Lathyrus sativus diamine oxidase counteracts histamine-induced cell proliferation, migration and pro-angiogenic mediators release in human colon adenocarcinoma cell line Caco-2.

Because histamine is a modulator of cancer cell proliferation and invasiveness, this study aimed at investigating the effect of Lathyrus sativus-derived diamine oxidase (LSAO) and its mechanism of action on Caco-2 cell line, considering that LSAO catalizes the oxidative deamination of histamine to the corresponding aldehyde, NH3 and H2 O2 . Histamine (0.01-1 µM) caused a proliferative effect on Caco-2 cells promoting cell migration, invasion and nitric oxide and vascular endothelial growth factor release. Histamine (1 µM) stimulus also down regulated occludin expression, favouring up regulation of pro-proliferative nuclear protein Ki67. Incubation with LSAO (0.004-0.4 µM) resulted in a significant inhibition of histamine-induced effects. LSAO rescued occludin expression and down regulated Ki67, and it inhibited histamine-induced increase of both MMP-2 and 9 expression. Histamine effects were mediated by RhoA-GTP down regulation and inversely related to phospho-p38MAPK/p50/65 up regulation. These effects were counteracted by LSAO incubation. Histamine catabolism by LSAO accounts for a significant down regulation of proliferation and invasiveness of Caco-2 cells. This study highlights the importance to control histamine levels in contrasting pro-angiogenic and metastatization capability of colon cancer cells and expands the knowledge about the diamine oxidase from L. sativus seeding as a phytotherapeutic approach for colon cancer.

HIV-1 Tat-induced diarrhea is improved by the PPARalpha agonist, palmitoylethanolamide, by suppressing the activation of enteric glia.

BACKGROUND: Diarrhea is a severe complication in HIV-1-infected patients with Trans-activator of transcription (HIV-1 Tat) protein being recognized as a major underlying cause. Beside its direct enterotoxic effects, Tat protein has been recently shown to affect enteric glial cell (EGC) activity. EGCs regulate intestinal inflammatory responses by secreting pro-inflammatory molecules; nonetheless, they might also release immune-regulatory factors, as palmytoilethanolamide (PEA), which exerts anti-inflammatory effects by activating PPARα receptors. We aimed at clarifying whether EGCs are involved in HIV-1 Tat-induced diarrhea and if PEA exerts antidiarrheal activity. METHODS: Diarrhea was induced by intracolonic administration of HIV-1 Tat protein in rats at day 1. PEA alone or in the presence of peroxisome proliferator-activated receptor (PPAR) antagonists was given intraperitoneally from day 2 to day 7. S100B, iNOS, NF-kappaB, TLR4 and GFAP expression were evaluated in submucosal plexi, while S100B and NO levels were measured in EGC submucosal plexi lysates, respectively. To verify whether PEA effects were PPARα-mediated, PPARα-/- mice were also used. After 7 days from diarrhea induction, endogenous PEA levels were measured in submucosal plexi homogenates deriving from rats and PPARα-/- mice. RESULTS: HIV-1 Tat protein induced rapid onset diarrhea alongside with a significant activation of EGCs. Tat administration significantly increased all hallmarks of neuroinflammation by triggering TLR4 and NF-kappaB activation and S100B and iNOS expression. Endogenous PEA levels were increased following HIV-1 Tat exposure in both wildtype and knockout animals. In PPARα-/- mice, PEA displayed no effects. In wildtype rats, PEA, via PPARα-dependent mechanism, resulted in a significant antidiarrheal activity in parallel with marked reduction of EGC-sustained neuroinflammation. CONCLUSIONS: EGCs mediate HIV-1 Tat-induced diarrhea by sustaining the intestinal neuroinflammatory response. These effects are regulated by PEA through a selective PPARα-dependent mechanism. PEA might be considered as an adjuvant therapy in HIV-1-induced diarrhea.

Endocannabinoid-related compounds in gastrointestinal diseases.

The endocannabinoid system (ECS) is an endogenous signalling pathway involved in the control of several gastrointestinal (GI) functions at both peripheral and central levels. In recent years, it has become apparent that the ECS is pivotal in the regulation of GI motility, secretion and sensitivity, but endocannabinoids (ECs) are also involved in the regulation of intestinal inflammation and mucosal barrier permeability, suggesting their role in the pathophysiology of both functional and organic GI disorders. Genetic studies in patients with irritable bowel syndrome (IBS) or inflammatory bowel disease have indeed shown significant associations with polymorphisms or mutation in genes encoding for cannabinoid receptor or enzyme responsible for their catabolism, respectively. Furthermore, ongoing clinical trials are testing EC agonists/antagonists in the achievement of symptomatic relief from a number of GI symptoms. Despite this evidence, there is a lack of supportive RCTs and relevant data in human beings, and hence, the possible therapeutic application of these compounds is raising ethical, political and economic concerns. More recently, the identification of several EC-like compounds able to modulate ECS function without the typical central side effects of cannabino-mimetics has paved the way for emerging peripherally acting drugs. This review summarizes the possible mechanisms linking the ECS to GI disorders and describes the most recent advances in the manipulation of the ECS in the treatment of GI diseases.

Cannabidiol restores intestinal barrier dysfunction and inhibits the apoptotic process induced by Clostridium difficile toxin A in Caco-2 cells.

BACKGROUND: Clostridium difficile toxin A is responsible for colonic damage observed in infected patients. Drugs able to restore Clostridium difficile toxin A-induced toxicity have the potential to improve the recovery of infected patients. Cannabidiol is a non-psychotropic component of Cannabis sativa, which has been demonstrated to protect enterocytes against chemical and/or inflammatory damage and to restore intestinal mucosa integrity. OBJECTIVE: The purpose of this study was to evaluate (a) the anti-apoptotic effect and (b) the mechanisms by which cannabidiol protects mucosal integrity in Caco-2 cells exposed to Clostridium difficile toxin A. METHODS: Caco-2 cells were exposed to Clostridium difficile toxin A (30 ng/ml), with or without cannabidiol (10-7-10-9 M), in the presence of the specific antagonist AM251 (10-7 M). Cytotoxicity assay, transepithelial electrical resistence measurements, immunofluorescence analysis and immunoblot analysis were performed in the different experimental conditions. RESULTS: Clostridium difficile toxin A significantly decreased Caco-2 cells' viability and reduced transepithelial electrical resistence values and RhoA guanosine triphosphate (GTP), bax, zonula occludens-1 and occludin protein expression, respectively. All these effects were significantly and concentration-dependently inhibited by cannabidiol, whose effects were completely abolished in the presence of the cannabinoid receptor type 1 (CB1) antagonist, AM251. CONCLUSIONS: Cannabidiol improved Clostridium difficile toxin A-induced damage in Caco-2 cells, by inhibiting the apoptotic process and restoring the intestinal barrier integrity, through the involvement of the CB1 receptor.

HIV-1 Tat-induced diarrhea evokes an enteric glia-dependent neuroinflammatory response in the central nervous system.

Despite the effectiveness of combined anti-retroviral therapy, human immunodeficiency virus (HIV) infected-patients frequently report diarrhea and neuropsychological deficits. It is claimed that the viral HIV-1 Trans activating factor (HIV-1 Tat) protein is responsible for both diarrhea and neurotoxic effects, but the underlying mechanisms are not known. We hypothesize that colonic application of HIV-1 Tat activates glial cells of the enteric nervous system (EGCs), leading to a neuroinflammatory response able to propagate to the central nervous system. We demonstrated that HIV-1 Tat-induced diarrhea was associated with a significant activation of glial cells within the colonic wall, the spinal cord and the frontal cortex, and caused a consistent impairment of the cognitive performances. The inhibition of glial cells activity by lidocaine, completely abolished the above-described effects. These observations point out the role of glial cells as putative effectors in HIV-1 Tat-associated gastrointestinal and neurological manifestations and key regulators of gut-brain signaling.

Rifaximin Improves Clostridium difficile Toxin A-Induced Toxicity in Caco-2 Cells by the PXR-Dependent TLR4/MyD88/NF-κB Pathway.

BACKGROUND: Clostridium difficile infections (CDIs) caused by Clostridium difficile toxin A (TcdA) lead to severe ulceration, inflammation and bleeding of the colon, and are difficult to treat. AIM: The study aimed to evaluate the effect of rifaximin on TcdA-induced apoptosis in intestinal epithelial cells and investigate the role of PXR in its mechanism of action. METHODS: Caco-2 cells were incubated with TcdA and treated with rifaximin (0.1-10 µM) with or without ketoconazole (10 µM). The transepithelial electrical resistance (TEER) and viability of the treated cells was determined. Also, the expression of zona occludens-1 (ZO-1), toll-like receptor 4 (TLR4), Bcl-2-associated X protein (Bax), transforming growth factor-ß-activated kinase-1 (TAK1), myeloid differentiation factor 88 (MyD88), and nuclear factor-kappaB (NF-κB) was determined. RESULTS: Rifaximin treatment (0.1, 1.0, and 10 µM) caused a significant and concentration-dependent increase in the TEER of Caco-2 cells (360, 480, and 680% vs. TcdA treatment) 24 h after the treatment and improved their viability (61, 79, and 105%). Treatment also concentration-dependently decreased the expression of Bax protein (-29, -65, and -77%) and increased the expression of ZO-1 (25, 54, and 87%) and occludin (71, 114, and 262%) versus TcdA treatment. The expression of TLR4 (-33, -50, and -75%), MyD88 (-29, -60, and -81%) and TAK1 (-37, -63, and -79%) were also reduced with rifaximin versus TcdA treatment. Ketoconazole treatment inhibited these effects. CONCLUSION: Rifaximin improved TcdA-induced toxicity in Caco-2 cells by the PXR-dependent TLR4/MyD88/NF-κB pathway mechanism, and may be useful in the treatment of CDIs.

Rifaximin, a non-absorbable antibiotic, inhibits the release of pro-angiogenic mediators in colon cancer cells through a pregnane X receptor-dependent pathway.

Activation of intestinal human pregnane X receptor (PXR) has recently been proposed as a promising strategy for the chemoprevention of inflammation-induced colon cancer. The present study was aimed at evaluating the effect of rifaximin, a non-absorbable antibiotic, in inhibiting angiogenesis in a model of human colorectal epithelium and investigating the role of PXR in its mechanism of action. Caco-2 cells were treated with rifaximin (0.1, 1.0 and 10.0 µM) in the presence or absence of ketoconazole (10 µM) and assessed for cell proliferation, migration and expression of proliferating cell nuclear antigen (PCNA). The release of vascular endothelial growth factor (VEGF) and nitric oxide (NO), expression of Akt, mechanistic target of rapamycin (mTOR), p38 mitogen activated protein kinases (MAPK), nuclear factor κB (NF-κB) and metalloproteinase-2 and -9 (MMP-2 and -9) were also evaluated. Treatment with rifaximin 0.1, 1.0 and 10.0 µM caused significant and concentration-dependent reduction of cell proliferation, cell migration and PCNA expression in the Caco-2 cells vs. untreated cells. Treatment downregulated VEGF secretion, NO release, VEGFR-2 expression, MMP-2 and MMP-9 expression vs. untreated cells. Rifaximin treatment also resulted in a concentration-dependent decrease in the phosphorylation of Akt, mTOR, p38MAPK and inhibition of hypoxia-inducible factor 1-α (HIF-1α), p70S6K and NF-κB. Ketoconazole (PXR antagonist) treatment inhibited these effects. These findings demonstrated that rifaximin causes PXR-mediated inhibition of angiogenic factors in Caco-2 cell line and may be a promising anticancer tool.

Palmitoylethanolamide Modulates Inflammation-Associated Vascular Endothelial Growth Factor (VEGF) Signaling via the Akt/mTOR Pathway in a Selective Peroxisome Proliferator-Activated Receptor Alpha (PPAR-α)-Dependent Manner.

BACKGROUND AND AIM: Angiogenesis is emerging as a pivotal process in chronic inflammatory pathologies, promoting immune infiltration and prompting carcinogenesis. Ulcerative Colitis (UC) and Crohn's Disease (CD) represent paradigmatic examples of intestinal chronic inflammatory conditions in which the process of neovascularization correlates with the severity and progression of the diseases. Molecules able to target the angiogenesis have thus the potential to synergistically affect the disease course. Beyond its anti-inflammatory effect, palmitoylethanolamide (PEA) is able to reduce angiogenesis in several chronic inflammatory conditions, but no data about its anti-angiogenic activity in colitis have been produced, yet. METHODS: The effects of PEA on inflammation-associated angiogenesis in mice with dextran sulphate sodium (DSS)-induced colitis and in patients with UC were assessed. The release of Vascular Endothelial Growth Factor (VEGF), the hemoglobin tissue content, the expression of CD31 and of phosphatidylinositol 3-kinase/Akt/mammalian-target-of-rapamycin (mTOR) signaling axis were all evaluated in the presence of different concentrations of PEA and concomitant administration of PPAR-α and -γ antagonists. RESULTS: Our results demonstrated that PEA, in a selective peroxisome proliferator activated receptor (PPAR)-α dependent mechanism, inhibits colitis-associated angiogenesis, decreasing VEGF release and new vessels formation. Furthermore, we demonstrated that the mTOR/Akt axis regulates, at least partly, the angiogenic process in IBD and that PEA directly affects this pathway. CONCLUSIONS: Our results suggest that PEA may improve inflammation-driven angiogenesis in colonic mucosa, thus reducing the mucosal damage and potentially affecting disease progression and the shift towards the carcinogenesis.

Palmitoylethanolamide Exerts Antiproliferative Effect and Downregulates VEGF Signaling in Caco-2 Human Colon Carcinoma Cell Line Through a Selective PPAR-α-Dependent Inhibition of Akt/mTOR Pathway.

Palmitoylethanolamide (PEA) is a nutraceutical compound that has been demonstrated to improve intestinal inflammation. We aimed at evaluating its antiproliferative and antiangiogenic effects in human colon adenocarcinoma Caco-2 cell line. Caco-2 cells were treated with increasing concentrations of PEA (0.001, 0.01 and 0.1 µM) in the presence of peroxisome proliferator-activated receptor-a (PPAR-α) or PPAR-γ antagonists. Cell proliferation was evaluated by performing a MTT assay. Vascular endothelial growth factor (VEGF) release was estimated by ELISA, while the expression of VEGF receptor and the activation of the Akt/mammalian target of rapamycin (mTOR) pathway were evaluated by western blot analysis. PEA caused a significant and concentration-dependent decrease of Caco-2 cell proliferation at 48 h. PEA administration significantly reduced in a concentration-dependent manner VEGF secretion and VEGF receptor expression. Inhibition of Akt phosphorylation and a downstream decrease of phospho-mTOR and of p-p70S6K were observed as compared with untreated cells. PPAR-α, but not PPAR-γ antagonist, reverted all effects of PEA. PEA is able to decrease cell proliferation and angiogenesis. The antiangiogenic effect of PEA depends on the specific inhibition of the AkT/mTOR axis, through the activation of PPAR-α pathway. If supported by in vivo models, our data pave the way to PEA co-administration to the current chemotherapeutic regimens for colon carcinoma. Copyright © 2016 John Wiley & Sons, Ltd.