Multiplicity Of Aberrant Crypt Foci Research Articles

Low-dose dimethylfumarate attenuates colitis-associated cancer in mice through M2 macrophage polarization and blocking oxidative stress

Colitis-associated cancer (CAC) is an aggressive subtype of colorectal cancer that can develop in ulcerative colitis patients and is driven by chronic inflammation and oxidative stress. Current chemotherapy for CAC, based on 5-fluorouracil and oxalipltin, is not fully effective and displays severe side effects, prompting the search for alternative therapies. Dimethylfumarate (DMF), an activator of the nuclear factor erythroid 2–related factor 2 (NRF2), is a potent antioxidant and immunomodelatrory drug used in the treatment of multiple sclerosis and showed a strong anti-inflammatory effect on experimental colitis. Here, we investigated the chemotherapeutic effect of DMF on an experimental model of CAC. Male NMRI mice were given two subcutaneous injections of 1,2 Dimethylhydrazine (DMH), followed by three cycles of dextran sulfate sodium (DSS). Low-dose (DMF30) and high-dose of DMF (DMF100) or oxaliplatin (OXA) were administered from the 8th to 12th week of the experiment, and then the colon tissues were analysed histologically and biochemically. DMH/DSS induced dysplastic aberrant crypt foci (ACF), oxidative stress, and severe colonic inflammation, with a predominance of pro-inflammatory M1 macrophages. As OXA, DMF30 reduced ACF multiplicity and crypt dysplasia, but further restored redox status, and reduced colitis severity by shifting macrophages towards the anti-inflammatory M2 phenotype. Surprisingly, DMF100 exacerbated ACF multiplicity, oxidative stress, and colon inflammation, likely through NRF2 and p53 overexpression in colonic inflammatory cells. DMF had a dual effect on CAC. At low dose, DMF is chemotherapeutic and acts as an antioxidant and immunomodulator, whereas at high dose, DMF is pro-oxidant and exacerbates colitis-associated cancer.

Panx1 knockout promotes preneoplastic aberrant crypt foci development in a chemically induced model of mouse colon carcinogenesis.

Colorectal cancer, which is the third leading cause of cancer-related deaths worldwide, is a multistep disease, featuring preneoplastic aberrant crypt foci (ACF) as the early morphological manifestation. The roles of hemichannel-forming transmembrane Pannexin 1 (Panx1) protein have not been investigated in the context of colon carcinogenesis yet, although it has contrasting roles in other cancer types. Thus, this study was conducted to examine the effects of Panx1 knockout (Panx1-/- ) on the early events of chemically induced colon carcinogenesis in mouse. Wild type (WT) and Panx1-/- female C57BL6J mice were submitted to a chemically induced model of colon carcinogenesis by receiving six intraperitoneal administrations of 1,2-dimethylhydrazine (DMH) carcinogen. Animals were euthanized 8 h (week 7) or 30 weeks (week 37) after the last DMH administration in order to evaluate sub-acute colon toxicity outcomes or the burden of ACF, respectively. At week 7, Panx1 genetic ablation increased DMH-induced genotoxicity in peripheral blood cells, malondialdehyde levels in the colon, and apoptosis (cleaved caspase-3) in colonic crypts. Of note, at week 37, Panx1-/- animals showed an increase in aberrant crypts (AC), ACF mean number, and ACF multiplicity (AC per ACF) by 56%, 57% and 20%, respectively. In essence, our findings indicate that Panx1 genetic ablation promotes preneoplastic ACF development during chemically induced mouse colon carcinogenesis, and a protective role of Panx1 is postulated.

Capsaicin lacks tumor-promoting effects during colon carcinogenesis in a rat model induced by 1,2-dimethylhydrazine.

Capsaicin (CPS, 8-methyl-N-vanillyl-trans-6-nonenamide), a pungent alkaloid from chili peppers, has contradictory effects in both experimental and human carcinogenesis. Thus, we evaluated the modifying effects of chronic CPS during the promotion and progression stages of rat colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH). Male Wistar rats were given four subcutaneous injections of DMH (40 mg/body weight (b.w.)) twice a week, for 2 weeks. After DMH-induced tumor initiation, the animals were treated with CPS at 5 or 50 mg/kg b.w. by gavage for 24 weeks (three times a week). High-dose CPS reduced both cell proliferation in adjacent "normal-appearing" colonic crypts and the total number of preneoplastic aberrant crypt foci (ACF) but did not change the number of dysplastic ACF or ACF multiplicity. Although the proportion of adenomas was increased, and tubular adenocarcinomas decreasedin high-dose CPS, both CPS interventions exerted no effects on total tumor incidence, volume, multiplicity, cell proliferation (Ki-67), and apoptosis (caspase-3). In accordance, high-dose CPS treatment had discrete effects on gene expression in colon tumors, as only 3/94 (3.19%) genes were significantly modified (downregulation of Cebpd and Fasl, and upregulation of Jag1). The findings of the present study show that CPS does not impact on the promotion/progression stages of rat colon carcinogenesis. Therefore, CPS at a high-dose intervention showed to be a safe food ingredient.

Decrypting the Molecular Mechanistic Pathways Delineating the Chemotherapeutic Potential of Ruthenium-Phloretin Complex in Colon Carcinoma Correlated with the Oxidative Status and Increased Apoptotic Events.

To explore fresh strategies in colorectal cancer (CRC) chemotherapy, we evaluated the capability of the ruthenium-phloretin complex in exterminating colon cancer by effectively addressing multiple apoptotic mechanisms on HT-29 cancer cells together with an animal model of colorectal cancer activated by 1,2-dimethylhydrazine and dextran sulfate sodium. Our current approach offers tangible evidence of the application of the ruthenium-phloretin complex in future chemotherapy. The complex triggers intrinsic apoptosis triggered by p53 and modulates the Akt/mTOR pathway along with other inflammatory biomarkers. The ruthenium-phloretin complex has been synthesized and successfully characterized by numerous spectroscopic methodologies accompanied by DPPH, FRAP, and ABTS assays assessing its antioxidant potential. Studies conducted in human cell lines revealed that the complex improved levels of p53 and caspase-3 while diminishing the activities of VEGF and mTOR, triggers apoptosis, and induces fragmentation of DNA in the HT-29 cells. Toxicity studies were conducted to identify the therapeutic doses of the novel complex in animal models. The outcomes of the in vivo report suggest that the complex was beneficial in repressing multiplicity of aberrant crypt foci as well as hyperplastic lesions and also promoted increased levels of CAT, SOD, and glutathione. In addition, the ruthenium-phloretin complex was able to control cell proliferation and boosted apoptotic outbursts in cancer cells associated with the increase in cellular response towards Bax while diminishing responses towards Bcl-2, NF-κB, and MMP-9. Our observations from the experiments deliver testament that the ruthenium-phloretin complex has the potential to act as a promising chemotherapeutic agent in colorectal cancer because it can affect the growth of ACF and hyperplastic abrasions in the colon tissues by evoking cell death.

Probiotic Lactobacillus rhamnosus modulates the gut microbiome composition attenuates preneoplastic colorectal Aberrant crypt foci

Lactobacillus rhamnosus (LGG) is well documented strain with wide clinical applications. We investigated herein the possible protection role of LGG in against early preneoplastic colon carcinogenesis Aberrant crypt foci (ACF) in rat model. Sprague Dawley rats were pre-treated with or without LGG via oral gavage, followed by ACF induction with Dimethylhydrazine (DMH). LGG treated groups showed significant decrease in total number of ACF’s, and increased Bax, P53, and Caspase-3 proteins expression with significant attenuation of NFkB, Cox2 and β catenin expression compared to DMH group. Serum cytokines array revealed significant elevation of IL-2, IL-6, and IFN-γ, and attenuated level of IL-10 in LGG+DMH treated animals. Finally, the 16S rRNA fecal microbiome genomic analysis showed that LGG alters the gut microbiome structure, composition and functions at phylum, family and genus level. Collectively, LGG reduces colon ACFs incidence and multiplicity via inhibition of pro-carcinogenesis biomarkers and modulate the gut microbiome.

Lyophilized açaí pulp (Euterpe oleracea Mart) attenuates colitis-associated colon carcinogenesis while its main anthocyanin has the potential to affect the motility of colon cancer cells

This study evaluated the possible protective effects of lyophilized açaí pulp (AP) in a colitis-associated carcinogenesis (CAC) rat model and the modifying effect of cyanidin 3-rutinoside (C3R) on the motility of RKO colon adenocarcinoma cells, using the wound healing assay. Male Wistar rats were induced to develop CAC using 1,2-dimethylhydrazine (DMH) and 2,4,6-trinitrobenzene acid (TNBS). Animals were randomly assigned to different groups that received basal diet or basal diet supplemented with 5.0% or 7.5% lyophilized AP. The findings indicate: 1) C3R (25 μM) has the potential to reduce RKO cell motility in vitro; 2) ingestion of lyophilized AP reduces the total number of aberrant crypt foci (ACF), ACF multiplicity, tumor cell proliferation and incidence of tumors with high grade dysplasia; 3) AP increases the gene expression of negative regulators of cell proliferation such as Dlc1 and Akt3, as well as inflammation (Ppara). Thus, lyophilized AP could exert a potential antitumor activity.

Effects of Watermelon Powder and l-arginine Supplementation on Azoxymethane-Induced Colon Carcinogenesis in Rats

Diets high in fruits and vegetables may help prevent colorectal cancer (CRC). Watermelon consumption may reduce CRC risk due to its concentration of l-citrulline and its role in endothelial nitric oxide (NO) production. Research suggests that increased NO levels have tumoricidal effects. The purpose of this study was to determine the effects of watermelon powder supplementation on aberrant crypt foci (ACF) formation, precancerous lesions, and expression of genes associated with colon carcinogenesis. Thirty-two male Sprague-Dawley rats were assigned into three groups: control, 0.36% l-arginine, or 0.5% watermelon powder and injected with azoxymethane (15 mg/kg body weight). Both l-arginine and watermelon powder groups exhibited lower total numbers of ACF and high multiplicity ACF (P < 0.01). The watermelon powder group exhibited higher NO levels and lower 8-hydroxyguanosine DNA damage (P < 0.05). Watermelon powder and l-arginine downregulated 8-oxoguanine DNA glycosylase gene expression and upregulated O6-methylguanine DNA methyltransferase gene expression (P < 0.05). Cyclooxgenase-2 gene expression was lower for rats fed with watermelon powder (P < 0.05). These results suggest that watermelon powder or l-arginine supplementation may reduce the risk of colon cancer by suppressing ACF formation through lowering oxidative DNA damage and inflammation, modulating DNA repair enzyme expression, and/or enhancing NO production.

Aberrant crypt foci are regionally affected by zinc treatment in a 1,2-dimethylhydrazine induced colon carcinogenesis model

Zinc is a trace element widely known for its marked antioxidant properties. To gain more insight into the site- and time- specific mechanisms by which it induces chemoprevention, this study was elaborated over a pre-cancerous model of colon carcinogenesis.Colon cancer was induced by 1,2-dimethylhydrazine (DMH) in mice (20 mg/kg for 2 weeks) and groups of animals were supplemented with or without zinc sulfate (ZnSO4, 200 mg/L) in drinking water for 4, 10 or 14 weeks. Colon tissues were collected for pathological observation, analyzing aberrant crypt (AC) and aberrant crypt foci (ACF) formations, multiplicity and distribution. Similarly, histological assessment and mucin production, as well as oxidative stress markers estimation was performed for the different groups.Results showed a significant increase in ACF and AC numbers, ACF multiplicity and demonstrated stronger distal occurrence than in the proximal after DHM administration. Histopathological analysis presented marked structural alterations and mucin loss in the distal than the proximal colons. A significant increase in myeloperoxidase (MPO), nitric oxide (NO), L-ornithine and malondialdehyde (MDA) levels was observed followed by a significant decrease in antioxidant markers (superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH)). Oral ZnSO4 supplementation (continuous or partial) induced significant decrease in ACF, AC numbers and multiplicity, restored histological architecture and mucin production, and a significant decrease in proinflammatory markers while it reduced antioxidants to normal levels.From this study, insight was obtained on the use of ZnSO4 as a chemopreventive agent and shed light on its potential, as a supplement in nutraceutical approaches.

Abstract 182: A new anticancer agent derived from decenoic acid for the treatment of colon cancer

Abstract The final goal of the current study is to create a promising lead compound that targets human colon carcinoma. After discovering in vitro antiproliferative activity of a naturally occurring compound, 10-hydroxy-2-decenoic acid, we designed and synthesized over 150 derivatives. The initial screening resulted in the discovery of three primary lead compounds, we then proceeded with modifications in side chain and eventually obtained a second-generation lead compound, named PPI (patented in 2014), whose IC50 was 0.3 μM. We focused on the transcription factor STAT3 that is able to drive carcinogenesis, apoptosis, and angiogenesis-related genes. In silico docking simulation exhibited that PPI can bind to SH2 domain of STAT3, suggesting that this drug blocks dimerization of STAT3, thereby PPI inactivates function of STAT3. PPI inhibited transcriptional activity of STAT3 when SW480, SW837, and HT29 human colon carcinoma cells were treated with 3.7 to 5 μM PPI for 24h. Flowcytometry analysis indicated that when HT29 cells were treated with 1 μM PPI, the percentage of cells in G1 increased by 5% and this was associated with a concomitant decrease of cells in the S and G2-M phases of the cell cycle. The results indicate that PPI causes carcinoma cells to arrest in the G1 phase. When HT29 cells were treated with 10 μM PPI, subG1 fraction and DNA fragmentation were seen, indicating induction of apoptosis. We then performed western blot analysis to determine whether treatment of carcinoma cells with PPI alters cellular levels of the G1 cell cycle control protein cyclin D1 and apoptosis/angiogenesis-related molecules. When HT29 cells were treated with PPI for 24 to 48h, there was a marked decrease in the levels of expression of the Bcl-2, Bcl-xL, and VEGF proteins and a marked increase in the levels of expression of the cleaved caspase 3, 7, 8, 9, and PARP proteins. PPI also inhibited expression levels and nuclear tanslocation of pSTAT3. To see if PPI exerts preventive effect on tumor promotion, F344 rats received sc injections of a carcinogen azoxymethane and then treated with soybean oil or PPI. There was a dose dependent decrease in multiplicity of aberrant crypt foci (ACF) in the colon. PPI inhibited the occurrence of larger ACF consisting of more than 4 aberrant crypts, indicating inhibitory effect of PPI on tumor promotion. In a mouse xenograft model, PPI decreased the size and number of blood vessels in the tumor. All mice survived without causing significant body weight loss during experiment. No histopathological abnormality was found in any of organ site. To confirm the effects of PPI on angiogenesis, we performed chorioallantoic membrane assays and found a dose-dependent decrease in average number of blood vessels. Inhibition of STAT3 by PPI may affect the function of molecules that are related to apoptosis, angiogenesis, and cell cycle progression and eventually contributes to PPI-induced growth inhibition. Citation Format: Masumi Suzui, Saeko Ando, Harutoshi Matsumoto, Katsumi Fukamachi, Mitsuru Futakuchi, Naoki Yoshimi. A new anticancer agent derived from decenoic acid for the treatment of colon cancer [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 182. doi:10.1158/1538-7445.AM2017-182

Effect of Watermelon Powder Supplementation on Azoxymethane‐Induced Colon Carcinogenesis in Rats

Colorectal cancer (CRC) is a public health concern and one of the most common cancers in men and women globally. Given the substantial morbidity and mortality associated with this disease, methods for prevention, in part through a diet high in fruits and vegetables, are under investigation. Specifically, watermelon may be a practical method for reducing CRC risk factors due to its high concentration of L‐citrulline, a precursor to L‐arginine, which plays a crucial role in endothelial nitric oxide (NO) production. Evidence suggests that increased levels of NO have tumoricidal effects; therefore, the purpose of this study was to determine the effects of watermelon powder supplementation on aberrant crypt foci (ACF) formation, precancerous lesions, and biomarkers that are associated with colon carcinogenesis in rats. Thirty‐two male Sprague‐Dawley rats, at 21 days old, were divided into three groups: control, 0.36% L‐arginine, or 0.5% watermelon powder. The 0.5% watermelon powder diet provided 0.36% of L‐arginine+L‐citrulline. Rats were injected subcutaneously with azoxymethane (dose of 15 mg/kg body weight), a carcinogenic agent, at the beginning of weeks 3 and 4. The watermelon group exhibited higher blood NO levels compared to the control group (P &lt; 0.05). Both the L‐arginine and watermelon powder groups exhibited lower total numbers of ACF and high multiplicity aberrant crypt foci (HMACF, &gt; 4 aberrant crypts/focus) (P &lt; 0.01). The watermelon powder group exhibited lower 8‐hydroxyguanosine DNA damage in the blood (P &lt; 0.05) compared to controls. Watermelon powder and L‐arginine downregulated 8‐oxoguanine DNA glycosylase (OGG1) gene expression (P &lt; 0.05) and upregulated O6‐methylguanine DNA methyltransferase (MGMT) gene expression (P &lt; 0.01). Cyclooxgenase‐2 (COX‐2) gene expression in the colon mucosa of the watermelon group was lower compared to the control group (P &lt; 0.05). These results suggest that watermelon powder or L‐arginine consumption may reduce the risk of colon cancer by suppressing ACF formation through lowering oxidative DNA damage and inflammation, modulating DNA repair enzyme expression, and enhancing NO production.Support or Funding InformationThe study was funded by The US National Watermelon Promotion Board.

Early Life and Postnatal Western Diet Feeding and Susceptibility to Chemically Induced Colonic Aberrant Crypt Foci in Male Rats Offspring

ABSTRACTThe modifying effects of a Western diet (WD) during early life on the susceptibility to colon carcinogenesis induced by dimethylhydrazine (DMH) were examined in male rats as later adults. Three groups were studied: a lifetime control diet-fed group, a test group fed WD since pregnancy from dams until postnatal day (PND) 42, and a group fed WD at adulthood. At PND 70, all groups received the carcinogen DMH and were euthanized 10 wk later. Colonic aberrant crypt foci (ACF) were scored (number and crypt multiplicity) and the altered pattern of β-catenin expression was evaluated in the colonic lesions. ACF multiplicity (≥4 crypts) was significantly higher in the group fed WD at early life than in the group fed the control diet. ACF number, crypt multiplicity, and the number of high-grade dysplastic lesions were significantly higher in the group fed WD at adulthood than in the groupfed the control diet. The number of lesions with altered β-catenin expression was higher in the groups receiving WD at early life or at adulthood than in the lifetime control-diet-fed group. These findings indicate that WD exposure at early life increased the susceptibility to colon carcinogenesis at adulthood.

Ethanolic Extract of Bark from Salix aegyptiaca Ameliorates 1,2-dimethylhydrazine-induced Colon Carcinogenesis in Mice by Reducing Oxidative Stress

ABSTRACTWe have previously shown that ethanolic extract from bark (EEB) of Salix aegyptiaca (Musk Willow) can inhibit proliferation and motility and induce apoptosis in colon cancer cells. Tandem mass spectrometry revealed EEB to be rich in catechin, catechol, and salicin. The present study investigated the chemopreventive effect of HPLC-fingerprinted EEB on 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) formation in mice. DMH (20 mg/kg body weight) was weekly injected subcutaneously to mice for the first 2 weeks. EEB (100 and 400 mg/kg body weight) was provided orally from the 7th to 14th week, after which colon tissues were evaluated histologically and biochemically. DMH treatment induced high number of ACF; EEB significantly reduced the number and multiplicity of ACF, along with a restoration in goblet cells and mucin accumulation. EEB supplementation improved the markers of inflammation (myeloperoxidase and neutrophil infiltration) and oxidative stress. More importantly, EEB amplified apoptosis of neoplastic cells in the colon mucosa of DMH-treated mice. It also lowered levels of markers for early transformation events such as EGFR, nuclear β-catenin, and COX-2 in colon cancer cell lines HT-29 and HCT-116. The innocuity of EEB (up to 1600 mg/kg) to mice reinforces its potential as a chemopreventive agent.

Effect of Rumex Abyssinicus on preneoplastic lesions in dimethylhydrazine induced colon carcinogenesis in rats.

BackgroundCancer as a multistage process can be reversed or blocked by using chemopreventive agents. Colon cancer chemoprevention has been widely investigated using cyclooxygenase inhibitors and many other chemicals of synthetic or natural origin. This particular study was carried out to assess the colon cancer chemopreventive effect of hydro-methanol extract of Rumex abyssinicus rhizome on rats.MethodColon cancer chemopreventive potential of hydro-methanol extract of Rumex abyssinicus rhizome was determined based on the number and multiplicity of aberrant crypt foci (ACF). Fifteen DMH (1, 2-dimethylhydrazine) treated and five untreated Wistar female rats were used. DMH was administered subcutaneously 30 mg/kg, after its pH was adjusted to 6.5–7. Treatment groups started receiving extract after six weeks of weekly DMH injections. The rats were divided in to four groups: Group 1 received only oral normal saline, Group 2 received DMH and normal saline, Group 3 and 4 received DMH plus 250 mg/kg and 500 mg/kg extract, respectively. Specific phytoconstituents of the plant, which were reviewed from original articles, were virtually evaluated for cyclooxygenase-2 (COX-2) inhibition. The binding energies and interactions of the phytochemicals from Rumex abyssinicus against COX-2 were determined by Autodock4.2.ResultsThere was a statistically significant reduction (p-value < 0.05) in the number of aberrant crypt (AC) and aberrant crypt foci (ACF) at both administered doses. However, significant association (p-value > 0.05) was not observed in reducing crypt multiplicity. The docking process resulted in estimated binding energies [−6.83 kcal/mol to −7.9 kcal/mol] which are closer to the positive controls or Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) [−4.55 kcal/mol to −10.84 kcal/mol]. The phytochemical-COX-2 interaction indicated the involvement of key amino acid residues in inhibition of cyclooxygenase like ARG120, TYR355, TYR385, SER530 and GLY526.ConclusionsRumex abyssinicus had demonstrated a chemopreventive potential at post-initiation stage. As the virtual screening data suggested, COX-2 inhibition by the anthraquinones in the extract could be one mechanism for the observed chemopreventive effect.

Protective effects of spray-dried açaí (Euterpe oleracea Mart) fruit pulp against initiation step of colon carcinogenesis

The present study aims to evaluate whether açaí (Euterpe oleracea Mart) fruit pulp powder (AP) feeding attenuates the initiation step of chemically-induced mouse colon carcinogenesis. Male Swiss mice were fed low fat diet containing 2.5% or 5.0% of AP (weeks 1 to 4) produced by a spray-drying system. All groups received an intraperitoneal (i.p.) injection of colon carcinogen azoxymethane (AOM, 15mg/kg of body weight) at week 3. After the first administration of carcinogen, blood samples were collected to perform Single Cell Gel Electrophoresis Assay. Some mice were euthanized at week 3 (n=5 mice/group) and liver samples were collected for immunohistochemical and glutathione analysis. Other mice received a second i.p. injection of AOM at week 4 and were fed a high-fat diet to accelerate the development of preneoplastic aberrant crypt foci (ACF) until week 14 (n=10 mice/group). Cyanidin 3-glucoside and cyanidin 3-rutinoside, lutein, α-carotene and β-carotene were identified as the main anthocyanins and carotenoids in AP, respectively. At week 3, both dietary AP interventions (2.5% or 5.0%) reduced (p<0.001) peripheral blood cell DNA damage induced by AOM. Moreover, dietary 5.0% AP increased (p=0.002) hepatic total glutathione. At week 14, 5.0% AP intake reduced (p<0.05) ACF multiplicity. The findings indicate that AP feeding attenuates chemically-induced mouse colon carcinogenesis by increasing total GSH and attenuating DNA damage and preneoplastic lesion development.

Abstract 4575: Açaí intake reduces the aberrant crypt foci development in a colitis-associated carcinogenesis model in male Wistar rats

Abstract Colorectal cancer is a main cause of cancer death in the western world. In Brazil, it is the third more common neoplasia in both men and women (INCA, 2014). In our previous study (Fragoso et al., Food Chem Toxicol., 2013), we showed that spray-dried açaí (Euterpe oleraceae Mart.) pulp intake retarded the development of colon cancer in male Wistar rats. Therefore, the objective of this research was to evaluate the potential protection of dietary freeze-dried açaí pulp using a chemically-induced colon carcinogenesis and colitis-associated model in male Wistar rats. The animals were randomly distributed into four groups: G1, DMH [dimethylhydrazine] + TNBS [2,4,6-trinitrobenzene sulfonic acid] + basal diet; G2, DMH + TNBS + basal diet with 5% açai pulp; G3, DMH + TNBS + basal diet with 7.5% açai pulp; G4, DMH + TNBS + basal diet with 0.2% of NAC [N-acetylcysteine]. The acute colitis was induced with the administration of 10 mg of TNBS dissolved in 0.25 ml 50% ethanol (v/v) by means of a Teflon cannula inserted 8 cm into the anus. At the end of the 25th week, animals were sacrificed for evaluation of Aberrant Crypt Foci (ACF) in the colon. Unsenctioned and fixed colons were analyzed for total aberrant crypt (AC) and total ACF and ACF multiplicity under methylene blue staining. The results were analyzed by ANOVA followed by post hoc Tukey test. A significant reduction in total ACF and AC for G2 when compared to G1 (p ≤0.001) was seen. Regarding the ACF multiplicity, it was observed a significant reduction in ACF with 4-10 aberrant crypts for G2 and G4 and for ACF with &amp;gt;10 aberrant crypts for G2-G4 groups in relation to the G1 group (p = ≤0.001, respectively). Also, it was observed a significant reduction in the number of total AC in the distal colon from the G2 and G4 groups compared to G1 group (p = 0.003) and in the medial colon from the G2 group compared to G1 group (p = 0.016). These results suggest that açaí intake may reduce the number of AC and ACF and the number of ACF with more than four aberrant crypts in this chemically-induced colon carcinogenesis and colitis-associated model in rats. Citation Format: MARIANA F. FRAGOSO, GUILHERME R. ROMUALDO, CLAUDIA H. PELLIZZON, LUIS F. BARBISAN. Açaí intake reduces the aberrant crypt foci development in a colitis-associated carcinogenesis model in male Wistar 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 4575. doi:10.1158/1538-7445.AM2015-4575

Abstract 3240: Proximal human aberrant crypt foci as surrogate markers of colorectal cancer risk

Abstract Despite effective use of screening colonoscopy, colorectal cancer (CRC) remains the second leading cause of cancer-related deaths. ‘Interval’ cancers, or those occurring between screening colonoscopy procedures, are often found within the proximal (right) colon, underscoring the need for advanced screening approaches using high-definition chromoendoscopy. To better define the colonic mucosa at-risk, we have focused considerable effort on identification and molecular analysis of aberrant crypt foci (ACF), an early macroscopically detectable lesion commonly found in the distal colon. ACF may serve as a surrogate marker of cancer risk, but have rarely been studied within context of the right colon. We hypothesize that proximal ACF may associate with risk factors for CRC and act as a surrogate marker for CRC risk. While distal ACF are frequent (approximately 13 per patient) proximal ACF are not (&amp;lt;1 per patient). We report that subjects with at least one proximal ACF are significantly more likely to have higher ACF multiplicity (p = 0.0002) and more importantly, are more likely to harbor synchronous colonic neoplasia (OR=2.38 [1.24-4.59], p = 0.0087). Histologically, proximal ACF are more frequently dysplastic (41%) compared to distal colon ACF (8%). ACF (n=49) were analyzed for a panel of oncogenes and tumor suppressors using mass spectrometry (MS)-based genotyping (Sequenom). Among the 112 targets analyzed, after KRAS (20%) and BRAF (22%), APC mutations (12%) were most common and specifically associated with dysplasia. Due to their diminutive size and flat morphology, proximal ACF are almost certainly missed during routine colonoscopy. However, our findings suggest that the identification, removal and analysis of proximal ACF may be especially important during screening colonoscopy of high-risk individuals. Citation Format: David A. Drew, Matthew P. Hanley, Allen Mo, Gyuhyeong Goh, Nicole A. Horelik, Thomas J. Devers, Joel Levine, Richard G. Stevens, James J. Grady, Daniel W. Rosenberg. Proximal human aberrant crypt foci as surrogate markers of colorectal cancer risk. [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 3240. doi:10.1158/1538-7445.AM2014-3240

Chemopreventive Effect of Amorphophallus campanulatus (Roxb.) blume tuber against aberrant crypt foci and cell proliferation in 1, 2-dimethylhydrazine induced colon carcinogenesis

Colorectal cancer is one of the leading causes of cancer death, both in men and women. This study investigated the effects of Amorphophallus campanulatus tuber methanolic extract (ACME) on aberrant crypt foci (ACF) formation, colonic cell proliferation, lipid peroxidative damage and the antioxidant status in a long term preclinical model of 1, 2-dimethylhydrazine (DMH) induced colon carcinogenesis in rats. Male Wistar rats were divided into six groups, viz., group I rats served as controls; group II rats treated as drug controls receiving 250 mg/ kg body weight of ACME orally; group III rats received DMH (20 mg/kg body weight) subcutaneously once a week for the first 15 weeks; groups IV, V and VI rats received ACME along with DMH during the initiation, post- initiation stages and the entire period of the study, respectively. All the rats were sacrificed at the end of 30 weeks and the intestinal and colonic tissues from different groups were subjected to biochemical and histological studies. Administration of DMH resulted in significant (p ≤ 0.05) intestinal and colonic lipid peroxidation (MDA) and reduction of antioxidants such as catalase, glutathione peroxidase, glutathione reductase, glutathione-S- transferase and reduced glutathione. Whereas the supplementation of ACME significantly (p ≤ 0.05) improved the intestinal and colonic MDA and reduced glutathione levels and the activities of antioxidant enzymes in DMH intoxicated rats. ACME administration also significantly suppressed the formation and multiplicity of ACF. In addition, the DMH administered rats showed amplified expression of PCNA in the colon and decreased expression of this proliferative marker was clearly noted with initiation, post-initiation and entire period of ACME treatment regimens. These results indicate that ACME could exert a significant chemopreventive effect on colon carcinogenesis induced by DMH.

Modulating effect of d‐carvone on 1,2‐dimethylhydrazine‐induced pre‐neoplastic lesions, oxidative stress and biotransforming enzymes, in an experimental model of rat colon carcinogenesis

The present study has aimed to evaluate chemopreventive potential of d-carvone on oxidative stress markers, biotransforming enzymes, incidence of colonic polyps and aberrant crypt foci (ACF) in 1,2-dimethylhydrazine (DMH)-induced experimental colon carcinogenesis. Rats were randomly divided into six groups, with group I serving as control. Group II animals received d-carvone every day orally (20mg/kg body weight) for 16weeks; groups III-VI received subcutaneous injections of DMH (20mg/kg body weight) once a week, for the first 4weeks. In addition, groups IV-VI received different doses of d-carvone (5, 10 and 20mg/kg body weight everyday orally) along with DMH injections. Our results revealed that supplementation with d-carvone significantly reduced incidence of polyps/ACF and ACF multiplicity in DMH-exposed rats compared to DMH-alone-exposed rats. Moreover, our results showed reduced activities of liver and circulatory antioxidants and increased levels of lipid peroxidation by products in DMH-exposed animals, which were significantly reversed on supplementation with d-carvone. In addition, colonic antioxidants and lipid peroxidation were significantly diminished in DMH-exposed rats, which were significantly elevated on supplementation with d-carvone. Furthermore, we also determined activities of biotransforming enzymes, which were found to be altered in DMH-exposed rats, but reversed on d-carvone supplementation. All these observations of changes were supported by histochemical findings. Overall, results obtained from this study suggest that d-carvone at 10mg/kg body weight provided optimum protection and could be used as an effective chemopreventive agent against colon carcinogenesis induced by DMH.

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.

A novel NSAID derivative, phospho-ibuprofen, prevents AOM-induced colon cancer in rats

The cancer chemopreventive properties and gastrointestinal toxicity of ibuprofen are well documented. Modification of existing NSAIDs has improved on the chemopreventive efficacy of this agent and reduced its toxicity. In this study, ibuprofen and a modified derivative (phospho-modified ibuprofen or p-ibuprofen) were used in a chemically induced model of colon cancer. Fisher 344 rats were injected with azoxymethane then treated with either ibuprofen (500 ppm) or p-ibuprofen (900 ppm) for 20 weeks to observe aberrant crypt foci (ACF) or 40 weeks to evaluate tumor incidence and multiplicity. β-catenin and p65 were measured in colonic tissues by immunofluorescence staining. Equal molar doses of ibuprofen (75 and 670 mg/kg) and p-ibuprofen (135 and 1,215 mg/kg) were administered to rats for 7 days to assess acute toxicity. The in vitro effect of p-ibuprofen on COX-2 and PGE2 synthesis, β-catenin expression and NF-κB activity were examined in RAW 264.7 macrophage and HCT 116 colon cancer cells. At week 20, p-ibuprofen and ibuprofen significantly reduced the multiplicity of ACF compared with control (p<0.05); 31.2 and 37.9%, respectively. At week 40, p-ibuprofen and ibuprofen reduced the multiplicity of colon tumors compared with control (p<0.01) by 47.2 and 56.6%, respectively. Equal molar concentrations of ibuprofen (670 mg/kg) and p-ibuprofen (1,215 mg/kg) resulted in stomach ulceration in 85.7% (6 out of 7) and 14.3% (1 out of 7) of rats, respectively, with p<0.01. Immunofluoresence staining and western blot analysis demonstrated that both ibuprofen and p-ibuprofen suppressed β-catenin nuclear translocation in colon cancer cells. In addition, p-ibuprofen but not ibuprofen inhibited NF-κB activation in colon cancer cells. Collectively, these results suggest that p-ibuprofen is a potential effective novel drug for long-term use in colon cancer prevention.