Shakir M. Saud

Fluorescence endoscopic detection of murine colitis-associated colon cancer by topically applied enzymatically rapid-activatable probe

Objectives Screening colonoscopy to monitor for early colitis-associated colon cancer (CAC) is difficult due to the aberrant mucosal patterns associated with long-standing colitis. The aim of this study was to develop a rapid fluorescent detection method for use during colonoscopy for improving the detection of CAC utilising a topically applied enzymatically activatable probe (gGlu-HMRG) which fluoresces in the presence of γ-glutamyltranspeptidase (GGT), an enzyme associated with cancer. Methods Expression of GGT in colon cell lines was examined with fluorescence microscopy and flow cytometry. A mouse model (azoxymethane/dextran sulphate sodium) of CAC was used and mice were examined with white light and fluorescence colonoscopy before and after topical gGlu-HMRG administration. Results Expression of GGT, although variable, was higher in human colon cancer cells than normal human colon cells. Using fluorescence colonoscopy in mice, gGlu-HMRG fluorescent lesions were detected 5 min after topical administration and fluorescence persisted for at least 30 min. Fluorescence guided biopsy revealed all fluorescent lesions that contained cancer or dysplasia (n=16), whereas three out of 12 non-fluorescent lesions contained low grade dysplasia and others did not contain neoplastic histology. Microscopic inflammatory infiltration also had variable fluorescence but in general was much lower (∼10-fold) in signal than cancer. Repeat fluorescence endoscopy allowed individual tumours to be monitored. Conclusion These results suggest that gGlu-HMRG can improve endoscopic detection of CAC with a higher target to background ratio than conventional white light colonoscopy. This could be of benefit to patients with long-standing colitis who must undergo repeated screening colonoscopies.

Chemopreventive activity of plant flavonoid isorhamnetin in colorectal cancer is mediated by oncogenic Src and β-catenin

Analysis of the Polyp Prevention Trial showed an association between an isorhamnetin-rich diet and a reduced risk of advanced adenoma recurrence; however, the mechanism behind the chemoprotective effects of isorhamnetin remains unclear. Here, we show that isorhamnetin prevents colorectal tumorigenesis of FVB/N mice treated with the chemical carcinogen azoxymethane and subsequently exposed to colonic irritant dextran sodium sulfate (DSS). Dietary isorhamnetin decreased mortality, tumor number, and tumor burden by 62%, 35%, and 59%, respectively. MRI, histopathology, and immunohistochemical analysis revealed that dietary isorhamnetin resolved the DSS-induced inflammatory response faster than the control diet. Isorhamnetin inhibited AOM/DSS-induced oncogenic c-Src activation and β-catenin nuclear translocation, while promoting the expression of C-terminal Src kinase (CSK), a negative regulator of Src family of tyrosine kinases. Similarly, in HT-29 colon cancer cells, isorhamnetin inhibited oncogenic Src activity and β-catenin nuclear translocation by inducing expression of csk, as verified by RNA interference knockdown of csk. Our observations suggest the chemoprotective effects of isorhamnetin in colon cancer are linked to its anti-inflammatory activities and its inhibition of oncogenic Src activity and consequential loss of nuclear β-catenin, activities that are dependent on CSK expression.

Resveratrol prevents tumorigenesis in mouse model of Kras activated sporadic colorectal cancer by suppressing oncogenic Kras expression

Sporadic and non-hereditary mutations account for the majority of colorectal cancers (CRC). After the loss of adenomatous polyposis coli (APC) function and activation of the β-catenin/LEF signaling pathway, activating mutations in Kras are major drivers of sporadic CRC. Preventing the outgrowth of cells that develop sporadic mutations will decrease CRC. Resveratrol, a naturally occurring polyphenolic compound has anti-inflammatory, anti-oxidant and anti-cancer activities. We used a genetically engineered mouse model for sporadic CRC where the APC locus is knocked out and Kras is activated specifically in the distal colon to determine the effects of resveratrol on preventing and treating CRC. Feeding mice a diet supplemented with 150 or 300 ppm resveratrol (105 and 210mg daily human equivalent dose, respectively) before tumors were visible by colonoscopy resulted in a 60% inhibition of tumor production. In the 40% of mice that did develop tumors Kras expression was lost in the tumors. In a therapeutic assay where tumors were allowed to develop prior to treatment, feeding tumor bearing mice with resveratrol resulted in a complete remission in 33% of the mice and a 97% decrease in tumor size in the remaining mice. Analysis of miRNA expression in non-tumoral and tumoral colonic tissue of resveratrol treated mice showed an increased expression of miR-96, a miRNA previously shown to regulate Kras translation. These data indicate that resveratrol can prevent the formation and growth of colorectal tumors by downregulating Kras expression.

Diallyl Disulfide (DADS), a Constituent of Garlic, Inactivates NF-κB and Prevents Colitis-Induced Colorectal Cancer by Inhibiting GSK-3β

There is a strong belief that garlic has medicinal properties and may even reduce the risk of developing certain cancers including those of the gastrointestinal tract. The chemopreventive effects of garlic may be attributed to the anti-inflammatory properties of the sulfur-containing constituents of garlic, which includes diallyl disulfide (DADS). Here, we demonstrate that DADS prevented colorectal tumorigenesis in a mouse model of colitis-induced colorectal cancer. Supplementation with 85 ppm of DADS (60 mg daily human equivalent dose) in the diet of FVB/N mice treated with chemical carcinogen azoxymethane (AOM) and colonic irritant dextran sodium sulfate (DSS) resulted in the reduction in tumor incidence, tumor number, and tumor burden by 21.54%, 47.3%, and 66.4%, respectively. Further analysis revealed that mice fed the DADS-supplemented diet resolved the initial DSS-induced inflammation faster than those on the control diet, preventing prolonged inflammation and cellular transformation. Subsequent mechanistic studies in vitro suggest that DADS chemopreventive effects are mediated through NF-κB signaling. When SW480 colorectal cancer cells were treated with DADS, NF-κB nuclear localization and activity were diminished. Interestingly, NF-κB suppression was found to be dependent on DADS inhibition of GSK-3β, a positive regulator of NF-κB. Inhibition of GSK-3β and loss of nuclear NF-κB activity were also observed in vivo in AOM/DSS-treated mice fed a diet supplemented with 85 ppm DADS. Our results indicate that DADS can prevent tumorigenesis by suppressing inflammation, a process largely involving GSK-3β inhibition and consequential reduction in NF-κB nuclear localization. Cancer Prev Res; 9(7); 607–15. ©2016 AACR.

Abstract A54: Changes in fatty acid profile indicate a chemo-preventive response to navy bean extract in an inflammation-associated colorectal cancer mouse model

Introduction: Consumption of dry beans and their fractions decrease colorectal neoplasia; however, the underlying molecular mechanisms are unclear. Aim of the study was to identify response indicators of dietary attenuation of colorectal tumorigenesis using metabolic profiling. Methods: The study had a 2 x 2 factorial design. After being either induced or not with azoxymethane/dextran sodium sulfate (AOM/DSS), male FVB/N mice were fed an AIN93G diet containing either 0 (Control) or 10% navy bean ethanol extract (BE) for 6 weeks. Colon tissue was collected and analyzed for colitis, aberrant cell proliferation, and tumor, whereas serum, feed, and fecal samples were analyzed for metabolite levels. Results: Dietary BE attenuated AOM/DSS-induced chronic colitis, aberrant epithelial cell proliferation, fecal blood (heme), and tumorigenesis (-40%; P=0.02) in the colon. Similar changes were observed for fecal medium-chain fatty acids (hexanoate, octonoate) and plant phenolics (vanillate) and serum markers of fatty acid oxidation (carnitine, hexanoylcarnitine), and the pentose phosphate pathway (sedophetulose-7-phosphate). Moreover, dietary BE increased fecal markers of apoptosis (ribose, uracil, pseudouridine, xanthine, hypoxanthine) and attenuated the AOM/DSS-induced decrease in serum glycerophosphocholine. Conclusions: Dietary BE may inhibit survival and proliferation of premalignant colorectal epithelial cells by promoting fatty acid oxidation and resolving inflammation. Citation Format: Thushanthi H. Perera, Matthew R, Young, Shakir M. Saud, Christopher R. Dextras, Yava L. Jones-Hall, Edward D. Karoly, Brante P. Sampey, Young S. Kim, Nancy H. Colburn, Gerd Bobe. Changes in fatty acid profile indicate a chemo-preventive response to navy bean extract in an inflammation-associated colorectal cancer mouse model. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A54.

Abstract 4118: To dine with red wine: Systems approach to studying resveratrol's protection against inflammatory colorectal cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The relationship between inflammation and the development of colorectal cancer has received considerable attention over the last decade. Novel agents with anti-inflammatory properties appear to have beneficial effects in reducing the risk of developing colorectal cancer. Resveratrol, a naturally occurring polyphenolic compound, has been studied extensively with considerable amounts of evidence demonstrating its efficacy as an anti-inflammatory and anti-cancer agent. Given the number and diversity of the reported protective roles of resveratrol, we used an unbiased systems approach to elucidate the underlying mechanisms of resveratrols protection against colitis-induced colorectal cancer. Utilizing genomics, metabolomics and microbiome analysis in a pre-clinical model of colitis-induced colorectal carcinogenesis, we have linked the anti-inflammatory actions of resveratrol to both known targets and other novel potential molecular mechanisms. In a study, we treated mice with chemical carcinogen azoxymethane (AOM) and then exposed the mice to colonic irritant dextran sodium sulfate (DSS) to induce colitis-associated colon carcinogenesis. We found that a diet enriched in resveratrol (300ppm) prevented polyp formation, as well as, prevented polyps from progressing to adenomacarcinomas. Resveratrol treatment promoted the early recovery of DSS-induced inflammation with less severity of leukocyte infiltration and lower levels of IL-6, IL-8, and IL-10. Interestingly, the level of IL-22, which contributes to host defense at mucosal surfaces, was also significantly reduced with resveratrol. The metabolomic profile of DSS-treated control animals showed an elevation of microbial derived metabolites in the serum, which was ameliorated with resveratrol treatment. Together, resveratrol appears to preserve the intestinal barrier during early inflammation. Prior studies have shown resveratrols role in inhibiting several key enzymes active during tumor progression. We have correlated such findings with metabolomic biomarkers. We found a significant reduction in arachidonic acid, the limiting reagent of pro-inflammatory enzyme Cyclooxygenase-2 (COX-2). We also saw a reduction in spermidine. This polyamine is a product of Ornithine decarboxylase (ODC) activity that promotes proliferation. We have linked some of these metabolomic changes to changes in gene expression. These pre-clinical studies are consistent with that of previous studies and demonstrate the efficacy of resveratrol in preventing colitis associated colorectal cancer. A systems approach to study the chemopreventive efficacy of anti-inflammatory compounds like resveratrol may be an important paradigm for discovering the chemopreventive mechanisms of these agents. Citation Format: Shakir M. Saud, Amiran Dzutsev, Giorgio Trinchieri, Nancy Colburn, Matthew R. Young, Young Kim. To dine with red wine: Systems approach to studying resveratrols protection against inflammatory colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4118. doi:10.1158/1538-7445.AM2014-4118

Abstract 198: Resveratrol's Metabolomic biomarkers of efficacy: the missing link to chemoprevention.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Chemoprevention has received considerable attention over the last decade. The notion of using bioactive food components to reduce the risk of developing, or the recurrence of cancer has long been recognized. Resveratrol (a naturally occurring polyphenolic compound) has been studied extensively with considerable evidence demonstrating its efficacy as an anti-inflammatory and anti-cancer agent, with the most evidence for its role in colon cancer prevention. However, Resveratrol intervention studies in clinical trials still remains controversial, due to the lack of biomarkers of efficacy. Utilizing Metabolomic analysis we have identified biomarkers of efficacy in a pre-clinical model of inflammation-induced colorectal carcinogenesis. Metabolomics is the process of identifying metabolites present in biological specimens. Our study has demonstrated the chemopreventative activity of Resveratrol in mice treated with the chemical carcinogen azoxymethane (AOM) and then exposed to colonic irritant dextran sodium sulfate (DSS) to induce inflammation-associated colon carcinogenesis. We found a diet enriched in Resveratrol resulted in a reduction in tumor incidence in AOM/DSS treated mice by 29 percent. Furthermore, we identified several biomarkers that are indicative of Resveratrol chemopreventive activities. Prior studies have shown Resveratrols role in inhibiting several key enzymes important during the carcinogenesis process. We have correlated such findings with Metabolomic biomarkers. We found a significant reduction in Arachidonic acid, the limiting reagent of pro-inflammatory enzyme COX-2, and a reduction in spermidine a product of Ornithine decarboxylase, an enzyme that promotes proliferation. More intriguingly, we found biomarkers representing changes in bile acids by Resveratrol, which highlight chemopreventative activities that were not previously described. We found that Resveratrol treatment causes a reduction in tumorigenic secondary bile acids, deoxycholic acid and lithocholic acid, which are metabolic byproducts of intestinal bacteria. These data suggested that dietary supplementation with Resveratrol results in a healthy microbiome. These pre-clinical studies should easily be translated into clinical studies designed to ascertain chemopreventive efficacy for bioactive compounds like Resveratrol, all with the objective to identify patients that will benefit from an intervention. Citation Format: Shakir Saud, Matthew Young, Ed Karoly, Chris Bernard, Nancy Colburn, Young Kim. Resveratrols Metabolomic biomarkers of efficacy: the missing link to chemoprevention. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 198. doi:10.1158/1538-7445.AM2013-198

Abstract A76: Diet versus drug paradigm for the prevention of colorectal cancer: Isorhamnetin and resveratrol versus sulindac and DFMO

Increase in polyamine synthesis and inflammation is associated with increased risk in colon carcinogenesis. In patients with a high risk for colon cancer a combination treatment with difluoromethylornithine (DFMO), a selective inhibitor of polyamine synthesis, and sulindac, a nonsteroidal anti-inflammatory drug, resulted in a synergistic reduction in adenoma recurrence. However, this drug combination has some toxicities and should be limited to those individuals with a positive benefit to risk ratio. Bioactive food components have been shown to modulate inflammation and polyamine synthesis and can influence the risk of developing colon cancer with less toxicity. In this study we demonstrated that the chemopreventive activity of isorhamnetin (a plant flavonoid), and resveratrol (a naturally occurring polyphenolic compound) may mimic the effects of sulindac and DFMO. In a mouse model for colitis-associated colon carcinogenesis where mice are treated with the chemical carcinogen azoxymethane (AOM) and then exposed to colonic irritant dextran sodium sulfate (DSS), a diet enriched in isorhamnetin or resveratrol resulted in a reduction in tumor incidence by 22 and 29 percent, respectively. Furthermore, analysis of biomarkers revealed both anti-proliferative and anti-inflammatory properties of both isorhamnetin and resveratrol in vivo. More intriguingly, we found that isorhamnetin mimics sulindac activity by inhibiting Src kinase activity and interfering with Wnt/beta catenin signaling both in culture and in mice. In addition, our metabolomic analyses suggest that resveratrol mimics DFMO activity by inhibiting ornithine decarboxylase (ODC) activity in the mouse colon. Inhibition of ODC by resveratrol was confirmed in culture. It will be interesting to see if the combination of isorhamnetin and resveratrol will have a synergistic effect on prevention of colon cancer. These findings suggest that isorhamnetin and resveratrol could be used as a safer alternative to sulindac and DMFO for the prevention of colon cancer. Citation Format: Shakir Saud, Young Kim, Gerd Bobe, Nancy H. Colburn, Matthew R. Young. Diet versus drug paradigm for the prevention of colorectal cancer: Isorhamnetin and resveratrol versus sulindac and DFMO. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A76.