Plecanatide and dolcanatide,novel guanylate cyclase-C agonists,ameliorate gastrointestinal inflammation in experimental models of murine colitis

AIM: To evaluate the effect of orally administeredplecanatide or dolcanatide, analogs of uroguanylin, on amelioration of colitis in murine models.METHODS: The cyclic guanosine monophosphate(cG MP) stimulatory potency of plecanatide and dolcanatide was measured using a human colon carcinoma T84 cellbased assay. For animal studies all test agents were formulated in phosphate buffered saline. Sulfasalazine or 5-amino salicylic acid(5-ASA) served as positive controls. Effect of oral treatment with test agents on amelioration of acute colitis induced either by dextran sulfate sodium(DSS) in drinking water or by rectal instillation of trinitrobenzene sulfonic(TNBS) acid, was examined in BALB/c and/or BDF1 mice. Additionally, the effect of orally administered plecanatide on the spontaneous colitis in T-cell receptor alpha knockout(TCRα-/-) mice was also examined. Amelioration of colitis was assessed by monitoring severity of colitis, disease activity index and by histopathology. Frozen colon tissues were used to measure myeloperoxidase activity.RESULTS: Plecanatide and dolcanatide are structurally related analogs of uroguanylin, which is an endogenous ligand of guanylate cyclase-C(GC-C). As expected from the agonists of GC-C, both plecanatide and dolcanatide exhibited potent cG MP-stimulatory activity in T84 cells. Once-daily treatment by oral gavage with either of these analogs(0.05-0.5 mg/kg) ameliorated colitis in both DSS and TNBS-induced models of acute colitis, as assessed by body weight, reduction in colitis severity(P 0.05) and disease activity index(P 0.05). Amelioration of colitis by either of the drug candidates was comparable to that achieved by orally administered sulfasalazine or 5-ASA. Plecanatide also effectively ameliorated colitis in TCRα-/- mice, a model of spontaneous colitis. As dolcanatide exhibited higher resistance to proteolysis in simulated gastric and intestinal juices, it was selected for further studies. CONCLUSION: This is the first-ever study reporting the therapeutic utility of GC-C agonists as a new class of orally delivered and mucosally active drug candidates for the treatment of inflammatory bowel diseases.     

Oral treatment with plecanatide or dolcanatide attenuates visceral hypersensitivity via activation of guanylate cyclase-C in rat models

AIM To investigate the effects of plecanatide and dolcanatide on maintenance of paracellular permeability, integrity of tight junctions and on suppression of visceral hypersensitivity. METHODS Transport of fluorescein isothiocyanate(FITC)-dextran was measured to assess permeability across cell monolayers and rat colon tissues. Effects of plecanatide and dolcanatide on the integrity of tight junctions in Caco-2 and T84 monolayers and on the expression and localization of occludin and zonula occludens-1(ZO-1) were examined by immunofluorescence microscopy. Anti-nociceptive activity of these agonists was evaluated in trinitrobenzene sulfonic acid(TNBS)-induced inflammatory as well as in non-inflammatory partial restraint stress(PRS) rat models. Statistical significance between the treatment groups in the permeability studies were evaluated using unpaired t-tests.RESULTS Treatment of T84 and Caco-2 monolayers with lipopolysaccharide(LPS) rapidly increased permeability, which was effectively suppressed when monolayers were also treated with plecanatide or dolcanatide. Similarly, when T84 and Caco-2 monolayers were treated with LPS, cell surface localization of tight junction proteins occludin and ZO-1 was severely disrupted. When cell monolayers were treated with LPS in the presence of plecanatide or dolcanatide, occludin and ZO-1 were localized at the cell surface of adjoining cells, similar to that observed for vehicle treated cells. Treatment of cell monolayers with plecanatide or dolcanatide without LPS did not alter permeability, integrity of tight junctions and cell surface localization of either of the tight junction proteins. In rat visceral hypersensitivity models, both agonists suppressed the TNBS-induced increase in abdominal contractions in response to colorectal distension without affecting the colonic wall elasticity, and both agonists also reduced colonic hypersensitivity in the PRS model. CONCLUSION Our results suggest that activation of GC-C signaling might be involved in maintenance of barrier function, possibly through regulating normal localization of tight junction proteins. Consistent with these findings, plecanatide and dolcanatide showed potent antinociceptive activity in rat visceral hypersensitivity models. These results imply that activation of GC-C signaling may be an attractive therapeutic approach to treat functional constipation disorders and inflammatory gastrointestinal conditions.     

Muc2-deficient mice spontaneously develop colitis, indicating that MUC2 is critical for colonic protection

BACKGROUND & AIMS: Expression of mucin MUC2, the structural component of the colonic mucus layer, is lowered in inflammatory bowel disease. Our aim was to obtain insight in the role of Muc2 in epithelial protection. METHODS: Muc2 knockout (Muc2(-/-)) and Muc2 heterozygous (Muc2(+/-)) mice were characterized and challenged by a colitis-inducing agent, dextran sulfate sodium (DSS). We monitored clinical symptoms, intestinal morphology, and differences in intestine-specific protein and messenger RNA levels. RESULTS: The Muc2(-/-) mice showed clinical signs of colitis (as of 5 weeks), aggravating as the mice aged. Microscopic analysis of the colon of Muc2(-/-) mice showed mucosal thickening, increased proliferation, and superficial erosions. Colonic goblet cells in the Muc2(-/-) mice were negative for Muc2, but trefoil factor 3 was still detectable. In Muc2(-/-) mice, transient de novo expression of Muc6 messenger RNA was observed in the distal colon. On day 2 of DSS treatment, the histologic damage was more severe in Muc2(+/-) versus wild-type (Muc2(+/+)) mice, but the disease activity index was not yet different. By day 7, the disease activity index and histologic score were significantly elevated in Muc2(+/-) versus Muc2(+/+) mice. The disease activity index of the Muc2(-/-) mice was higher (versus both Muc2(+/+) and Muc2(+/-) mice) throughout DSS treatment. The histologic damage in the DSS-treated Muc2(-/-) mice was different compared with Muc2(+/+) and Muc2(+/-) mice, with many crypt abscesses instead of mucosal ulcerations. CONCLUSIONS: This study shows that Muc2 deficiency leads to inflammation of the colon and contributes to the onset and perpetuation of experimental colitis.     

Claudin-2 Regulates Colorectal Inflammation via Myosin Light Chain Kinase-Dependent Signaling

Background

Claudins have been demonstrated to be associated with inflammatory bowel disease (IBD), but the specific role of claudin-2 in colorectal inflammation remains undefined.

Aims

We aimed to determine the role of claudin-2 in TNFα-induced colorectal inflammation.

Methods

We used claudin-2 (?/?) mice to assess the role of claudin-2 in colon. The mice were intraperitoneally injected with 3 μg of recombinant murine TNFα, and the NF-κB signaling and mRNA expression levels of proinflammatory cytokines and myosin light chain kinase (MLCK) were evaluated. Moreover, in claudin-2 (?/?) mice, colitis was induced by the administration of dextran sodium sulfate (DSS). The involvement of claudin-2 in colorectal inflammation was also investigated using the Caco-2 human colon adenocarcinoma cell line, and the expression of claudin-2 was downregulated using claudin-2 siRNA.

Results

TNFα-induced colorectal inflammation via NF-κB signaling activation was enhanced in claudin-2 (?/?) mice compared with that in claudin-2 (+/+) mice. MLCK expression level in the colon tissue of claudin-2 (?/?) mice treated with TNFα was enhanced in comparison to that of the claudin-2 (+/+) mice. DSS-induced colitis was more severe in the claudin-2 (?/?) mice than in the claudin-2 (+/?) mice. In in vitro experiments, the decreased expression of claudin-2 enhanced the expressions of IL-6, IL-1β and MLCK.

Conclusions

Our findings concerning the role of claudin-2 in epithelial inflammatory responses enrich our collective understanding of mucosal homeostasis and intestinal diseases such as IBD. Furthermore, the results of this study indicate that claudin-2 and MLCK are potential therapeutic targets for treatments against intestinal disease.     

Therapeutic effect of nimesulide on colorectal carcinogenesis in experimental murine ulcerative colitis

BACKGROUND: Patients with ulcerative colitis (UC) exhibit an increased risk for the development of cancer of the colon and rectum. Cyclooxygenase (COX)-2 inhibitors are known to suppress sporadic colorectal cancer, but it is unknown whether selective COX-2 inhibitors exhibit a preventive effect in UC-associated neoplasia. This study investigated the preventive effect of nimesulide, a selective COX-2 inhibitor, on colorectal carcinogenesis in an experimental model of murine UC. METHODS: Chronic colitis was induced in mice by administration of four cycles of dextran sulfate sodium (DSS) (each cycle: 5% DSS for 7 days and then distilled water for 14 days). The mice were killed 120 days after the completion of the fourth cycle. The mice were divided into the following five groups: group A served as a disease control; group B received a diet mixed with 400 p.p.m. of nimesulide during the whole period; group C received nimesulide during the four cycles of DSS administration (active phase); group D received nimesulide for 120 days from the end of the fourth cycle (remission phase); group E received no agents including DSS and served as a normal control. RESULTS: The incidence of dysplasia and/or cancer was 28%, 15%, 11.8%, 6.7% and 0% in groups A-E, respectively. In group D, nimesulide significantly suppressed the occurrence of dysplasia and/or cancer (P < 0.05). Strong COX-2 expression was detected by immunohistochemistry in cancer and dysplastic lesions while diffusely weak COX-2 expression was also found in the residual colon (i.e. lesion-free colon). The mucosal concentration of prostaglandin E(2) was significantly lower in groups B and D than in group A. CONCLUSIONS: The administration of the selective COX-2 inhibitor nimesulide (especially during the remission phase) exerts a suppressive effect on the development of dysplasia and/or cancer in a murine model of DSS-induced colitis. These findings may have relevance to long-standing UC in humans.     

G protein-coupled receptor kinase 6 controls chronicity and severity of dextran sodium sulphate-induced colitis in mice

BACKGROUND: Infiltration of inflammatory cells into the colon plays an important role in the onset and course of inflammatory bowel disease. G-protein-coupled receptor kinase 6 (GRK6) is an intracellular kinase that regulates the sensitivity of certain G-protein-coupled receptors, including those involved in the migration of inflammatory cells. Therefore, it is hypothesised that GRK6 plays a role in determining the course of inflammation. AIM: To analyse the role of GRK6 in the course of dextran sodium sulphate (DSS)-induced colitis. METHODS: Colitis was induced by administering 1% DSS in drinking water to GRK6(-/-), GRK6(+/-) and wild-type (WT) mice for 6 days. The severity of colitis was assessed on the basis of clinical signs, colon length and histology. Moreover, keratinocyte-derived chemokine (KC) levels, granulocyte infiltration, interleukin 1beta (IL1beta), CD4, CD8 and forkhead box protein P3 (FoxP3) expression in the colon were determined. In addition, regulatory T cell function in WT and GRK6(-/-) mice was analysed. The chemotactic response of granulocytes to colon culture supernatants was assessed using a transendothelial migration assay. RESULTS: The severity of colitis was increased in GRK6(-/-) and GRK6(+/-) mice and was accompanied by increased KC levels and increased granulocyte infiltration. Moreover, the chemotactic response of GRK6(-/-) granulocytes to supernatants of colon cultures was enhanced. Interestingly, the WT mice completely recovered from colitis, whereas the GRK6(-/-) and GRK6(+/-) mice developed chronic colitis, which was accompanied by increased IL1beta and CD4 expression and decreased FoxP3 expression. Moreover, regulatory T cell function was impaired in the GRK6(-/-) mice. CONCLUSIONS: The intracellular level of GRK6 is an important factor in determining the onset, severity and chronicity of DSS-induced colitis.     

Wuchereria bancrofti macrophage migration inhibitory factor-2 (rWbaMIF-2) ameliorates experimental colitis

Immunomodulatory molecules produced by helminth parasites are receiving much attention recently as novel therapeutic agents for inflammation and autoimmune diseases. In this study, we show that macrophage migration inhibitory factor (MIF) homologue from the filarial parasite, Wuchereria bancrofti (rWbaMIF-2), can suppress inflammation in a dextran sulphate sodium salt (DSS)-induced colitis model. The disease activity index (DAI) in DSS given mice showed loss of body weight and bloody diarrhoea. At autopsy, colon of these mice showed severe inflammation and reduced length. Administration of rWbaMIF-2 significantly reduced the DAI in DSS-induced colitis mice. rWbaMIF-2-treated mice had no blood in the stools, and their colon length was similar to the normal colon with minimal inflammation and histological changes. Pro-inflammatory cytokine genes (TNF-α, IL-6, IFN-γ, IL-1β, IL-17A and NOS2) were downregulated in the colon tissue and peritoneal macrophages of rWbaMIF-2-treated mice. However, there were significant increases in IL-10-producing Treg and B1 cells in the colon and peritoneal cavity of rWbaMIF-2-treated mice. These findings suggested that rWbaMIF-2 treatment significantly ameliorated the clinical symptoms, inflammation and colon pathology in DSS given mice. This immunomodulatory effect of rWbaMIF-2 appeared to be by promoting the infiltration of Treg cells into the colon.     

T helper 1-inducing property of IL-27/WSX-1 signaling is required for the induction of experimental colitis

BACKGROUND: WSX-1, a component of the interleukin (IL)-27 receptor, is a novel class I cytokine receptor with homology to the IL-12 receptor beta2 chain. Initially, WSX-1 signaling was reported to play an important role in the promotion of T helper-1 responses, but recent reports have revealed an anti-inflammatory property in WSX-1 signaling. In the present study, we investigated the role of IL-27/WSX-1 signaling in a murine colitis model, dextran sulfate sodium (DSS) colitis, by using WSX-1 knockout (KO) mice. METHODS: First, we observed whether WSX-1 KO mice developed colitis spontaneously. Second, we induced DSS colitis in WSX-1 KO and wild-type (WT) mice. RESULTS: WSX-1 KO mice were observed not to develop colitis spontaneously. The severity of DSS colitis was decreased in WSX-1 KO mice in comparison with WT mice in association with a reduced production of interferon-gamma, IL-6, and tumor necrosis factor-alpha by lamina propria mononuclear cells from WSX-1 KO mice and the absence of T-bet expression in the colon from WSX-1 KO mice. CONCLUSIONS: This study revealed the inflammatory property of IL-27/WSX-1 signaling in intestinal inflammation. As a result, IL-27/WSX-1 signal pathway may thus be a promising candidate for the therapeutic intervention of human inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.     

Clinical and histopathological features of dextran sulfate sodium induced acute and chronic colitis associated with dysplasia in rats

We examined the clinical and histopathological features of the dextran sulfate sodium (DSS) induced acute and chronic colitis in rats as a model for studying basic biology of the inflamed colonic mucosa. Acute colitis was induced in male Wistar rats by 4 days (AI) or 7 days (AII) of oral 5% (wt/vol) DSS (mol. wt. 54,000) in their drinking water. Chronic colitis was induced in 8 experimental groups: CI=7 days DSS followed by 10 days water (=one cycle); CII=two cycles; CIII to CVIII (three to eight cycles) received only 4 days 5% DSS followed by 10 days drinking water. The entire colons were examined histologically; dysplasia was graded as: indefinite/probably negative for dysplasia, indefinite/probably positive for dysplasia, low-grade dysplasia, or high-grade dysplasia. The earliest clinical findings in the acute colitis group over 4 days occurred on day 2 (hemoccult positive stools, loose stools or diarrhea and weight loss). The maximal disease activity was noted on day 7 accompanied by a 53% mortality rate. The histological inflammation scores were significantly higher on day 7 than on day 4. All rats had extensive ulcerations predominantly in the rectum and cecum. The number of rats having ulcerations was markedly lower in the chronic colitis groups. The majority (75%) of the crypt lesions suspicious for dysplasia were classified as mucosa indefinite/probably negative for dysplasia. We classified 18 crypt lesions as low-grade dysplasia and one lesion as high-grade dysplasia (after eight cycles). No invasive carcinoma was observed. Most low-grade dysplasias (83%) occurred after five cycles of DSS/water, located mostly in the rectum (44%) and colon transversum (33%). Our findings suggest that the DSS colitis model in rats may be an interesting model for studying the sequence chronic inflammation-dysplasia in human ulcerative colitis. Further long-term studies with the present DSS colitis model in rats might also prove it as a reliable model to study the sequence high-grade dysplasia and colitis associated-cancer.     

Transforming growth factor-alpha (TGF-alpha) is not needed for malignant transformation in experimental colitis

BACKGROUND: Transforming growth factor-alpha (TGF-alpha) is a key mediator of colonic mucosal protection and/or repair mechanisms in orally induced acute dextran sodium sulphate (DSS) colitis. However, it also has been suggested that TGF-alpha may contribute to malignant transformation in the colon. The aim of the studies was to determine whether TGF-alpha is needed for malignant transformation in orally induced chronic DSS colitis using TGF-alpha deficient mice (wa-1) and Balb/c mice, a strain competent in TGF-alpha. METHODS: Chronic colitis was induced by oral administration of DSS (5%) for 7 days followed by drinking water for 10 days in wa-1 and Balb/c mice (n = 20, per group). In the two subsequent cycles (7 days DSS, 10 days water) 3% DSS-water was utilized due to a high mortality in the wa-1 group. Mucosal injury severity was assessed histologically and graded (three grades). A crypt damage score (CDS) reflecting all three grades of mucosal pathology was calculated. Mucosal dysplasia and cancerous lesions were noted. RESULTS: Seven per cent of the entire colonic mucosa was completely destroyed in wa-1 animals compared to 3% in Balb/c mice (P < 0.05). The CDS was 10.2 +/- 0.4 and 4.8 +/- 0.3 in wa-1 and Balb/c mice, respectively (P < 0.05). Fifteen incidences of mucosal dysplasia were found in the 10 surviving wa-1 animals and 31 incidences were found in 20 Balb/c animals. In both groups, one fully developed adenomatous cancerous lesion was present. CONCLUSIONS: The markedly increased severity of mucosal injury in chronic induced DSS colitis in TGF-alpha deficient wa-1 mice compared to Balb/c mice further substantiates that endogenous TGF-alpha is a pivotal mediator of protection and/or healing mechanisms in the colon. The appearance of dysplastic and cancerous lesions in TGF-alpha deficient animals suggests that TGF-alpha per se is not essential for malignant mucosal cell transformation in colitis.     

Increased expression of HIP/PAP and regenerating gene III in human inflammatory bowel disease and a murine bacterial reconstitution model

Although microorganisms play a role in gut inflammation, it remains uncertain which epithelial genes are expressed in response to luminal flora and whether these molecules are also involved in pathologic mucosal inflammation. Germ-free mice were orally challenged with a bacterial suspension prepared from conventionally housed mice (bacterial reconstitution). Thereafter, the differential gene expression in gut epithelial cells was identified by differential display. The expression of the identified genes was also examined in dextran sulfate sodium (DSS)-induced colitis and human inflammatory bowel disease (IBD) epithelial cells. Regenerating gene III (Reg III) was strongly induced in gut epithelial cells following bacterial reconstitution, as well as in the colitis initiated by DSS. The mRNA expression of hepatocarcinoma-intestine-pancreas/pancreatic associated protein (HIP/PAP), a human counterpart of Reg III, was enhanced in colonic epithelial cells of patients with IBD. Reg III mRNA expression was localized in the epithelial cells including goblet cells and columnar cells in mice; on the other hand, HIP/PAP-expressing cells were correlated with Paneth cell metaplasia in human colon. Epithelial expression of Reg III or HIP/PAP was induced under mucosal inflammation initiated by exposure to commensal bacteria or DSS as well as inflamed IBD colon.     

Plecanatide, an Oral Guanylate Cyclase C Agonist Acting Locally in the Gastrointestinal Tract, Is Safe and Well-Tolerated in Single Doses

Purpose

Plecanatide, an analogue of uroguanylin, activates the guanylate cyclase C (GC-C) receptor found on the GI mucosal epithelial cells, leading to secretion of fluid, facilitating bowel movements. Plecanatide is being investigated as a potential treatment for constipating GI disorders. The aim of this investigation was to assess the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of single doses of plecanatide in healthy volunteers.

Methods

A total of 72 healthy volunteers at a single site were randomized in 9 cohorts to receive oral plecanatide or placebo from 0.1 to 48.6 mg. Plasma PK samples were collected pre-dose and post-dose. PD assessments included time to first stool, stool frequency, and stool consistency using the Bristol Stool Form Scale. All adverse events were documented.

Results

Plecanatide was safe and well-tolerated at all dose levels. A total of 17 of 71 subjects (23.9 %) reported 25 treatment-emergent adverse events (TEAEs) during the study. The number of TEAEs reported by subjects who received plecanatide or placebo was comparable (24.5 vs. 22.2 %, respectively). There were no dose-related increases in TEAEs or any SAEs reported. No measurable systemic absorption of oral plecanatide was observed at any of the oral doses studied, utilizing an assay sensitive down to 1 ng/mL.

Conclusions

Plecanatide, an oral GC-C agonist, acting locally within the GI tract without measurable systemic exposure, was safe and well-tolerated in single doses up to 48.6 mg. The study was not powered for statistical analyses, but trends in PD parameters supported continued clinical development.     

Protective effects of panax notoginseng saponin on dextran sulfate sodium-induced colitis in rats through phosphoinositide-3-kinase protein kinase B signaling pathway inhibition

BACKGROUND Intestinal inflammation is a common digestive tract disease, which is usually treated with hormone medicines. Hormone medicines are effective to some extent, but long-term use of them may bring about many complications.AIM To explore the protective effects of panax notoginseng saponin(PNS) against dextran sulfate sodium(DSS)-induced intestinal inflammatory injury through phosphoinositide-3-kinase protein kinase B(PI3K/AKT) signaling pathway inhibition in rats.METHODS Colitis rat models were generated via DSS induction, and rats were divided into control(no modeling), DSS, DSS + PNS 50 mg/k, and DSS + PNS 100 mg/kg groups. Then, the intestinal injury, oxidative stress parameters, inflammatory indices, tight junction proteins, apoptosis, macrophage polarization, and TLR4/AKT signaling pathway in colon tissues from rats in each of the groups were detected. The PI3 K/AKT signaling pathway in the colon tissue of rats was blocked using the PI3K/AKT signaling pathway inhibitor, LY294002.RESULTS Compared with rats in the control group, rats in the DSS group showed significantly shortened colon lengths, and significantly increased disease activity indices, oxidative stress reactions and inflammatory indices, as well as significantly decreased expression of tight junction-associated proteins. In addition, the DSS group showed significantly increased apoptotic cell numbers,and showed significantly increased M1 macrophages in spleen and colon tissues.They also showed significantly decreased M2 macrophages in colon tissues, as well as activation of the PI3K/AKT signaling pathway(all P 0.05). Compared with rats in the DSS group, rats in the DSS + PNS group showed significantly lengthened colon lengths, decreased disease activity indices, and significantly alleviated oxidative stress reactions and inflammatory responses. In addition, this group showed significantly increased expression of tight junction-associated proteins, significantly decreased apoptotic cell numbers, and significantly decreased M1 macrophages in spleen and colon tissues. This group further showed significantly increased M2 macrophages in colon tissues, and significantly suppressed activation of the PI3K/AKT signaling pathway, as well as a dose dependency(all P 0.05). When the PI3K/AKT signaling pathway was inhibited, the apoptosis rate of colon tissue cells in the DSS + LY294002 group was significantly lower than that of the DSS group(P 0.05).CONCLUSION PNS can protect rats against DSS-induced intestinal inflammatory injury by inhibiting the PI3K/AKT signaling pathway, and therefore may be potentially used in the future as a drug for colitis.     

Geranylgeranylacetone protects mice from dextran sulfate sodium-induced colitis

Objective. Geranylgeranylacetone (GGA) has recently been reported to induce heat shock protein (HSP) 70, which has a protective function against inflammation. We investigated the therapeutic effects of oral administration of GGA on dextran sulfate sodium (DSS)-induced colitis in mice.Material and methods. BALB/c mice were given 3% DSS solution orally for 7?days to induce colitis. The disease activity of colitis was assessed clinically every day, and histology in the colon was evaluated at 7?days post-DSS. The levels of myeloperoxidase (MPO) activity, tumor necrosis factor (TNF)-α and interferon (IFN)-γ in the colon tissues were also examined. In addition, expression of HSPs 25, 40, 70 and 90 in the colon tissue was determined by Western blot analysis. Mice were orally administered GGA (50–500?mg/kg) when treatment of DSS started.Results. It was found that GGA significantly reduced the clinical severity of colitis and suppressed the levels of MPO activity, TNF-α and IFN-γ induced by DSS in the colon. On the other hand, GGA enhanced the expression of HSP70 in the colon of mice given DSS. HSP70-positive cells were identified in the epithelial cells of the colon from mice treated with GGA and DSS.Conclusions. Taken together, these results suggest that GGA is a new anti-inflammatory drug that could be useful in the treatment of colitis such as inflammatory bowel disease.     

Protective action of NADPH oxidase inhibitors and role of NADPH oxidase in pathogenesis of colon inflammation in mice

AIM: To investigate the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in colon epithelial cells in the pathogenesis of acute and chronic colon inflammation in a mouse model of dextran sulphate sodium (DSS)-induced colitis.METHODS: Balb/c mice were divided into three groups: 8 mice with acute DSS-induced colitis (3.5% DSS solution; 7 d), 8 mice with chronic DSS-induced colitis (3.5% DSS solution for 5 d + water for 6 d; 4 cycles; total: 44 d) and 12 mice without DSS supplementation as a control group. Primary colonic epithelial cells were isolated using chelation method. The cells were cultivated in the presence of mediators (lipopolysaccharide (LPS), apocynin or diphenyleneiodonium). Viability of cells was assessed by fluorescent microscopy. Production of reactive oxygen species (ROS) by the cells was measured fluorometrically using Amplex Red. Production of tumour necrosis factor-alpha (TNF-α) by the colonic epithelial cells was analysed by ELISA. Nox1 gene expression was assessed by real-time PCR.RESULTS: Our study showed that TNF-α level was increased in unstimulated primary colonic cells both in the acute and chronic colitis groups, whereas decreased viability, increased ROS production, and expression of Nox1 was characteristic only for chronic DSS colitis mice when compared to the controls. The stimulation by LPS increased ROS generation via NADPH oxidase and decreased cell viability in mice with acute colitis. Treatment with NADPH oxidase inhibitors increased cell viability and decreased the levels of ROS and TNF-α in the LPS-treated cells isolated from mice of both acute and chronic colitis groups.CONCLUSION: Our study revealed the importance of NADPH oxidase in the pathogenesis of both acute and chronic inflammation of the colon.     

Comparative clinicopathological and immunohistochemical study of ras and p53 in flat and polypoid type colorectal tumours.

Mutations in oncogenes and tumour suppressor genes may have an important oncogenic role. Although flat type tumours have been frequently detected in recent years, ras and p53 expressions have not been studied in these tumours. Using a monoclonal and polyclonal antibody to the ras p21 and p53 product, paraffin wax embedded sections of 98 colorectal tumours (43 cases of the flat type colorectal tumour and 55 cases of polypoid type tumour) were stained using the immunoperoxidase technique. Staining was evaluated by light microscopic examination. Positive staining rate of ras p21 for the flat type was 0%; for the polypoid type, it was 60% in cancer with submucosal invasion, 82% in adenoma with high grade dysplasia, and 0% in adenoma with low grade dysplasia. The positive staining rate of p53 for the flat type was 50% in submucosal cancer, 9% in adenoma with high grade dysplasia, and 0% in adenoma with low grade dysplasia. For the polypoid type, it was 40% in submucosal cancer, 12% in adenoma with high grade dysplasia, and 0% in adenoma with low grade dysplasia. The intermediate staining rate of p53 in the polypoid type was 20% in submucosal cancer and 41% in adenoma with high grade dysplasia. It was seen that p53 was commonly expressed in both flat and polypoid lesions, p21 was not expressed in flat lesions, whereas it was commonly expressed in polypoid neoplasms. In the flat type cancer, a genetic change different from that of the polypoid type cancer is suggested.     

Effect of adenovirus-mediated PTEN gene on ulcerative colitis-associated colorectal cancer

Purpose

This study investigated the expression pattern of PTEN and its effect on carcinogenesis of ulcerative colitis-associated colorectal cancer, leading to insights into the underlying molecular mechanism.

Methods

We established a mouse model of ulcerative colitis-associated colorectal cancer by treating the animals with azoxymethane (AOM) and dextran sulphate sodium (DSS), and investigated the inflammation–dysplasia–carcinoma sequence. Expression patterns of PTEN, p-Akt and Ki-67 were shown by immunohistochemistry; western blotting techniques were used to detect protein expression of PTEN, p-Akt and caspase 3; TUNEL assay was used to measure apoptosis in colon epithelial cells; and colorimetric analysis was able to determine MPO activity in colon tissues.

Results

During the inflammation–dysplasia–carcinoma sequence, PTEN expression gradually decreased, while p-Akt expression increased; PTEN and p-Akt levels were negatively correlated. Compared to the AOM-DSS and Ad-0 groups, Ad-PTEN mice had longer colons, fewer tumours (P?<?0.01) and smaller tumour sizes (P?<?0.05). After injecting Ad-PTEN, expression of p-Akt, Ki-67 and MPO activity decreased dramatically, whereas PTEN increased. The TUNEL assay showed increased apoptotic cells and caspase 3 expression in the Ad-PTEN group.

Conclusion

PTEN plays an important role in the inflammation–dysplasia–carcinoma sequence and may be a new molecular target in preventing and treating ulcerative colitis-associated colorectal cancer.     

Colorectal carcinoma with special reference to growth pattern classifications: clinicopathologic characteristics and genetic changes

Background: Background: Many colorectal carcinomas are known to develop from preexisting polypoid adenomas; however, they can also develop from so-called “flat adenomas”. To elucidate the growth patterns of flat- or depressed-derived colorectal carcinomas, we investigated the clinicopathologic characteristics and genetic changes of invasive carcinomas. Methods: Seventy-five colorectal carcinomas were classified into three groups: 46 upward growth (UG) type, 22 downward growth (DG) type, and 7 lateral growth (LG) type. All of them had histologically infiltrated the submucosa (SM) and muscularis propria (MP). Ki-ras mutation was examined by polymerase chain reaction-single-strand conformation polymorphism analysis, and overexpression of p53 protein was analyzed by immunohistochemistry. Results: No DG or LG carcinomas histologically demonstrated an adenomatous remnant, whereas UG carcinomas did (SM, 19 of 26; 73%; MP, 3 of 20; 15%). The percentage of tumors existing in the right colon was significantly higher in LG carcinomas (71%) than in the UG type (28%; P= 0.037). The frequency of Ki-ras mutation was significantly higher in the UG carcinomas than in the DG and LG carcinomas (52% vs 0%; P < 0.0001; and vs 0%; P= 0.014). However, the frequency of this mutation in SM-UG carcinomas with an adenomatous remnant (9 of 19; 47%) did not differ significantly from that in SM-UG carcinomas without an adenomatous remnant (3 of 7; 43%). The frequency of p53 overexpression did not differ among UG (57%), LG (57%), and DG (50%) carcinomas. Conclusions: These results suggest that UG carcinomas develop on the basis of the adenoma-carcinoma sequence, while the development of DG and LG carcinomas is different from that of UG carcinomas. Received: April 9, 2001 / Accepted: September 28, 2001     

Western-style diets induce macrophage infiltration and contribute to colitis-associated carcinogenesis

Background and Aim: A Western‐style diet (WD) is known to play an important role in inflammatory bowel disease and colon carcinogenesis. The purpose of this study was to understand the role of macrophages in WD‐induced colitis associated with carcinogenesis. Methods: Male BALB/c mice were fed a WD or a control diet (CD) for 4 weeks and exposed to azoxymethane (AOM) followed by 2% dextran sulfate sodium (DSS) for 7 days. Results: The WD increased susceptibility to DSS‐induced inflammation and accelerated the infiltration of macrophages. The incidence and multiplicity of colon tumors were higher in mice fed the WD than in those fed the CD (P < 0.05). Levels of prostaglandin‐endoperoxide synthase (PTGS) 2 and prostaglandin (PG) E₂ in the colon were higher after treatment with AOM and DSS in mice fed the WD than in those fed the CD. In addition, WD consumption increased the DNA binding activity of nuclear factor‐kappaB and the serum concentration of tumor necrosis factor (TNF)‐α. Mice fed the WD had higher numbers of F4/80‐positive cells surrounding cancer cells compared with mice fed the CD. These cells expressed PTGS2, TNF‐α and β‐catenin, which are up‐regulated by the WD. We also found that the WD increased unphosphorylated β‐catenin accumulation in the cytoplasm and nucleus of colon cancer cells. Conclusions: A WD increases the susceptibility to DSS‐induced inflammation and accelerates the infiltration of macrophages. In turn, this resulted in the development and progression of colon cancer.     

Flat-elevated and depressed, subtypes of flat early colorectal cancers, should be distinguished by their pathological features

Flat-type colorectal tumors have are being detected with increasing frequency. It has become clear that these flat lesions contain two subtypes; flat-elevated and depressed lesions. However, their clinicopathological features and roles in colorectal carcinogenesis remain obscure. We classified colorectal adenomas and submucosal invasive cancers into three types: polypoid, flat-elevated, and depressed types. A clinicopathological study of 2505 colorectal tumors (2407 adenomas, 98 submucosal invasive cancers) was then performed. Furthermore, 64 tumors (25 adenomas with high-grade dysplasia, 39 submucosal invasive cancers) from which DNA was extracted were examined for K-ras gene mutation. The percentages of each configuration in the resected materials were 62.0%, 36.4%, and 1.6% of the polypoid, flat-elevated, and depressed types, respectively. The rate of submucosal invasive cancer in the depressed type was always high regardless of size. In the polypoid and flat-elevated types, lesions of larger size showed higher rates of invasion. Analysis of submucosal invasive cancers revealed no adenomatous components in any of the depressed-type lesions; in the polypoid and flat-elevated types the frequencies of cancer with adenomatous components were 83.6% and 77.8%, respectively. The flat-elevated type was more frequently located (77.8%) in the proximal colon than the other types (polypoid type 16.4%, depressed type 25.0%). The incidence of K-ras gene mutation was 47.2%, 18.2%, and 0% in the polypoid, flat-elevated, and depressed types, respectively. These findings suggest that the flat-elevated and depressed types are similar in that they are both morphologically flat and have infrequent incidences of K-ras gene mutation, but these two lesions differ in their pathological features. Especially, depressed type lesions have a tendency to invade the submucosal layer even when they are small. Therefore one should always be aware of this type of lesion during colonoscopic examination.