Repositioning of simvastatin for diabetic colon cancer: role of CDK4 inhibition and apoptosis.

There is increased risk of colon cancer in both men and women having diabetes. The objective of the study was to evaluate the role of simvastatin in colon cancer associated with type 2 diabetes mellitus. Diabetes was induced by administering high fat diet with low dose streptozotocin model. 1,2 dimethylhydrazine (25 mg/kg, sc) was used for colon cancer induction. MTT assay, scratch assay, clonogenic assay and annexin V-FITC assay using flow cytometry were performed on HCT-15 cell line. Simvastatin controlled diabetes and colon cancer in animal models and reduced mRNA expression of CDK4 in colon tissues. In vitro studies revealed that simvastatin showed a decrease in cell viability and produced dose dependent decrease in clone formation. There was decrease in the rate of migration with increase in concentration of simvastatin in scratch assay. Moreover, simvastatin induced apoptosis as depicted from annexin V-FITC assay using flow cytometry as well as that revealed by tunnel assay. Our data suggest that simvastatin exhibits protective role in colon cancer associated with diabetes mellitus and acts possibly via down regulation of CDK4 and induction of apoptosis and hence can be considered for repositioning in diabetic colon cancer.

Selective Role of TNFα and IL10 in Regulation of Barrier Properties of the Colon in DMH-Induced Tumor and Healthy Rats.

Recently it has been reported that the tumor adjacent colon tissues of 1,2-dymethylhydrazine induced (DMH)-rats revealed a high paracellular permeability. We hypothesized that the changes might be induced by cytokines. Colorectal cancer is accompanied by an increase in tumor necrosis factor alpha (TNFα) and interleukin 10 (IL10) that exert opposite regulatory effects on barrier properties of the colon, which is characterized by morphological and functional segmental heterogeneity. The aim of this study was to analyze the level of TNFα and IL10 in the colon segments of DMH-rats and to investigate their effects on barrier properties of the proximal and distal parts of the colon in healthy rats. Enzyme immunoassay analysis showed decreased TNFα in tumors in the distal part of the colon and increased IL10 in proximal tumors and in non-tumor tissues. Four-hour intraluminal exposure of the colon of healthy rats with cytokines showed reduced colon barrier function dependent on the cytokine: TNFα decreased it mainly in the distal part of the colon, whereas IL10 decreased it only in the proximal part. Western blot analysis revealed a more pronounced influence of IL10 on tight junction (TJ) proteins expression by down-regulation of the TJ proteins claudin-1, -2 and -4, and up-regulation of occludin only in the proximal part of the colon. These data may indicate a selective role of the cytokines in regulation of the barrier properties of the colon and a prominent role of IL10 in carcinogenesis in its proximal part.

Probing the chemoprevention potential of the antidepressant fluoxetine combined with epigallocatechin gallate or kaempferol in rats with induced early stage colon carcinogenesis.

The enhanced chemopreventive action against 1,2 Dimethylhydrazine (DMH)-induced preneoplastic lesion in rats could be achieved via simultaneous administration of the antidepressant fluoxetine (FLX) with two natural polyphenolic compounds viz., kaempferol (KMP) and/or epigallocatechin-gallate (EGCG). The obtained results revealed that single FLX pre-treatment possess a significant apoptotic effect by increasing the activity of serum and colon tissue caspase 3. It also attenuated the DMH driven increase in, colon tissue MDA, NO, PCNA and COX-2 expression as well as serum and colon tissue ß-catenin, with a decrease in the multiplicity of ACF and number of MPLs. The combination of FLX with either KMP or EGCG improved the antioxidant, anti-inflammatory and antiproliferating activities but with higher apoptotic activity in case of KMP. Eventually, histopathological assessment of colon tissues exposed that while sole pre-treatment can improve DMH-induced hyperplasia with only moderate inflammatory infiltration, tissues from the combined pre-treatment regimens groups exhibited almost a normal colonic architecture with slight submucosal edema. The study proved that single FLX administration prior to DMH exerts a chemopreventive effect and that the investigated combined pre-treatment regimens demonstrated more potent chemopreventive and antiproliferative actions.

Quercetin ameliorates reactive oxygen species generation, inflammation, mucus depletion, goblet disintegration, and tumor multiplicity in colon cancer: Probable role of adenomatous polyposis coli, ß-catenin.

1,2 Dimethyl hydrazine (DMH), a cogent environmental toxicant, targets the colon. Previous reports suggest that DMH-mediated dysregulation of the Wnt/ß-catenin pathway plays a vital role in the initial events of colon carcinogenesis. Our study was designed to investigate the effect of quercetin on DMH-mediated colon cancer by targeting adenomatous polyposis coli (APC) and ß-catenin in Wistar rats. Animals were pretreated orally with quercetin at doses of either 25 or 50 mg/kg bodyweight (bw) and DMH at a dose of 20 mg/kg bw subcutaneously up to the 15th week and sacrificed after the 30th week. DMH administration leads to reactive oxygen species generation, resulting in an imbalance in redox homeostasis and causing membrane lipid peroxidation, which is also partly due to the decrease in the level of tissue antioxidant machinery. Increased inflammatory and proliferative proteins were observed in DMH-induced colon cancerous rats. DMH treatment also led to dysregulation in the apoptotic pathway with decreased Bax:Bcl-2 ratio. Quercetin pretreatment ameliorates DMH-induced proliferation, activities of detoxifying enzymes, and putative early markers (mucin depletion and goblet cell disintegration) in colonic tissue. It also significantly regulates APC and ß-catenin expression and inhibits tumor incidence and multiplicity. Histological results further confirm the beneficial role of quercetin in averting DMH-induced pathological alterations.

Effects of 1,2-Dimethylhydrazine on Barrier Properties of Rat Large Intestine and IPEC-J2 Cells.

Colon cancer is accompanied by a decrease of epithelial barrier properties, which are determined by tight junction (TJ) proteins between adjacent epithelial cells. The aim of the current study was to analyze the expression of TJ proteins in a rat model of 1,2-dimethylhydrazine (DMH)-induced colorectal cancer, as well as the barrier properties and TJ protein expression of IPEC-J2 cell monolayers after incubation with DMH. Transepithelial electrical resistance and paracellular permeability for sodium fluorescein of IPEC-J2 were examined by an epithelial volt/ohm meter and spectrophotometry. The expression and localization of TJ proteins were analyzed by immunoblotting and immunohistochemistry. In the colonic tumors of rats with DMH-induced carcinogenesis, the expression of claudin-3 and -4 was significantly increased compared to controls. The transepithelial electrical resistance of IPEC-J2 cells increased, while paracellular permeability for sodium fluorescein decreased, accompanied by an increased expression of claudin-4. The increase of claudin-4 in rat colon after chronic DMH exposure was consistent with the acute effect of DMH on IPEC-J2 cells, which may indicate an essential role of this protein in colorectal cancer development.

Protective Role of Vanillic Acid against Diethylnitrosamine- and 1,2-Dimethylhydrazine-Induced Hepatocarcinogenesis in Rats.

This study aimed to evaluate the cancer chemopreventive activity of vanillic acid (VA) in diethylnitrosamine- and 1,2-dimethylhydrazine-induced liver and colon carcinogenesis in rats. VA did not induce the formation of hepatic glutathione S-transferase placental form (GST-P) positive foci and colonic aberrant crypt foci, demonstrating no carcinogenic activity. VA (75 mg kg-1 body weight) could significantly reduce the number and areas of hepatic GST-P positive foci when administered before carcinogen injections, but no such effect was seen when it was administered after carcinogen injection. No protection was seen in the colon when VA was treated before or after carcinogen injection. Immunohistochemical studies demonstrated the decreased expression of proliferating cell nuclear antigen and the induction of apoptosis. Mechanistic studies showed that VA significantly induced the expression of GSTA-5 and Nrf-2 genes, which are associated with the detoxification system. Likewise, the antiproliferative effect was noticed by the reduction of Cyclin D1 expression. The apoptotic activity may be due to the upregulation of Caspase-3 and Bad levels and downregulation of the Bcl-2 level. These data suggest that VA exhibited significant protection against diethylnitrosamine- and 1,2-dimethylhydrazine-induced hepatocarcinogenesis, which might be related to the induction of the detoxifying enzyme, the reduction of proliferation and the induction of apoptosis.

Inhibitory Effect of Thai Purple Rice Husk Extract on Chemically Induced Carcinogenesis in Rats.

This study investigated the cancer chemopreventive effects of an acidic methanol extract of purple rice husk on chemically induced carcinogenesis in rats. This purple rice husk extract (PRHE) had high polyphenol contents. Vanillic acid was a major phenolic compound in PRHE. Three major anthocyanins found in PRHE were malvidin-3-glucoside, peonidin-3-glucoside and cyanidin-3-glucoside. PRHE was not toxic and clastogenic in rats. The LD50 of PRHE was greater than 2000 mg kg-1 body weight (BW). The oral administration of 300 or 1000 mg kg-1 BW of PRHE for 28 days significantly decreased the number of micronucleated hepatocytes in diethylnitrosamine-initiated rats. The inhibitory mechanisms were associated with the reduction of cytochrome P450 2E1 expression and induction of some detoxifying enzymes in the liver. In addition, treatment with 500 mg kg-1 BW of PRHE for eight weeks did not induce preneoplastic lesions in the liver and colon. It significantly inhibited hepatic glutathione-S-transferase positive foci formation induced by diethylnitrosamine and 1,2-dimethylhydrazine by suppression of hepatocyte proliferation and induction of apoptosis. In conclusion, PRHE did not present toxicity, clastogenicity or carcinogenicity in rats. It exhibited cancer chemopreventive properties against chemically induced early stages rat hepatocarcinogenesis. Anthocyanins and vanillic acid might be candidate anticarcinogenic compounds in purple rice husk.

Chronic stress decreases ornithine decarboxylase expression and protects against 1,2-dimethylhydrazine-induced colon carcinogenesis.

Biological response to stress depends on the type, timing, and severity of the stressor. Acute stressful environments may positively activate molecular and cellular mechanisms to favor adaptation; however, chronic stress is often associated with detrimental health effects. Colon cancer (CC) is one of the leading causes of death associated with cancer and has been mentioned as a stress-related disease. In the present work, the effect of chronic stress on the initial phase of CC was evaluated, and special emphasis was placed on ornithine decarboxylase (ODC) expression and polyamines for their role in hyperproliferative diseases. BALB/c mice (n = 5/group) were administered the pro-carcinogen 1,2-dimethylhydrazine (DMH) for 8 weeks (20 mg/kg body weight/week) to induce colon carcinogenesis, and then exposed for 4 weeks to two physical stressors: restraint and forced-swimming. Distal colon inflammatory lesions and histomorphological changes were evaluated by hematoxylin-eosin staining; plasma corticosterone levels, colon ODC expression, and urinary polyamines were determined by competitive ELISA, RT-qPCR, Western Blot, and HPLC, respectively. The short-term exposure to DMH triggered colon inflammation, initiated colon carcinogenesis and increased ODC expression; meanwhile, the exposure to chronic stress activated the hypothalamic-pituitary-adrenal (HPA) axis, elicited the production of plasmatic corticosterone, and decreased ODC expression. The exposure of DMH-treated mice to chronic stress counteracted the inflammatory effect of DMH and maintained ODC homeostasis. In early phase of carcinogenesis, the exposure of DMH-treated mice to chronic stress had a positive effect against colon inflammation and maintained ODC homeostasis. The cross-talk between corticosterone, ODC expression, and inflammation in a tumor environment is discussed.

Gallic acid prevents 1, 2-Dimethylhydrazine induced colon inflammation, toxicity, mucin depletion, and goblet cell disintegration.

1,2-Dimethylhydrazine (DMH), an environmental toxicant specifically targets the colon. The present study was aimed to evaluate the efficacy of gallic acid (GA) against colon toxicity induced by DMH in Wistar rats. GA, a phenolic acid has numerous beneficial properties, which include antiviral, antifungal and antioxidant properties which help cells to overcome oxidative stress and balance the redox homeostasis. GA was administered orally at two doses (25 and 50 mg/kg body weight) once daily for 14 days and a single dose (40 mg/kg body weight) of DMH was administered subcutaneously on 14th day. Animals were sacrificed on the 15th day and we could find that GA at both the doses significantly ameliorates DMH-induced increased toxicity markers and also substantially increases the glutathione content level and activities of detoxifying enzymes. It also ameliorates the expression of proliferation, inflammation, apoptosis, goblet cell disintegration, and mucin depletion in the colon that was elevated upon administration of DMH. Histological alterations provide further confirmation of the protective role of GA against DMH-induced colon toxicity. The results of this study clearly indicate supplementation of GA is beneficial in ameliorating DMH-induced oxidative stress, inflammation, proliferation, apoptosis, mucin depletion, and goblet cell disintegration in colon of Wistar rats.

State of humoral immunity and cytokine profile in rats under experimental carcinogenesis with applying enterosorporation chemotherapeutic factors.

The number of cancer patients is growing in all countries. Due to the malignant progression of cancer, inflammatory processes are observed, which are inextricably linked with the response of the immune system. AIM: The aim of this research was to study the effectiveness of the cytostatic Vincristine on the background of sorption correction of disorders by the carbon sorbent AUT-M (activated carbon tissue material) in adenocarcinoma of the colon. MATERIALS AND METHODS: To simulate carcinogenesis, 1,2-dimethylhydrazine (DMH) was administered subcutaneously to 76 rats for 30 weeks at a dose of 7.2 mg / kg body weight. The evolution of adenocarcinoma of the colon in experimental animals was confirmed by histological examination. After simulation of colon cancer, the animals were intragastrically administered entorosorbent at a dose of 1 ml of suspension (corresponding to 0.2 g of net weight of the drug) per 100 g of body weight of the animal, during 21 days. After detoxification therapy, rats with simulated carcinogenesis were administered the daily cytostatic Vincristine at a dose of 0.23 mg/kg for 14 days. In the above groups of experimental rats, the state of the humoral part of the immune system was studied by the content of serum IgA, IgM, IgG and circulating immune complexes. According to the content of pro- and anti-inflammatory cytokines, inflammatory processes in experimental animals with induced carcinogenesis of the colon on the background of enterosorption and cytostatic corrections were studied. The changes are confirmed by parametric and nonparametric methods of evaluation of the obtained data. RESULTS: It was found that long-term administration of the carcinogen is accompanied by changes in the humoral part of the immune system, as evidenced by the increase in serum IgA, IgM, IgG and CEC level. The results of the research showed that the introduction of 1,2- dimethylhydrazine to animals is accompanied by a change in the cytokine profile, increases the level of pro-inflammatory IL-6 and decreases the level of anti-inflammatory IL-4. CONCLUSIONS: Simultaneous use of enterosorption and cytostatic therapy helped to restore the cytokine balance and indicators of the humoral part of immunological protection.

Augmentation of diethylnitrosamine-induced early stages of rat hepatocarcinogenesis by 1,2-dimethylhydrazine.

Diethylnitrosamine (DEN) and 1,2-dimethylhydrazine (DMH) are classical carcinogens used in experimental rodent carcinogenesis. However, the interaction effects of these carcinogens on biochemical and molecular changes during carcinogenesis have not been investigated. Therefore, the effect of DEN and DMH co-administration on preneoplastic lesion formation and its molecular mechanism in rats were determined. Triple intraperitoneal administrations of DEN were made before, during or after double subcutaneous injections of DMH. At week 8 of the experiment, the preneoplastic hepatic glutathione-S-transferase placental form (GST-P) positive foci and colonic aberrant crypt foci (ACF) were analyzed. The combined treatment of these carcinogens increased toxicity to rats. Administration of DMH alone did not induce hepatic GST-P positive foci, while co-treatment with DMH enhanced hepatic GST-P positive foci formation. However, DEN did not influence the size or number of colonic ACF. The treatment with DMH alone induced CYP2E1 and P450 reductase, demonstrating that DMH enhanced DEN metabolism in DEN- and DMH-treated rats. These findings were related to increases in hepatic O6-methylguanine DNA adducts and hepatotoxicity, which are associated with the induction of cell proliferation and liver cancer development. DEN-induced early stages of rat hepatocarcinogenesis were synergistically promoted by DMH via metabolic enzyme induction leading to enhanced DNA mutation and hepatocarcinogenicity.

Bone Marrow Cell Therapy on 1,2-Dimethylhydrazine (DMH)-Induced Colon Cancer in Rats.

BACKGROUND/AIMS: Stem cell based therapies are being under focus due to their possible role in treatment of various tumors. Bone marrow stem cells believed to have anticancer potential and are preferred for their activities by stimulating the immune system, migration to the site of tumor and ability for inducting apoptosis in cancer cells. The current study was aimed to investigate the tumor suppressive effects of bone marrow cells (BMCs) in 1,2-dimethylhydrazine (DMH)-induced colon cancer in rats. METHODS: The rats were randomly allocated into four groups: control, BMCs alone, DMH alone and BMCs with DMH. BMCs were injected intrarectally while DMH was injected subcutaneously at 20 mg/kg body weight once a week for 15 weeks. Histopathological examination and gene expression of survivin, ß-catenin and multidrug resistance-1 (MDR-1) by real-time reverse transcription-polymerase chain reaction (RT-PCR) in rat colon tissues. This is in addition to oxidative stress markers in colon were performed across all groups. RESULTS: The presence of aberrant crypt foci was reordered once histopathological examination of colon tissue from rats which received DMH alone. Administration of BMCs into rats starting from zero-day of DMH injection improved the histopathological picture which showed a clear improvement in mucosal layer, few inflammatory cells infiltration periglandular and in the lamina propria. Gene expression in rat colon tissue demonstrated that BMCs down-regulated survivin, ß-catenin, MDR-1 and cytokeratin 20 genes expression in colon tissues after colon cancer induction. Amelioration of the colon status after administration of MSCs has been evidenced by a major reduction of lipid peroxidation, nitric oxide, and increasing of glutathione content and superoxide dismutase along with catalase activities. CONCLUSION: Our findings demonstrated that BMCs have tumor suppressive effects in DMH-induced colon cancer as evidenced by down-regulation of survivin, ß-catenin, and MDR-1 genes and enhancing the antioxidant activity.

Capecitabine lipid nanoparticles for anti-colon cancer activity in 1,2-dimethylhydrazine-induced colon cancer: preparation, cytotoxic, pharmacokinetic, and pathological evaluation.

The cornerstone of this investigation is to determine the pharmacokinetic and histopathological behavior of solid lipid nanoparticles of capecitabine (CB-SLNs) in 1,2-dimethylhydrazine (DMH) induced colon cancer. The nanoparticles were prepared by microemulsion method. CB-SLNs were characterized for an optimal system. The cytotoxicity of CB-SLNs was evaluated by using MTT assay method. Further, pharmacokinetic and histopathological behavior of SLNs were studied in DMH induced colon cancer rats. The optimized nanoparticles have the particle size, zeta potential, and entrapment efficiency of 145.6 ± 3.6 nm, -26.9 ± 2.7 mV, and 88.33 ± 3.74%, respectively. Particles of CB were nearly spherical in shape and converted to amorphous form revealed by SEM and DSC, XRD studies. The nanoparticles showed dose-dependent cytotoxicity activity from 10 to 125 µg/mL compared with suspension. Pharmacokinetic studies revealed that 2.7-folds enhancement in the oral bioavailability and in aberrant crypt foci number, apoptotic index comparison with suspension formulation.

Protective effect of metformin in an aberrant crypt foci model induced by 1,2-dimethylhydrazine: Modulation of oxidative stress and inflammatory process.

Colorectal Cancer (CRC) is the third most frequent type of cancer worldwide. In the past few years, studies have revealed a protective effect of metformin (MET-an anti-hyperglycemic drug, used to treat type 2 diabetes), against CRC. The protective effect of MET has been associated with AMPK activation (and mTOR inhibition), resulting in suppressed protein synthesis, and reduced cell proliferation in malignant transformed cells. To elucidate new mechanisms for the protective effect of metformin, we evaluated the oxidative stress and inflammatory process modulation, since these processes are strictly involved in colorectal carcinogenesis. The present study evaluated the protective effect of MET in a CRC model induced by 1,2-dimethylhydrazine (DMH) in Balb/c female mice. The simultaneous/continuous treatment (administration of MET and DMH simultaneously), revealed protective activity of MET, preventing the formation of aberrant crypt foci (ACF) in 71.4% at distal colon sections, and was able to restore basal labeling of apoptosis. Treatment with MET also reduced the inflammatory process induced by DMH, resulting in of the reduction of oxidative stress and nitric oxide related parameters. © 2016 Wiley Periodicals, Inc.

Asiatic acid attenuates pre-neoplastic lesions, oxidative stress, biotransforming enzymes and histopathological alterations in 1,2-dimethylhydrazine-induced experimental rat colon carcinogenesis.

Asiatic acid (AA), a pentacyclic triterpenoid, derived from the tropical medicinal plant Centella asiatica is known to exhibit numerous pharmacological properties. We hypothesized that AA will have chemopreventive potential against 1,2-dimethylhydrazine (DMH)-induced experimental colon carcinogenesis in male Wistar rats. Rats were arbitrarily divided into six groups. Group I rats were processed as control. Group II rats received AA (8 mg/kg b.w., p.o.) and groups III-VI rats received subcutaneous injections of DMH (20 mg/kg b.w.) once a week, for the first four weeks. In addition, groups IV-VI rats received AA at the doses of 2, 4 and 8 mg/kg b.w., respectively, for 16 weeks. Our results discovered that supplementation with AA to the DMH-exposed rats significantly decreased the incidence of polyps and Aberrant crypt foci (ACF) as compared to the DMH-alone-exposed rats. Moreover, in the AA-supplemented DMH-exposed rats, we ascertained increased activities of the antioxidants and decreased levels of lipid peroxidation (LPO) in the liver and circulation and enhanced levels of both LPO and antioxidants in the colon, which were altered in the DMH-alone-exposed rats. Furthermore, we also observed altered activities of vitamins C and E and biotransforming enzymes in DMH-alone-exposed rats, which were reversed on AA supplementation. All the observations were supported by our histological findings. Thus, we can conclude that, AA could be used as an effective chemopreventive agent against DMH-induced colon carcinogenesis.

Thymoquinone and its therapeutic potentials.

Herbal medicine has attracted great attention in the recent years and is increasingly used as alternatives to chemical drugs. Several lines of evidence support the positive impact of medicinal plants in the prevention and cure of a wide range of diseases. Thymoquinone (TQ) is the most abundant constituent of the volatile oil of Nigella sativa seeds and most properties of N sativa are mainly attributed to TQ. A number of pharmacological actions of TQ have been investigated including anti-oxidant, anti-inflammatory, immunomodulatory, anti-histaminic, anti-microbial and anti-tumor effects. It has also gastroprotective, hepatoprotective, nephroprotective and neuroprotective activities. In addition, positive effects of TQ in cardiovascular disorders, diabetes, reproductive disorders and respiratory ailments, as well as in the treatment of bone complications as well as fibrosis have been shown. In addition, a large body of data shows that TQ has very low adverse effects and no serious toxicity. More recently, a great deal of attention has been given to this dietary phytochemical with an increasing interest to investigate it in pre-clinical and clinical researches for assessing its health benefits. Here we report on and analyze numerous properties of the active ingredient of N. sativa seeds, TQ, in the context of its therapeutic potentials for a wide range of illnesses. We also summarize the drug's possible mechanisms of action. The evidence reported sugests that TQ should be developed as a novel drug in clinical trials.

Wild-type K-ras has a tumour suppressor effect on carcinogen-induced murine colorectal adenoma formation.

K-ras mutations are found in ~40% of human colorectal adenomas and carcinomas and contribute to colorectal tumour formation at an early stage. Wild-type K-ras has been reported to be deleted in some tumours, but the consequences of changes in wild-type K-ras copy number for experimental colorectal carcinogenesis have not been investigated. To characterize the effects of K-ras copy number changes on formation of carcinogen-induced colorectal neoplasms in mice, wild-type (K-ras(+/+) ) and heterozygous K-ras exon 1 knockout (K-ras(+/-) ) mice were given 10 weekly treatments of 1, 2-dimethylhydrazine (DMH) to induce colorectal tumours. Colorectal expression levels of K-ras 4A and 4B transcripts in K-ras(+/-) mice were ~50% decreased compared with K-ras(+/+) mice. One year after DMH treatment, survival of K-ras(+/-) mice decreased from 88 to 82% compared with wild-type mice. Colorectal adenomas significantly increased from 0.52 ± 0.15 in K-ras(+/+) mice to 0.87 ± 0.14 in K-ras(+/-) mice (mean ± SEM per mouse, P < 0.01); total tumour volume increased 2.13-fold (P < 0.05). Comparing K-ras(+/+) with K-ras(+/-) murine adenomas, Ki-67-positive proliferating tumour cells significantly increased from 7.77 ± 0.64% to 9.15 ± 0.92% and cleaved caspase-3-positive apoptotic tumour cells decreased from 1.40 ± 0.37% to 0.80 ± 0.22% (mean ± SEM, P < 0.05 for both). No K-ras or B-raf mutations were detected in the adenomas. Immunohistochemical studies showed no significant changes in extracellular signal regulating kinase/mitogen-activated protein kinase (Erk/MapK) or PI3K/Akt pathway activation in the adenomas. In conclusion, the data collectively show that a 50% reduction in K-ras gene dosage and RNA expression promoted experimental colorectal tumourigenesis, consistent with wild-type K-ras having a tumour suppressor effect on carcinogen-induced murine colorectal adenoma formation.

Modulatory effect of troxerutin on biotransforming enzymes and preneoplasic lesions induced by 1,2-dimethylhydrazine in rat colon carcinogenesis.

Colon cancer is the third most global oncologic problem faced by medical fraternity. Troxerutin, a flavonoid present in tea, coffee, cereal grains, and a variety of fruits and vegetables, exhibits various pharmacological and biological activities. This study was carried out to investigate the effect of troxerutin on xenobiotic metabolizing enzymes, colonic bacterial enzymes and the development of aberrant crypt foci (ACF) during 1,2-dimethylhydrazine (DMH) induced experimental rat colon carcinogenesis. Male albino Wistar rats were randomly divided into six groups. Group 1 served as control. Group 2 received troxerutin (50 mg/kg b.w., p.o. every day) for 16 weeks. Groups 3-6 received subcutaneous injections of DMH (20 mg/kg b.w.) once a week, for the first four weeks. In addition, groups 4-6 received different doses of troxerutin (12.5, 25, 50 mg/kg b.w., p.o. every day respectively) along with DMH injections. Our results reveal that DMH treated rats exhibited elevated activities of phase I enzymes such as cytochrome P450, cytochrome b5, cytochrome P4502E1, NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase and reduced activities of phase II enzymes such as glutathione-S-transferase (GST), DT-diaphorase (DTD) and uridine diphospho glucuronyl transferase (UDPGT) in the liver and colonic mucosa of control and experimental rats. Furthermore, the activities of fecal and colonic mucosal bacterial enzymes, such as ß-glucronidase, ß-glucosidase, ß-galactosidase and mucinase were found to be significantly higher in DMH alone treated rats than those of the control rats. On supplementation with troxerutin to DMH treated rats, the alterations in the activities of the biotransforming enzymes, bacterial enzymes and the pathological changes were significantly reversed, the effect being more pronounced when troxerutin was supplemented at the dose of 25 mg/kg b.w. Thus troxerutin could be considered as a good chemopreventive agent against the formation of preneoplastic lesions in a rat model of colon carcinogenesis.

Oral Administration of Carotenoid-Rich Dunaliella salina Powder Inhibits Colon Carcinogenesis via Modulation of Wnt/ß-catenin Signaling Cascades in a Rat Model.

The present study aims to investigate the oral therapeutic and molecular role of carotenoid-rich Dunaliella salina powder (DSP) against 1,2-dimethylhydrazine (DMH)-triggered colon carcinogenesis. In this study, thirty six male Wistar rats were categorized into six distinct groups (G1-G6): G1 group with no intervention, G2 group received only DSP (1000 mg/kg), G3 group received only DMH carcinogen (20 mg/kg), and G4-G6 group received both DMH and DSP at various phases (pre-initiation, post-initiation and entire phases) for 32 weeks. Body weight, tumor incidence, tumor volume, histopathological examination, antioxidants, and detoxification enzymes activities were analyzed in the experimental rats. In addition, the protein expression profile of components involved in the Wnt/ß-catenin signaling pathway was determined by western blot analysis. Matrix metalloproteinases (MMP-7 and MMP-9), proliferation marker (PCNA), and pro-apoptotic (Bcl-2 and Bax) proteins were analyzed using immunohistochemistry. Colorimetric assay was used to determine the levels of anti-inflammatory (iNOS and COX-2) and apoptotic proteins (Caspase-3 and Caspase-9). Results showed that concomitant administration of DSP with DMH significantly reduced tumor progression and prevented colon carcinogenesis in rats. However, treatment with DSP before or after DMH exposure did not significantly prevent colon carcinogenesis. DMH and DSP treatment group showed increased activities of antioxidant enzymes with significant reduction in the oxidative stress. Additionally, the detoxification enzymes and colonic histopathology of those rats were restored to that of control rats. The administration of DSP to rats exposed to DMH exhibited antitumor effects via inhibition of the Wnt/ß-catenin signaling pathway with induced apoptosis through the Bcl-2/Bax/caspases signaling cascades. Moreover, the same group also showed significant anti-inflammatory activity via regulating iNOS and COX-2 biomarkers. Our findings revealed molecular chemopreventive activity of carotenoid-rich DSP through regulating Wnt/beta-catenin and intrinsic apoptotic pathways. Thus, DSP is propound to function as a potent antioxidant, anti-proliferative, and anti-inflammatory therapeutic agent against colon carcinogenesis.

Repair and removal of azoxymethane-induced O6-methylguanine in rat colon by O6-methylguanine DNA methyltransferase and apoptosis.

Azoxymethane (AOM) is an alkylating agent that generates mutagenic and carcinogenic O(6)-methylguanine (O(6)meG) adducts in DNA. O(6)meG has been detected in human colonic DNA; hence, understanding the innate cellular events occurring in response to the formation of O(6)meG is important in developing preventive strategies for colorectal cancer. We explored the time-course, dose-response, and kinetics of O(6)meG formation and its removal by the DNA repair protein, O(6)-methylguanine DNA methyltransferase (MGMT), and apoptosis. In rats given AOM (10 mg/kg), the formation of O(6)meG occurs within 2 h of exposure, accompanied by rapid depletion of MGMT activity and followed by the induction of an acute apoptotic response that peaks at 6-8 h. MGMT repair and apoptosis are dependent on AOM dose and O(6)meG load. Apoptosis is initiated only when a high O(6)meG load is present and MGMT activity is fully depleted. AOM, 10 mg/kg, overwhelms MGMT repair for about 96 h and renewed MGMT activity is only observed once O(6)meG is no longer detectable. A threshold for apoptosis is observed at 6 h after 6 mg/kg AOM, when a high O(6)meG persists and MGMT activity is very low. These data suggest that apoptosis is probably triggered by O(6)meG, but only once the capacity of MGMT to repair O(6)meG is exhausted. In the colonic epithelium, apoptosis may be complementary to MGMT, in terms of minimising potentially mutagenic events and maintaining a healthy genome.