Colonic Epithelial Cell Proliferation Research Articles

Inflammation-induced tumorigenesis in the colon is regulated by caspase-1 and NLRC4

Chronic inflammation is a known risk factor for tumorigenesis, yet the precise mechanism of this association is currently unknown. The inflammasome, a multiprotein complex formed by NOD-like receptor (NLR) family members, has recently been shown to orchestrate multiple innate and adaptive immune responses, yet its potential role in inflammation-induced cancer has been little studied. Using the azoxymethane and dextran sodium sulfate colitis-associated colorectal cancer model, we show that caspase-1-deficient (Casp1(-/-)) mice have enhanced tumor formation. Surprisingly, the role of caspase-1 in tumorigenesis was not through regulation of colonic inflammation, but rather through regulation of colonic epithelial cell proliferation and apoptosis. Consequently, caspase-1-deficient mice demonstrate increased colonic epithelial cell proliferation in early stages of injury-induced tumor formation and reduced apoptosis in advanced tumors. We suggest a model in which the NLRC4 inflammasome is central to colonic inflammation-induced tumor formation through regulation of epithelial cell response to injury.

Abstract A105: The extracellular calcium sensing receptor regulates the proliferation of colonic epithelial cells

Abstract The progression of colorectal cancer (CRC) from normal colonic epithelium to the malignant phenotype is associated with diverse genetic and epigenetic changes, including mutations of APC or beta-catenin genes. Extensive experimental and epidemiological evidence also suggest that differences in diet contribute to more variation in CRC incidence than any other factor. For example, several animal experimental studies support the notion that dietary Ca2+ prevents the development of CRC. The precise mechanism(s) mediating the chemopreventive properties of Ca2+ remain incompletely understood. The extracellular Ca2+-sensing receptor (CaR), a GPCRs originally cloned from parathyroid chief cells, plays a major role in the control of extracellular Ca2+ concentration by regulating parathyroid hormone secretion. However, subsequent studies demonstrated that the CaR is also expressed in other tissues and organs, including the entire gastrointestinal (GI) tract. Interestingly, CaR expression in colonic carcinomas is greatly reduced or lost, suggesting that the suppression of its expression may be associated with abnormal differentiation and malignant progression. Here, we assessed the role of the CaR in colonic epithelial cells proliferation. In order to elucidate the mechanisms involved, we examined in vitro changes in beta-catenin phosphorylation and intracellular distribution and cellular proliferation triggered by CaR stimulation. We found that CaR stimulation promotes, in human epithelial cells derived from normal colon and from colorectal adenocarcinoma, a decrease of up to 60% in beta-catenin Ser-552 phosphorylation, a residue involved in beta-catenin nuclear import and transcriptional activity regulation. This change in phosphorylation was accompanied by a redistribution of beta-catenin from the cytoplasm and nuclei to the plasma membrane and by a marked proliferation inhibition. To determine the role of the CaR in the proliferation of colonic cells in vivo, we employed a novel mouse model where the expression of the CaR was specifically knocked out in the GI tract. The results show that the crypts in the distal colon are deeper in knock out (KO) mice (389 ± 7.39 Arbitrary Units, mean ± SEM) than controls littermates (268 ± 6.35 AU) P < 0.0001. Similarly, the crypts in the proximal colon are deeper in KO mice (289 ± 7.02 AU) than controls (232 ± 5.36 AU) P < 0.0001. We also found significant morphological differences in the crypt’ structure of several regions in the proximal colon of CaR KO mice, including the absence of a well-organized surface layer of epithelial cells and the presence of disorganized crypts and numerous enlarged globet cells. Further analysis of the colon of KO mice showed that the number of proliferating cells, as revealed by Ki-67 proliferation marker immunostaining, was significantly larger than control littermates. Specifically, proliferating cells in the KO mice were detected in the base of the crypt and up to 60% of the crypt column. In contrast, in control littermates the proliferating cells were fewer and present mostly at the base of the crypt. We hypothesize that pathways emanating from the CaR lead to proliferation inhibition of colon-derived epithelial cells by a mechanism that involves beta-catenin Ser-552 dephosphorylation and that the genetic ablation of this receptor promotes hyperproliferation of colonic crypts. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A105.

The Mechanisms of Sai-rei-to, a Herbal Drug, in the Treatment of Experimental Colitis

Purpose: Current medical management of IBD consists of aminosalicylats (ASAs), steroids, and immunosuppressant therapies including azathioprine, and recently TNF-α antibody therapy has also been used. But treatment with these drugs reduces immunity which may be a trigger for adverse events including infection. Sai-rei-to (SRT), a type of kampo medicine (Japanese herbal medicine), is a compound drug made from the crude extracts of various medicinal plants. These plants and extracts have been reported to have an antiinflammatory action. Recently in Japan, the administration of SRT was shown to reduce the amount of steroid administration required for IBD patients. But the mechanisms of the effect of this drug for IBD are unknown. In this study, we demonstrated the effect of SRT on colonic epithelial cell proliferation and apoptosis in rats with TNBS-induced experimental colitis. Methods: SRT, prednisolone (PSL) and 5-ASA (ASA) were administered in various combinations to TNBS rats for 4 weeks. After the rats were sacrificed, any inflammatory change of the colonic mucosa was studied using Matt's classification. The cell proliferation of colonic mucosa was studied the BrdU uptake, and apoptosis cells were stained by Tunel method. Results: The treatment of SRT, PSL and ASA alone significantly reduced colonic inflammation (TNBS control:39.75±1.26, vs. SRT:34.33±1.09, PSL:31.11±1.89, ASA:33.57±1.26) and there was no significant difference on the inflammation between these drugs. PSL administration significantly decreased the number of BrdU-labeled cells (TNBS control:4.66±1.1, vs. PSL:3.44±2.1), but SRT and ASA did not influence the number of BrdU-labeled cells (SRT:4.63±1.1, ASA:4.64±3.2). Both ASA and SRT administration increased the number of total cells in colonic pits (TNBS control:42.61±21.8, ASA:57.11±13.7, SRT:74.34±19.9). In the SRT+ASA group, administration of SRT further increased the number of total cells which had already been increased by ASA treatment (SRT+ASA:72.64±22.7). SRT and ASA treatment alone increased the number of cells above the proliferative zone (TNBS control:24.8±17.3, vs. SRT:56.63±26.3, ASA:39.19±17.8). ASA, PSL and SRT alone significantly decreased the number of apoptosis cells (TNBS control:9.53±2.17, vs. ASA:1.52±0.29, PSL:1.62±0.6, SRT:0.4±0.18). In the SRT+ASA group (ASA:1.52±0.29, vs. SRT+ASA:0.36±0.05) administration of SRT further decreased apoptosis cells which had been decreased by ASA. Conclusion: The treatment of SRT alone decreased colonic inflammation similar to PSL and ASA. SRT reduced both the colonic cells above the PZ zone and the apoptosis cells. From these data, the effect of SRT treatment for IBD is not an inducer of proliferation, and reduces and protects the desquamated colonic epithelial cells.

Heme induces DNA damage and hyperproliferation of colonic epithelial cells via hydrogen peroxide produced by heme oxygenase: A possible mechanism of heme‐induced colon cancer

Epidemiological and animal model studies have suggested that high intake of heme, present in red meat, is associated with an increased risk of colon cancer. However, the mechanisms underlying this association are not clear. This study aimed to investigate whether heme induces DNA damage and cell proliferation of colonic epithelial cells via hydrogen peroxide produced by heme oxygenase (HO). We examined the effects of zinc protoporphyrin (ZnPP; a HO inhibitor) and catalase on DNA damage, cell proliferation, and IL-8 production induced by the addition of hemin (1-10 microM) to human colonic epithelial Caco-2 cells. DNA damage was determined with a comet assay, and cell proliferation was evaluated with 5-bromo-2'-deoxyuridine incorporation assay. Both ZnPP and exogenous catalase inhibited the hemin-induced DNA damage and cell hyperproliferation dose-dependently. IL-8 messenger RNA expression and IL-8 production in the epithelial cells increased following the hemin treatment, but the production was inhibited by ZnPP and catalase. These results indicate that hemin has genotoxic and hyperproliferative effects on Caco-2 cells by HO and hydrogen peroxide. The mechanism might explain why a high intake of heme is associated with increased risk of colon cancer.

W1744 The Extracellular Calcium-Sensing Receptor Regulates the Proliferation of Normal Colonic Epithelial Cells and Reverses the Malignant Phenotype of Colon Cancer Cells

Background: EGFR family members are all thought to signal through the MAPK and PI3K/ Akt pathways. Upregulation of EGFR, HER2, HER3 and MAPK signaling is well documented in colorectal tumors. Similarly, PI3K activity is elevated in the vast majority of colorectal cancers and over-expression of Akt isoforms has also been detected in CRC. We previously reported that the sulfide and sulfone metabolites of the non-steroidal anti-inflammatory drug (NSAID) sulindac downregulate the expression of EGFR, inhibit EGFR signaling through the MAPK pathway, and induce apoptotic cell death in CRC cells. Goals: 1) To examine the effects of sulindac metabolites (SMs) on protein levels and phosphorylation status of the other EGFR family members, HER2 and HER3, and the downstream signaling effector, Akt and 2) To determine if downregulation of EGFR family members is required for the apoptotic effects of SMs. Results: SMs downregulated both total and phosphorylated forms of HER2 and HER3 in a manner similar to their effects on EGFR. This downregulation was seen by 12h after treatment and persisted through at least 48h. Unexpectedly bothmetabolites significantly increased Akt phosphorylation as early as 1h after treatment in all cell lines tested. SW620 CRC cells which lack EGFR and do not signal through HER2 remained sensitive to both the apoptotic effects and the pAkt stimulatory effects of SMs. siRNA treatment resulted in a 76% knockdown of HER3 expression in HT29 (EGFR+) cells and 84% in SW620 (EGFR-) cells. HER3 knockdown failed to induce significant levels of apoptosis in either cell line but it did not prevent the apoptotic effect or the pAkt induction by SMs. Pre-treatment of HT29 cells with LY294002 prevented Akt phosphorylation by SMs and potentiated the growth inhibitory effects of sulindac sulfide in this cell line. Conclusion: These results demonstrate that I) EGFR, HER2, and HER3 appear to be similarly downregulated by SMs; II) Downregulation of EGFR family members does not appear to be sufficient for the apoptotic effect of sulindacmetabolites; III) Sulindacmetabolites induce an unexpected stimulation of Akt phosphorylation through an EGFR family member-independent effect and IV) Prevention of NSAID-induced AKT phosphorylation might potentiate the growth inhibitory activity of these drugs.

Carbachol induces TGF-alpha expression and colonic epithelial cell proliferation in sensory-desensitised rats

Signals for the expression of the peptide growth factors epidermal growth factor and transforming growth factor-alpha (TGFalpha) in the gastrointestinal mucosa are largely unknown. We have shown earlier that extrinsic afferents in the gastrointestinal tract induce TGFalpha expression in colonic mucosa via the deliberation of neurotransmitters substance P and calcitonin gene-related peptide. The aim of our present study was to determine the effects of carbachol on mucosal TGFalpha expression and epithelial cell proliferation in vivo. Rats were divided in three groups. Group 1 was treated with vehicle only, group 2 received one single subcutaneous injection of 250 microg/kg of carbachol and animals in group 3 were sensory-desensitised prior to the injection of 250 microg/kg carbachol. TGFalpha expression and epithelial cell proliferation was evaluated by polymerase chain reaction, Western blot analysis and bromodeoxyuridine staining. Carbachol induced a significant increase in mucosal epithelial cell proliferation and TGFalpha expression. Sensory desensitisation did neither abolish the increased TGFalpha expression nor the increase in epithelial cell proliferation. Parasympathetic pathways are involved in the control of TGFalpha expression in gastrointestinal mucosa as well as in epithelial cell proliferation.

Genetic reduction of circulating insulin‐like growth factor‐1 inhibits azoxymethane‐induced colon tumorigenesis in mice

High levels of insulin-like growth factor-1 (IGF-1) have been associated with a significant increase in colon cancer risk. Additionally, IGF-1 inhibits apoptosis and stimulates proliferation of colonic epithelial cells in vitro. Unfortunately, IGF-1 knockout mice have severe developmental abnormalities and most do not survive, making it difficult to study how genetic ablation of IGF-1 affects colon tumorigenesis. To test the hypothesis that inhibition of IGF-1 prevents colon tumorigenesis, we utilized a preexisting mouse model containing a deletion of the igf1 gene in the liver through a Cre/loxP system. These liver-specific IGF-1 deficient (LID) mice display a 50-75% reduction in circulating IGF-1 levels. We conducted a pilot study to assess the impact of liver-specific IGF-1 deficiency on azoxymethane (AOM)-induced colon tumors. LID mice had a significant inhibition of colon tumor multiplicity in the proximal area of the colon compared to their wild-type littermates. We examined markers of proliferation and apoptosis in the colons of the LID and wild-type mice to see if these were consistent with tumorigenesis. We observed a decrease in proliferation in the colons of the LID mice and an increase in apoptosis. Finally, we examined cytokine levels to determine whether IGF-1 interacts with inflammatory pathways to affect colon tumorigenesis. We observed a significant reduction in the levels of 7 out of 10 cytokines that were measured in the LID mice as compared to wild-type littermates. Results from this pilot study support the hypothesis that reductions in circulating IGF-1 levels may prevent colon tumorigenesis and affect both proliferation and apoptosis. Future experiments will investigate downstream genes of the IGF-1 receptor.

Effect of Omeprazole-Induced Hypergastrinaemia on the Proliferation of Colonic Mucosal Epithelial Cells in the Rat

In the present study we have investigated the effects of proglumide (PRO) and somatostatin analog (SMS 201-995) on the mitotic activity of colonic mucosa in rats under basal conditions and after omeprazole (OM). The stathmokinetic method was used. Proglumide and OM were administered for 5 days, twice daily, SMS 201-995 on the 5th day of the experiment only, also twice daily. It was found that: 1) OM enhanced the proliferation of colonic mucosa, as well as increased serum gastrin, when compared to controls, 2) OM and PRO, when applied jointly, decreased the mitotic activity of the colonic epithelium, 3) PRO alone increased the colonic cell proliferation.

Effectiveness of IκB kinase inhibitors in murine colitis-associated tumorigenesis

Nuclear factor kappaB (NF-kappaB) activation is involved in various inflammatory illnesses, for example inflammatory bowel disease, and is thought to be a key factor in the tumor-promoting mechanism of inflammation-associated tumorigenesis. This study investigated whether inhibitors of IKKbeta, which is a critical kinase for NF-kappaB activation, reduce colitis-associated tumorigenesis in mice. We used a mouse model of the disease whereby administration of azoxymethane (AOM) followed by repeated dextran sulfate sodium (DSS) ingestion causes severe colonic inflammation and the subsequent development of multiple tumors. Effects of IKKbeta inhibitors, NBD peptide, and IMD-0354 were examined. In a colitis-associated cancer model, treatment with the IKKbeta inhibitors NBD peptide and IMD-0354 significantly reduced the number of tumors compared with the untreated group. The tumors were also significantly smaller in the inhibitor-treated mice than in the untreated mice. Macrophage and neutrophil infiltration decreased with the inhibitor treatment. NF-kappaB activation and the expression of Cox-2 and iNOS were observed in tumor tissues, and the inhibitors ameliorated their expression. These inhibitors blocked NF-kappaB activation and the expression of proinflammatory cytokines mediated by the culture supernatant of inflamed colon in murine primary macrophages. In-vitro and in-vivo experiments showed that these drugs, especially NBD peptide, could also inhibit the proliferation of colonic epithelial cells. These results imply that IKKbeta-targeted NF-kappaB blockade is an attractive therapeutic approach for the prevention of colitis-associated tumors.

Dietary calcium and colonic epithelial cell proliferation.

Dietary Calcium and Colonic Epithelial Cell Proliferation Get access Nutrition Reviews, Volume 44, Issue 8, August 1986, Pages 264–265, https://doi.org/10.1111/j.1753-4887.1986.tb07650.x Published: 01 August 1986

Ethanol Does Not Promote MeIQx-initiated Rat Colon Carcinogenesis Based on Evidence from Analysis of a Colon Cancer Surrogate Marker

Epidemiological studies suggest that alcohol consumption increases the risk of developing colorectal cancer. However, the data are confounded by numerous cosegregating variables. To cast further light on the relationships between alcohol intake and colon cancer development, 21-day-old male F344/DuCrj rats were fed 200 ppm 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in their diet for 8 weeks and doses of 0, 0.1, 0.3, 1, 3, 10 and 20% of ethanol in their drinking water ad libitum for 16 weeks thereafter. The rats were sacrificed after 24 weeks of experiment, and aberrant crypt foci (ACF), surrogate lesions for colon cancer, were examined under a light microscope at low magnification. Ethanol was found not to affect the ACF formation at any dose compared with the initiated-controls. Furthermore, ethanol did not alter colon epithelial cell proliferation. These data, obtained by analysis of a colon cancer surrogate marker lesion, indicate that ethanol lacks promotion activity for MeIQx-initiated rat colon carcinogenesis.

A novel mTOR inhibitor is efficacious in a murine model of colitis

Ulcerative colitis is an autoimmune-inflammatory disease characterized by increased proliferation of colonic epithelial cells, dysregulation of signal transduction pathways, elevated mucosal T cell activation, increased production of proinflammatory cytokines, and enhanced leukocyte infiltration into colonic interstitium. Several compounds that possess antiproliferative properties and/or inhibit cytokine production exhibit a therapeutic effect in murine models of colitis. Mammalian target of rapamycin (mTOR), a protein kinase regulating cell proliferation, is implicated in colon carcinogenesis. In this study, we report that a novel haloacyl aminopyridine-based molecule (P2281) is a mTOR inhibitor and is efficacious in a murine model of human colitis. In vitro studies using Western blot analysis and cell-based ELISA assays showed that P2281 inhibits mTOR activity in colon cancer cells. In vitro and in vivo assays of proinflammatory cytokine production revealed that P2281 diminishes induced IFN-gamma production but not TNF-alpha production, indicating preferential inhibitory effects of P2281 on T cell function. In the dextran sulfate sodium (DSS) model of colitis, 1) macroscopic colon observations demonstrated that P2281 significantly inhibited DSS-induced weight loss, improved rectal bleeding index, decreased disease activity index, and reversed DSS-induced shortening of the colon; 2) histological analyses of colonic tissues revealed that P2281 distinctly attenuated DSS-induced edema, prominently diminished the leukocyte infiltration in the colonic mucosa, and resulted in protection against DSS-induced crypt damage; and 3) Western blot analysis showed that P2281 blocks DSS-induced activation of mTOR. Collectively, these results provide direct evidence that P2281, a novel mTOR inhibitor, suppresses DSS-induced colitis by inhibiting T cell function and is a potential therapeutic for colitis. Given that compounds with anticancer activity show promising anti-inflammatory efficacy, our findings reinforce the cross-therapeutic functionality of potential drugs.

Activation of Apoptosis by 1-Hydroxy-5,7-Dimethoxy-2-Naphthalene-Carboxaldehyde, a Novel Compound from Aegle marmelos

We have identified a natural compound that activates apoptosis of epithelial cancer cells through activation of tumor necrosis factor-alpha (TNF-alpha), TNF receptor (TNFR)-associated death domain (TRADD), and caspases. The molecule 1-hydroxy-5,7-dimethoxy-2-naphthalene-carboxaldehyde (HDNC, marmelin) was isolated and characterized from ethyl acetate fraction of extracts of Aegle marmelos. HDNC treatment inhibited the growth of HCT-116 colon cancer tumor xenografts in vivo. Immunostaining for CD31 showed that there was a significant reduction in microvessels in the HDNC-treated animals, coupled with decreased cyclooxygenase-2, interleukin-8, and vascular endothelial growth factor mRNA. Using hexoseaminidase assay, we determined that HDNC inhibits proliferation of HCT-116 colon and HEp-2 alveolar epithelial carcinoma cells. Furthermore, the cancer cells showed increased levels of activated caspase-3 and induced G(1) cell cycle arrest, which was suppressed by caspase-3 inhibitors. HDNC induced TNF-alpha, TNFR1, and TRADD mRNA and protein expression. Moreover, caspase-8 and Bid activation, and cytochrome c release, were observed, suggesting the existence of a cross-talk between death receptor and the mitochondrial pathways. HDNC inhibited AKT and extracellular signal-regulated kinase phosphorylation both in cells in culture and in tumor xenografts. In addition, electrophoretic mobility shift assay and luciferase reporter assays showed that HDNC significantly suppressed TNF-alpha-mediated activation and translocation of nuclear factor-kappaB (NF-kappaB). This was further confirmed by Western blot analysis of nuclear extracts wherein levels of RelA, the p65 component of NF-kappaB, were significantly less in cells treated with HDNC. Together, the data suggest that the novel compound HDNC (marmelin) is a potent anticancer agent that induces apoptosis during G(1) phase of the cell cycle and could be a potential chemotherapeutic candidate.

Endothelin-3 stimulates survival of goblet cells in organotypic cultures of fetal human colonic epithelium

Cells within the normal human colonic epithelium undergo a dynamic cycle of growth, differentiation, and death. The organotypic culture system of human fetal colonic epithelial cells seeded on top of collagen gels with embedded colonic fibroblasts allowed prolonged culture of the colonic epithelial cells (Kalabis J, Patterson MJ, Enders GM, Marian B, Iozzo RV, Rogler G, Gimotty PA, Herlyn M. FASEB J 17: 1115-1117, 2003). Herein, we have evaluated the role of endothelin-3 (ET3) and both cognate endothelin receptors (ETRA, ETRB) for human colonic epithelial cell growth and survival. ET3 was produced continuously by the fibroblasts as a result of adenovirus-mediated gene transfer. The presence and function of the endothelin receptors (ETRs) in epithelial cells was evaluated by [(3)H]thymidine incorporation using primary epithelial cells in monoculture and by immunohistochemistry on human fetal and adult paraffin-embedded tissues. In organotypic culture, ET3 increased the number of goblet cells but not of enteroendocrine cells. The increase in goblet cells was caused by prolonged cell survival and differentiation. The inhibition of both ETRA and ETRB significantly decreased the number of goblet cells and proliferation in epithelial cells, whereas the number of enteroendocrine cells remained unchanged. ET3 induced activation of IkappaB and MAPK in the epithelial cells, suggesting that these signaling pathways mediate its proproliferation and prosurvival activities. Our results demonstrate that ET3 is involved in regulating human colonic epithelial cell proliferation and survival, particularly for goblet cells, and may be an important component of colonic homeostasis.

A Novel mTOR Inhibitor Is Efficacious in a Murine Model of Colitis

Purpose: Ulcerative colitis is an autoimmune-inflammatory disease characterized by increased proliferation of colonic epithelial cells, dysregulation of signal transduction pathways, elevated mucosal T-cell activation, increased production of pro-inflammatory cytokines (e.g., TNF-α, IFN-γ), and enhanced leukocyte infiltration into colonic interstitium. Several compounds that possess anti-proliferative activities (e.g., rapamycin and its analog everolimus; mammalian target of rapamycin (mTOR) inhibitors) and/or inhibit IFN-γ production (NCX-1015, a nitric oxide derivative of prednisolone) exhibit a therapeutic effect in murine models of colitis. In this study, we report that a novel aminopyridine based molecule (P2281) is a mTOR inhibitor and is efficacious in a murine model [the dextran sulfate sodium (DSS) model] of human colitis. Methods: Cell-based ELISAs, Western blot analysis and DSS-model of colitis were employed. Results: In vitro studies using western blot analysis and cell-based ELISA assays each showed that P2281 inhibits mTOR activity (69% inhibition of mTOR phosphorylation at 10 μM in ELISA assays). In vitro and in vivo assays of pro-inflammatory cytokine production revealed that P2281 diminishes induced IFN-γ synthesis/release (IC50: ∼20 μM) but not TNF-α synthesis/release (IC50: > 100 μM). In the disease model of colitis (i) macroscopic colon observations revealed that P2281, administered intraperitoneally, significantly inhibited DSS-induced weight loss (5.70 ± 2.4% for P2281-treated mice vs. 17.75 ± 3.07% for control mice), improved rectal bleeding index (0.42 ± 0.08 for P2281-treated mice vs. 0.67 ± 0.17 for control mice), decreased disease activity index (3.92 ± 0.8 for P2281-treated mice vs. 6.83 ± 0.76 for control mice) and reversed DSS-induced shortening of the colon (colon lengths: 11.15 ± 0.3, 6.85 ± 0.39, and 7.88 ± 0.3 cms for naïve, control and P2281-treated mice, respectively); (ii) histological analyses of colonic tissues revealed that P2281 attenuated DSS-induced edema, prominently diminished the leukocyte infiltration in the colonic mucosa and resulted in protection against DSS-induced crypt damage. Conclusion: These results provide direct evidence that P2281, a novel mTOR inhibitor, suppresses DSS-induced colitis by inhibiting synthesis/release of pro-inflammatory mediators (e.g, IFN-γ) and leukocyte infiltration, and is a potentially attractive therapeutic for colitis.

Substance P-Mediated Expression of the Pro-Angiogenic Factor CCN1 Modulates the Course of Colitis

Substance P (SP) regulates important intestinal functions, such as mucosal permeability, motility, chloride secretion, and inflammation via the neurokinin-1 receptor (NK-1R). Previous reports showed that vascularization and expression of angiogenic factors are evident in the colonic mucosa of rats with colitis and patients with inflammatory bowel disease. Here we determined whether SP is associated with angiogenesis. Human NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) and mice with dextran sodium sulfate-induced colitis were used. We found that expression of the angiogenic factor CCN1 was increased in the colons of patients with Crohn's disease and ulcerative colitis. Mucosal extracts from inflammatory bowel disease patients induced human intestinal microvascular endothelial cell migration that was inhibited by blockade of CCN1 and its receptor integrin alphavbeta3. Both the degree of angiogenesis and CCN1 expression were elevated in the colons of mice with dextran sodium sulfate-induced colitis, which was reduced by treatment with the NK-1R antagonist CJ-12255. SP also increased CCN1 expression in NCM460-NK-1R colonocytes. SP exposure to human intestinal microvascular endothelial cells co-cultured with NCM460-NK-1R cells induced angiogenic activity that was inhibited by CCN1 silencing. In addition, intracolonic overexpression of CCN1 induced angiogenesis in mouse colon. Thus, SP mediates angiogenesis via CCN1 during colitis.

Calcium and calcium sensing receptor modulates the expression of thymidylate synthase, NAD(P)H:quinone oxidoreductase 1 and survivin in human colon carcinoma cells: Promotion of cytotoxic response to mitomycin C and fluorouracil

Ca(2+) and the cell-surface calcium sensing receptor (CaSR) constitute a novel and robust ligand/receptor system in regulating the proliferation and differentiation of colonic epithelial cells. Here we show that activation of CaSR by extracellular Ca(2+) (or CaSR agonists) enhanced the sensitivity of human colon carcinoma cells to mitomycin C (MMC) and fluorouracil (5-FU). Activation of CaSR up-regulated the expression of MMC activating enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO-1) and down-regulated the expression of 5-FU target, thymidylate synthase (TS) and the anti-apoptotic protein survivin. Cells that were resistant to drugs expressed little or no CaSR but abundant amount of survivin. Disruption of CaSR expression by shRNA targeting the CaSR abrogated these modulating effects of CaSR activation on the expression of NQO1, TS, survivin and cytotoxic response to drugs. It is concluded that activation of CaSR can enhance colon cancer cell sensitivity to MMC and 5-FU and can modulate the expression of molecules involved in the cellular responses to these cytotoxic drugs.

Genetic Ablation of M3 Muscarinic Receptors Attenuates Murine Colon Epithelial Cell Proliferation and Neoplasia

Colon epithelial cells express and most colon cancers overexpress M(3) muscarinic receptors (M(3)R). In human colon cancer cells, post-M(3)R signaling stimulates proliferation. To explore the importance of M(3)R expression in vivo, we used the azoxymethane-induced colon neoplasia model. Mice treated with weekly i.p. injection of saline [10 wild-type (WT) mice] or azoxymethane (22 WT and 16 M(3)R(-/-) mice) for 6 weeks were euthanized at 20 weeks. At week 20, azoxymethane-treated WT mice weighed approximately 16% more than M(3)R(-/-) mice (33.4 grams +/- 1.0 grams versus 27.9 grams +/- 0.5 grams; mean +/- SE, P < 0.001). In azoxymethane-treated M(3)R(-/-) mice, cell proliferation (BrdUrd staining) was reduced 43% compared with azoxymethane-treated WT mice (P < 0.05). Whereas control mice (both WT and M(3)R(-/-)) had no colon tumors, azoxymethane-treated WT mice had 5.3 +/- 0.5 tumors per animal. Strikingly, azoxymethane-treated M(3)R(-/-) mice had only 3.2 +/- 0.3 tumors per mouse (P < 0.05), a 40% reduction. Tumor volume in azoxymethane-treated M(3)R(-/-) mice was reduced 60% compared with azoxymethane-treated WT mice (8.1 mm(3) +/- 1.5 mm(3) versus 20.3 mm(3) +/- 4.1 mm(3); P < 0.05). Compared with WT, fewer M(3)R(-/-) mice had adenomas (6% versus 36%; P = 0.05), and M(3)R(-/-) mice had fewer adenocarcinomas per mouse (0.6 +/- 0.1 versus 1.7 +/- 0.4; P < 0.05). Eleven of 22 WT but no M(3)R(-/-) mice had multiple adenocarcinomas (P < 0.001). Compared with WT, azoxymethane-treated M(3)R-deficient mice have attenuated epithelial cell proliferation, tumor number, and size. M(3)R and post-M(3)R signaling are novel therapeutic targets for colon cancer.

618 Genetic Ablation of M 3 Muscarinic Receptors Attenuates Both Colon Epithelial Cell Proliferation and Neoplasia

618 Genetic Ablation of M 3 Muscarinic Receptors Attenuates Both Colon Epithelial Cell Proliferation and Neoplasia

Pleurotus ostreatus inhibits proliferation and modulates expression of cell cycle regulatory proteins in human breast and colon cancer cells

Oyster mushroom (Pleurotus ostreatus) is one of the edible mushrooms consumed in the US. The anticancer effects of P. ostreatus were demonstrated in vitro and in animal studies. However, the molecular mechanism(s) responsible for the biological activities of P. ostreatus remain to be elucidated. In the present study we evaluated the effect of an extract from Oyster mushroom (OME) on the proliferation of human breast and colon cancer cells. We found strong inhibitory effects of OME on the human colon cancer cells (HCT-116, HT-29) and breast cancer cells (MDA-MB-231, MCF-7) in a dose- and time-dependent manner. Interestingly, proliferation of non-tumorigenic colon (FHC) or mammary (MCF10A) epithelial cells was not affected by OME. Flow cytometry demonstrated that P. ostreatus induced cell cycle arrest at G0/G1 phase in colon HT-29 and breast MCF-7 cancer cells. cDNA microarray analysis revealed that OME changes expression of cell cycle regulatory proteins which expression was confirmed by western blot analysis. Therefore, our data demonstrate that OME induces cell cycle arrest of HT-29 by the up-regulation of expression of p-21, whereas cell cycle arrest of MCF-7 is induced by the up-regulation of expression of p-53 and p-21. In conclusion, our data suggest that the consumption of the dietary mushroom P. ostreatus could specifically inhibit growth of colon and breast cancer cells without any effect on normal cells.