Vittorio Cosenza

Possible endocannabinoid control of colorectal cancer growth

BACKGROUND & AIMS The endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit cancer cell proliferation by acting at cannabinoid receptors (CBRs). We studied (1). the levels of endocannabinoids, cannabinoid CB(1) and CB(2) receptors, and fatty acid amide hydrolase (FAAH, which catalyzes endocannabinoid hydrolysis) in colorectal carcinomas (CRC), adenomatous polyps, and neighboring healthy mucosa; and (2). the effects of endocannabinoids, and of inhibitors of their inactivation, on human CRC cell proliferation. METHODS Tissues were obtained from 21 patients by biopsy during colonoscopy. Endocannabinoids were measured by liquid chromatography-mass spectrometry (LC-MS). CB(1), CB(2), and FAAH expression were analyzed by RT-PCR and Western immunoblotting. CRC cell lines (CaCo-2 and DLD-1) were used to test antiproliferative effects. RESULTS All tissues and cells analyzed contain anandamide, 2-AG, CBRs, and FAAH. The levels of the endocannabinoids are 3- and 2-fold higher in adenomas and CRCs than normal mucosa. Anandamide, 2-AG, and the CBR agonist HU-210 potently inhibit CaCo-2 cell proliferation. This effect is blocked by the CB(1) antagonist SR141716A, but not by the CB(2) antagonist SR144528, and is mimicked by CB(1)-selective, but not CB(2)-selective, agonists. In DLD-1 cells, both CB(1) and CB(2) receptors mediate inhibition of proliferation. Inhibitors of endocannabinoid inactivation enhance CaCo-2 cell endocannabinoid levels and block cell proliferation, this effect being antagonized by SR141716A. CaCo-2 cell differentiation into noninvasive cells results in increased FAAH expression, lower endocannabinoid levels, and no responsiveness to cannabinoids. CONCLUSIONS Endocannabinoid levels are enhanced in transformed colon mucosa cells possibly to counteract proliferation via CBRs. Inhibitors of endocannabinoid inactivation may prove useful anticancer agents.

Up-regulation of anandamide levels as an endogenous mechanism and a pharmacological strategy to limit colon inflammation

Direct stimulation of cannabinoid CB1 receptors exerts a protective function in animal models of inflammatory bowel diseases (IBDs). However, it is not known whether endocannabinoids are up‐regulated during IBDs in animals or humans, nor whether pharmacological elevation of endocannabinoid levels can be exploited therapeutically in these disorders. In this study we addressed these questions. Colon inflammation was induced in mice and rats with 2,4dinitrobenzene‐ and 2,4,6‐trinitrobenzene sulfonic acids (DNBS and TNBS), respectively. DNBS‐treated mice were treated chronically (for 3 or 7 days) with inhibitors of anandamide enzymatic hydrolysis (N‐arachidonoyl‐serotonin, AA‐5‐HT) or reuptake (VDM11), 10 or 5 mg/kg, s.c., or with 5‐amino‐salicilic acid (5‐ASA, 1.4 mg/kg, i.r.). Endocannabinoids (anandamide and 2‐arachidonoylglycerol, 2‐AG) were quantified in mouse colon, or in rat colon mucosa and submucosa, and in bioptic samples from the colon of patients with untreated ulcerative colitis, by liquid chromatography‐mass spectrometry. A strong elevation of anandamide, but not 2‐AG, levels was found in the colon of DNBS‐treated mice, in the colon submucosa of TNBS‐treated rats, and in the biopsies of patients with ulcerative colitis. VDM‐11 significantly elevated anandamide levels in the colon of DNBS‐treated mice and concomitantly abolished inflammation, whereas AA‐5‐HT did not affect endocannabinoid levels and was significantly less efficacious at attenuating colitis. 5‐ASA also increased anandamide levels and abolished colitis. Thus, anandamide is elevated in the inflamed colon of patients with ulcerative colitis, as well as in animal models of IBDs, to control inflammation, and elevation of its levels with inhibitors of its cellular reuptake might be used in the treatment of IBDs.

Direct evidence of oxidative damage in acute and chronic phases of experimental colitis in rats

During inflammatory colitis in man and experimental animals, the production of free radicals increases. This study evaluated the histological pattern and biochemical parameters of oxidative damage during acute and chronic colitis induced by 2,4,-trinitrobenzenesulfonic acid + ethanol in rats. On the samples of scraped mucosa of six groups of rats, one not treated, one killed after 1 hr, and those killed one, two, four, and eight weeks after the induced-damage, we determined the histological and superoxide dismutase activity and the concentration of lipoperoxides, malonyldialdheyde, and reduced glutathione. After 1 hr, the mucosal damage and superoxide dismutase activity were slight; glutathione, lipoperoxides, and malonyldialdheyde were significantly increased. At one week, the histological damage was severe, decreasing progressively, and significantly correlated to superoxide dismutase activity. Lipoperoxides and malonyldialdheyde were high throughout the study. Glutathione was significantly increased at one and two weeks and dramatically decreased thereafter. Therefore, in experimental colitis the cascade of free-radical production induces a constant self-maintaining lipoperoxidation and consumes the cellular antioxidant capability.

Butyrate, mesalamine, and factor XIII in experimental colitis in the rat: Effects on transglutaminase activity

BACKGROUND/AIMS Butyrate and factor XIII may improve ulcerative colitis; they also affect tissue and serum transglutaminase levels. We investigated the therapeutic potential of sodium butyrate and factor XIII and the role of transglutaminase during mucosal repair in experimental colitis. METHODS Rats with induced colitis were treated with sodium butyrate, mesalamine, sodium butyrate plus mesalamine, or saline enemas. Thromboxane B2 was monitored as index of inflammation. In a fifth group, the effectiveness of intravenous Factor XIII was assessed. RESULTS Sodium butyrate, alone or plus mesalamine, reduced histological activity from 13.7 +/- 1.7 (saline) to 2.5 +/- 1.3 and 2.3 +/- 1.1 (P < 0.01), respectively. Transglutaminase, reduced in the colons of the saline group (783 +/- 157 vs. normal 1800 +/- 192 mU/g; P < 0.01), returned toward normal values in the sodium butyrate or sodium butyrate plus mesalamine groups (1390 +/- 228 and 1226 +/- 172 mU/g, respectively; P < 0.01 vs. saline). Furthermore, sodium butyrate plus mesalamine reduced thromboxane B2 levels by day 5 (0.92 +/- 0.16 vs. saline 1.85 +/- 0.34 ng/mL; P < 0.05). Factor XIII therapy improved the histological picture (2.7 +/- 2.1 vs. saline 13.8 +/- 1.7; P < 0.01) and increased transglutaminase levels both in serum (2.81 +/- 0.11 vs. saline 1.45 +/- 0.09 mU/mL; P < 0.01) and in colon (1503 +/- 127 vs. saline 747 +/- 103). CONCLUSIONS Sodium butyrate and factor XIII improve colitis, sodium butyrate plus mesalamine reduce early thromboxane B2 synthesis, and transglutaminase(s) plays a role in ulcer healing.

Glutathione supplementation improves oxidative damage in experimental colitis

BACKGROUND The pathogenesis of inflammatory bowel disease is due, in part, to enhanced free-radical production and reduced antioxidant potential in mucosa cells. AIM We evaluated in a rat model of trinitrobenzensulphonic acid (TNBS) colitis to see whether parenteral administration of glutathione is able to improve mucosal oxidative damage at onset (study A) and during chronic phases of colitis (study B). METHODS In study A, the rats were injected with a single dose of glutathione (200 mg/kg, i.p.) or saline (0,2 ml, i.p.) 1 h before colitis induction and killed 1 h later. In study B, rats with induced colitis were treated with daily injection of glutathione (50 mg/kg, i.p.) or saline (0,2 ml, i.p.), and killed at 1, 2, 4 and 8 weeks. We evaluated on mucosal samples the macroscopic and histological damage and the oxidative stress assessed by the mucosal levels of lipoperoxides, malonyldialdehyde, glutathione and cysteine. RESULTS In study A, colitis induction caused a significant increase to the total histological score (p<0.05), lipoperoxide and malonyldialdehyde levels (p<0.001), but did not affect glutathione and cysteine content. Glutathione pre-treatment decreased both total histological score (p<0.05) and lipoperoxide and malonyldialdehyde values (p<0.001). In study B, the extensive macroscopic and histological colonic damage induced by TNBS was accompanied by a reduction of glutathione and cysteine mucosal levels (p<0.01) and increased lipid peroxidation. Glutathione supplementation significantly improved colonic damage (p<0.01), restored glutathione and cysteine levels, and decreased, and even, if not totally, abolished lipid peroxidation (p<0.001). CONCLUSION This paper further supports the pathogenic role of the imbalance in oxidant/antioxidant content in inducing mucosal colonic damage.

Transglutaminases in Crohn's disease.

Transglutaminases are a family of Ca-dependent enzymes involved in various biological events. Circulating transglutaminase (factor XIIIa) is decreased in blood of patients with inflammatory bowel diseases. There is evidence that factor XIIIa and tissue type transglutaminase, present in cell cytosol, bind to various proteins of the extracellular matrix. This study examined the value of serum transglutaminase assay in the treatment and follow up of Crohns disease and then investigated the intestinal location of both forms of transglutaminases by immunohistochemistry in normal and abnormal tissues. Serum transglutaminase activity was assayed in 36 patients with active Crohns disease (CDAI > 150). Eighteen patients were studied prospectively from relapse into remission. A significant inverse correlation (p < 0.001) was found between circulating transglutaminase and Crohns disease activity index; a correlation was also found between serum transglutaminase and serum orosomucoid (p < 0.01) and C reactive protein (p < 0.01). Patients were prospectively studied until clinical remission showed improvement in both their CDAI score mean (SD) (230 (46) to 72 (34), p < 0.01) and transglutaminase activity mean (SD) (0.61 (0.12) to 0.93 (0.13) mU/ml, p < 0.01). The immunohistochemistry assessment showed a colocalisation of factor XIIIa and tissue transglutaminase to the extracellular matrix of damaged tissues. In conclusion, these data confirm the value of serum transglutaminase assay as marker of Crohns disease activity, extend the utility of serum transglutaminase assay to follow up of the disease, and emphasised the role of different types of transglutaminases in extracellular matrix assembly in the damaged tissues.

Recombinant Factor XIII Improves Established Experimental Colitis in Rats

Factor XIII (FXIII) is the plasma-borne transglutaminase involved in fibrin clot stabilization and wound healing. FXIII levels in the plasma of patients with inflammatory bowel diseases are lower than normal and there is a significant inverse correlation of FXIII levels with clinical severity. Moreover, uncontrolled studies report beneficial effects of FXIII supplementation in patients resistant to conventional therapies. We investigated the effects of intravenous recombinant FXIII (rFXIII) treatment in experimentally induced rat colitis to verify that FXIII was the active agent in plasma FXIII concentrates and to better understand the potential therapeutic use of this protein. Colitis was induced by instillation of 12% 2,4,6-trinitrobenzenesulfonic acid (TNBS) in 50% ethanol into the colon of male Wistar rats. Rats were treated with 0.65 mg/kg rFXIII or vehicle (intravenously) daily for 10 days. Treatment was started either immediately after TNBS/EtOH instillation (to evaluate effects on developing lesions) or seven days later (to evaluate effects on established lesions). In both cases rats were killed either immediately after the end of treatment (to evaluate immediate effects) or 17 days later (to evaluate long-lasting effects). The effects of rFXIII were compared to positive (5-amino-2-hydroxybenzoic acid) control over a 35-day time course. The severity of lesions was determined by colon weight and macroscopic and histologic scores. Transglutaminase activity was measured in both colon tissue and serum. rFXIII treatment reduced lesion severity significantly not only in developing but also in established lesions. Improvements in healing persisted at least 18 days after treatment was discontinued. Serum and tissue transglutaminase levels were restored by rFXIII treatment. In conclusion, pure rFXIII is as effective as plasma FXIII concentrates in a rat model of experimental colitis. In addition, rFXIII significantly improves the healing of preexisting lesions, a characteristic useful in treatment of human inflammatory bowel diseases.

Differential expression of multiple transglutaminases in human colon: impaired keratinocyte transglutaminase expression in ulcerative colitis

Background and aims: Ulcerative colitis (UC) is characterised by refractory inflammatory ulceration and damage to the colon. The mechanisms underlying impaired healing have yet to be defined. As transglutaminase expression resulting in matrix protein cross linking is associated with increased wound healing in a rat model of colitis, we hypothesised that different types of transglutaminase might also play a role in UC. Patients and methods: Endoscopic and histological indices were studied in 26 patients with UC (10 active and 16 inactive) and in 20 normal controls undergoing colonoscopy. Transglutaminase activity was evaluated in plasma (factor XIIIa) by a radioenzymatic method. Factor XIIIa, tissue and keratinocyte transglutaminase protein content, and mRNA expression in the colon were evaluated by western blot analysis and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Colonic location of transglutaminases and their reaction products, the ε-(γ-glutamyl)lysine bonds, was evaluated by immunohistochemistry using specific monoclonal antibodies. Results: Transglutaminase activity was significantly lower in the plasma of patients with active UC (4.2 (2.4) mU/ml; p<0.05 v controls) than in those with inactive UC and controls (10.6 (2.2) and 12.1 (1.7) mU/ml). As shown by western blot, protein levels of tissue transglutaminase and factor XIIIa were unchanged in active UC compared with inactive disease and controls, while the keratinocyte form was reduced in active UC. Tissue transglutaminase and factor XIIIa immunostaining was strongly present in damaged areas colocalising with isopeptide bonds. In contrast, the keratinocyte form was almost absent in active UC and localised in the upper part of the crypts in normal subjects. RT-PCR showed upregulation of tissue transglutaminase mRNA in active UC (320% compared with controls) while keratinocyte transglutaminase gene expression was downregulated in active UC. Conclusions: The results of the present study support the concept that, in the damaged colon, transglutaminases are needed in response to chronic injury and underline the key role of these enzymes in mucosal healing.

Factor XIII Improves Gastric Stress Lesions in Rats

Background/Aims: Tissue transglutaminase has been reported to be involved in the healing of experimental gastric ulcer; nevertheless, other type(s) of transglutaminase could be involved. The present experiments aimed at examining whether plasma transglutaminase (factor XIIIa) contributes to such healing and at evaluating whether factor XIII supplementation improves gastric mucosal lesions. Methods: The healing effect of 200 U/kg of factor XIII administered intravenously was examined using a water immersion restraint rat model of stress gastric damage. The rats were sacrified 0, 2, 4, and 12 h after stress. The gastric mucosa was examined macroscopically and microscopically, and the transglutaminase activities were assayed in serum and gastric mucosa. Factor XIIIa and tissue transglutaminase protein levels in the gastric mucosa were analyzed by immunoblot. Immunohistochemistry was used to identify the location of tissue transglutaminase, factor XIIIa, and fibronectin in the gastric mucosa. Results: The transglutaminase activity, reduced by stress in the gastric mucosa, increased up to 12 h after stress, peaking at 4 h, when the ulcer index significantly decreased. The serum transglutaminase level was low at all time points. Exogenous administration of factor XIII allowed a faster reduction of the ulcer index that was coincident with an increased transglutaminase activity in the mucosa. Both tissue transglutaminase and factor XIIIa protein levels were reduced by 6 h of stress and increased after factor XIII administration. Immunohistochemistry showed a colocalization of both factor XIIIa and tissue transglutaminase with fibronectin in the extracellular matrix of the damaged area. Conclusions: Two forms of transglutaminase are involved in the healing of stress-induced gastric erosions, and factor XIII administration allows faster gastric mucosa healing.

Transglutaminase in azoxymethane-induced colon cancer in the rat

A widespread form of transglutaminase, tissue transglutaminase, has been identified in a number of mammalian cell types, both normal and transformed cells; its biological role is not well understood. We investigated the effect of experimentally induced colon cancer on transglutaminase activity in the rat. Azoxymethane (15 mg/kg for six weeks), given by a course of weekly intraperitoneal injections, produces tumors almost exclusively confined to the intestinal tract. Transglutaminase activity was assayed on tissue homogenates both during the period of treatment and, when the cancer had developed, on tumor tissue and on macroscopically uninjured adjacent tissue. A transient proliferative phase was present in the intestine during azoxymethane treatment: in this phase we found a coincidentally increased transglutaminase levels. Transglutaminase activity in tumors of both small and large intestine was significantly higher than in adjacent tissue. Immunohistochemistry revealed higher levels of transglutaminase in tumors, mainly localized in the extracellular matrix, than in adjacent tissues, where it was widely distributed. The present study shows that transglutaminase, besides its potential role in intracellular process during early proliferative phase of carcinogenesis, may also play an important role in matrix processing during tumor growth and differentiation.