated to drive microbial translocation, and direct evidence of intestinal epithelial breakdown has been reported in untreated pathogenic SIV infection of rhesus macaques. To assess and characterize the extent of epithelial cell damage in virally-suppressed HIV-infected patients, we analyzed intestinal biopsy tissues for changes in the epithelium at the cellular and molecular level. The intestinal epithelium in the HIV gut is grossly intact, exhibiting no decreases in the relative abundance and packing of intestinal epithelial cells. We found no evidence for structural and subcellular localization changes in intestinal epithelial tight junctions (TJ), but observed significant decreases in the colonic, but not terminal ileal, transcript levels of TJ components in the HIV+ cohort. This result is confirmed by a reduction in TJ proteins in the descending colon of HIV+ patients. In the HIV+ cohort, colonic TJ transcript levels progressively decreased along the proximal-to-distal axis. In contrast, expression levels of the same TJ transcripts stayed unchanged, or progressively increased, from the proximal-to-distal gut in the healthy controls. Non-TJ intestinal epithelial cell-specific mRNAs reveal differing patterns of HIV-associated transcriptional alteration, arguing for an overall change in intestinal epithelial transcriptional regulation in the HIV colon. These findings suggest that persistent intestinal epithelial dysregulation involving a reduction in TJ expression is a mechanism driving increases in colonic permeability and microbial translocation in the ART-treated HIV-infected patient, and a possible immunopathogenic factor for non-AIDS related complications.

Cholera toxin notches epithelial junctions.

Cholera toxin (CT) is the factor responsible for watery diarrhea associated with Vibrio cholerae infection. In this issue, Guichard et al. (2013) report that CT compromises intestinal epithelium barrier function via cyclic AMP (cAMP)-induced disruption of Rab11- and exocyst-dependent delivery of endocytic recycling cargo to cell-cell junctions.

Cholera toxin disrupts barrier function by inhibiting exocyst-mediated trafficking of host proteins to intestinal cell junctions.

Cholera toxin (CT), a virulence factor elaborated by Vibrio cholerae, is sufficient to induce the severe diarrhea characteristic of cholera. The enzymatic moiety of CT (CtxA) increases cAMP synthesis in intestinal epithelial cells, leading to chloride ion (Cl(-)) efflux through the CFTR Cl(-) channel. To preserve electroneutrality and osmotic balance, sodium ions and water also flow into the intestinal lumen via a paracellular route. We find that CtxA-driven cAMP increase also inhibits Rab11/exocyst-mediated trafficking of host proteins including E-cadherin and Notch signaling components to cell-cell junctions in Drosophila, human intestinal epithelial cells, and ligated mouse ileal loops, thereby disrupting barrier function. Additionally, CtxA induces junctional damage, weight loss, and dye leakage in the Drosophila gut, contributing to lethality from live V. cholerae infection, all of which can be rescued by Rab11 overexpression. These barrier-disrupting effects of CtxA may act in parallel with Cl(-) secretion to drive the pathophysiology of cholera.