Dysplastic Aberrant Crypt Foci Research Articles

Methylation study of tumor suppressor genes in human aberrant crypt foci, colorectal carcinomas, and normal colon.

Aberrant crypt foci (ACF) are the earliest preneoplastic lesions in human colon, identifiable on chromoendoscopic screening. Our objective was to evaluate the %methylation of APC, CDKN2A, MLH1, RASSF1, MGMT, and WIF1 tumor suppressor genes (TSG) in ACF, corresponding colorectal carcinomas (CRC), and normal colonic mucosal controls. In this study, macroscopically normal-appearing mucosal flaps were sampled 5-10 cm away from the tumor mass from 302 fresh colectomy specimens to identify ACF-like lesions. Thirty-five cases with multiple ACFs were selected (n 35) as the main study group, with corresponding sections from CRC (n 35) as disease controls, and mucosal tissue blocks from 20 colectomy specimens (normal controls), operated for non-neoplastic pathologies. Genomic DNA was extracted, and methylation-specific polymerase chain reaction (PCR) was performed on a customized methylation array model. %Methylation data were compared among the groups and with clinicopathological parameters. Selected target mRNA and protein expression studies were performed. %Methylation of TSGs in ACF was intermediate between normal colon and CRC, although a statistically significant difference was observed only for the WIF1 gene (P < 0.01). Also, there was increased nuclear β-catenin expression and upregulation of CD44-positive cancer-stem cells in ACF and CRCs than in controls. Right-sided ACFs and dysplastic ACFs had a higher %methylation of CDKN2A (P < 0.01), whereas hyperplastic ACFs had a higher %methylation of RASSF1 (P 0.04). The topographic characteristics of ACFs did not correlate with TSG %methylation. Early epigenetic methylation of WIF1 gene is one of the mechanisms for ACF development in human colon.

The Effect of Low Doses of Acetylsalicylic Acid on the Occurrence of Rectal Aberrant Crypt Foci.

Background and Objectives: Aberrant crypt foci (ACF) are one of the earliest putative preneoplastic and, in some cases, neoplastic lesions in human colons. Many studies have confirmed the reduction of ACFs and colorectal adenomas after treatment with acetylsalicylic acid (ASA) commonly referred to as ASA; however, the minimum effective dose of ASA and the duration of use has not been fully elucidated. The objective of our study was to assess the significance of low dose ASA (75-mg internally once daily) to study the chemopreventive effect of ASA in ACF and adenomas development in patients taking this drug for a minimum period of 10 years. Materials and&amp;nbsp;Methods: Colonoscopy, combined with rectal mucosa staining with 0.25% methylene blue, was performed on 131 patients. The number of rectal ACF in the colon was divided into three groups: ACF &lt; 5; ACF 5-10; and ACF &gt; 10. Patients were divided into two groups: the "With ASA" group (the study group subjects taking ASA 75-mg daily for 10 years); and "Without ASA" group (control group subjects not taking ASA chronically). The incidence of different types of rectal ACF and colorectal polyps in both groups of subjects was analysed and ascertained. Results: Normal ACF was found in 12.3% in the study group vs. 87.7% control group, hyperplastic 22.4% vs. 77.6%, dysplastic 25% vs. 75%, mixed 0% vs. 100%. Treatment with ASA affects the occurrence of colorectal adenomas. The amount of dysplastic ACFs was lower in the study group than in the control group. The increase in dysplastic ACFs decreases with age in both groups, with the increase greater in those not taking ASA. Conclusions: Patients who take persistent, chronic (&gt;10 years) low doses of ASA have a lower total number of all types of rectal ACFs and adenomas compared to the control group.

The Epithelial-Stromal Microenvironment in Early Colonic Neoplasia.

Stromal cells play a central role in promoting the progression of colorectal cancer. Here, we analyze molecular changes within the epithelial and stromal compartments of dysplastic aberrant crypt foci (ACF) formed in the ascending colon, where rapidly developing interval cancers occur. We found strong activation of numerous neutrophil/monocyte chemokines, consistent with localized inflammation. The data also indicated a decrease in interferon signaling and cell-based immunity. The immune checkpoint and T-cell exhaustion gene PDCD1 was one of the most significantly upregulated genes, which was accompanied by a decrease in cytotoxic T-cell effector gene expression. In addition, CDKN2A expression was strongly upregulated in the stroma and downregulated in the epithelium, consistent with diverse changes in senescence-associated signaling on the two tissue compartments. IMPLICATIONS: Decreased CD8 T-cell infiltration within proximal colon ACF occurs within the context of a robust inflammatory response and potential stromal cell senescence, thus providing new insight into potential promotional drivers for tumors in the proximal colon.

Heme oxygenase – 1 Expression in Liver and Colon of Rats Exposed to Oxidative stress and Dysplasia by a Carcinogen Diethylnitrosamine and the Possible Therapeutic Effects of Probiotic Versus Pyridazine Derivative and Chemotherapy

Background: Diethylnitrosamine (DENA) is dietary carcinogen. It is known as cancer initiator in various organs. The present study investigated the destructive changes of DENA in liver and colon and the possible therapeutic effects of doxorubicin (DOX); pyridazine derivative (MDP) and lactobacillus casei (LAB) against DENA induced dysplasia in liver and colon.Methods: Lactobacillus casei were tested for their probiotic properties and prepared for rat administration. Sixty adult male albino rats were divided into six groups. A normal control group received the vehicle; DENA group was injected intraperitoneally (ip) with 55mg/kg body weight twice per week for six weeks. DENA+MDP group received MDP at a dose of 10mg/kg (ip) twice per week for the next 4 weeks after DENA administration; DENA+DOX group received DOX at a dose of 10mg/kg (ip) twice per week for the next 4 weeks after DENA administration; DENA+LAB received LAB orally at a dose of (1.5 x 109 CFU) twice per week for the next 4 weeks after DENA administration. DENA+MDP+DOX group received both MDP and DOX as the aforementioned before. Sera, liver and colon were obtained after the end of experiment. Serum aspartate transaminase and alanine transaminase were detected as well as glutathione peroxidase (GSHPX), nitric oxide, tumor necrosis factor (TNF-α), alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA). Histo-pathological studies and immune-histochemical examination of heme oxygenase-1 (HO-1) were done. Morphometric study was performed. All measurements were followed by statistical analysis.Results: DENA induced significant increase in liver enzymes with significant increase in oxidation and inflammation biomarkers and AFP and CEA. Histologically, DENA showed degenerative changes in hepatocytes and dysplastic aberrant crypt foci in colon. Liver and colon displayed increased cytoplasmic and nuclear immune-expression of HO-1. Therapeutic groups showed partial improvement in biochemical parameters and histological structure. However, Lactobacillus casei showed the best result in attenuating pathological and biochemical changes in liver and colon. Conclusion: Lactobacillus casei displayed a potential anti-tumorigenic activity against DENA in liver and colon. This may be exerted via HO-1 modulation and suppression of oxidation and inflammation.

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.

Dysplastic Aberrant Crypt Foci: Biomarkers of Early Colorectal Neoplasia and Response to Preventive Intervention.

The discovery of aberrant crypt foci (ACF) more than three decades ago not only enhanced our understanding of how colorectal tumors form, but provided new opportunities to detect lesions prior to adenoma development and intervene in the colorectal carcinogenesis process even earlier. Because not all ACF progress to neoplasia, it is important to stratify these lesions based on the presence of dysplasia and establish early detection methods and interventions that specifically target dysplastic ACF (microadenomas). Significant progress has been made in characterizing the morphology and genetics of dysplastic ACF in both preclinical models and humans. Image-based methods have been established and new techniques that utilize bioactivatable probes and capture histologic abnormalities in vivo are emerging for lesion detection. Successful identification of agents that target dysplastic ACF holds great promise for intervening even earlier in the carcinogenesis process to maximize tumor inhibition. Future preclinical and clinical prevention studies should give significant attention to assessing the utility of dysplastic ACF as the earliest identifiable biomarker of colorectal neoplasia and response to therapy.See all articles in this Special Collection Honoring Paul F. Engstrom, MD, Champion of Cancer Prevention.

Rectal aberrant crypt foci (ACF) as a predictor of benign and malignant neoplastic lesions in the large intestine

BackgroundThe importance of ACF is not fully explained, however, their number may be a good predictor of synchronous and metachronic adenoma or other polyps whose removal reduces the risk of CRC. Due to the epidemiological and genetic association of ACF with pre-cancer lesions, they may be a potential CRC biomarker. The aim of our study was to show that the number and type of rectal ACF may be a good predictive factor for the presence of polyps located proximally from the splenic flexure and that the type and number of ACF can correlate with the number and specific types of polyps in the large intestine.MethodsThe study included 131 patients who underwent colonoscopy combined with rectal mucosa staining with 0.25% methylene blue. The number of rectal ACF was determined and bioptats were sampled for histopathological examination to assess the type of ACF. Endoscopic ACF assessment criteria given by L. Roncucci were used. The obtained material was subjected to statistical analysis using probability distribution, U-test, t-student test, and chi 2 as well as the Statistica 7.1 software package.ResultsThe study population was divided into three subgroups according to the number of ACF observed, i.e. ACF < 5, 5–10 and > 10. ACF < 5 were found in 35 patients (29.41%), 5–10 ACF in 70 (58.82%) and ACF > 10 in 14 individuals (11.76%).The study revealed the presence of normal ACF (p = 0.49), hyperplastic ACF (p = 0.34), dysplastic ACF (p = 0.11), and mixed ACF (p = 0.06). A single type of ACF was most commonly observed (n = 88, p = 0.74). In the researched group a larger number of ACF is concurrent with adenomas and hyperplastic polyps. The number of ACF clearly correlates with the dysplasia advancement in the adenoma and the number of polyps found.ConclusionsRectal ACF are a useful marker for the presence of cancerous lesions in the proximal and distal sections of the large intestine.

Apiaceous and Cruciferous Vegetables Fed During the Post-Initiation Stage Reduce Colon Cancer Risk Markers in Rats

Vegetable consumption reduces colon cancer risk when fed in the initiation stage of carcinogenesis; however, the effect of vegetable consumption during the post-initiation stage has rarely been examined. We investigated the chemopreventive effects of feeding apiaceous and cruciferous vegetables on colon cancer risk in the post-initiation stage. Thirty male Wistar rats (∼5 wk, 92 g) were subcutaneously injected with 1,2-dimethylhydrazine 1 time/wk for 2 wk. One week after the last dose, rats were randomly assigned to 3 groups: the basal diet, an apiaceous vegetable-containing diet (API; 21% fresh wt/wt), or a cruciferous vegetable-containing diet (CRU; 21% fresh wt/wt). All diets contained ∼20% protein, 7% fat, and 63% digestible carbohydrate. Experimental diets were fed for 10 wk, after which colons were harvested. CRU reduced aberrant crypt foci (ACF) number compared to the basal group (P=0.014) and API (P=0.013), whereas API decreased the proportion of dysplastic ACF relative to the basal group (P<0.05). Both CRU and API reduced doublecortin-like kinase 1-positive marker expression relative to basal by 57.9% (P=0.009) and 51.4% (P<0.02). The numbers of CD44-positive ACF did not differ between the groups. We identified 14 differentially expressed microRNAs (miRNAs). Of these, expression of 6 miRNAs were greater or tended to be greater (P≤0.10) in one or both vegetable-containing groups compared to the basal group. Bioinformatic analysis of these expression changes in miRNA predicted a change in WNT/β-catenin signaling, indicating downregulation of β-catenin in the vegetable-fed groups. Consistent with this bioinformatics analysis, β-catenin-accumulated ACF were decreased in CRU (93.1%, P=0.012), but not in API (54.4%, P=0.125), compared to the basal group. Both apiaceous and cruciferous vegetables, fed post-initiation, reduce colonic preneoplastic lesions as well as cancer stem cell marker expression in rats, possibly by suppressing oncogenic signaling through changes in miRNA expression.

Detection of aberrant crypt foci with image-enhanced endoscopy.

Background and study aims Conventional detection of aberrant crypt foci (ACF) with dye-spraying and magnifying observation is labor- and skill-intensive. We performed a prospective non-inferiority study to investigate the utility of image-enhanced endoscopy (IEE) for detection of ACF. Patients and methods Patients with a history of colorectal neoplasm were eligible. The number of ACF in the lower rectum was counted first using IEE magnification with narrow-band imaging (NBI) or blue-laser imaging (BLI), and subsequently using the methylene blue method. The primary endpoint was the ACF detection rate with IEE, i. e., the number of ACF detected with IEE relative to the number of ACF detected with methylene blue. The secondary endpoints were bowel preparation time, ACF detection time, and the detection rate with NBI or BLI. Results A total of 40 patients were enrolled (NBI 20 and BLI 20). The overall detection rate for ACF with IEE was 81.7 % (503/616; 95 %CI 78.8 – 84.6 %), meeting the primary endpoint. The detection rate for ACF with BLI (84.9 %, 258/304) was significantly higher than with NBI (78.5 %, 245/312; P < 0.05). Both bowel preparation time and ACF detection time were significantly shorter with IEE versus the methylene blue method ( P < 0.01, respectively). The detection rates for dysplastic and non-dysplastic ACF with IEE were 84.4 % (27/32) and 80.3 % (469/584), respectively. Conclusion IEE is able to detect ACF during colonoscopy with sensitivity non-inferior to that of the conventional methylene blue method. IEE is simpler than the methylene blue method and is therefore a potentially useful new tool for ACF detection.

Protective effect of p-coumaric acid against 1,2 dimethylhydrazine induced colonic preneoplastic lesions in experimental rats

Oxidative stress and gut microbial enzymes are intricately linked to the onset of colon carcinogenesis. Phytochemicals that modulate these two factors hold promise for the development of such agents as anticancer drugs. The present study evaluates the chemopreventive potential of p-coumaric acid (p-CA) – a phenolic acid in rats challenged with the colon specific procarcinogen DMH (1,2 di-methyl hydrazine). Rats were randomized into six groups (n=7/group). Group 1 (control); Group 2 (p-CA 200mg/kg b.w.); Group 3 (DMH 40mg/kg b.w.); Groups 4 (DMH+p-CA 50mg/kg b.w.) and Group 5 (DMH+p-CA 100mg/kg b.w.) and Group 6 (DMH+p-CA 200mg/kg b.w.). After the experimental duration of 15 weeks’ rats were subjected to necropsy and tissues were collected for the histological and biochemical investigations. DMH induced colonic preneoplastic lesions viz., aberrant crypt foci (ACF), dysplastic ACF (DACF), mucin depleted foci (MDF) and beta catenin accumulated crypts (BCAC) were significantly suppressed by p-CA supplementation. Glucuronide conjugation of DMH in liver and its subsequent deconjugation mediated by microbes in the colon induced the formation of colonic preneoplastic lesions. p-CA inhibited these lesions and protected the rat colon against genotoxic insult by scavenging the free radicals via its strong antioxidant response and detoxification mechanism as measured by TBARS and enzymic antioxidants in control and experimental rats. Of the three tested doses, p-CA at a dose of 100mg/kg body weight is found to exhibit a significant optimum effect compared to the other two doses 50mg/kg body weight and 200mg/kg body weight.

Abstract A25: Inhibition or delay of microadenoma development by atorvastatin, naproxen and ED-71 in mice genetically predisposed to colorectal adenomas

Abstract Previous studies by this group demonstrate the ability of chemopreventive agents to differentially inhibit morphological subtypes (flat and polypoid) of colorectal adenomas. Recent efforts have focused on the response of microadenomas, a lesion not routinely evaluated by others, to therapeutic intervention. Microadenomas, or dysplastic aberrant crypt foci (ACFs), are direct precursors of colorectal adenomas and represent the earliest microscopic aberration that can be detected prior to colon tumor formation. The goal of the present study was to: 1) characterize the incidence and multiplicity of microadenomas (≤4 crypts) in Apc+/Min-FCCC mice that spontaneously develop multiple colorectal adenomas, and 2) compare the effect of three classes of chemopreventive agents on microadenoma formation. Animals for baseline histological characterization were subjected to colonoscopic examinations at 7 weeks of age (standard time of treatment initiation) and categorized as tumor-free or tumor-bearing. Forty five percent of the Apc+/Min-FCCC mice without gross colon tumors (N=11) possessed microadenomas at baseline, with an overall average of 0.63 ± 0.29 (Mean ± SEM) per mouse. Among mice with gross tumors at baseline (N=10), 30% had microadenomas (0.3 ± 0.16). Results from treatment studies demonstrate that several agents have the ability to modulate microadenoma formation or progression when administered chronically for 14 weeks. First, atorvastatin (100 ppm), a cholesterol-lowering agent, completely eliminated the formation of microadenomas in mice that were free of gross tumors at baseline (as compared to controls, P = 0.007). In addition, a 33% reduction in tumor incidence was observed among atorvastatin-treated animals at 14 weeks as compared to controls (atorvastatin vs. controls: 55.6% vs. 88.5%, P = 0.017). Second, administration of the non-steroidal anti-inflammatory drug naproxen (400 ppm) caused a significant reduction (89.3%) in the multiplicity of microadenomas as compared to that of animals receiving unsupplemented control diet (0.06 ± 0.06 vs. 0.56 ± 0.24, respectively). Inhibition of microadenomas by naproxen translated into a significant reduction in tumor incidence, (naproxen - 64.7%, controls - 100%, P = 0.0076). Third, ED-71 (eldecalcitol; 1α,25-dihydroxy-2β;-(3-hydroxypropyloxy) Vitamin D3), a novel analog of calcitriol, at a dose of 0.1 μg/kg bw, unexpectedly increased the multiplicity of microadenomas in drug-treated mice as compared to controls (0.9 ± 0.22 vs. 0.5 ± 0.25 P = 0.06, respectively). In contrast, ED-71 reduced the mean incidence of adenomas (&amp;gt; 4 crypts) by 47% (ED-71 - 45.5%, controls 92.3%; P = 0.02) and adenoma multiplicity by 47.6% (ED-71 - 1.1 ± 0.39, controls - 2.1 ± 0.72; P &amp;gt; 0.05) in mice that were free of gross tumors at baseline. These data suggest that ED-71 is effective in preventing the progression of microadenomas to adenomas. In summary, these findings demonstrate the importance of evaluating the response of microadenomas to therapy and suggest that microadenomas represent a promising target for chemopreventive intervention. Supported by CA129467, N01CN43309 and HHSN2610005. * Equal contribution to the abstract. Citation Format: Wen-Chi L. Chang, Harry S. Cooper, Harvey Hensley, Tianyu Li, Karthik Devarajan, Margie L. Clapper. Inhibition or delay of microadenoma development by atorvastatin, naproxen and ED-71 in mice genetically predisposed to colorectal adenomas. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A25.

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.

Autophagy is upregulated during colorectal carcinogenesis, and in DNA microsatellite stable carcinomas.

Cancer cells are exposed to a wide range of stress sources, such as nutrient deprivation and hypoxia, as well as cytotoxic chemotherapy and radiotherapy. Certain forms of stress can also promote survival activating the metabolic autophagy pathway in cancer cells. Autophagy is dramatically increased in cancer cells. In these conditions, it is becoming evident that autophagy protects cells, by providing an alternative energy source and by eliminating dysfunctional organelles or proteins. Its role in tumorigenesis is more controversial and both the presence and the absence of autophagy have been implicated. Autophagy is known to be associated with the poor outcome of patients with various types of cancers, and its effectiveness as a prognostic marker in colorectal cancer was demonstrated by several studies. The inhibition of autophagy may be a potential therapeutic target in colorectal cancer. In vitro experiments have shown that the inhibition of autophagy increases 5-FU-induced apoptosis. There are two trials currently investigating the addition of chloroquine to 5-FU-based chemotherapy and bevacizumab. In the present study, we evaluated the expression of LC3B-II in samples of human colorectal microadenomas (i.e., dysplastic aberrant crypt foci) and carcinomas compared to normal mucosa. Furthermore, the expression pattern of LC3B-II was assessed in carcinomas classified as DNA microsatellite stable (MSS) and unstable (MSI). Thus, immunofluorescence techniques coupled with confocal microscopy and immunoblot experiments were performed. The results clearly showed a significant increase in expression of the autophagic key factor in microadenomas and carcinomas with respect to normal mucosa. In MSS carcinomas, the level of LC3B-II expression was higher than that in the MSI carcinomas.

Combined therapeutic efficacy of carvacrol and X-radiation against 1,2-dimethyl hydrazine-induced experimental rat colon carcinogenesis.

Colon cancer is one of the most commonly diagnosed cancers, and is a major cause of cancer morbidity and mortality worldwide. The objective of the present study is to evaluate the combined therapeutic efficacy of carvacrol (CVC) and X-radiation against 1,2-dimethylhydrazine-induced colon cancer. Male albino Wistar rats were randomly divided into six groups. Group 1 served as control; group 2 received 40 mg/kg b.wt of CVC orally everyday throughout the experimental period (32 weeks); groups 3-6 received subcutaneous injections of DMH (20 mg/kg b.wt), once a week for the first 15 weeks; group 4 received a single dose of X-radiation at the 31st week; group 5 received CVC (40 mg/kg b.wt) two days after the last injection of DMH and continued everyday till the end of the experimental period; group 6 received CVC as in group 5 and radiation as in group 4. DMH-treated rats showed increased incidence of aberrant crypt foci (ACF), dysplastic aberrant crypt foci (DACF), mast cell number, argyrophilic nucleolar organizer regions; elevated activities of phase I enzymes, decreased activities of phase II enzymes, decreased mucin content and altered colonic and liver histology as compared to control rats. Though the individual treatments with CVC and X-radiation to DMH-treated rats reversed the above changes, the combined treatment with both CVC and X-radiation showed a marked effect. Our findings emphasize the potential role of combined therapeutic effect of CVC and X-radiation against DMH-induced colon carcinogenesis.

Abstract 2721: Induction of aberrant crypt foci in the colon of mice exposed to tobacco carcinogen 2-amino-9H-pyrido[2,3-b]indole

Abstract Epidemiological studies indicate that tobacco smoking is a risk factor for colon cancer. However, the etiological agents in tobacco smoke remain uncertain. Heterocyclic aromatic amines (HAAs) that are produced during high-temperature cooking of the proteinaceous food are associated with the elevated risk of colon cancer. Tobacco smoking also produces HAAs, the most abundant one being 2-amino-9H-pyrido[2,3-b]indole (AαC). The carcinogenic potency of AαC is unknown in humans, but AαC forms DNA adducts in multiple organs, including liver and colon, as well as being carcinogenic in rodents. In this study, AαC-induced colon cancer risk was assessed by determining DNA adducts and putative colonic precancerous lesions, aberrant crypt foci (ACF) and dysplastic ACF. For comparison, the HAA, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), a liver and colon carcinogen in mice, and azoxymethane (AOM), a potent colon carcinogen in rodents, were also administered. AαC (400 ppm) or MeIQ (300 ppm) were fed in a high-fat diet to A/J mice (n = 12 per group) for 4 weeks. AOM was injected intraperitoneally to another 12 mice at 5 mg/kg body weight weekly for 6 weeks. At the end of carcinogen treatment, liver and colon were harvested from 4 mice from each group for DNA adduct measurement. dG-C8-AαC, dG-C8-MeIQ, and O6-methyl-dG DNA adducts were measured by liquid chromatography/mass spectrometry. The remaining 8 mice from each group were then fed a carcinogen-free diet for additional 8 weeks, and then colons harvested. Colonic total and dysplastic ACF were counted by light microscopy after appropriate staining. AαC feeding induced the greatest number of DNA adducts in liver, followed by AOM and MeIQ. AOM administration and AαC feeding induced similar numbers of DNA adducts in the colon, both of which were significantly greater than MeIQ. However, AOM administration induced a significantly greater number of total and dysplastic ACF in the colons than did feeding either AαC or MeIQ, which had similar numbers of ACF and dysplastic ACF. The high levels of AαC DNA adducts and induced dysplastic ACF in this animal model, provide biological plausibility for a causal role for AαC in tobacco-induced colorectal carcinogenesis. However, DNA adduct numbers induced by these three carcinogens was not proportional to the colonic ACF or dysplastic ACF number, suggesting that different types of DNA adducts differ in their ability to promote colonic precancerous lesions. Citation Format: Sangyub Kim, Jingshu Guo, Sabrina P. Trudo, Daniel D. Gallaher, Robert J. Turesky. Induction of aberrant crypt foci in the colon of mice exposed to tobacco carcinogen 2-amino-9H-pyrido[2,3-b]indole. [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 2721. doi:10.1158/1538-7445.AM2015-2721

Protective effect of p-methoxycinnamic acid, an active phenolic acid against 1,2-dimethylhydrazine-induced colon carcinogenesis: modulating biotransforming bacterial enzymes and xenobiotic metabolizing enzymes.

Objective of the study is to evaluate the modifying potential of p-methoxycinnamic acid (p-MCA), an active rice bran phenolic acid on biotransforming bacterial enzymes and xenobiotic metabolizing enzymes in 1,2-dimethylhydrazine-induced rat colon carcinogenesis. 48 male albino wistar rats were divided into six groups. Group1 (control) received modified pellet diet and 0.1 % carboxymethylcellulose; group2 received modified pellet diet along with p-MCA (80 mg/kg b.wt. p.o.) everyday for 16 weeks; groups 3-6 received 1,2-dimethylhydrazine (DMH) (20 mg/kg b.wt.) subcutaneous injection once a week for the first 4 weeks, while groups 4-6 received p-MCA at three different doses of 20, 40 and 80 mg/kg b.wt. p.o. everyday for 16 weeks. A significant increase in carcinogen-activating enzymes (cytochrome P450, cytochrome b5, cytochrome P4502E1, NADH-cytochrome-b5-reductase and NADPH-cytochrome-P450 reductase) with concomitant decrease in phaseII enzymes, DT-Diaphorase, glutathione S-transferase, UDP-glucuronyl-transferase and gamma glutamyltransferase were observed in group3 compared to control. DMH treatment significantly increased the activities of feacal and colonic bacterial enzymes (β-glucosidase, β-galactosidase, β-glucuronidase, nitroreductase, sulphatase and mucinase). p-MCA supplementation (40 mg/kg b.wt) to carcinogen exposed rats inhibited these enzymes, which were near those of control rats. The formation of dysplastic aberrant crypt foci in the colon and the histopathological observations of the liver also supports our biochemical findings. p-MCA (40 mg/kg b.wt.) offers remarkable modulating efficacy of biotransforming bacterial and xenobiotic metabolizing enzymes in colon carcinogenesis.

Colon epithelial proliferation and carcinogenesis in diet‐induced obesity

Colorectal cancer is the third leading cause of cancer death in Japan and the United States and is strongly associated with obesity, especially visceral obesity. Several metabolic mediators, such as adiponectin, have been suspected to play a role in obesity-related carcinogenesis. In a previous human study, the existence of a significant correlation between the number of human dysplastic aberrant crypt foci (ACF) and the visceral fat area was demonstrated, and also that of a significant inverse correlation between the number of dysplastic ACF and the plasma adiponectin level. Other studies have investigated the effect of adiponectin under the normal and high-fat diet conditions in a mouse model of azoxymethane-induced colon cancer. Enhanced formation of both ACF and tumors was observed in the adiponectin-deficient mice, as compared with that in the wild-type, under the high-fat diet condition but not under the normal diet condition. Furthermore, that the 5'-AMP-activated kinase/mammalian target of rapamycin pathway is involved in the promotion of colorectal carcinogenesis in adiponectin-deficient mice under the high-fat diet condition was shown. Therefore, that the 5'-AMP-activated kinase/mammalian target of rapamycin signaling pathway may play an important role in colorectal carcinogenesis was speculated. Metformin, a biguanide derivative widely used in the treatment of diabetes mellitus, has been shown to exert a suppressive effect on ACF formation in both mouse models and humans. Therefore, metformin might be a promising candidate as a safe drug for chemoprevention of colorectal carcinogenesis. Further studies with high evidence levels, such as randomized, controlled studies, are needed to clarify these relationships.

Pomegranate polyphenolics suppressed azoxymethane-induced colorectal aberrant crypt foci and inflammation: possible role of miR-126/VCAM-1 and miR-126/PI3K/AKT/mTOR

The antitumorigenic activities of polyphenols such as ellagitannins and anthocyanins in pomegranate (Punica granatum L.) have been previously studied where cytotoxic, anti-inflammatory and antioxidant effects were evident in various cancer models. The objective of this study was to investigate the role of miR-126/vascular cell adhesion molecule 1 (VCAM-1) and miR-126/phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) in pomegranate-mediated anti-inflammatory and anticarcinogenic effects in vivo and in vitro. Sprague-Dawley rats (n = 10 per group) received pomegranate juice (2504.74 mg gallic acid equivalents/l) or a polyphenol-free control beverage ad libitum for 10 weeks and were injected with azoxymethane (AOM) subcutaneously (15mg/kg) at weeks 2 and 3. Consumption of pomegranate juice suppressed the number of aberrant crypt foci (ACF) and dysplastic ACF by 29 and 53.5% (P = 0.05 and 0.04), respectively, and significantly lowered proliferation of mucosa cells. Pomegranate juice significantly downregulated proinflammatory enzymes nitric oxide synthase and cyclooxygenase-2 messenger RNA (mRNA) and protein expression. In addition, it suppressed nuclear factor-κB and VCAM-1 mRNA and protein expression in AOM-treated rats. Pomegranate also inhibited phosphorylation of PI3K/AKT and mTOR expression and increased the expression of miR-126. The specific target and functions of miR-126 were investigated in HT-29 colon cancer cell lines. In vitro, the involvement of miR-126 was confirmed using the antagomiR for miR-126, where pomegranate reversed the effects of the antagomiR on the expression of miR-126, VCAM-1 and PI3K p85β. In summary, therapeutic potentials of pomegranate in colon tumorigenesis were due in part to targeting miR-126-regulated pathways, which contributes in the underlying anti-inflammatory mechanisms.

Th Inducing POZ-Kruppel Factor (ThPOK) Is a Key Regulator of the Immune Response since the Early Steps of Colorectal Carcinogenesis

We purposed to evaluate the role of Th inducing POZ-Kruppel Factor (ThPOK), a transcriptional regulator of T cell fate, in tumour-induced immune system plasticity in colorectal carcinogenesis. The amounts of CD4+, CD8+ and CD56+ and ThPOK+ cells infiltrate in normal colorectal mucosa (NM), in dysplastic aberrant crypt foci (microadenomas, MA), the earliest detectable lesions in colorectal carcinogenesis, and in colorectal carcinomas (CRC), were measured, and the colocalization of ThPOK with the above-mentioned markers of immune cells was evaluated using confocal microscopy. Interestingly, ThPOK showed a prominent increase since MA. A strong colocalization of ThPOK with CD4 both in NM and in MA was observed, weaker in carcinomas. Surprisingly, there was a peak in the colocalization levels of ThPOK with CD8 in MA, which was evident, although to a lesser extent, in carcinomas, too. In conclusion, according to the data of the present study, ThPOK may be considered a central regulator of the earliest events in the immune system during colorectal cancer development, decreasing the immune response against cancer cells.

PLZF Expression during Colorectal Cancer Development and in Normal Colorectal Mucosa according to Body Size, as Marker of Colorectal Cancer Risk

Promyelocytic leukemia zinc finger protein (PLZF) is a protein involved in various signaling, growth regulatory, and differentiation pathways, including development/function of some T cells. Here, we aimed at the detection of PLZF during colorectal carcinogenesis, using immunofluorescence, and at the evaluation of the colocalization of PLZF with CD2 and CD56 positive cells (T, γ δ, NK, and NKT cells), using confocal-microscopy, along colorectal carcinogenesis, since its earliest stages, that is, dysplastic aberrant crypt foci (ACF). Furthermore, we analyzed PLZF in the normal colonic mucosa (NM) according to anthropometric parameters of the subject. NM exhibited strong CD56 fluorescent staining. This infiltration was lost in both ACF and colorectal carcinoma (CRC), while PLZF presence increased from NM to ACF and CRC. Strong association was found between CD56+ colonic mucosa cell infiltration and body mass index. Interestingly, an increased stromal PLZF-reactivity was present in NM of obese subjects. This study shows that overexpression of PLZF and exclusion of NK cells in dysplastic microenvironment are very early events in the stepwise sequence leading to CRC and that lower levels of CD56+ cells in NM, together with increased levels of PLZF+ cells, can be a reflection of colon cancer risk due to obesity.