Caffeic acid 3,4-dihydroxyphenethyl ester prevents colorectal cancer through inhibition of multiple cancer-promoting signal pathways in 1,2-Dimethylhydrazine/dextran sodium sulphate mouse model.

OBJECTIVE: To elucidate the potential feature and mechanism of the caffeic acid 3,4-dihydroxyphenethyl ester (CADPE) molecule, which can prevent colorectal cancer (CRC) in the 1,2-Dimethylhydrazine (DMH)/dextran sodium sulphate (DSS)-induced mouse model. METHODS: Institute of cancer research (ICR) male mice were injected with 20 mg/kg DMH for a week. After that, 2% DSS was administered in the drinking water for another 7 d. The CADPE treatment was given to the DMH/DSS induced male mice at three different periods until their sacrifice. Histopathological examination was used for observing the CRC development at colonic mucosa. Immunohistochemistry (IHC), blood cells smearing and crypt damage scoring methods were used for investigating the anti-inflammation feature of CADPE related to CRC. The reversing targets searching method was applied with artificial intelligence (AI), computer-aided drug designing (CADD) and Ingenuity Pathway Analysis (IPA) techniques for predicting the potential targets and mechanism of CADPE highly related to CRC. RESULTS: The data indicated that CADPE inhibited CRC tumor development in the colitis-associated DMH/DSS induced mouse model after giving the early treatment. CADPE also impeded the acute inflammation by decreasing the infiltration of neutrophils significantly during the initial stage of CRC development. Finally, our data showed that CADPE prevented CRC by blocking active sites of three pivotal protein targets including epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) in two major cancer development pathways. CONCLUSIONS: CADPE effectively prevented CRC at early stage of tumor germination in the DMH/DSS mouse model highly likely due to its anti-acute inflammation characteristic and the ability of blocking EGFR, ERK and mTOR activities in two highly related CRC developing pathways.

Alterations in mitochondrial membrane in chemopreventive action of fish oil.

Mitochondria are major regulators of pathways related to tumorigenesis; therefore, mitochondrial membrane characteristics and associated cell signaling events were evaluated with different ratios of fish oil (FO) and corn oil (CO) in experimental colon carcinogenesis. Treatment with carcinogen 1,2-dimethylhydrazine (DMH) altered reactive oxygen species (ROS), Ca(2+), and membrane characteristics, which resulted in an elevation in apoptosis in initiation phase and reduction in post-initiation phase. FO+CO(2.5:1)+DMH treatment, however, altered mitochondrial membrane parameters, ROS, and Ca(2+) to increase apoptosis in both phases, whereas FO+CO(1:1)+DMH treatment enhanced apoptosis only in post-initiation phase suggesting that FO supplementation in higher ratio has better chemopreventive efficacy.

Bimodal visualization of colorectal uptake of nanoparticles in dimethylhydrazine-treated mice.

AIM: To investigate colorectal uptake of solid lipid nanoparticles (SLNs) in mice receiving different doses of 1,2-dimethylhydrazine (DMH) using magnetic resonance (MR) and laser-scanning confocal fluorescence microscope (LSCFM) imaging. METHODS: Eight mice were sacrificed in a pilot study to establish the experimental protocol and to visualize colorectal uptake of SLNs in normal mice. Gadopentetate dimeglumine and fluorescein isothiocyanate (FITC)-loaded SLN (Gd-FITC-SLN) enemas were performed on mice receiving DMH for 10 wk (group 1, n = 9) or 16 wk (group 2, n = 7) and FITC-SLN enema was performed on 4 DMH-treated mice (group 3). Pre- and post-enema MR examinations were made to visualize the air-inflated distal colorectum. Histological and LSCFM examinations were performed to verify colorectal malignancy and to track the distribution of SLNs. RESULTS: Homogeneous enhancement and dense fluorescence (FITC) deposition in colorectal wall were observed in normal mice and 1 DMH-treated mouse (group 1) on fluid attenuated inversion recovery (FLAIR) and LSCFM images, respectively. Heterogeneous mural enhancement was found in 6 mice (4 in group 1; 2 in group 2). No visible mural enhancement was observed in the other mice. LSCFM imaging revealed linear fluorescence deposition along the colorectal mucosa in all groups. Nine intraluminal masses and one prolapsed mass were detected by MR imaging with different enhancement modes and pathologies. Interstitial FITC deposition was identified where obvious enhancement was observed in FLAIR images. Bladder imaging agent accumulations were observed in 11 of 16 DMH-treated mice of groups 1 and 2. CONCLUSION: There are significant differences in colorectal uptake and distribution of SLNs between normal and DMH-treated mice, which may provide a new mechanism of contrast for MR colonography.

In vitro ¹4C-labeled amino acid uptake changes and surface abnormalities in the colon after 1,2-dimethylhydrazine-induced experimental carcinogenesis: protection by zinc.

The present study explored the regulatory role of zinc on the in vitro uptake of ¹4C-glucose and ¹4C-labeled amino acids and on colonic surface abnormalities after 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis. Rats were segregated into four groups: control, DMH-treated, zinc-treated, and DMH + zinc-treated. Colon carcinogenesis was induced through weekly subcutaneous injections of DMH (30 mg/kg body weight) for 16 weeks. Zinc (in the form of zinc sulfate) was given to rats at a dose level of 227 mg/L in their drinking water. DMH treatment caused a significant decrease in the activities of disaccharidases (sucrase, lactase, and maltase), but a significant increase in the activity of alkaline phosphatase. In vitro uptake of ¹4C-D-glucose and the amino acids ¹4C-glycine, ¹4C-alanine, ¹4C-lysine, and ¹4C-leucine were significantly higher in the colons of DMH-treated rats. Zinc supplementation of DMH-treated rats resulted in regulating the altered intestinal enzyme activities and in vitro uptake of ¹4C-amino acids and ¹4C-glucose. Scanning electron microscopy revealed drastic alterations in the colon surface morphology after DMH treatment, which were restored after zinc supplementation. Our results confirm a beneficial effect of zinc against DMH-induced alterations in the colons of rats.

Effect of functional buffalo cheese on fatty acid profile and oxidative status of liver and intestine of mice.

The objective of this study was to determine the influence of administration of buffalo dairy products on lipid content and conjugated linoleic acid (CLA) incorporation on liver and intestine of mice. Buffalo cheeses were selected according to nutritional properties and CLA content. Cheeses were previously manufactured using as adjunct culture bacteria with probiotic or technological properties. BALB/c mice were fed for 28 days, and then a single dose of 1,2-dimethylhydrazine (DMH) as oxidant agent was administered before the influence of diet and DMH on antioxidant status in tissues was evaluated. Mice fed buffalo cheese showed the highest body weight gain (P < .05). Polyunsaturated fatty acid (PUFA) content in foods was very different, but total PUFA incorporation was similar in mouse tissues. CLA was only detected in fat tissues of mice fed dairy products, with cis-9, trans-11 being the major isomer. A higher linolenic (C(18:3)) acid content was found in tissues of mice fed commercial diet (control group), and it was partially replaced by CLA in groups receiving buffalo milk or cheese. Lipoperoxides (thiobarbituric acid-reactive substances) were higher in tissues of the control group with or without DMH administration, and DMH had a cytotoxic effect on colon cells (P < .05). Superoxide dismutase (SOD) and catalase activities in liver and intestine were similar among animals, with a slight increase of SOD detected after DMH treatment. Consumption of buffalo dairy products did not affect the oxidative status of mice tissues even after DMH application. In the present study, a protective effect of buffalo cheese and milk on intestine cells was determined.

Inhibition of 1, 2-dimethylhydrazine-induced mucin-depleted foci and O6-methylguanine DNA adducts in the rat colorectum by boiled garlic powder.

The scavenging capacity of reactive oxygen species, such as hydroxyl radicals, is reported not to decrease in boiled garlic (an odorless garlic preparation). We therefore examined the modifying effect of boiled garlic powder (BGP) on 1,2-dimethylhydrazine-induced mucin-depleted foci (MDF) and aberrant crypt foci (ACF), preneoplastic lesions, in the rat colorectum. Male F344 rats (5 weeks old) were fed a basal diet, or experimental diets containing 5% or 1% BGP for 5 weeks. One week later, all rats were injected s.c. with DMH (40 mg/kg, once weekly for 2 weeks). At 10 weeks of age, all the rats were sacrificed, and the colorectum was evaluated for MDF and ACF. In rats given DMH and the 5% or 1% BGP diets (Groups 2 and 3), the numbers of MDF decreased significantly in a dose-dependent manner, compared with the DMH and basal diet value (Group 1) (p<0.01). The numbers of ACF in Group 2, but not Group 3, showed a non-significant tendency to decrease. Next, the effects of BGP on the formation of DMH-induced O6-methylguanine (O6-MeG) DNA adducts in rats were studied. Male F344 rats (5 weeks old) were fed the basal diet, or 10% BGP diet for 5 weeks. All rats were injected i.p. once with 40 mg/kg DMH at the end of week 5. The animals were sacrificed 6 hours after DMH injection to analyze the O6-MeG DNA adducts in the colorectal mucosa. Dietary administration of BGP significantly inhibited the O6-MeG DNA adduct levels in the colorectal mucosa, compared with the controls (p<0.01). These results suggested that BGP may exert chemopreventive effects against colon carcinogenesis at least in the initiation stage.

Membrane fluidity and surface changes during initiation of 1,2 dimethylhydrazine-induced colon carcinogenesis: protection by zinc.

The present study evaluated the modulatory effects of zinc on colonic membrane fluidity and surface abnormalities following 1,2 dimethylhydrazine (DMH)-induced colon carcinogenesis. Rats were segregated into four groups: normal control, DMH treated, zinc treated, DMH + zinc treated. Colon carcinogenesis was initiated through weekly subcutaneous injections of DMH (30 mg/kg body weight) for 8 weeks. Zinc (in the form of zinc sulphate) was supplemented to rats at a dose level of 227 mg/L in drinking water, ad libitum, for the entire duration of the study. Brush border membranes (BBM) were isolated from the colon of rats and the fluidity parameters were assessed by steady-state fluorescence polarization technique using the membrane extrinsic fluorophore 1,6-diphenyl-1,3,5-hexatriene (DPH). The translational diffusion was measured by using the excimer formation of pyrene incorporated in the membrane. The results demonstrated a significant increase in the polarization and anisotropy, accompanied by an increase in order parameter in the membrane preparations from the colon of DMH-injected rats. Further, studies with pyrene fluorophore indicated a marked decrease in membrane microviscosity following DMH treatment. However, the alterations in membrane fluorescence polarization and the fluidity parameters were completely restored following zinc treatment. Drastic alterations in colon surface were noticed after 8 weeks of DMH treatment. However, zinc treatment to DMH-treated rats greatly restored normalcy in the colonic surface. The study concludes that zinc has a strong membrane stabilizing effect and thus has a positive beneficial effect against chemically induced colonic preneoplastic progression in rats.

Effect of Neem (Azadirchta indica) on serum glycoprotein contents of rats administered 1,2 dimethylhydrazine.

The present study was designed to investigate the effects of aqueous Azadirchta Indica leaf extract (AAILE) on serum glycoprotein contents and tumor incidence rate in colon of rats subjected to Dimethylhydrazine (DMH) treatment. Forty rats were divided equally and randomly into four groups viz., Group I (normal control), Group II (DMH-treated), Group III (AAILE) and Group IV (DMH + AAILE treated). Group II and IV animals were injected subcutaneously every week with DMH (30 mg/kg b.wt.) for two durations of 10 and 20 weeks. AAILE was given orally three times a week on alternate days (100 mg/kg b.wt.) to animals belonging to groups III and IV. Blood samples were drawn from all the animals by ocular vein puncture every month for the estimation of Total Sialic Acid (TSA) and Lipid Bound Sialic Acid (LSA), which served as markers for the cancer. No incidence of tumor was recorded in the animals given DMH treatment for 10 weeks. However, DMH treatment for 20 weeks showed 100% tumor incidence. Animals treated with DMH for both the time durations showed a significant increase in the levels of TSA in comparison to normal control, which however were decreased significantly following AAILE supplementation. There was no significant difference between LSA levels of DMH-treated animals and normal controls. The present study suggested that supplementation of AAILE in cancer-bearing animals attenuates considerably the molecular events that initiate the development of tumors.

Inhibitory effects of high temperature- and pressure-treated garlic on formation of 1,2-dimethylhydrazine-induced mucin-depleted foci and O(6)-methylguanine DNA adducts in the rat colorectum.

High temperature- and pressure-treated garlic (HTPG) has been reported to have enhanced antioxidative and cytotoxic activities. However, there have been no reports on chemopreventive effects using animal cancer models. This study first examined the modifying effects of HTPG on 1,2-dimethylhydrazine (DMH)-induced mucin-depleted foci (MDF) and aberrant crypt foci (ACF), preneoplastic lesions in the rat colorectum. Male F344 rats (5 weeks old) were fed basal diet, or experimental diets containing 1% or 3% HTPG for 5 weeks. One week later, all rats were injected s.c.with DMH (40 mg/kg, once weekly for 2 weeks). At 10 weeks of age, all the rats were sacrificed, and the colorectum was evaluated for MDF and ACF. In rats given DMH and 3% HTPG, the numbers of MDF were decreased significantly as compared with those of rats given DMH alone (p< 0.01), and the numbers of ACF showed a tendency to decrease, although not significantly. Next, the effects of HTPG on the formation of DMH-induced O6-methylguanine (O6-MeG) DNA adducts in rats were studied. Male F344 rats (5 weeks old) were fed the basal diet or 10% HTPG diet for 5 weeks. All rats were injected i.p. once with 40 mg/kg DMH at the end of week 5. The animals were sacrificed 6 hours after DMH injection to analyze the O6-MeG DNA adducts in the colorectal mucosa and liver. Dietary administration of HTPG significantly reduced the adduct levels in the colorectal mucosa and liver, compared with the controls (both p< 0.01). The activities of some detoxification enzymes in the liver of DMH-treated rats were also measured. HTPG significantly reduced the activity of cytochrome P450 (CYP) 2E1, known to be responsible for activation of DMH in rat liver (p< 0.05). In contrast, HTPG significantly enhanced the activities of phase 2 enzymes, quinone reductase (QR) and glutathione S-transferase (GST), in rat liver (both p< 0.05). These results suggested that HTPG might have chemopreventive effects against colon carcinogenesis, at least in the initiation stage.

Marine phosphatidylcholine suppresses 1,2-dimethylhydrazine-induced colon carcinogenesis in rats by inducing apoptosis.

In vitro and animal studies indicate that n-3 polyunsaturated fatty acids (PUFAs) suppress carcinogenesis. This study presents a new insight on effectiveness of marine phospholipids for suppression of colon carcinogenesis. The purpose of this study was to investigate growth inhibition and apoptosis inducing effects of n-3 PUFA in the form of marine phosphatidylcholine (PC) on chemically induced (1,2-dimethylhydrazine) colon cancer in rats. Growth inhibition of Caco-2 cells was determined by colorimetric sodium 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) dye reduction assay. For animal studies, the rats were fed 5 different diets containing docosahexaenoic acid (DHA)-ethyl ester, eicosapentaenoic acid (EPA)-ethyl ester, squid meal PC (rich in DHA), starfish PC (rich in EPA), and corn oil. The 1,2-dimethylhydrazine (30 mg/kg) or saline was injected 48 hours before the experiment. Rats were anesthetized, and apoptotic as well as mitotic cells in crypt were counted based on morphological criteria in isolated crypts. Squid meal and starfish PC potently inhibited the growth of Caco-2 cells. The experimental diets containing n-3 PUFA suppressed colon cancer in rats. Rats that consumed diets containing DHA-ethyl ester, EPA-ethyl ester, squid meal PC, and starfish PC showed increased apoptosis (P < .01) and suppressed proliferation. These results suggest that marine PC-containing diets might be an effective dietary protective factor against colon cancer.

Effects of VPS extract of Coriolus versicolor on cancer of the large intestine using a serial sacrifice technique.

VPS, a hot water extract of the Coriolus versicolor mushroom, was given at a 2% dose level in the diet of female Swiss Webster CFW outbred mice in a serial sacrifice experiment. The mice were also administered either 1,2-dimethylhydrazine dihydrochloride (1,2-DMH) as ten weekly subcutaneous (s.c) injections of 20 microg/g body weight or physiological saline (PS) as ten weekly (s.c) injections of 0.01 ml/g body weight. The animals were sacrificed at 26 weeks or 35 weeks after the first injection of 1,2-DMH or PS. The number of mice with large intestinal tumors and the total number of these tumors were: Group I (1,2-DMH), 29 and 438; Group 2 (VPS + 1,2-DMH), 29 and 344; Group 3 (VPS + PS), 0 and 0; and Group 4 (PS), I and 1, in the mice sacrificed at 26 weeks. The corresponding tumor incidences in mice sacrificed at 35 weeks were: Group 1 (1,2-DMH), 30 and 323; Group 2 (VPS + 1,2-DMH), 29 and 521; Group 3 (VPS + PS), 1 and 2; and Group 4 (PS), 0 and 0. Histopathologically, the tumors were diagnosed as polypoid adenomas and adenocarcinomas of the cecum, colon and rectum. Contrary to expectations, the VPS treatment enhanced the development of large intestinal tumors induced by 1,2-DMH in animals sacrificed at 35 weeks after the first injection of the carcinogen.

A high-fat diet generates alterations in nuclear receptor expression: prevention by vitamin A and links with cyclooxygenase-2 and beta-catenin.

Epidemiologic studies suggest that intake of high energy from fat, inducing overweight, increases the risk of cancer development and promotes colon carcinogenesis. It is therefore important to understand which parameters are affected early on by a high-fat diet in order to devise and improve protective nutritional strategies. We investigated the effect of high energy/fat intake on colon mucosa of male Wistar rats induced by a single 1,2-dimethylhydrazine (DMH) injection. Aberrant crypt foci (ACF) were numbered and modifications in cyclooxygenase-2 (COX-2) and beta-catenin levels assessed. Peroxisome proliferator- and retinoic acid-activated receptors (PPAR and RAR, RXR) are key transcription factors regulating gene expression in response to nutrient-activated signals. A short-term study was designed to evaluate whether alterations in mRNA expression of nuclear receptors can be detected at the beginning of the weight gain phase induced by an appetizing hyperlipidic diet (HLD). HLD consumption induced early downregulation of PPARgamma (-33.1%) and RARbeta (-53.1%) mRNA expression concomitant with an increase in levels of COX-2 (+45.5%) and beta-catenin (+84.56%) and in the number of ACF (191.56 +/- 88.60 vs. 21.14 +/- 11.64, p < 0.05). These findings suggest that HLD increases ACF occurrence, possibly through alterations in the mRNA expression profile of nuclear receptors. Moreover, the use HLD rich in retinyl esters or supplemented with all-trans retinoic acid led to a reduction in the number of ACF. Vitamin A also prevented HLD-induced alterations and the increase in levels of COX-2 and beta-catenin. The present observations show a protective role for vitamin A against disturbances associated with HLD exposure in induced colon carcinogenesis.

Prevention of development of N,N'-dimethylhydrazine-induced colon tumors by a water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi) mycelia in male ICR mice.

The protective effects of a dietary water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi or Mannentake) mycelia (designated as MAK) against development of colon tumors were investigated in male ICR mice. The animals were given weekly injections of N,N'-dimethylhydrazine (DMH, 10 mg/kg body weight) for the initial 10 weeks to induce colon carcinogenesis, and then fed on diet with or without 5% MAK for 10 weeks. There were no significant differences in incidence and the total number of colon tumors between the groups. However, the MAK diet group demonstrated significantly reduced sizes of tumors in comparison with the MF diet group. Moreover, this was linked to a lowered PCNA positive index and shortening of the germinal region in the colon. beta-catenin positive tumor cell nuclei were also significantly decreased in the MAK group. The present results thus indicate that dietary MAK could act as a potent chemopreventive agent for colon carcinogenesis.

Supplementation of Bifidobacterium longum to a high-fat, low-calcium diet lowers cytolytic activity of fecal water in rats injected with 1,2-dimethylhydrazine dihydrochloride.

The effects of supplementing Bifidobacterium longum SBT 2928 and Lactobacillus acidophilus SBT 2062 to a high-fat, low-calcium diet on bile acid concentration, fatty acid concentration, cytolytic activity and intestinal alkaline phosphatase (ALP) activity of fecal water in rats injected with and without 1,2-dimethylhydrazine dihydrochloride (DMH) were examined. Male Wistar rats at 8 weeks of age were fed a diet containing 18% coconut oil, 2% corn oil and 0.1% calcium for 15 d. Lyophilized cultures were supplemented to test diets at a concentration of 1%. The feeding of a high-fat, low-calcium diet elevated the bile acid concentration, cytolytic activity and ALP activity of fecal water as compared to the AIN-76A diet, whereas the fatty acid concentration was not changed. None of the cultures had any effect on these parameters. Furthermore, 8 week-old rats were given a single subcutaneous injection of DMH at 40 mg/kg body weight, and fed the same diets for 15 d. The DMH injection had no effect on the bile acid concentration but increased the fatty acid concentration and cytolytic activity of fecal water. In contrast, ALP activity was lower in the DMH-treated rats than in the non-treated rats. The ingestion of B. longum lowered cytolytic activity but had no effect on the bile acids, fatty acids and ALP activity of fecal water. L. acidophilus had no effect on these parameters.