AOM-induced Aberrant Crypt Foci Research Articles

Mangiferin (mango) attenuates AOM-induced colorectal cancer in rat’s colon by augmentation of apoptotic proteins and antioxidant mechanisms

Mangiferin (MF) is a natural C-glucosylxantone compound that has many substantial curative potentials against numerous illnesses including cancers. The present study's goal is to appraise the chemo preventive possessions of MF on azoxymethane (AOM)-mediated colonic aberrant crypt foci (ACF) in rats. Rats clustered into 5 groups, negative control (A), inoculated subcutaneously with normal saline twice and nourished on 0.5% CMC; groups B-E injected twice with 15 mg/kg azoxymethane followed by ingestion of 0.5% CMC (B, cancer control); intraperitoneal inoculation of 35 mg/kg 5-fluorouracil (C, reference rats) or nourished on 30 mg/kg (D) and 60 mg/kg (E) of MF. Results of gross morphology of colorectal specimens showed significantly lower total colonic ACF incidence in MF-treated rats than that of cancer controls. The colon tissue examination of cancer control rats showed increased ACF availability with bizarrely elongated nuclei, stratified cells, and higher depletion of the submucosal glands compared to MF-treated rats. Mangiferin treatment caused increased regulation of pro-apoptotic (increased Bax) proteins and reduced the β-catenin) proteins expression. Moreover, rats fed on MF had significantly higher glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), and lower malondialdehyde (MDA) concentrations in their colonic tissue homogenates. Mangiferin supplementation significantly down-shifted pro-inflammatory cytokines (transforming growth factor-α and interleukine-6) and up-shifted anti-inflammatory cytokines (interleukine-10) based on serum analysis. The chemo-protective mechanistic of MF against AOM-induced ACF, shown by lower ACF values and colon tissue penetration, could be correlated with its positive modulation of apoptotic cascade, antioxidant enzymes, and inflammatory cytokines originating from AOM oxidative stress insults.

Changes in the Fecal Metabolome Accompany an Increase in Aberrant Crypt Foci in the Colon of C57BL/6 Mice Fed with a High-Fat Diet.

High-fat diet (HFD)-induced obesity is a risk factor for colon cancer. Our previous data show that compared to an AIN-93 diet (AIN), a HFD promotes azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) formation and microbial dysbiosis in C57BL/6 mice. To explore the underlying metabolic basis, we hypothesize that AOM treatment triggers a different fecal metabolomic profile in C57BL/6 mice fed the HFD or the AIN. We found that 65 of 196 identified metabolites were significantly different among the four groups of mice (AIN, AIN + AOM, HFD, and HFD + AOM). A sparse partial least squares discriminant analysis (sPLSDA) showed that concentrations of nine fecal lipid metabolites were increased in the HFD + AOM compared to the HFD, which played a key role in overall metabolome group separation. These nine fecal lipid metabolite concentrations were positively associated with the number of colonic ACF, the cell proliferation of Ki67 proteins, and the abundance of dysbiotic bacteria. These data suggest that the process of AOM-induced ACF formation may increase selective fecal lipid concentrations in mice fed with a HFD but not an AIN. Collectively, the accumulation of these critical fecal lipid species may alter the overall metabolome during tumorigenesis in the colon.

Azoxymethane Alters the Plasma Metabolome to a Greater Extent in Mice Fed a High-Fat Diet Compared to an AIN-93 Diet.

Consumption of a high-fat diet (HFD) links obesity to colon cancer in humans. Our data show that a HFD (45% energy fat versus 16% energy fat in an AIN-93 diet (AIN)) promotes azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) formation in a mouse cancer model. However, the underlying metabolic basis remains to be determined. In the present study, we hypothesize that AOM treatment results in different plasma metabolomic responses in diet-induced obese mice. An untargeted metabolomic analysis was performed on the plasma samples by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). We found that 53 of 144 identified metabolites were different between the 4 groups of mice (AIN, AIN + AOM, HFD, HFD + AOM), and sparse partial least-squares discriminant analysis showed a separation between the HFD and HFD + AOM groups but not the AIN and AIN + AOM groups. Moreover, the concentrations of dihydrocholesterol and cholesterol were inversely associated with AOM-induced colonic ACF formation. Functional pathway analyses indicated that diets and AOM-induced colonic ACF modulated five metabolic pathways. Collectively, in addition to differential plasma metabolomic responses, AOM treatment decreases dihydrocholesterol and cholesterol levels and alters the composition of plasma metabolome to a greater extent in mice fed a HFD compared to the AIN.

Azoxymethane Alters the Plasma Metabolome to a Greater Extent in Mice Fed a High Fat Diet Compared to AIN-93 Diet

Abstract Objectives Consumption of a high fat diet (HFD) links obesity to colon cancer in humans. Consistent with this observation, our data show that a HFD (45% energy fat versus 16% energy fat in AIN-93 diet, AIN) promotes azoxymethane (AOM) induced colonic aberrant crypt foci (ACF) formation in a mouse cancer model. However, the underlying metabolic basis remains to be determined. We hypothesize that AOM treatment results in different plasma metabolomic responses in AOM mouse colon cancer model fed with a HFD compared to the AIN. Methods Four-week-old male C57BL/6 mice were fed the AIN or HFD in the context of with or without AOM treatment for 16 weeks. At the end of this study, plasma samples were extracted and derivatized by silylation and methyloximation then analyzed by time-of-flight mass spectrometry using an untargeted metabolomic technique. Results Untargeted metabolomic profiling showed that 53 of 144 identified metabolites were different between the 4 groups of mice (AIN only, AIN + AOM; HFD only, HFD + AOM). Sparse partial least squares discriminant analysis (two-dimensional plots) showed a separation between the HFD and HFD + AOM groups but not AIN and AIN + AOM groups. Major metabolites responsible for this separation were beta-sitosterol, dihydrocholesterol, alpha-tocopherol, methylphosphate, citric acid, cholesterol, isocitric acid, myristic acid, arachidonic acid, and nicotinamide. Importantly, the levels of dihydrocholesterol and cholesterol were inversely associated with AOM-induced colonic ACF formation. Functional pathway and enrichment analyses indicated that diets and AOM-induced colonic ACF modulated 5 metabolic pathways: (a) citrate cycle (TCA cycle), (b) arginine biosynthesis, (c) aminoacyl-tRNA biosynthesis, (d) alanine, aspartate and glutamate metabolism, (e) glyoxylate and dicarboxylate metabolism. Conclusions Collectively, we demonstrate that AOM decreases dihydrocholesterol and cholesterol levels, and alters the composition of plasma metabolome to a greater extent in mice fed a HFD compared to the AIN. Funding Sources This work was supported by U.S. Department of Agriculture, Agricultural Research Service, research project 3062-51000-056-00D.

Gum arabic and red propolis protecteting colorectal preneoplastic lesions in a rat model of azoxymethane1.

PurposeTo evaluate red propolis, gum arabic and L-lysine activity on colorectal preneoplastic lesions induced by azoxymethane (AOM). Methods The study featured 4 control groups (I-IV) and 4 experimental groups (V-VIII), totaling 48 rats. Once a week for 2 weeks, animals on control groups received saline, while animals in experimental groups received azoxymethane (15 mg/kg i.p.). The follow up along 16 weeks included daily oral gavage to administer water (I and V), L-lysine (150 mg/kg)(II and VI), própolis (100mg/5ml/kg)(III and VII), or gum arabic (5ml/kg)(IV and VIII). Was performed surgery on the animals in the end of this time in order to collect blood for biological assays (TBARS, GSH), followed by their sacrifice to tissue extract. Results Oxidative stress (TBARS) and the number of aberrant crypt foci (ACF) in distal colon were lower using própolis (p<0.01 for both parameters). Gum arabic reduced preneoplastic lesions (ACF ≤ 4 crypts) on distal colon and on the entire colon (p<0.05). Conclusions Red propolis reduced AOM-induced oxidative stress (TBARS) and total number of ACF in the distal colon. L-lysine neither protected against nor enhanced AOM-induced ACF. Gum arabic reduced the number of ACF.

Chemopreventive effects of Ku-jin tea against AOM-induced precancerous colorectal lesions in rats and metabolomic analysis

Ku-jin tea (KJT) is a health beverage prepared from the leaves of the plant Acer tataricum subsp. ginnala that has been consumed in some regions of China for thousands of years. KJT contains high levels of anti-inflammatory and antioxidative compounds such as ginnalins, but little is known about the chemopreventive effect of KJT on colon cancer. In this study, we investigated the preventive effects of KJT on colon carcinogenesis using the azoxymethane (AOM)-induced precancerous colorectal lesion model in rats. The results showed that the number of aberrant crypts, aberrant crypt foci (ACF) and crypts/focus in rats of the KJT + AOM group were significantly decreased compared with rats of the AOM group (p < 0.01). Further exploration of the prevention mechanism of KJT by UPLC-QTOF/MS-based urinary metabolomics showed that 5 metabolic pathways were modulated, including purine metabolism and amino acid metabolism, in the group with KJT. In addition, the levels of the immunomodulatory cytokines IL-1α and IL-10 were significantly decreased, and the levels of IL-2 in the serum of AOM rats increased after KJT treatment. Our present data suggest that KJT can inhibit AOM-induced colonic ACF formation and might be a useful chemopreventive agent against colorectal carcinogenesis.

Sphingosine Kinase 1 expression in peritoneal macrophages is required for colon carcinogenesis.

Accumulating evidence suggests that the sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate (S1P) pathway plays a pivotal role in colon carcinogenesis. Our previous studies indicate that the SphK1/S1P pathway mediates colon carcinogenesis at least by regulating cyclooxygenase 2 (COX-2) expression and prostaglandin E2 (PGE2) production. However, the mechanisms by which this pathway regulates colon carcinogenesis are still unclear. First, we show that SphK1 deficient mice significantly attenuated azoxymethane (AOM)-induced colon carcinogenesis as measured by colon tumor incidence, multiplicity, and volume. We found that AOM activates peritoneal macrophages to induce SphK1, COX-2, and tumor necrosis factor (TNF)-α expression in WT mice. Interestingly, SphK1 knockout (KO) mice revealed significant reduction of COX-2 and TNF-α expression from AOM-activated peritoneal macrophages, suggesting that SphK1 regulates COX-2 and TNF-α expression in peritoneal macrophages. We found that inoculation of WT peritoneal macrophages restored the carcinogenic effect of AOM in Sphk1 KO mice as measured by aberrant crypt foci (ACF) formation, preneoplastic lesions of colon cancer. In addition, downregulation of SphK1 only in peritoneal macrophage by short hairpin RNA (shRNA) reduced the number of ACF per colon induced by AOM. Intraperitoneal injection of sphingolipids demonstrates that S1P enhanced AOM-induced ACF formation, while ceramide inhibited. Finally, we show that SphK inhibitor SKI-II significantly reduced the number of ACF per colon. These results suggest that SphK1 expression plays a pivotal role in the early stages of colon carcinogenesis through regulating COX-2 and TNF-α expression from activated peritoneal macrophages.

Abstract 4791: Cystathionine-β-Synthase (CBS) and the progression of colorectal carcinogenesis

Abstract Introduction: We recently demonstrated that cystathionine-β-synthase (CBS) stimulates colon cancer bioenergetics, migration, invasion and angiogenesis and is increased in colorectal cancer compared to normal colonic mucosa. The role of CBS in carcinogenesis is still, however, unknown. Here we compare CBS protein levels and immunohistochemistry in human colonic biopsies at different stages of colorectal carcinogenesis. In addition, we used Azoxymethane (AOM) in CBS knockdown mice to study the development of aberrant crypt foci (ACF), a model of sporadic colorectal carcinogenesis. Methods: CBS protein expression was assessed in human biopsies of normal mucosa, tubular adenoma and carcinoma in situ by Western blotting. Immunohistochemical (IHC) staining was used to assess CBS levels in formalin-fixed/paraffin-embedded specimens of normal colonic mucosa, adenomatous polyps and colon adenocarcinoma. To assess the effect of CBS gene dosage on azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation CBS heterozygous mice (CBS+/-) and wild-type (CBS+/+) controls were treated with the mutagen AOM at 10mg/kg via intraperitoneal injection once per week for 5 weeks. At the end of 16 weeks, the colons were harvested and ACF visualized with methylene blue staining by 3 independently blinded observers. Significance (p ≤ 0.05) was determined using GraphPad Prism 7 software. Results: Consistent with our previously reported findings CBS levels were relatively low in biopsies of normal mucosa. By comparison, it was elevated in polyps exhibiting both low- and high-grade dysplasia. IHC staining of normal mucosa and hyperplastic polyps revealed CBS immunoreactivity in a small number of cells located along the basal lamina aspect of the colonic crypts in both normal and hyperplastic polyps. A sight increase in cytoplasmic CBS staining also was noted in the epithelial cells of hyperplastic polyps. In contrast, the epithelial cells of tubular adenoma specimens exhibited higher levels of diffuse cytoplasmic CBS staining with frequent focal areas of intense staining adjacent to mucin-containing vesicles. Sections of adenocarcinoma exhibited diffuse CBS staining throughout the cytoplasm of the cancer cells. AOM treatment induced significantly less ACF in the colons of CBS(+/-) mice compared to wild-type controls. The loss of one allele of CBS reduced the number of AOM-induced ACF by half. The mean number of ACF ± SD for CBS(+/+) and CBS(+/-) were 10.9 ± 5.2 and 5.6 ± 2.5, respectively (p &amp;lt; 0.05). Conclusion: CBS protein levels increase and the pattern of expression changes with progression from benign hyperplastic polyps to invasive adenocarcinoma, suggesting the enzyme may play a functional role in colorectal carcinogenesis. In addition, CBS single allele knockdown significantly reduces ACF formation. CBS, and its product H2S, may, therefore, be a driver of carcinogenesis. Citation Format: John R. Zatarain, Ketan Thanki, Michael E. Nicholls, Manjit Maskey, Muhammad U. Jawad, Ches'Nique Phillips, Katalin Modis, Csaba Szabo, Celia Chao, Mark R. Hellmich. Cystathionine-β-Synthase (CBS) and the progression of colorectal carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4791. doi:10.1158/1538-7445.AM2017-4791

Chemopreventive effect of Phaleria macrocarpa on colorectal cancer aberrant crypt foci in vivo

Ethnopharmacological relevanceNatural products are important ingredients for pharmaceutical applications specifically new entities for treating cancer and other diseases. Phaleria macrocarpa is native of Indonesia and considered as a prolific source of bioactive substances useful for chemoprevention. Aim of the studyTo investigate the chemopreventive properties of Phaleria macrocarpa on azoxymethane (AOM)-induced aberrant crypt foci (ACF) in rats. MethodsThe biological activities of the ethanol extract of P. macrocarpa fruits were evaluated both in vitro and in vivo. First the extract was investigated for its in vitro antioxidant activity by the total phenolic content and ferric reducing antioxidant power assay. Then the chemopreventive effect of P. macrocarpa was performed on AOM-induced aberrant crypt foci as colorectal carcinoma model in rats. Resultthe crude ethanolic extract of P. macrocarpa has high antioxidant activity and modulated the oxidative stress as proved by the up-regulation of glutathione-s-transferase and superoxide dismutase. Immunohistochemical staining of the treated sections showed overexpression of PCNA and Bax, reduced crypt sizes and numbers, indicating the characteristic feature of apoptotic cancer cells. PCNA is a landmark of cell damage and turn-over and can be associated with clinical cancer mutation. The most potent doses were 250mg/kg and 500mg/kg as compared to 35mg/kg 5-fluorouracil. ConclusionIn this sense, the potential modulation of the colorectal pathophysiological pathway by P. macrocarpa natural compounds mostly flavonoids offer a great possibility for the discovery of new leads towards the colorectal cancer.

Plum polyphenols inhibit colorectal aberrant crypt foci formation in rats: potential role of the miR-143/protein kinase B/mammalian target of rapamycin axis

The nutritional prevention of aberrant crypt foci by polyphenols may be a crucial step to dietary cancer prevention. The objective of this study was to determine the underlying mechanisms that contribute to the anti-inflammatory and antitumorigenic properties of plum (Prunus salicina L.) polyphenols, including chlorogenic acid and neochlorogenic acid, in azoxymethane (AOM)-treated rats. The hypothesis was that plum polyphenolics suppress AOM-induced aberrant crypt foci formation through alterations in the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway and relative micro-RNA expressions. Sprague-Dawley rats (n=10/group) received plum beverage (1346mg gallic acid equivalents/L) or a control beverage ad libitum for 10 weeks with subcutaneous injections of AOM (15mg/kg) at weeks 2 and 3. Results show that the consumption of the plum beverage decreased the number of dysplastic aberrant crypt foci by 48% (P<.05) and lowered proliferation of mucosal cells by 24% (P<.05). The plum beverage decreased the activity of glutathione peroxidase, superoxide dismutase, and catalase in mucosal scrapings, as well as the superoxide dismutase activity in serum. The results were accompanied by a down-regulation of proinflammatory enzymes nuclear factor κB, nitric oxide synthase, cyclooxygenase-2, and vascular cell adhesion molecule 1 messenger RNA. Plum inhibited the expression of AKT and mTOR messenger RNA, phosphorylated AKT, mTOR, and hypoxia-inducible factor-1α protein levels, and the ratio of the phosphorylated/total protein expression of mTOR. Also, the plum beverage increased the expression of miR-143, which is involved in the regulation of AKT. These results suggest that plum polyphenols may exhibit a chemopreventive potential against colon carcinogenesis by impacting the AKT/mTOR pathway and miR-143.

Abstract 907: Chemopreventive effects of a non-camellia tea against azoxymethane -induced precancerous colorectal lesions in male rats

Abstract Tea (the leaves from Camellia plants) is one of the most popular beverages in the world; substantial evidence supports its success in the prevention of carcinogenesis and other chronic diseases in animal models. Plants other than Camellia have been widely used as tea as well in various cultures. We have systematically collected and identified over 30 species of frequently used Non-Camellia teas, which have been consumed at least 300 years in China or other nations. Recently, non-Camellia teas have attracted much attentions due to high potentials of preventing and treating metabolic and inflammatory diseases, as well as cancers. Epidemiologic studies report that tea consumption significantly reduces risk of colorectal cancer (CRC). We here reported in vivo preventive effects of the Ku-jin-cha (KJC) tea for CRC. KJC tea is a kind of “Non-Camellia tea” prepared from leaves of the plant Acer ginnala and popularly used in some Chinese regions for thousands of years. KJC contains high levels of anti-inflammatory and anti-oxidative compounds such as ginnalins. However, the potential chemopreventive effect of KJC remains unknown. Here, we investigated modulatory effects of KJC on colon carcinogenesis using the rat azoxymethane (AOM) induced precancerous colorectal lesion model. Wistar rats (male, 4∼5 weeks old) were randomly divided into 4 groups (Control, KJC, AOM and AOM+KJC, 10 rats/group) and were fed either normal water or experimental water containing KJC for 12 weeks. After one week of water or KJC drinking, all rats except the Control Group were injected with 15 mg/kg AOM (s.c., once weekly for 3 weeks). At 16∼17 weeks of age, urine and sera were collected for metabolomic analysis using UPLC-QTOF/MS (ultra-HPLC coupled with time-of-flight mass spectrometry). Rats were sacrificed at the end of KJC administration, and colorectums collected for evaluation of aberrant crypt (AC), aberrant crypt foci (ACF) and Crypts/focus. Spleens, thymuses and bloods were also taken for immunological analysis. Our results show that the numbers of AC, ACF and Crypts/focus in rats of the KJC+AOM Group were significantly decreased, versus rats of the AOM Group (p &amp;lt; 0.01). No ACF was found in rats without AOM injection. In addition, significant metabolic alterations were observed in the groups with KJC. Although KJC administration did not cause marked weight changes of spleens and thymuses, the effect of KJC on levels of inflammatory cytokines and mediators were also examined. Our results indicated that KJC significantly inhibited AOM-induced colonic ACF formation, suggesting that KJC might have a chemopreventive effect against colon carcinogenesis at least in the initiation stage of CRC. Furthermore, KJC may exert the chemopreventive effects on ACF formation by changing metabolic pathways and modulating immunity and inflammatory responses. This warrants further studies on the cellular and molecular mechanisms of KJC tea. Citation Format: Chunnian He, Wu Bi, L. Harris Zhang, Li Cao, Pei-Gen Xiao. Chemopreventive effects of a non-camellia tea against azoxymethane -induced precancerous colorectal lesions in male rats. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 907. doi:10.1158/1538-7445.AM2015-907

P0026 The chemopreventive potential of Curcuma purpurascens rhizome in reducing azoxymethane-induced aberrant crypt foci in rats

Background Curcuma purpurascens rhizome, a member of the Zingiberaceae family, is a popular spice in Indonesia and is traditionally used for assorted remedies. Dichloromethane extract of C. purpurascens rhizome (DECPR) has previously been shown to have apoptosis-inducing effect on colon cancer cells. Methods We examined the potential of DECPR to prevent colon cancer development in rats treated with azoxymethane (AOM, 15 ml/kg) by the incidence of aberrant crypt foci (ACF). Starting from the day immediately after AOM treatment, the rats were orally administered once a day for 2 months with 10% Tween-20 (5 ml/kg, cancer control), DECPR (250 mg/kg, low dose) and DECPR (500 mg/kg, high dose). Meanwhile, the control group was intra-peritoneally injected with fluorouracil (35 mg/kg) for 5 days consecutively. After euthanising the rats, the number of ACF was enumerated in colon tissues. Bax, Bcl-2, and PCNA protein levels were examined using immunohistochemical and western blot analyses. Antioxidant enzymatic activity was measured in colon tissue homogenates and associated with malondialdehyde level. Findings DECPR exposure at both doses significantly decreased AOM-induced ACF formation, which was accompanied with the reduced expression of PCNA. Up-regulation of Bax and down-regulation of Bcl-2 suggested the involvement of apoptosis in the chemopreventive effect of DECPR. In addition, the oxidative stress resulting from AOM treatment was significantly attenuated after administration of DECPR, which was shown by the elevated antioxidant enzymatic activity and reduced malondialdehyde level. Interpretation DECPR significantly inhibits ACF formation in AOM-treated rats and may offer protection against colon cancer development.

The chemopreventive potential of Curcuma purpurascens rhizome in reducing azoxymethane-induced aberrant crypt foci in rats

Curcuma purpurascens BI. rhizome, a member of the Zingiberaceae family, is a popular spice in Indonesia that is traditionally used in assorted remedies. Dichloromethane extract of C. purpurascens BI. rhizome (DECPR) has previously been shown to have an apoptosis-inducing effect on colon cancer cells. In the present study, we examined the potential of DECPR to prevent colon cancer development in rats treated with azoxymethane (AOM) (15 mg/kg) by determining the percentage inhibition in incidence of aberrant crypt foci (ACF). Starting from the day immediately after AOM treatment, three groups of rats were orally administered once a day for 2 months either 10% Tween 20 (5 mL/kg, cancer control), DECPR (250 mg/kg, low dose), or DECPR (500 mg/kg, high dose). Meanwhile, the control group was intraperitoneally injected with 5-fluorouracil (35 mg/kg) for 5 consecutive days. After euthanizing the rats, the number of ACF was enumerated in colon tissues. Bax, Bcl-2, and proliferating cell nuclear antigen (PCNA) protein expressions were examined using immunohistochemical and Western blot analyses. Antioxidant enzymatic activity was measured in colon tissue homogenates and associated with malondialdehyde level. The percentage inhibition of ACF was 56.04% and 68.68% in the low- and high-dose DECPR-treated groups, respectively. The ACF inhibition in the treatment control group was 74.17%. Results revealed that DECPR exposure at both doses significantly decreased AOM-induced ACF formation, which was accompanied by reduced expression of PCNA. Upregulation of Bax and downregulation of Bcl-2 suggested the involvement of apoptosis in the chemopreventive effect of DECPR. In addition, the oxidative stress resulting from AOM treatment was significantly attenuated after administration of DECPR, which was shown by the elevated antioxidant enzymatic activity and reduced malondialdehyde level. Taken together, the present data clearly indicate that DECPR significantly inhibits ACF formation in AOM-treated rats and may offer protection against colon cancer development.

Antioxidant and Anti-inflammatory Effects of Yam (Dioscorea batatas Decne.) on Azoxymethane-induced Colonic Aberrant Crypt Foci in F344 Rats.

Yam (Dioscorea batatas Decne.) has long been used as a health food and oriental folk medicine because of its nutritional fortification, tonic, anti-diarrheal, anti-inflammatory, antitussive, and expectorant effects. Reactive oxygen species (ROS), which are known to be implicated in a range of diseases, may be important progenitors of carcinogenesis. The aim of this study was to investigate the modulatory effect of yam on antioxidant status and inflammatory conditions during azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats. We measured the formation of aberrant crypt foci (ACF), hemolysate antioxidant enzyme activities, colonic mucosal antioxidant enzyme gene expression, and colonic mucosal inflammatory mediator gene expression. The feeding of yam prior to carcinogenesis significantly inhibited AOM-induced colonic ACF formation. In yam-administered rats, erythrocyte levels of glutathione, glutathione peroxidase (GPx), and catalase were increased and colonic mucosal gene expression of Cu/Zn-superoxide dismutase (SOD), Mn-SOD, and GPx were up-regulated compared to the AOM group. Colonic mucosal gene expression of inflammatory mediators (i.e., nuclear factor kappaB, inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor alpha, and interleukin-1beta) was suppressed by the yam-supplemented diet. These results suggest that yam could be very useful for the prevention of colon cancer, as they enhance the antioxidant defense system and modulate inflammatory mediators.

Evaluation of chemopreventive effects of Acanthus ilicifolius against azoxymethane-induced aberrant Crypt Foci in the rat colon.

Background Acanthus ilicifolius, a mangrove medicinal plant, is traditionally used to treat a variety of diseases. The aim of this research is to assess the chemoprotective outcomes of A. ilicifolius ethanolic extract against azoxymethane (AOM) induced colonic aberrant crypt foci (ACF) in rats.Methodology/Principal FindingsIn our study, rats were arranged in to five groups. Rats in the normal control group were given subcutaneous injections of normal saline once weekly for 2 weeks. The AOM control, reference and treatment groups were given subcutaneous injection of AOM, 15 mg/kg body weight, once weekly for 2 weeks each. The reference group was treated with 35 mg/kg 5-Fluorouracil via intraperitoneal injection once weekly for 8 weeks, and the treatment groups were administered by gavage with 250 and 500 mg/kg A. ilicifolius extract daily for 8 weeks. Both normal and AOM control groups received the vehicle; 10% Tween-20 only.Rats treated with 250 mg/kg and 500 mg/kg of A. ilicifolius extracts showed a decrease in the mean number of ACF by 65% and 53%, respectively. Those fed with A. ilicifolius showed significantly decreased multiplicity of ACF formations when compared with the results from the AOM control group. The 250 mg/kg A. ilicifolius treatment group showed significant decreases in lipid peroxidation MDA levels when compared with the AOM control group. In immunohistochemistry staining, the proliferating nuclear cell antigen (PCNA)-positive cells were significantly higher in the AOM control group than in the A. ilicifolius-treated groups. RT-PCR showed that A. ilicifolius caused a change in the regulation of apoptosis-related genes expression.Conclusion/SignificanceThe results of the current study show that AOM-treated rats receiving oral exposure to A. ilicifolius demonstrated a significant decrease in the number of ACF in the colon when compared to AOM-treated rats receiving vehicle only. A ilicifolius may be an effective herbal approach for the prevention of AOM-induced ACF in the rat colon.

Role of Hepatic and Intestinal P450 Enzymes in the Metabolic Activation of the Colon Carcinogen Azoxymethane in Mice

P450-mediated bioactivation of azoxymethane (AOM), a colon carcinogen, leads to the formation of DNA adducts, of which O6-methylguanine (O6-mG) is the most mutagenic and contributes to colon tumorigenesis. To determine whether P450 enzymes of the liver and intestine both contribute to AOM bioactivation in vivo, we compared tissue levels of AOM-induced DNA adducts, microsomal AOM metabolic activities, and incidences of colonic aberrant crypt foci (ACF) among wild-type (WT), liver-specific P450 reductase (Cpr)-null (LCN), and intestinal epithelium-specific Cpr-null (IECN) mice. At 6 h following AOM treatment (at 14 mg/kg, s.c.), O6-mG and N7-mG levels were highest in the liver, followed by the colon, and then small intestine in WT mice. As expected, hepatic adduct levels were significantly lower (by >60%) in LCN mice but unchanged in IECN mice, whereas small-intestinal adduct levels were unchanged or increased in LCN mice but lower (by >50%) in IECN mice compared to that in WT mice. However, colonic adduct levels were unchanged in IECN mice compared to that in WT mice and increased in LCN mice (by 1.5–2.9-fold). The tissue-specific impact of the CPR loss in IECN and LCN mice on microsomal AOM metabolic activity was confirmed by rates of formation of formaldehyde and N7-mG in vitro. Furthermore, the incidence of ACF, a lesion preceding colon cancer, was similar in the three mouse strains. Thus, AOM-induced colonic DNA damage and ACF formation is not solely dependent on either hepatic or intestinal microsomal P450 enzymes. P450 enzymes in both the liver and intestine likely contribute to AOM-induced colon carcinogenesis.

Amelioration of azoxymethane induced-carcinogenesis by reducing oxidative stress in rat colon by natural extracts

BackgroundAzoxymethane (AOM) is a potent carcinogenic agent commonly used to induce colon cancer in rats; the cytotoxicity of AOM is considered to mediate oxidative stress. This study investigated the chemopreventive effect of three natural extracts [pomegranate peel extract (PomPE), papaya peel extract (PapPE) and seaweed extract (SE)] against AOM-induced oxidative stress and carcinogenesis in rat colon.MethodsEighty Sprague–Dawley rats (aged 4 weeks) were randomly divided into 8 groups (10 rats/group). Control group was fed a basal diet; AOM-treated group was fed a basal diet and received AOM intraperitonial injections for two weeks at a dose of 15 mg/kg bodyweight, whereas the other six groups were received oral supplementation of PomPE, PapPE or SE, in the presence or absence of AOM injection. All animals were continuously fed ad-libitum until aged 16 weeks, then all rats were sacrificed and the colon tissues were examined microscopically for pathological changes and aberrant crypt foci (ACF) development, genotoxicity (induced micronuclei (MN) cells enumeration), and glutathione and lipid peroxidation.ResultsOur results showed that AOM-induced ACF development and pathological changes in the colonic mucosal tissues, increased bone marrow MN cells and oxidative stress (glutathione depletion, lipid peroxidation) in rat colonic cells. The concomitant treatment of AOM with PomPE, PapPE or SE significantly ameliorated the cytotoxic effects of AOM.ConclusionsThe results of this study provide in-vivo evidence that PomPE, PapPE and SE reduced the AOM-induced colon cancer in rats, through their potent anti-oxidant activities.

Chemopreventive effects of PBI-Se, a selenium-containing analog of PBIT, on AOM-induced aberrant crypt foci in F344 rats

Inducible nitric oxide synthase (iNOS) is a potential target for the treatment of inflammation and cancer. Previously, we showed that the selective iNOS inhibitor S,S'-1,4-phenylenebis(1,2-ethanediyl)bis-isothiourea (PBIT) caused significant inhibition of colon carcinogenesis induced by azoxymethane (AOM), although it did not completely abrogate NO production due to the exogenous bioavailability of NO and NO generation by eNOS in tumor tissues. To create an iNOS-targeting molecule that may have additional benefits, a novel isosteric analog of PBIT, PBI-Se, was developed, in which sulfur was replaced with selenium. Chemopreventive efficacy of PBI-Se was evaluated in an AOM-induced rat colon carcinogenesis model using aberrant crypt foci (ACF) as the endpoint. At 7 weeks of age, rats (12/group) were fed the control diet (AIN 76A) and then colonic ACF were induced with two AOM treatments. Three days later, rats were fed diets containing PBI-Se (0-20ppm) for 8 weeks, and then ACF were evaluated histopathologically. Dietary administration of 10 or 20ppm of PBI-Se significantly suppressed AOM-induced total colonic ACF formation (32 or 41%, p<0.002-0.0003), and multi-crypt (4 or more) aberrant foci (29 or 47%, p<0.01-0.0004), respectively. The inhibition by PBI-Se was dose-dependent and was half the dose of PBIT for inhibiting total ACF in rats. Both PBIT and PBI-Se induced dose-dependent apoptosis in CaCo2 cells and caused a significant decrease in the cell cycle proteins cyclin D1 (70%, p<0.0001) and iNOS (99%, p<0.0001). Treatment with PBIT (30 and 60µM) and PBI-Se (2 and 4 µM) significantly decreased the LPS-induced cytokine interleukin-6 level. Incorporation of selenium into the structure of PBIT provided the agent with additional novel cytotoxic and immunologic properties. Results from the invitro and invivo bioassays suggest that PBI-Se could be developed further for the prevention and treatment of colon cancer.

Abstract 4392: Targeting HADC-2: Chemopreventive effect of OSU-HDAC42 on rat colon carcinogenesis and APCmin/+ Mice intestinal tumorigenesis.

Abstract Epigenetic modulators particularly histone deacetylases (HDACs) are valid targets cancer for prevention and Therapy. Recent studies from our laboratory and others suggest that HDAC-2 over-expression is associated with colon tumor progression and potential target for colon cancer prevention. The present study explored the chemopreventive effects of novel HDAC-2 inhibitor, OSU-HDAC42, in a mouse model of familial adenomatous polyposis (FAP). We used a rat colon carcinogenesis model assay, inhibition of aberrant crypt foci (ACF) development, as measure of the dose-response and chemopreventive effects. Colonic ACF were induced by azoxymethane (AOM) (15 mg/kg BW once weekly for 2 weeks by s.c. injection at the age of 8 and 9 weeks). One week after AOM-treatment groups of rats were fed AIN-76A diet containing 0, 75, 150 and 300 ppm OSU-HDAC42 for eight-weeks, and then colonic ACF were evaluated. Six week-old male C57Bl/6J-APCmin/+ and wild type mice were fed AIN-76A diet containing 150 ppm OSU-HADC42 or 300 ppm SAHA for 80 days. At ∼120 days of age, all mice were killed, and the intestines were evaluated for polyps. Dietary administration of OSU-HDAC42 produced a dose-dependent inhibition of AOM-induced colonic ACF formation (by 13-50%, p&amp;lt;0.05 -0.001) and reduced the number of foci with ≥4 crypts/focus (by 25-57%, P&amp;lt;0.05-0.0001) in F344 rats. 150 ppm OSU-HDAC42 significantly inhibited development of small intestinal (&amp;gt;46%, p&amp;lt;0.001) and colon tumors (&amp;gt;26%, P≥0.05) in APCmin/+ mice. Whereas 300 ppm SAHA showed limited non-significant inhibition of intestinal polyp formation in APCmin/+ mice. Further, OSU-HDAC42 showed inhibition in small intestinal polyps size measuring 1-2-mm (P&amp;lt;0.001). Importantly, mice fed with 150ppm OSU-HDAC42 showed significant reduction in HDAC-2, PCNA, CyclinA, CDK2, CDC25C expression pattern and increased p53 expression levels. These observations demonstrate that novel HDAC inhibitor, OSU-HDAC42 potential chemopreventive against the chemically-induced and polyposis models of colon tumorigenesis. {This work is supported by NCI-N01-CN53300} Citation Format: Durgadevi Ravillah, Li Qian, Misty Brewer, Yuting Zhang, Laura Biddick, Venkateshwar Madka, Jagan M.R. Patlolla, Altaf Mohammed, Vernon Steele, Chintalapally V. Rao. Targeting HADC-2: Chemopreventive effect of OSU-HDAC42 on rat colon carcinogenesis and APCmin/+ Mice intestinal tumorigenesis. [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 4392. doi:10.1158/1538-7445.AM2013-4392

Hesperidin alleviates oxidative stress and downregulates the expressions of proliferative and inflammatory markers in azoxymethane-induced experimental colon carcinogenesis in mice

Colon cancer is a common malignant neoplasm causing huge morbidity and mortality worldwide. Current therapeutic interventions are unsatisfying, which necessitates novel chemopreventive strategies. The present study was intended to elucidate the chemopreventive efficacy of hesperidin against azoxymethane (AOM)-induced mouse colon carcinogenesis. Swiss albino mice were subjected to intraperitoneal injections of AOM once a week for 3 consecutive weeks. Hesperidin treatments were provided in the initiation or post-initiation phases. The number and multiplicity of aberrant crypt foci (ACF), tumor incidence and antioxidant status were determined. Histopathological analyses, proliferating cell nuclear antigen (PCNA) index and modulations in the expression of inflammatory markers such as nuclear factor kappa B (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were studied. Hesperidin treatments significantly inhibited the number and multiplicities of AOM-induced ACF and tumor incidence. Hesperidin reduced oxidative stress parameters and enhanced antioxidant status. A marked decrease in the PCNA index was evident on hesperidin administration. Hesperidin treatments caused a prominent downregulation of NF-κB and its target molecules iNOS and COX-2, thereby combating inflammation. This study proves the chemopreventive efficacy of hesperidin against the deleterious traits of colon carcinogenesis including accelerated proliferation, inflammation and persistent oxidative stress.