Aberrant Crypt Foci In Rats Research Articles

Chemoprotective effect of arbutin on azoxymethane-induced aberrant crypt foci in rat colon via modulation of PCNA/Bax protein.

Arbutin is utilized in traditional remedies to cure numerous syndromes because of its anti-microbial, antioxidant, and anti-inflammatory properties. This study aimed to evaluate chemopreventive effects of arbutin on azoxymethane (AOM)-induced colon aberrant crypt foci (ACF) in rats. Five groups of rats were used: normal control group (rats injected hypodermically with sterile phosphate-buffered saline once per week for two weeks) and groups 2-5, which were subcutaneously inoculated with 15 mg/kg AOM once a week for two weeks. AOM control and 5-fluorouracil (5-FU) control groups were fed 10% Tween orally daily for 8 weeks using a feeding tube. The treated groups were fed 30 and 60 mg/kg arbutin every day for 2 months. ACF from the AOM control group had aberrant nuclei in addition to multilayered cells and an absence of goblet cells. The negative control group displayed spherical cells and nuclei in basal positions. Histological examination revealed a reduced number of AFC cells from colon tissues of the 5-FU reference group. Arbutin-fed animals showed down-regulation of proliferating cell nuclear antigen (PCNA) and up-regulation of Bax protein compared to AOM control. Rats fed with arbutin displayed a significant increase of superoxide dismutase (SOD) and catalase (CAT) activities in colon tissue homogenates compared to the AOM control group. In conclusion, arbutin showed therapeutic effects against colorectal cancer, explained by its ability to significantly decrease ACF, down-regulate PCNA protein, and up-regulate Bax protein. In addition, arbutin significantly increased SOD and CAT, and decreased malondialdehyde (MDA) levels, which might be due to its anti-proliferative and antioxidant properties.

Pinostrobin attenuates azoxymethane-induced colorectal cytotoxicity in rats through augmentation of apoptotic Bax/Bcl-2 proteins and antioxidants

Objectives: Pinostrobin (5-hydroxy-7-methoxyflavanone; PN) is a natural active ingredient with numerous biological activities extensively utilized in tumour chemotherapy. The present study investigates the chemo-preventive potentials of PN on azoxymethane-mediated colonic aberrant crypt foci in rats. Methods: Sprague Dawley rats clustered into five groups, normal control (A) and cancer controls were subcutaneously injected with normal saline and 15 mg/kg azoxymethane, respectively, and nourished on 10% tween 20 and fed on 10% tween 20; reference control (C), injected with 15 mg/kg azoxymethane and injected (intraperitoneal) with 35 mg/kg 5-fluorouracil (5-FU); D and E rat groups received a subcutaneous injection of 15 mg/kg azoxymethane and nourished on 30 and 60 mg/kg of PN, respectively. Results: The acute toxicity trial showed a lack of any abnormal signs or mortality in rats ingested with 250 and 500 mg/kg of PN. The gross morphology of colon tissues revealed significantly lower total colonic aberrant crypt foci incidence in PN-treated rats than that of cancer controls. Histological examination of colon tissues showed increased aberrant crypt foci availability with bizarrely elongated nuclei, stratified cells and higher depletion of the submucosal glands in cancer controls. PN treatment caused positive modulation of apoptotic (Bax and Bcl-2) proteins and inflammatory cytokines (TNF-α, IL-6 and IL-10). Moreover, rats fed on PN had significantly higher antioxidants (superoxide dismutase) and lower malondialdehyde concentrations in their colon tissue homogenates. Conclusion: The chemoprotective efficiency of PN against azoxymethane-induced aberrant crypt foci is shown by lower aberrant crypt foci values and higher aberrant crypt foci inhibition percentage, possibly through augmentation of genes responsible for apoptotic cascade and inflammations originating from azoxymethane oxidative stress insults.

The chemopreventive effects of Huangqin-tea against AOM-induced preneoplastic colonic aberrant crypt foci in rats and omics analysis.

Despite that colorectal cancer (CRC) is a severe global health problem, effective chemopreventive strategies against CRC are still lacking. Huang-qin tea (HQT), a healthy herbal tea, is prepared from the aerial parts of Scutellaria baicalensis Georgi and has been consumed in China for thousands of years. HQT contains abundant flavonoids, which display potent anticancer effects, but no research studies have investigated the cancer-preventive effects of HQT on CRC in vivo. Here, we found that HQT inhibits azoxymethane-induced aberrant crypt foci (ACF) formation in a preneoplastic colonic ACF rat model. The essential role of the gut microbiota in the chemopreventive effect of HQT on CRC in a pseudo-germ-free rat model was confirmed. Besides, HQT modulates inflammatory cytokine expression by significantly decreasing IL-1β, IL-6, IL-10, and TNF-α expression, and elevating IFN-γ production. 16S rDNA sequencing analysis indicated that HQT regulated the gut microbiota by increasing the abundance of beneficial bacteria (Lachnoclostridium, Alistipes, Roseburia, and Lactococcus) and reducing the levels of Bacteroides, Parasutterella, and unidentified_Clostridiales. Fecal metabolomics showed that HQT modulated the AOM-induced metabolomic disorder, and these altered metabolites were almost involved in the lipid metabolic pathways. The Spearman correlation analysis revealed a correlation between the gut microbiota and fecal metabolites. Collectively, these results suggested that HQT exerted beneficial effects on host health by inhibiting inflammation, and by regulating the gut microbiota profile and certain metabolic pathways. In conclusion, HQT inhibits AOM-induced ACF formation by modulating the gut microbiota composition and improving metabolomic disorders, indicating the potential of HQT as a functional beverage candidate for the prevention and treatment of CRC.

Inhibition of Formation of Azoxymethane-induced Colonic Aberrant Crypt Foci in Rats by Edible Green Algae Capsosiphon fulvescens and Brown Algae Hizikia fusiforme.

Capsosiphon fulvescens (green seaweed) and Hizikia fusiforme (brown seaweed) are marine algae consumed as food supplements, especially in Japan, China and Korea, and are considered traditional medicinal tonics for certain ailments. The aim of this study was to investigate the possible inhibitory effects of dietary C. fulvescens and H. fusiforme on azoxymethane (AOM)-induced colorectal cancer (CRC) in rats. F344 male rats (5 weeks, 150 g) were divided into six groups as follows. Group 1: Injected with normal saline solution and fed control diet (untreated control). Group 2: Injected with AOM and fed control diet (treated control). Group 3: Injected with AOM and fed 1% C. fulvescens diet. Group 4: Injected with AOM and fed 2% C. fulvescens diet. Group 5: Injected with AOM and fed 2% H. fusiforme diet. Group 6: Injected with AOM and fed 6% H. fusiforme diet. Test animals received subcutaneous injections of AOM (15 mg/1 ml/kg body weight) once a week for 2 weeks to induce aberrant crypt foci (ACF) in treated control and experimental groups. We evaluated the effects of dietary C. fulvescens and H. fusiforme at two different dose levels: 1 and 2% C. fulvescens, and 2 and 6% H. fusiforme, on colonic carcinogenesis by AOM in rats. Our results suggest that body weights were not significantly different amongst groups. We found that feeding C. fulvescens and H. fusiforme with a control diet significantly (p<0.05) inhibited the development of ACF in experimental groups. C. fulvescens and H. fusiforme in food also significantly (p<0.05) reduced the proliferating cell nuclear antigen labeling index in the colonic tissues of experimental groups. These results demonstrate the chemopreventive potential of C. fulvescens and H. fusiforme against CRC in an AOM-induced rats.

Anticarcinogenic Effects of Dried Citrus Peel in Colon Carcinogenesis Due to Inhibition of Oxidative Stress

ABSTRACTColorectal cancer is one of the leading causes of death worldwide. Reactive oxygen species produce oxidative stress and contribute to colorectal carcinogenesis. Because dietary citrus has been shown to reduce oxidative stress, we investigated the effects of citrus peel extract at dilutions of 1/200–1/500 on the activity of oxidative-stress-related transcription factors, including AP-1, NF-κB, NRF2, p53, and STAT3, in human colon cancer cell line HCT116 cells using a luciferase reporter gene assay. NRF2 transcriptional activities were 1.8- to 2.0-fold higher than the untreated control value. In addition, NF-κB, p53, and STAT3 transcriptional activities were 12–26% lower than the untreated control value. Administration of dried citrus peel in the diet of F344 rats at a dose of 1,000 ppm prevented the formation of azoxymethane-induced precancerous aberrant crypt foci (ACF) in the colon. The total number of ACF in rats fed with dried citrus peel was reduced to 75% of the control value. Moreover, the levels of oxidative-stress-related markers, reactive carbonyl species, in the serum of F344 rats were significantly reduced following the administration of dried citrus peel. These data suggest that citrus peel possesses an ability to suppress cellular oxidative stress through induction of NRF2, thereby preventing azoxymethane-induced colon carcinogenesis.

Risk factors for colorectal cancer in man induce aberrant crypt foci in rats: Preliminary findings

ABSTRACTEpidemiological studies have demonstrated clear associations between specific dietary and environmental risk factors and incidence of colorectal cancer, but the mechanisms responsible for these associations are not known. An animal model could facilitate such an understanding. Both genotoxic and nongenotoxic carcinogens induce aberrant crypt foci (ACF) in the colons of F344 rats. F344 rats were provided with diets that contained putative risk factors for CRC: low calcium and low vitamin D, high iron, high fructose, and decreased light (UV) exposure or a control diet for 14 wk. The rats were then assessed with biochemical measures and by topological examination for evidence of colon abnormalities. Circulating ionized calcium was decreased from 2.85 to 1.69 mmol/L, and ACF were increased from 0.7 to 13.6 lesions/colon (both P < 0.001). Rats exposed to the multiple environmental conditions associated with colon cancer, developed ACF similar to the heterogeneous or ill-defined ACF in the human colon. Heterogeneous ACF are the most frequently seen in humans and are also seen in rats shortly after exposure to the non-genotoxic colon carcinogen, dextransulfate sodium. The rodent model could be used to assess the pathways from diet and environment to colon cancer and to provide guidance for clinical studies.

Chemopreventive effects of Strobilanthes crispus leaf extract on azoxymethane-induced aberrant crypt foci in rat colon.

In this work, microscopic and histological studies suggest that Strobilanthes crispus ethanol extract reduce azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in rats. S. crispus is considered a traditional medicine and used as an antioxidant. Its leaf contains a large amount of phenolic compounds to which its radical scavenging role is attributed and enhance its ability to eradicate oxidative stress reactions. The study was designed to determine the chemopreventive effect of S. crispus ethanol extract in vivo and in vitro by elucidating the effect of the extract on intermediate biomarkers which can be used as effective predictors of colon cancer. S. crispus was analyzed for DPPH free radical scavenging, nitric oxide (NO) and ferric acid reduction. The results indicated that S. crispus oral administration significantly inhibited colorectal carcinogenesis induced by AOM as revealed by the reduction in the number of ACF. S. crispus down-regulated the expression of PCNA, Bcl2 and β-catenin. Additionally, it exerted a pronounced inhibitory effect on MDA and NO levels and stimulatory effect on CAT and GPx activities. These results demonstrate that S. crispus is a chemopreventive agent for colorectal cancer through the suppression of early and intermediate carcinogenic phases that may be related to its flavonoid content.

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.

The chemopotential effect of Annona muricata leaves against azoxymethane-induced colonic aberrant crypt foci in rats and the apoptotic effect of Acetogenin Annomuricin E in HT-29 cells: a bioassay-guided approach.

Annona muricata has been used in folk medicine for the treatment of cancer and tumors. This study evaluated the chemopreventive properties of an ethyl acetate extract of A. muricata leaves (EEAML) on azoxymethane-induced colonic aberrant crypt foci (ACF) in rats. Moreover, the cytotoxic compound of EEAML (Annomuricin E) was isolated, and its apoptosis-inducing effect was investigated against HT-29 colon cancer cell line using a bioassay-guided approach. This experiment was performed on five groups of rats: negative control, cancer control, EEAML (250 mg/kg), EEAML (500 mg/kg) and positive control (5-fluorouracil). Methylene blue staining of colorectal specimens showed that application of EEAML at both doses significantly reduced the colonic ACF formation compared with the cancer control group. Immunohistochemistry analysis showed the down-regulation of PCNA and Bcl-2 proteins and the up-regulation of Bax protein after administration of EEAML compared with the cancer control group. In addition, an increase in the levels of enzymatic antioxidants and a decrease in the malondialdehyde level of the colon tissue homogenates were observed, suggesting the suppression of lipid peroxidation. Annomuricin E inhibited the growth of HT-29 cells with an IC50 value of 1.62 ± 0.24 μg/ml after 48 h. The cytotoxic effect of annomuricin E was further substantiated by G1 cell cycle arrest and early apoptosis induction in HT-29 cells. Annomuricin E triggered mitochondria-initiated events, including the dissipation of the mitochondrial membrane potential and the leakage of cytochrome c from the mitochondria. Prior to these events, annomuricin E activated caspase 3/7 and caspase 9. Upstream, annomuricin E induced a time-dependent upregulation of Bax and downregulation of Bcl-2 at the mRNA and protein levels. In conclusion, these findings substantiate the usage of A. muricata leaves in ethnomedicine against cancer and highlight annomuricin E as one of the contributing compounds in the anticancer activity of A. muricata leaves.

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 B46: Dietary acrylamide differentially modulates azoxymethane-induced F344 rat colon tumor formation and ectopic growth of human colon tumor cells in nude (nu/nu) mice

Abstract Acrylamide, a known rodent and a possible human carcinogen is ubiquitously formed in foods at high temperature. We earlier reported that dietary acrylamide at doses known to cause rodent tumors, did not increase the formation of azoxymethane (AOM)-induced colon aberrant crypt foci (ACF), putative precancerous lesions. On the contrary, we observed a significant decrease in ACF in rats receiving acrylamide at 10 and 50 mg/kg diet and a trend to increase ACF at the lowest dose (5mg acrylamide/kg diet) tested. In this study using two rodent models, we assessed if dietary acrylamide, at levels reflecting those in popular human foods, modulated colon tumor formation and progression. In Experiment-1, 4 groups of weanling male F344 rats received AIN-93G diets with acrylamide at 0.5, 1.0 or 2.0 mg/kg diet or without (0 mg/kg diet, control). After two weeks, rats in each diet group received either two weekly s.c. injections of AOM or saline. All rats were on the experimental diets ad libitum for 20 weeks post AOM/saline injections after which they were killed and their colons assessed for tumors. In Experiment-2, athymic nude (nu/nu) mice were s.c. injected with HT-29 human colon adenocarcinoma cells and tumors were allowed to grow for 3 weeks after which they received one of the 4 experimental diets; tumor growth was monitored bi-weekly and mice were killed 4 weeks after. None of the saline-injected rats had tumors in their colon. 23 rats that received acrylamide had blood in their stool at 19 weeks post-AOM injections, and hence the study was stopped in a timely manner at 20 weeks post-AOM injections. The colon tumor incidence was 54.2% in the control group and 66.67% in the highest acrylamide-treated group (2 mg/kg diet). Tumor multiplicity was similar across all diet groups. However, tumor size and burden were higher in the group that received 2 mg/kg acrylamide by comparison to the control. These results suggest acrylamide by itself is not a “carcinogen” at upper exposure levels commonly seen in popular foods, but acts as a “co-carcinogen” at the highest tested dose by exacerbating the risk of developing colon tumors by a known colon-specific carcinogen AOM. The nude mice study indicated no differences in ectopic human colon tumor growth between acrylamide-treated and control mice suggesting that acrylamide at the levels tested does not aid in the progression of established human tumors in this experimental model. Our data provides characterization of acrylamide as a possible colon cancer promoter in association with a known and possibly other environmental tumor initiators/promoters. This additional information will be useful in defining the health risks of acrylamide when exposed through the diet, and will assist regulators in developing a better understanding of the relevance to humans. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B46.

Dark chocolates suppress rat aberrant crypt foci formation by downregulation of inflammation involved gene expression

Colon cancer is the second leading cause of cancer death in the United States. Cocoa and chocolate products have among the highest concentrations of polyphenols compared to other food sources containing polyphenolic compounds. An increasing body of epidemiologic evidence supports the concept that diets rich in polyphenols promote health and attenuate or delay the onset of various diseases, including colon cancer. We determined whether dark chocolate protects against colon cancer by inhibiting aberrant crypt foci (ACF) formation. Forty eight male Spague Dawley rats were randomly assigned to receive diets containing no chocolate, regular dark chocolate or bloomed dark chocolate and injected subcutaneously with saline or azoxymethane (AOM, 2 times during wk 3 and 4). Relative to the control (no chocolate), dark chocolate diets, regardless regular or bloomed, lowered the total number of aberrant crypt (P=0.0124) and total number of ACF (P=0.0222). Chocolate diet‐fed animals downregulated the levels of cyclooxygenase 2 (P=0.0347) and RelA (P=0.0445) gene expression. These results suggest that dark chocolates reduced gene expression of inflammatory mediators which may contribute to the lower number of early preneoplastic lesions in colon carcinogenesis. This study was supported by CHNR08‐810 and SDSU NUTR 302L class.

Abstract A35: Chemoprevention of colon carcinogenesis in F344 rats by Se,Se′-1,4-phenylenebis(1,2-ethanediyl)bis-isoselenourea (PBISe) a novel analog of PBIT, an iNOS inhibitor

Abstract Inducible nitric oxide synthase (iNOS) is potential target for inflammation and cancer. Previously, we have shown that S,S′-1,4-Phenylenebis(1,2-ethanediyl)bisisothiourea (PBIT) inhibit colon carcinogenesis induced by azoxymethane (AOM). Although, colon cancer inhibitory efficacy of PBIT has been significant, selective iNOS inhibitors do not completely abrogate NO production due to the exogenous bioavailability and NO generation by eNOS in tumor tissues. To create an iNOS selective and multi-targeted molecule, we have developed a novel isosteric analogue of PBIT, namely PBISe in which sulfur was replaced with selenium. We examined the chemopreventive efficacy of PBISe on AOM-induced rat colon carcinogenesis model using aberrant crypt foci (ACF) as end point. At seven weeks of age, rats (12/group) were fed the control diet (AIN 76A) and colonic ACF were induced by AOM. Three days after second AOM treatment, rats were fed the diets containing 0, 10 and 20 ppm of PBI-Se and continued on the respective diets for 8 weeks before ACF were evaluated histopathologically. Furthermore, PBISe (0–12 µM) and PBIT (0 – 100 µM) were studied for the markers of growth inhibition and induction of apoptosis in the human colon cancer CaCo2 cell line. In addition, inhibitory effect of PBISe and PBIT on proinflammatory signaling molecules iNOS, IL-8 and IL-6 were studied by RT-PCR. Results suggest that dietary administration of 10 and 20ppm of PBISe significantly suppressed AOM-induced total colonic ACF formation (32 – 41%, p&amp;lt; 0.001), and multi crypt (4 or more) aberrant foci (29 – 47%, p&amp;lt; 0.001) respectively. Dietary PBISe suppressed AOM-induced colonic ACF in a dose-dependent manner and it required less than half the dose of PBIT to inhibit total ACFs in rats. Both PBIT and PBISe in CaCo2 cells induced dose-dependent apoptosis. PBIT and PBISe treatment on CaCo2 cells showed significant decrease in cell cycle protein cyclin D1 (∼70%, p&amp;lt;0.0001) and iNOS (∼99%, p&amp;lt;0.0001). Also, treatment with PBIT (30 and 60 M) significantly decreased cytokine induced IL-6 at both the doses whereas PBISe had no such effect. PBIT significantly attenuated the LPS-induced IL-6 and IL-8 production in a dose dependent manner. Whereas, PBISe significantly increased IL-8 and attenuated IL-6 production in CaCo2 cells suggesting differential modulatory effects on immune cells/inflammatory pathways. Incorporation of selenium into the structure of PBIT has provided the agent with additional novel cytotoxic and immunologic properties. Results from in vitro and in vivo bioassay clearly suggest that PBI-Se could be further developed for prevention and treatment of colon cancer. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A35.

Chemopreventive Effects of Frondanol A5, a Cucumaria frondosa Extract, against Rat Colon Carcinogenesis and Inhibition of Human Colon Cancer Cell Growth

Sea cucumber extracts have been widely used to treat individuals with inflammatory conditions in East Asia. The present study has been designed to test potential colon cancer-preventive properties of Frondanol A5, a glycolipid extract from the sea cucumber, Cucumaria frondosa, using in vivo and in vitro models of colon cancer. Chemopreventive efficacy of Frondanol A5 was evaluated on azoxymethane-induced rat colon carcinogenesis using colonic aberrant crypt foci (ACF) as efficacy marker. At 7 weeks of age, groups of rats (12 per group) were fed the AIN-76A diet, and ACFs were induced by azoxymethane (15 mg/kg body weight). Three days after azoxymethane treatment, rats were fed with the diets containing 0, 150, and 450 ppm of Frondanol A5 and continued on the diets for 8 weeks, at which time ACFs were evaluated. Expression levels of proliferating cell nuclear antigen and p21(WAF1/CIP1) were determined in ACFs. Further, Frondanol A5 (10-120 microg/mL) was studied for its growth-inhibitory and apoptotic effects in the HCT-116 cell line. Dietary administration of 150 and 450 ppm of Frondanol A5 significantly suppressed azoxymethane-induced total colonic ACF formation, approximately 34% to 55% (P < 0.01 to P < 0.0001), and multicrypt aberrant foci (48-68.5%, P < 0.0001) in a dose-dependent manner. ACFs in rats treated with Frondanol A5 showed significant upregulation of p21(WAF1/CIP1) and downregulation of proliferating cell nuclear antigen compared with control group. Frondanol A5 showed growth inhibition at S and G(2)-M phase with a decrease in Cdc25c and an increase in p21(WAF1/CIP) with significant apoptosis associated with H2AX phosphorylation and caspase-2 cleavage in HCT116 cells. Overall, Frondanol A5 exhibits potential chemopreventive properties for colon carcinogenesis, which suggests further development of this sea cucumber extract.

Conversion of the Common Food Constituent 5-Hydroxymethylfurfural into a Mutagenic and Carcinogenic Sulfuric Acid Ester in the Mouse in Vivo

5-Hydroxymethylfurfural (HMF), formed by acid-catalyzed dehydration and in the Maillard reaction from reducing sugars, is found at high levels in numerous foods. It was shown to initiate colon aberrant crypt foci in rats and skin papillomas and hepatocellular adenomas in mice. HMF is inactive in in vitro genotoxicity tests using standard activating systems but is activated to a mutagen by sulfotransferases. The product, 5-sulfoxymethylfurfural (SMF), is a stronger carcinogen than HMF. SMF has not been detected in the biotransfomation experiments conducted on HMF in humans and animals in vivo up to date. Here, we report pharmacokinetic properties of HMF and SMF in FVB/N mice. Sensitive assays for the quantification of HMF and SMF by LC-MS/MS multiple reaction monitoring were devised. SMF, intravenously injected (4.4 micromol/kg body mass), showed first-order elimination kinetics in blood plasma (t(1/2) = 7.9 min). HMF, injected intravenously (793 micromol/kg body mass), demonstrated biphasic kinetics in plasma (t(1/2) = 1.7 and 28 min for the initial and terminal elimination phases, respectively); the volume of distribution of the central compartment corresponded approximately to the total body water. The maximum SMF plasma level was observed at the first sampling time, 2.5 min after HMF administration. On the basis of these kinetic data, it was estimated that between 452 and 551 ppm of the initial HMF dose was converted to SMF and reached the circulation. It is likely that additional SMF reacted with cellular structures at the site of generation and thus is ignored in this balance. Our work supports the hypothesis that HMF-related carcinogenicity may be mediated by its reactive metabolite SMF.

Fatty acid synthase is over‐expressed in large aberrant crypt foci in rats treated with azoxymethane

Fatty acid synthase (FAS) is over-expressed in many human cancers including colon. High levels of FAS expression have also been observed in a number of lesions that are precursors to invasive colorectal cancer. However, FAS expression in aberrant crypt foci (ACF), the earliest identifiable lesions in colon cancer development, has not been investigated. In this study, we treated Fisher rats with a single dose of the colon carcinogen azoxymethane then evaluated ACF 100 days later. We showed that large ACF (>or=4 crypts/focus) have a significantly higher level of immunohistochemical staining for FAS than either small ACF (<or=3 crypts/focus) or normal crypts. Furthermore, the severity of nuclear atypia in ACF was positively associated with increased expression of FAS. These findings suggest that the genes associated with FAS over-expression are activated early in the stepwise development of colon cancer, though not until the ACF have reached a critical size with a level of nuclear atypia indicative of dysplasia.

Quercetin May Suppress Rat Aberrant Crypt Foci Formation by Suppressing Inflammatory Mediators That Influence Proliferation and Apoptosis1–3

The flavonoid quercetin suppresses cell proliferation and enhances apoptosis in vitro. In this study, we determined whether quercetin protects against colon cancer by regulating the protein level of phosphatidylinositol 3-kinase (PI 3-kinase) and Akt or by suppressing the expression of proinflammatory mediators [cyclooxygenase (COX)-1, COX-2, inducible nitric oxide synthase (iNOS)] during the aberrant crypt (AC) stage. Forty male rats were randomly assigned to receive diets containing quercetin (0 or 4.5 g/kg) and injected subcutaneously with saline or azoxymethane (AOM; 2 times during wk 3 and 4). The colon was resected 4 wk after the last AOM injection and samples were used to determine high multiplicity AC foci (HMACF; foci with >4 AC) number, colonocyte proliferation and apoptosis by immunohistochemistry, expression of PI 3-kinase (p85 and p85α subunits) and Akt by immunoblotting, and COX-1, COX-2, and iNOS expression by real time RT-PCR. Quercetin-fed rats had fewer (P = 0.033) HMACF. Relative to the control diet, quercetin lowered the proliferative index (P = 0.035) regardless of treatment and diminished the AOM-induced elevation in crypt column cell number (P = 0.044) and expansion of the proliferative zone (P = 0.021). The proportion of apoptotic colonocytes in AOM-injected rats increased with quercetin treatment (P = 0.014). Levels of p85 and p85α subunits of PI 3-kinase and total Akt were unaffected by dietary quercetin. However, quercetin tended to suppress (P < 0.06) the expression of COX-1 and COX-2. Expression of iNOS was elevated by AOM injection (P = 0.0001). In conclusion, quercetin suppresses the formation of early preneoplastic lesions in colon carcinogenesis, which occurred in concert with reductions in proliferation and increases in apoptosis. It is possible the effects on proliferation and apoptosis resulted from the tendency for quercetin to suppress the expression of proinflammatory mediators.

Lack of Chemopreventive Activity of Agaricus blazei Mushroom on the Development of 1,2-Dimethylhydrazine-Induced Colonic Aberrant Crypt Foci in Rats

Chemopreventive and immunomodulatory potential of methanolic (MET) and dichloromethanic (DCl) extracts of Agaricus blazei mushroom were investigated in the postinitiation stage of colon carcinogenesis in male Wistar rats. Animals were initiated with 1,2-dimethylhydrazine (DMH) and treated i.g. with DCl or MET extracts. After 9 wk, animals were sacrificed for evaluation of aberrant crypt foci (ACF) development, crypt cellular proliferation, preneoplastic liver lesions (GST-P), proliferative response of spleen cells to mitogen, and natural killer activity. Administration of DCl extracts did not suppress DMH-induced colonic ACF nor did it affect the crypt multiplicity, but the highest dose of MET significantly reduced the development of preneoplastic lesions in the colon and liver. Lymphoproliferative response was slightly decreased in the initiated control group, which was restored by treatment with MET. No toxicity from DCl and MET extracts was observed (groups MET and DCl).

Evaluation of Flutamide Genotoxicity in Rats and in Primary Human Hepatocytes

Flutamide, an effective competitive inhibitor of the androgen receptor used orally for palliative treatment of prostatic carcinoma and regulation of prostatic hyperplasia was evaluated for its genotoxic effects in the intact rat and in primary cultures of human hepatocytes. Negative responses were obtained in all the in vivo assays as well as in the in vitro assay. In rats given a single oral dose of 500 mg/kg flutamide, fragmentation and repair of liver DNA were absent, and no increase was observed in the frequency of micronucleated hepatocytes. In the liver of rats given flutamide as initiating agent at the dose of 500 mg/kg/week for 6 successive weeks, γ-glutamyltraspeptidase-positive foci were detected only in 3 of 10 rats. There was no evidence of a promoting effect on the development of aberrant crypt foci in rats given 100 mg/kg flutamide on alternate days for 8 successive weeks. In primary cultures of human hepatocytes from one male and one female donor DNA fragmentation as measured by the Comet assays, and DNA repair synthesis as revealed by quantitative autoradiography, were absent after a 20 hr exposure to flutamide concentrations ranging from 18 to 56 μM. Taken as a whole, our results seem to indicate that flutamide is a non-genotoxic drug.