Wen-Chi L. Chang

5-aminosalicylic acid inhibits colitis-associated colorectal dysplasias in the mouse model of azoxymethane/dextran sulfate sodium-induced colitis.

Background: The impact of the antiinflammatory agent 5‐aminosalicylic acid (5‐ASA) on the risk for colitis‐associated colorectal cancer remains controversial. The chemopreventive activity of 5‐ASA was evaluated in the Swiss Webster model of azoxymethane (AOM)/dextran sulfate sodium (DSS)‐induced colitis‐associated neoplasia. Methods: Mice were injected with AOM (7.4 mg/kg i.p.) and randomized to receive either vehicle or 5‐ASA (75, 150, and 225 mg/kg) for the remainder of the study. DSS treatment began at 9 weeks of age and continued for 3 cycles. At the time of sacrifice (18 weeks of age), the entire colon and rectum were processed for histopathologic examination. Results: An inverse trend was observed between dose and multiplicity of colonic dysplasias in all drug‐treated groups (P = 0.03), with animals receiving 75 mg/kg 5‐ASA exhibiting 56% of the number of dysplasias of the AOM/DSS controls (mean ± SEM: 7.6 ± 1.4 and 13.6 ± 2.7, respectively). Administration of 75 mg/kg 5‐ASA decreased both the mean multiplicity of flat dysplasias (1.8 ± 0.4 for drug‐treated versus 5.6 ± 1.2 for AOM/DSS control) and the burden of polypoid dysplasias (tumor burden: 6.7 ± 2.7 for drug‐treated versus 14.9 ± 3.9 units for AOM/DSS controls) significantly (P = 0.002 and 0.04, respectively). Inflammation was least severe in the 75 mg/kg group, which exhibited the fewest number of colorectal tumors. Conclusions: These data suggest that low‐dose 5‐ASA may be efficacious in preventing colitis‐associated dysplasias and provide strong support for optimizing this therapy for the prevention of colonic neoplasms in patients with ulcerative colitis.

Sulindac selectively inhibits colon tumor cell growth by activating the cGMP/PKG pathway to suppress Wnt/β-catenin signaling

Nonsteroidal anti-inflammatory drugs (NSAID) display promising antineoplastic activity for colorectal and other cancers, but toxicity from COX inhibition limits their long-term use for chemoprevention. Previous studies have concluded that the basis for their tumor cell growth inhibitory activity does not require COX inhibition, although the underlying mechanism is poorly understood. Here, we report that the NSAID sulindac sulfide inhibits cyclic guanosine 3′,5′-monophosphate phosphodiesterase (cGMP PDE) activity to increase intracellular cGMP levels and activate cGMP-dependent protein kinase (PKG) at concentrations that inhibit proliferation and induce apoptosis of colon tumor cells. Sulindac sulfide did not activate the cGMP/PKG pathway, nor affect proliferation or apoptosis in normal colonocytes. Knockdown of the cGMP-specific PDE5 isozyme by siRNA and PDE5-specific inhibitors tadalafil and sildenafil also selectively inhibited the growth of colon tumor cells that expressed high levels of PDE5 compared with colonocytes. The mechanism by which sulindac sulfide and the cGMP/PKG pathway inhibits colon tumor cell growth involves the transcriptional suppression of β-catenin to inhibit Wnt/β-catenin T-cell factor transcriptional activity, leading to downregulation of cyclin D1 and survivin. These observations suggest that safer and more efficacious sulindac derivatives can be developed for colorectal cancer chemoprevention by targeting PDE5 and possibly other cGMP-degrading isozymes. Mol Cancer Ther; 12(9); 1848–59. ©2013 AACR.

Sulindac Sulfone Is Most Effective in Modulating β‐Catenin‐Mediated Transcription in Cells with Mutant APC

Abstract: Sulindac sulfone (FGN‐1, Aptosyn), a metabolite of the nonsteroidal anti‐inflammatory drug sulindac, lacks cyclooxygenase inhibitory activity. Although its ability to inhibit tumorigenesis in both carcinogen‐treated animals and patients with familial adenomatous polyposis has been attributed to the induction of apoptosis, its complete mechanism of action remains unclear. The purpose of the present study was to determine the ability of sulindac metabolites to regulate cellular levels of β‐catenin and downstream targets of the adenomatous polyposis coli (APC)/β‐catenin pathway in vitro. Sulindac sulfone was consistently more potent than the sulfide metabolite in all analyses, significantly decreasing the expression of total cellular β‐catenin (50% of control), pro‐caspase 3 (49%), cyclin D1 (51%), and PPARδ (65%) in SW480 cells. No significant alteration in pro‐caspase 3 or β‐catenin expression was found in HCA7, LS174, or Caco‐2 cells treated with sulindac sulfone. A dose‐dependent reduction in TCF‐mediated transcriptional activity was also observed in SW480 cells. These data demonstrate that sulindac sulfone can modulate the APC/β‐catenin pathway in vitro and that its efficacy is dependent upon the mutational status of APC and β‐catenin.

Functional characterization of peroxisome proliferator‐activated receptor‐β/δ expression in colon cancer

This study critically examined the role of PPARβ/δ in colon cancer models. Expression of PPARβ/δ mRNA and protein was lower and expression of CYCLIN D1 protein higher in human colon adenocarcinomas compared to matched non‐transformed tissue. Similar results were observed in colon tumors from Apc+/Min‐FCCC mice compared to control tissue. Dietary administration of sulindac to Apc+/Min‐FCCC mice had no influence on expression of PPARβ/δ in normal colon tissue or colon tumors. Cleaved poly (ADP‐ribose) polymerase (PARP) was either increased or unchanged, while expression of 14‐3‐3ε was not influenced in human colon cancer cell lines cultured with the PPARβ/δ ligand GW0742 under conditions known to increase apoptosis. While DLD1 cells exhibited fewer early apoptotic cells after ligand activation of PPARβ/δ following treatment with hydrogen peroxide, this change was associated with an increase in late apoptotic/necrotic cells, but not an increase in viable cells. Stable over‐expression of PPARβ/δ in human colon cancer cell lines enhanced ligand activation of PPARβ/δ and inhibition of clonogenicity in HT29 cells. These studies are the most quantitative to date to demonstrate that expression of PPARβ/δ is lower in human and Apc+/Min‐FCCC mouse colon tumors than in corresponding normal tissue, consistent with the finding that increasing expression and activation of PPARβ/δ in human colon cancer cell lines inhibits clonogenicity. Because ligand‐induced attenuation of early apoptosis can be associated with more late, apoptotic/necrotic cells, but not more viable cells, these studies illustrate why more comprehensive analysis of PPARβ/δ‐dependent modulation of apoptosis is required in the future.

Detection and volume determination of colonic tumors in Min mice by magnetic resonance micro‐imaging

We applied MRI to the in vivo detection of spontaneous colorectal tumors in a unique mouse model, the Fox Chase Cancer Center (FCCC) ApcMIN mouse. Unlike other Min (multiple intestinal neoplasia) strains, FCCC ApcMIN animals develop an appreciable number of tumors in the large intestine, which makes them an appropriate mouse model for colon cancer in humans. We describe a method for marking the colon on MRI data sets that involves a bowel‐cleansing procedure and the insertion of a polyurethane tube (filled with an MRI contrast agent) fully into the colon. We found that tumors as small as 1.5 mm in diameter can be consistently identified from MRI datasets with a voxel size of 0.1 mm × 0.133 mm × 0.133 mm. Tumor volumes were determined from the MRM data sets with the use of a novel approach to planimetry in 3D data sets. We observed a correlation between tumor volume (as measured from the MRI datasets) and tumor weight of 0.942, and a P‐value of 0.008, based on Spearmans test. These data show that MRI can be used to accurately monitor tumor growth in mouse models of colorectal carcinogenesis. Magn Reson Med 52:524–529, 2004.

Endoscopic imaging and size estimation of colorectal adenomas in the multiple intestinal neoplasia mouse

BACKGROUND The scientific potential of animal models of carcinogenesis has not been fully realized because of our limited ability to monitor tumor growth in vivo. OBJECTIVE To develop an endoscopy-based protocol for the accurate estimation of adenoma size in vivo from images obtained during colonoscopy. DESIGN To compare estimates of lesion size acquired during endoscopy with those obtained from magnetic resonance imaging (MRI) and at necropsy. SETTING A small-animal imaging facility. SUBJECTS Adenomatous polyposis coli multiple intestinal metaplasia Fox Chase Cancer Center mice that develop multiple colorectal adenomas. METHODS The mice received colonoscopic examination by using a rigid endoscope, and high-resolution images of colon adenomas were captured by using a charge-coupled-device camera. Lesion size was estimated by comparing the dimensions of the adenoma relative to a reference rod by using a novel geometric construction. The resulting areas were compared with estimates from MRIs and validated at necropsy. MAIN OUTCOME MEASUREMENTS Cross-sectional area of colon adenomas. RESULTS The cross-sectional area of 20 adenomas was measured in vivo during colonoscopy and compared with the size as measured at necropsy, which yielded a Pearson correlation coefficient of 0.94 (P = 6.52 x 10(-9)). Assessment of interoperator variability, when using measurements from 11 adenomas, yielded a Pearson correlation coefficient of 0.85 (P = 4.35 x 10(-3)) and demonstrated excellent reproducibility. LIMITATIONS Only the distal colon could be viewed, and endoscopic measurements were 2-dimensional. CONCLUSIONS An endoscopic method for the reliable measurement of colorectal adenomas in vivo was established. The application of this technique to mouse models of colon carcinogenesis will provide unique insight into the dynamics of adenoma growth.

Plecanatide-mediated activation of guanylate cyclase-C suppresses inflammation-induced colorectal carcinogenesis in Apc+/Min-FCCC mice

AIM To evaluate the effect of orally administered plecanatide on colorectal dysplasia in Apc+/Min-FCCC mice with dextran sodium sulfate (DSS)-induced inflammation. METHODS Inflammation driven colorectal carcinogenesis was induced in Apc+/Min-FCCC mice by administering DSS in their drinking water. Mice were fed a diet supplemented with plecanatide (0-20 ppm) and its effect on the multiplicity of histopathologically confirmed polypoid, flat and indeterminate dysplasia was evaluated. Plecanatide-mediated activation of guanylate cyclase-C (GC-C) signaling was assessed in colon tissues by measuring cyclic guanosine monophosphate (cGMP) by ELISA, protein kinase G-II and vasodilator stimulated phosphoprotein by immunoblotting. Ki-67, c-myc and cyclin D1 were used as markers of proliferation. Cellular levels and localization of β-catenin in colon tissues were assessed by immunoblotting and immunohistochemistry, respectively. Uroguanylin (UG) and GC-C transcript levels were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). A mouse cytokine array panel was used to detect cytokines in the supernatant of colon explant cultures. RESULTS Oral treatment of Apc+/MinFCCC mice with plecanatide produced a statistically significant reduction in the formation of inflammation-driven polypoid, flat and indeterminate dysplasias. This anti-carcinogenic activity of plecanatide was accompanied by activation of cGMP/GC-C signaling mediated inhibition of Wnt/β-catenin signaling and reduced proliferation. Plecanatide also decreased secretion of pro-inflammatory cytokines (IL-6, IL1 TNF), chemokines (MIP-1, IP-10) and growth factors (GCSF and GMCSF) from colon explants derived from mice with acute DSS-induced inflammation. The effect of plecanatide-mediated inhibition of inflammation/dysplasia on endogenous expression of UG and GC-C transcripts was measured in intestinal tissues. Although GC-C expression was not altered appreciably, a statistically significant increase in the level of UG transcripts was detected in the proximal small intestine and colon, potentially due to a reduction in intestinal inflammation and/or neoplasia. Taken together, these results suggest that reductions in endogenous UG, accompanied by dysregulation in GC-C signaling, may be an early event in inflammation-promoted colorectal neoplasia; an event that can potentially be ameliorated by prophylactic intervention with plecanatide. CONCLUSION This study provides the first evidence that orally administered plecanatide reduces the multiplicity of inflammation-driven colonic dysplasia in mice, demonstrating the utility for developing GC-C agonists as chemopreventive agents.

Differential response of flat and polypoid colitis-associated colorectal neoplasias to chemopreventive agents and heterocyclic amines

Individuals with ulcerative colitis face an increased risk of developing colorectal cancer and would benefit from early chemopreventive intervention. Results from preclinical studies in the mouse model of dextran sulfate sodium-induced colitis demonstrate that flat and polypoid colitis-associated dysplasias arise via distinct genetic pathways, impacted by the allelic status of p53. Furthermore, flat and polypoid dysplasias vary in their response to induction by the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and inhibition by 5-aminosalicylic acid, a common therapy for the maintenance of colitis patients. These data suggest that use of combination therapy is essential for the optimal inhibition of colitis-associated colorectal cancer.

Differential preventive activity of sulindac and atorvastatin in Apc+/Min-FCCCmice with or without colorectal adenomas

Objective The response of subjects to preventive intervention is heterogeneous. The goal of this study was to determine if the efficacy of a chemopreventive agent differs in non-tumour-bearing animals versus those with colorectal tumours. Sulindac and/or atorvastatin was administered to Apc+/Min-FCCC mice with known tumour-bearing status at treatment initiation. Design Male mice (6–8 weeks old) underwent colonoscopy and received control chow or chow with sulindac (300 ppm), atorvastatin (100 ppm) or sulindac/atorvastatin. Tissues were collected from mice treated for 14 weeks (histopathology) or 7 days (gene expression). Cell cycle analyses were performed on SW480 colon carcinoma cells treated with sulindac, atorvastatin or both. Results The multiplicity of colorectal adenomas in untreated mice bearing tumours at baseline was 3.6-fold higher than that of mice that were tumour free at baseline (P=0.002). Atorvastatin completely inhibited the formation of microadenomas in mice that were tumour free at baseline (P=0.018) and altered the expression of genes associated with stem/progenitor cells. Treatment of tumour-bearing mice with sulindac/atorvastatin led to a 43% reduction in the multiplicity of colorectal adenomas versus untreated tumour-bearing mice (P=0.049). Sulindac/atorvastatin increased the expression of Hoxb13 and Rprm significantly, suggesting the importance of cell cycle regulation in tumour inhibition. Treatment of SW480 cells with sulindac/atorvastatin led to cell cycle arrest (G0/G1). Conclusions The tumour status of animals at treatment initiation dictates response to therapeutic intervention. Atorvastatin eliminated microadenomas in tumour-free mice. The tumour inhibition observed with Sul/Atorva in tumour-bearing mice was greater than that achieved with each agent.

Imaging Matrix Metalloproteases in Spontaneous Colon Tumors: Validation by Correlation with Histopathology

The use of fluorescent probes in conjunction with white-light colonoscopy is a promising strategy for improving the detection of precancerous colorectal lesions, in particular flat (sessile) lesions that do not protrude into the lumen of the colon. We describe a method for determining the sensitivity and specificity of an enzymatically activated near-infrared probe (MMPSense680) for the detection of colon lesions in a mouse model (APC+/Min-FCCC) of spontaneous colorectal cancer. Fluorescence intensity correlates directly with the activity of matrix metalloproteinases (MMPs). Overexpression of MMPs is an early event in the development of colorectal lesions. Although the probe employed serves as a reporter of the activity of MMPs, our method can be applied to any fluorescent probe that targets an early molecular event in the development of colorectal tumors.