AOM-induced Mouse Research Articles

Bekatul Beras Hitam Terfermentasi Memperbaiki Kondisi Kolon Mencit yang Diinduksi Senyawa Karsinogen Azoxymethane

Worldwide, colon cancer has become the fourth cause of death in terms of cancer. A high fiber and antioxidants diet help promote a healthy diet and prevent colon cancer. Black rice bran has high content both in fiber and phenolic. The aim of this research was to observe the potential of fermented black rice bran in improving colon conditions profiles of azoxymethane (AOM)-induced mice, comprising lactic acid bacteria (LAB) colony count, short-chain fatty acids (SCFAs) concentration, and malondialdehyde (MDA). Rhizopus oligosporus was used for fermenting the black rice bran. Five weeks old male Balb/c mice were divided into four groups (n=6) based on their diets. AOM was injected intraperitoneally and Dextran Sodium Sulphate was added to their drinking water, thus initiating inflammation in the colon. The number of LAB in faeces at the end of intervention in the groups of fermented rice bran group (FRB) (9.04±0.04 log CFU) and non-fermented rice bran group (NRB) (8.99±0.04 log CFU) were higher than that of the positive control group (8.33±0.06 log CFU/g) but fewer as compared to the negative control (9.63±0.05 log CFU). The concentrations of SCFAs (acetic acid, propionic acid and butyric acid) of the cecum content in the NRB group (11.92±0.00, 2.31±0.01 and 3.41±0.01 mM) were higher in the positive control group (8.90±1.30, 1.60±0.16 and 2.27±0.31 mM). As compared to the positive control group, the concentration of propionic acid of cecum content in the FRB group was higher (2.09±0.32 mM), but the concentrations of acetic acid and butyric acid were indifferent. The MDA level of the FRB group (1.41±0.03 µmol) was lower thanthat of the NRB group (1.88±0.05 µmol), and the MDA level of NRB was lower than that of the positive control group (2.03±0.09 µmol). The results showed that both FRB and NRB had a positive role in increasing the number of LAB, ALRP concentrations, and decreasing MDA levels in AOM-induced mice colons.

Polysaccharide of L. casei SB27 reduced colon cancer cell prognosis through mitochondrial damage by up-regulation of HINT2

Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system worldwide. This study aimed to explore the effects of Polysaccharide of L. casei SB27 in colon cancer and its possible mechanisms.Colon cancer was induced by giving dextran sulfate sodium and Azoxymethane. HCT-116 cells were used in to in vitro model in this experiment.Polysaccharide of L. casei SB27 reduced colon cancer in DSS + AOM-induced mice. Polysaccharide of L. casei SB27 reduced colon cancer prognosis in vitro model. Polysaccharide of L. casei SB27 reduced Short-chain fatty acids by Bacillus coli. Polysaccharide of L. casei SB27 promoted A mitochondrial damage by Calcium ion entry. Polysaccharide of L. casei SB27 induced HINT2/MCU signaling pathway. IP showed that HINT2 protein interlinked MCU protein. Polysaccharide of L. casei SB27 suppressed HINT2 Ubiquitination. The regulation of HINT2 affected the effects of L. casei SB27 Polysaccharide on colon cancer Prognosis and A mitochondrial damage by Calcium ion entry in vitro model.In conclusion, the present report demonstrated that Polysaccharide of L. casei SB27 reduced colon cancer cell Prognosis through A mitochondrial damage by up-regulation of HINT2. Polysaccharide of L. casei SB27 might be used for colon cancer treatment and could be helpful for personalized treatment.

Effect of Angiogenesis and Lymphangiogenesis in Diesel Exhaust Particles Inhalation in Mouse Model of LPS Induced Acute Otitis Media.

Lymphangiogenesis and angiogenesis might have significant involvement in the pathogenesis of otitis media with effusion. This study investigated the effect of diesel exhaust particles (DEP) on inflammation and lymphangiogenesis in a mouse model of acute otitis media (AOM). BALB/c mice were injected with LPS and exposed to 100 µg/m3 DEP. The mice were divided into four groups: control (no stimulation), AOM, AOM + DEP, and DEP + AOM.The effects of DEP inhalation pre- and post-DEP induction were estimated based on measurements of the auditory brainstem response, mRNA levels of lymphangiogenesis-related genes and cytokines, and histology of the middle ear. Cell viability of human middle ear epithelial cells decreased in a dose-response manner at 24 and 48 hours post-DEP exposure. DEP alone did not induce AOM. AOM-induced mice with pre- or post-DEP exposure showed thickened middle ear mucosa and increased expression of TNF-α and IL1-β mRNA levels compared to the control group, but increased serum IL-1β levels were not found in the AOM + Post DEP. The mRNA expression of TLR4, VEGFA, VEGFAC, and VEGFR3 was increased by pre-AOM DEP exposure.The expression of VEFGA protein was stronger in the AOM + Post DEP group than in any other group. The expression of CD31 and CD45 markers in the mouse middle ear tissue was higher in the Pre DEP + AOM group than in the AOM group. This result implies that pre-exposure to DEP more strongly increases inflammation and lymphangiogenesis in a mouse model of acute otitis media.

Abstracts

Abstracts

Abstract 1926: Selective induction of apoptosis by aqueous extract of Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusain HCT 116 colon cancer cells and CCD 841 CoN colon epithelial cells

Abstract Scutellaria barbata (SB) and Oldenlandia diffusa (OD) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors. These two herbs are included in most of the herbal cancer treatment formulas in Taiwan hospitals. We previously showed that aqueous extracts of SB and OD inhibited mutagenesis, DNA binding and metabolism of aflatoxin B1 and benzo(a)pyrene. They were also shown to inhibit foci formation in the colon of AOM-induced mice. Other researchers demonstrated their effects in regulation of various apoptotic enzymes and pathways. Ethanol extract of SB was shown to inhibit colorectal cancer growth by suppression of the Wnt/β-catenin signaling pathway while ethanol extract of OD was indicated in the inhibition of proliferation and induction of apoptosis of 5-FU resistant colorectal cancer cells via the regulation of PI3K/AKT signaling pathway. In this study, the effects of aqueous extracts of SB and OD on the induction and modulation of apoptosis in HCT 116 colon cancer cells and CCD 841 CoN normal color epithelial cells were assessed using TUNEL assay, Apoptosis/Necrosis Detection Kit, and Human Apoptosis Antibody Array - Membrane (43 Targets) test (Abcam). Our data demonstrated that both the 3-hours and 6 hours of 1.5 mg/mL SB treatments induced apoptosis in HCT 116 colon cancer cells significantly (77.5 ± 1.5%, 87.5± 3.5%) as compared to the negative control (13.5 ± 0.5%, 17.0 ± 6.0%), p < 0.05. The percent of apoptosis was not significantly different as compared to that of the positive control induced by 1µMol Staurosporine (99.5 ± 0.5%, 91.9 ± 9.0%); while there was no notable induction of apoptosis in normal CCD 841 CoN colon epithelial cells (18.5 ± 1.5%, 14.4 ± 4.0% separately). The majority of the normal cells remained non-induced (71.0 ± 1.0%, 68.5 ± 6.5%). A similar selective induction of apoptosis, in the two cell lines separately, was obtained from the 3-hours and 6 hours of 1.5 mg/mL OD treatments (HCT116 - 44.0 ± 4.0%, 83.5± 15.5%; CCD841 CoN - 6.5 ± 5.5%, 7.0 ± 2.0% respectively). Modulation of various apoptosis markers production such as up-regulation of Bax, BID, Bad, p27, p53, Caspases 8 and 3; and down-regulation of Bcl-2 and Bcl-w was also observed. These results suggested that SB and OD contain phytochemicals which selectively induce apoptosis in HCT 116 cancer cells by modulating these markers while not significantly affecting the normal CCD 841 CoN colon epithelial cells. Further study of their specific modulation effects and phytochemicals on apoptosis is warranted to reveal their potential chemopreventive and therapeutic properties against colon cancer and other cancers. Citation Format: Camille Kordas, Joshua Li, Kristin Ferrer, Yi Shan Jin, Nathaly Manrique, Min Seo Kang, Joon Seo, Sung Been Han, Jasmine Cha, Anthony Miller, Padma Tadi Uppala, Brian Yuen Wong. Selective induction of apoptosis by aqueous extract of Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusain HCT 116 colon cancer cells and CCD 841 CoN colon epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1926.

Abstract A46: Dietary weight loss and sulindac treatment each reverse obesity-associated inflammation and tumorigenesis in a mouse model of carcinogen-induced colon cancer

Abstract Purpose: We previously showed diet-induced obesity (DIO) increases (and calorie restriction decreases) systemic markers of inflammation and azoxymethane (AOM)-induced colon carcinogenesis in FVB/N mice. Our current study examines whether moderate weight loss via low-fat diet (LFD) and/or treatment with the nonsteroidal anti-inflammatory drug sulindac reverses the inflammatory and tumor-promoting effects of DIO in the AOM-induced mouse model of colon cancer. Methods: Male FVB/N mice were treated with AOM (10 mg/kg i.p.) for 5 weeks, then randomized to a control (10% kcal from fat) or DIO (60% kcal from fat) diet. After 15 weeks on diet, 5 mice/group were killed and their tissues collected for interim timepoint analysis. The remaining DIO mice were further randomized to either remain on DIO or switch to control diet to induce weight loss (formerly obese [FOb] mice), and within each diet group (control, DIO, and FOb), half the mice were randomized to receive sulindac treatment (140 ppm in the diet). Eight weeks later, all mice were killed and their serum, tumors, and other tissues collected for analysis. Results: The DIO mice, compared to controls, had significantly greater body weight and body fat (P<0.05), while FOb mice had intermediate levels of weight and adiposity. Sulindac did not affect adiposity in any diet group. At the interim timepoint, DIO mice had ~10-fold greater tumor multiplicity relative to control mice (p<0.01) and Fob mice (p<0.01). The control and FOb groups did not significantly differ from each other. At study termination sulindac significantly reduced tumor multiplicity in both control (P<0.05) and DIO (P<0.0001) mice, but not FOb mice. In the mice that received sulindac, tumor multiplicity did not significantly differ between diet groups. Tumor multiplicity in the DIO+sulindac, FOb, and FOb+sulindac groups was also significantly lower than the interim timepoint DIO mice (P<0.05), suggesting the interventions induced tumor regression. Serum levels of several inflammation-related proteins, including interleukin 6, chemokine (C-X-C motif) ligand 1, monocyte chemoattractant protein 1, and vascular endothelial growth factor, were elevated in DIO mice versus controls (P<0.05), but did not differ between FOb and control mice. Sulindac treatment significantly reduced serum levels of these proteins in DIO mice (P<0.01). Gene expression in paired colon tumor and mesenteric adipose tissue samples is currently being assessed by microarray analysis to investigate crosstalk between these tissues and inform the design of future studies on the mechanism(s) mediating sulindac’s effects. Conclusions: Both moderate weight loss and sulindac treatment reverse the effects of chronic obesity on AOM-induced colon tumorigenesis. The anticancer effects of sulindac were independent of any change in adiposity. Given the challenges of losing weight, our findings suggest that further investigation of the effects of NSAID treatment on colon cancer risk and/or progression in obese patients is warranted. Citation Format: Stephen D. Hursting, Laura W. Bowers, Arunima Punjala, Andrew J. Dannenberg. Dietary weight loss and sulindac treatment each reverse obesity-associated inflammation and tumorigenesis in a mouse model of carcinogen-induced colon cancer [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A46.

Effects of Lactobacillus acidophilus and Bifidobacterium bifidum probiotics on the serum biochemical parameters, and the vitamin D and leptin receptor genes on mice colon cancer.

Objective(s):The preclinical reports have shown that specific probiotics like Bifidobacterium bifidum (B. bifidum) and Lactobacillus acidophilus (L. acidophilus) can be applied as the biotherapeutic agents in the inhibition or therapy of colorectal cancer via the modification of gut bacteria. In the previous studies, we have assessed the impact of L. acidophilus and B. bifidum probiotics on gut bacteria concentration and also their chemo-protective impact on mice colon cancer. In the following, we assessed the effects of these probiotics on the gene expression of vitamin D receptor (VDR) and the leptin receptor (LPR) and the serum biochemical parameters on mice colon cancer. Materials and Methods:Thirty-six male BALB/c mice were equally shared into 4 groups; (i) health with routine dietary foods without any treatment, (ii) azoxymethane (AOM)-induced mice colon cancer with common dietary foods, (iii) and (iv) AOM-induced mice colon cancer with oral consumption of L. acidophilus and B. bifidum (1×109 cfu/g) for 5 months, respectively. Then, the serum total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), alanine transaminase, alkaline phosphatase, and albumin and also VDR and LPR genes expression were evaluated. Results:Oral consumption of L. acidophilus and B. bifidum probiotics significantly decreased the triglycerides, alkaline phosphatase, LDL, and also the VDR and LPR gene expression in mice colon cancer (P<0.005).Conclusion: L. acidophilus and B. bifidum probiotics with the modification of the biochemical parameters and the expression of the VDR and LPR genes can play a key role in the protection of mouse colon cancer.

Probiotic Pediococcus pentosaceus strain GS4 alleviates azoxymethane-induced toxicity in mice

Probiotic treatment has been gaining attention due to its remarkable effects in alleviating toxicity and carcinogenesis. The novel strain Pediococcus pentosaceus GS4 has been reported for probiotic, survivability in simulated gastrointestinal fluid, and antioxidative and biohydrogenation properties. Therefore, we hypothesize that this specific strain might be able to assuage the effect of azoxymethane (AOM)-induced toxicity in mice. Twenty-eight Swiss albino mice were divided into 4 groups and were studied for 32 weeks. Azoxymethane (10 mg/kg body weight) was administered intraperitoneally twice (0th and 14th days), and probiotic GS4 (1.1 × 109 colony-forming unit/mL) was given orally for the respective groups. Mice who served as the normal control received only normal saline. GS4-intervened AOM-induced mice showed marked improvement at the histopathologic level, in the liver and kidney. Moreover, probiotic GS4 intervention in AOM-induced mice exhibited a significant reduction in the liver function biomarker when compared with the AOM-induced mice. Probiotic GS4 intervention reduced the intestinal structural deformities as evident from the elevated brush border membrane–associated disaccharidases (sucrase, lactase) and intestinal alkaline phosphatase activities, which were found disrupted by AOM intoxication. Fecal bacterial load was found to be reduced in AOM-induced mice which were subsequently replenished by the probiotic GS4 intervention as apparent from the enhanced fecal bacterial load. There were no adverse effects observed in the probiotic control group. Conclusively, novel probiotic strain GS4 exhibited safe and beneficial effects against the toxicity threats posed by AOM. Thus, GS4 could be considered as a potential food supplement/additive for therapeutic purposes in gastrointestinal disorders related to inflammation and cancer.

Curcumin ameliorates the tumor-enhancing effects of a high-protein diet in an azoxymethane-induced mouse model of colon carcinogenesis

An increasing number of reports suggest that a high-protein diet (HPD) is associated with an increased risk for colorectal cancer (CRC). One of the proposed mechanisms is that an HPD increases the delivery of protein to the colon and generates various toxic metabolites that contribute to colon carcinogenesis. Curcumin was shown to exert significant preventive properties against CRC. We therefore hypothesized that curcumin can reverse the tumor-enhancing effects of an HPD. This study examined the effects of curcumin on the development of azoxymethane (AOM)-induced colorectal tumors in HPD-fed mice. A total of 30 female Balb/c mice were randomly divided into 3 groups: those fed a normal diet (20% casein), those fed an HPD (HPD; 50% casein), and those fed an HPD supplemented with curcumin (HPDC; 0.02% curcumin). The mice were subjected to an AOM–dextran sodium sulfate colon carcinogenesis protocol. Mice in the HPDC group exhibited a significant (40%) reduction in colorectal tumor multiplicity when compared with those in the HPD group. The expression of colonic inflammatory proteins (cyclooxygenase-2 and inducible nitric oxide synthase), the levels of plasma inflammatory markers (nitric oxide and tumor necrosis factor–α), fecal ammonia, short- and branched-chain fatty acid levels, and the rate of colonocyte proliferation were significantly lower in the HPDC than the HPD group. In conclusion, curcumin inhibited the development of colorectal tumors in an AOM-induced mouse model of colon carcinogenesis by attenuating colonic inflammation, proliferation, and toxic metabolite production. Curcumin might be useful in the chemoprevention of CRC in individuals consuming an HPD.

Protective Effects of Turbinaria ornata and Padina pavonia against Azoxymethane-Induced Colon Carcinogenesis through Modulation of PPAR Gamma, NF-κB and Oxidative Stress.

The aim of this study was to investigate the antiproliferative and protective effects of the brown seaweeds, Turbinaria ornata and Padina pavonia, against azoxymethane (AOM)-induced colon carcinogenesis in mice. Both algal extracts showed anti-proliferative effects on the human carcinoma cell line HCT-116 in vitro, with T. ornata demonstrating a more potent effect. Male albino Swiss mice received intraperitoneal injections of AOM (10 mg/kg) once a week for two consecutive weeks and 100 mg/kg of either T. ornata or P. pavonia extracts. AOM-induced mice exhibited alterations in the histological structure of the colon, elevated lipid peroxidation and nitric oxide, declined glutathione content and reduced activity of superoxide dismutase and glutathione peroxidase. In addition, AOM induced downregulation of peroxisome proliferator activated receptor gamma (PPARγ) and p53 mRNA expression, with concomitant upregulation of nuclear factor-kappa B (NF-κB) in colon tissue. Administration of either algal extract markedly alleviated the recorded alterations. In conclusion, the current study suggests that T. ornata and P. pavonia, through their antioxidant and anti-inflammatory effects, are able to attenuate colon inflammation by downregulating NF-κB expression. Furthermore, the protective effects of both algae against AOM-initiated carcinogenesis were attributed, at least in part, to their ability to upregulate colonic PPARγ and p53 expression.

Luteolin inhibits matrix metalloproteinase 9 and 2 in azoxymethane-induced colon carcinogenesis

The present investigation deals with the antimetastatic role of luteolin (LUT) by inhibiting matrix metalloproteinase (MMP)-9 and -2 in azoxymethane (AOM)-induced colon carcinogenesis in Balb/C mice. Animals received AOM at a dosage of 15 mg/kg body weight intraperitoneally once a week for 3 weeks. AOM-induced mice was treated with LUT (1.2 mg of LUT/kg body weight/day orally). After the experimental period, the tumor markers such as γ-glutamyl transferase (GGT), 5' nucleotidase (5'ND), cathepsin-D (Cat-D), and carcinoembroyonic antigen (CEA) were elevated upon induction with AOM. Subsequent treatment with LUT results in the reduction of the tumor markers was recorded. The expressions of MMP-9 and MMP-2 were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence methods. The expressions of MMP-9 and MMP-2 were increased during AOM induction and upon treatment with LUT reduced the expressions. RT-PCR analysis of tissue inhibitor of matrix metalloproteinase (TIMP)-2 was limited during AOM-induced colorectal cancer (CRC). Supplementation of LUT increased the expression of TIMP-2. To conclude, LUT acts as an antimetastatic agent by suppressing MMP-9 and MMP-2 productions and upregulating TIMP-2 expression, thereby suggesting that LUT can be a chemotherapeutic agent against CRC.

Inflammation-Associated Serum and Colon Markers as Indicators of Dietary Attenuation of Colon Carcinogenesis in ob/ob Mice

Although inflammatory cytokines and obesity-associated serum proteins have been reported as biomarkers of colorectal adenoma risk in humans, little is known of biomarkers of response to interventions that attenuate tumorigenesis. Dietary navy beans and their fractions attenuate colon carcinogenesis in carcinogen-induced genetically obese mice. We hypothesized that this attenuation would be associated with changes in inflammatory cytokines and obesity-related serum proteins that may serve as measures of efficacy. ob/ob mice (n = 160) were injected with the carcinogen azoxymethane (AOM) to induce colon cancer and randomly placed on one of four diets (control, whole navy bean, bean residue fraction, or bean extract fraction) for 26 to 28 wk. Serum was analyzed for 14 inflammation- or obesity-related proteins, and colon RNA was analyzed for expression of 84 inflammation-associated genes. Six of 14 serum proteins were increased [i.e., interleukin (IL)-4, IL-5, IL-6, IL-10, IFN gamma, granulocyte macrophage colony-stimulating factor] in hyperplastic/dysplastic stages of colon carcinogenesis. Bean-fed mice had significantly higher monocyte chemoattractant protein-1 and lower IL-6 levels in serum. In colon mucosa, 55 of 84 inflammation-associated genes differed between AOM-induced and noninduced mice. Of the 55 AOM-induced genes, 5 were counteracted by bean diets, including IL-6 whose increase in expression levels was attenuated by bean diets in AOM-induced mice. In summary, IL-6 emerged as a serum protein that was increased in hyperplastic/dysplastic stages of colon carcinogenesis, but attenuated with bean-based diet in serum and colon mucosa. Changes in a subset of inflammation-associated serum proteins and colon gene expression may serve as response indicators of dietary attenuation of colon carcinogenesis.

Protective role of luteolin on the status of lipid peroxidation and antioxidant defense against azoxymethane-induced experimental colon carcinogenesis

The modifying effect of dietary exposure to a flavonoid, luteolin (LUT) during the azoxymethane (AOM)-induced colon carcinogenesis was investigated in this study. Aberrant crypt foci (ACF), lipid peroxidation (LPO), enzymic and non-enzymic antioxidants and histopathological analysis were performed. Colon carcinogenesis was induced by injecting 15 mg/body kg weight of AOM, intraperitoneally (i.p.), once in a week for 3 weeks in male Balb/c mice. AOM-induced mice were treated with LUT (1.2 mg of LUT/kg body weight/day orally). After the experimental period, frequency of ACF, levels of thiobarbutaric acid reactive substances (TBARS) and hydroxy radical (OH˙) were found to be increased, whereas glutathione (GSH), Vitamins C, E and A were decreased in the plasma and colon of AOM-induced mice. However, LUT treatment to AOM-induced mice significantly decreased the incidence of ACF, levels of TBARS and OH˙ with a concordant increase in non-enzymic antioxidants in plasma and colon tissue. The activities of the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were found to be decreased due to the induction of colon cancer in mouse. LUT treatment ameliorated the activities of these antioxidant enzymes. The histological study revealed a significant increase in the enlarged nuclei and hyperchromatism of cells in AOM-induced mice whereas LUT significantly reduced the signs in the colon. The immunohistochemical expression of MDA-DNA adduct was studied. In AOM-induced group, the expression was increased and treatment with LUT decreased significantly. The present study depicts that LUT can act as an effective chemopreventive agent against colon cancer.

Inverse association between phospholipase A2 and COX-2 expression during mouse colon tumorigenesis.

Cytosolic phospholipase A(2) (cPLA(2)) releases arachidonic acid (AA) from intracellular phospholipids. We evaluated the status of cPLA(2) in azoxymethane (AOM)-induced mouse colon tumors. Despite increased expression of cyclooxygenase 2 (3.7-fold) and PGE(2) (3.4-fold) production in tumors, cPLA(2) mRNA levels and enzyme activity were significantly reduced (3.6- and 3-fold, respectively). Reduced levels of cPLA(2) were also observed in pre-neoplastic aberrant crypt foci (ACF), a distinct morphological alteration that represents an early stage in the pathogenesis of colon tumors. Furthermore, the reciprocal expression patterns of these two genes were found to occur in human colorectal cancers (CRC). Examination of the activity of the secretory phospholipases A(2) (sPLA(2)) and expression of the groups V and X sPLA(2)s revealed no compensatory increase in tumor tissue. As cPLA(2) has been shown to be involved in TNF-alpha-induced apoptosis in certain cell types, and TNF-alpha expression is significantly enhanced in AOM-induced tumors (2.8-fold), we examined the role of cPLA(2) in TNF-alpha-induced apoptosis of cultured mouse colonocytes (YAMC). The specific cPLA(2) inhibitor, AACOCF(3) (arachidonoyl trifluoromethyl ketone), was able to protect colonocytes from TNF-alpha-induced apoptosis in vitro. In summary, our data demonstrate an inverse relationship between COX-2 and cPLA(2) expression in both AOM-induced mouse colon tumors and human CRC and suggest that down regulation of cPLA(2) may attenuate TNF-alpha mediated apoptosis during tumorigenesis and facilitate tumor progression.

Frequent mutations of the β- catenin gene in mouse colon tumors induced by azoxymethane

The beta-catenin gene is frequently mutated at codons 33, 41 and 45 of the glycogen synthase kinase-3beta phosphorylation motif in human colon cancers in patients without APC mutations. Frequent mutations at codons 32 and 34, as well as 33 and 41, have been detected in rat colon tumors induced by azoxymethane (AOM), with the second G of CTGGA sequences being considered as a mutational hot-spot. In the present study, exon 3 of the beta-catenin gene in mouse colon tumors induced by AOM was amplified by PCR and mutations were detected by the single strand conformation polymorphism method, restriction enzyme fragment length polymorphism and direct sequencing. All 10 colon tumors tested were found to have beta-catenin mutations, four in codon 34, three in codon 33, two in codon 41 and one in codon 37, nine being G:C-->A:T transitions. However, no mutations were found in codon 32 of the mouse beta-catenin gene. On immmunostaining, beta-catenin was observed in the cytoplasm and nucleus of the tumor cells. The cytoplasmic staining was homogeneous, while both homogeneous and heterogeneous patterns were noted for the nuclei. Highly frequent mutations of the beta-catenin gene in AOM-induced mouse colon tumors suggest that consequent alterations in the stability and localization of the protein may play an important role in this colon carcinogenesis model.

Differential expression of p16(INK4a) in azoxymethane-induced mouse colon tumorigenesis.

Alterations in the p16(INK4a) gene have been implicated in the pathogenesis of different human cancers and animal tumors. We postulated that alterations in the p16(INK4a) gene may also be involved in mouse colon tumorigenesis induced by the chemical carcinogen azoxymethane (AOM). In the present study, p16(INK4a) deletion status and its expression were examined in an AOM-induced mouse colon tumor model. Polymerase chain reaction-based deletion analysis of p16(INK4a) exon 2 showed no deletions in the colon tumors. The expression and localization of p16(INK4a) and its gene product were examined by reverse transcription-polymerase chain reaction and immunohistochemical analyses, respectively. The p16(INK4a) mRNA levels were low, and in some cases undetectable, in control colon tissue. However, colon tumors exhibited an eightfold increase in p16(INK4a) mRNA level when compared with control colon tissue (P < 0.01). Whereas control colon epithelium was uniformly negative for p16(INK4a) immunoreactivity, p16(INK4a)-immunoreactive cells were markedly increased in preneoplastic lesions and adenomas isolated from AOM-treated mice. To further examine the p16(INK4a) regulatory pathway, the retinoblastoma tumor-suppressor protein (Rb) was also examined immunohistochemically in these tissues. A heterogeneous Rb immunostaining was observed in preneoplastic lesions and adenomas. Immunohistochemical analysis also showed a reciprocal relationship between p16(INK4a) and Rb protein expression. These findings suggest that alterations in the p16(INK4a)/Rb pathway may play an important role in AOM-induced mouse colon tumorigenesis. Mol. Carcinog. 28:139-147, 2000.

Altered expression of cyclin D1 and cyclin-dependent kinase 4 in azoxymethane-induced mouse colon tumorigenesis.

Alterations in the expression of the cell cycle regulators, cyclin D1 and cyclin-dependent kinase 4 (Cdk4), have been implicated in malignancies of both humans and experimental animal models. We hypothesize that altered expression of cyclin D1 and Cdk4 may also be involved in mouse colon tumorigenesis induced by the chemical carcinogen, azoxymethane (AOM). In the present study, SWR/J mice were given AOM by i.p. injection at a dose of 10 mg/kg once a week for 8 weeks, and colonic tissue and tumors were isolated 18 weeks later. The expression and localization of cyclin D1 and Cdk4 were examined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analyses. Cyclin D1 and Cdk4 mRNA levels in tumor samples were increased 1.3-fold (P < 0.01) and 1.2-fold (P < 0.01), respectively, when compared with control mouse colon tissue. Control colon epithelium was uniformly negative for cyclin D1 immunoreactivity, whereas minimal Cdk4 nuclear staining was confined to the lower portion of the crypts within the control tissue. Both cyclin D1 and Cdk4 immunoreactive cells were markedly increased in preneoplastic lesions and in adenomas isolated from AOM-treated mice. Furthermore, some morphologically normal colon crypts from AOM-treated mice showed positive cyclin D1 immunoreactivity. These findings suggest that overexpression of cyclin D1 and Cdk4 occurs early in the AOM-induced mouse colon tumorigenesis and may contribute to tumor progression in this model.

AOM-induced mouse colon tumors do not express full-length APC protein.

While evidence in both sporadic and inherited human colorectal cancer and MIN mice implicate the tumor suppressor gene, APC, in the causation of colorectal carcinogenesis, this gene has not been confirmed to be involved in rodent chemically-induced colon cancer models (RCCM). These experimental models are widely used to elucidate mechanisms involved in colon carcinogenesis (initiation, promotion and progression) as well as studies on chemoprevention (dietary and other) and intervention. To validate the RCCM as relevant models for sporadic human colorectal cancer, and to facilitate research on the role of the APC gene in colon carcinogenesis, we investigated the role of APC in azoxymethane (AOM)-induced colorectal tumors in mice. Using an antibody that recognizes the carboxy terminus of APC, we have characterized the pattern of staining observed in normal mouse intestinal tissue, in MIN mouse intestinal adenomas and in AOM-induced mouse colon tumors. The APC protein was localized in the cytoplasm of normal colonic epithelial cells. In the small intestine there was APC immunoreactivity along the villous and staining of the Paneth cells at the base of the glands. In the proximal and distal colonic crypts there appeared to be a gradient of staining which increased towards the luminal surface. This gradient was not as apparent in the small intestinal villi. Nuclei and mucus in the goblet cells showed no immunoreactivity. MIN mouse small bowel and colonic adenomas, known to have lost APC, stained negatively for APC. AOM-induced adenomas and carcinomas also consistently stained negatively using this antibody. This study demonstrates for the first time the loss of wild-type APC protein in AOM-induced mouse colon tumors and suggests that alterations in expression of this tumor suppressor gene, which is so commonly mutated in human colon cancer, is also involved in this animal model of colon cancer.