YYFZBJS inhibits colorectal tumorigenesis by remodeling gut microbiota and influence on M2 macrophage polarization in vivo and in vitro

Our previous studies indicated that the extract of Yi-Yi-Fu-Zi-Bai-Jiang-San (YYFZBJS) had potent anticancer activities by significantly inhibiting intestinal tumor development in Apc^Min/+ mice. However, knowledge regarding the mechanism and effect of YYFZBJS in the prevention of colorectal cancer is limited. In this study, we aim to investigate the preventive effects of YYFZBJS in enterotoxigenic Bacteroides fragilis (ETBF)-colonized mice with azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced tumorigenesis. First, the colonic tissues of the AOM/DSS mouse models were collected for biomedical analysis, and gut microbiota profiling was detected post YYFZBJS treatment using a 16S rRNA gene sequencing. Then, antibiotic solution (Abx) mice were acclimated with AOM/DSS treatment and then fed with ETBF with or without YYFZBJS for three cycles. As expected, the intragastric administration of YYFZBJS in the AOM/DSS mouse model significantly decreased the tumor load, the severity of disease activity index (DAI) scores, and the level of M2 macrophage markers such as CD206, Arg-1 and IL-10. Notably, the reverse of polarized macrophages induced by YYFZBJS could suppress CRC cell proliferation and infiltration, as demonstrated by the decrease of some tumor proliferation-related proteins in a dose-dependent manner. Importantly, ETBF dysbiosis can contribute to colon tumor development by stimulating p-STAT3 mediated M2 macrophages polarization to promote chronic inflammation and adenoma malignant transformation, which YYFZBJS can effectively limit. Altogether, we demonstrate that ETBF dysbiosis may contribute to M2 macrophages-promoted colon carcinogenesis and progression of CRC cells, while YYFZBJS could be a promising protective agent against ETBF-mediated colorectal cancer.     

Testosterone strongly enhances azoxymethane/dextran sulfate sodium-induced colorectal cancer development in C57BL/6 mice

Colorectal cancer (CRC) is known to occur more frequently in males than in females, with sex hormones reportedly influencing the development. The purpose of the study was to investigate whether orchiectomy in C57BL/6 male mice reduces colorectal tumorigenesis and whether testosterone administration increases tumorigenesis after orchiectomy in an azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model. Clinical symptoms, including colitis and tumor incidence, were evaluated in the absence or presence of testosterone in AOM/DSS-treated male, as well as orchiectomized (ORX) male and female mice. The levels of serum testosterone and colonic myeloperoxidase, interleukin (IL)-1β, and IL-6 were measured by ELISA. Target mRNA expression was assessed by quantitative real-time PCR. Orchiectomy significantly diminished the AOM/DSS-induced colitis indices, including disease activity index, colon shortening, and histological severity at week 2, and decreased tumor numbers and incidence rates in the distal part of the colon increased following AOM/DSS administration at week 13; this reduction was reversed by testosterone supplementation. Furthermore, it was confirmed that the ELISA level (MPO and IL-1β) and the mRNA expression of the inflammatory mediators (COX-2 and iNOS) were maintained at high levels in the tumors of the testosterone-treated group compared with AOM/DSS groups. Interestingly, both endogenous and exogenous testosterone administrations were associated with tumor development (> 2 mm in size) and submucosal invasive cancer. Based on multivariate logistic regression analysis, testosterone was identified as a reasonable hazard factor for the progression of submucosal invasive cancer of the distal colon. In conclusion, endogenous and exogenous testosterone presented a stimulating effect on AOM/DSS-induced colitis and carcinogenicity.     

Blockade of MK2 is protective in inflammation‐associated colorectal cancer development

Chronic inflammation is a risk factor for colorectal cancer. The MAPK‐activated protein kinase 2 (MK2) pathway controls multiple cellular processes including p38‐dependent inflammation. This is the first study to investigate the role of MK2 in development of colitis‐associated colon cancer (CAC). Herein, we demonstrate that MK2^?/? mice are highly resistant to neoplasm development when exposed to AOM/DSS, while wild type (WT) C57BL/6 develop multiple neoplasms with the same treatment. MK2‐specific cytokines IL‐1, IL‐6 and TNF‐α were substantially decreased in AOM/DSS treated MK2^?/? mouse colon tissues compared with WT mice, which coincided with a marked decrease in macrophage influx. Restoring MK2‐competent macrophages by injecting WT bone marrow derived macrophages into MK2^?/? mice led to partial restoration of inflammatory cytokine production with AOM/DSS treatment; however, macrophages were not sufficient to induce neoplasm development. These results indicate that MK2 functions as an inflammatory regulator to promote colonic neoplasm development and may be a potential target for CAC.     

Crucial involvement of the CCL3‐CCR5 axis‐mediated fibroblast accumulation in colitis‐associated carcinogenesis in mice

Patients with inflammatory bowel diseases often develop colon carcinoma. Combined treatment of azoxymethane (AOM) and dextran sulfate sodium (DSS) recapitulates colitis‐associated cancer in mice. AOM/DSS‐induced tumor formation was reduced in CCL3‐ or its specific receptor, CCR5‐deficient mice despite the presence of a massive infiltration of inflammatory cells. However, AOM/DSS‐induced type I collagen‐positive fibroblast accumulation in the colon was reduced in CCL3‐ or CCR5‐deficient mice. This was associated with depressed expression of heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF), which is expressed mainly by fibroblasts. Moreover in vitro, CCL3 induced fibroblasts to proliferate and to enhance HB‐EGF expression. Furthermore, CCR5 blockade reduced tumor formation together with reduced fibroblast accumulation and HB‐EGF expression, even when administered after the development of multiple colon tumors. Thus, CCL3‐CCR5‐mediated fibroblast accumulation may be required, in addition to leukocyte infiltration, to induce full‐blown colitis‐associated carcinogenesis. Our studies shed light on a therapeutic potential of CCR5 antagonist for patients with colitis‐associated cancer.     

The MUTYH base excision repair gene protects against inflammation-associated colorectal carcinogenesis

MUTYH DNA glycosylase removes mismatched adenine opposite 7, 8-dihydro-8-oxoguanine (8-oxoG), which is the major mutagenic lesion induced by oxidative stress. Biallelic mutations in MUTYH are associated with MUTYH-Associated polyposis (MAP) and increased risk in colorectal cancer (CRC). We investigated cancer susceptibility associated with MUTYH inactivation in a mouse model of inflammation-dependent carcinogenesis induced by azoxymethane (AOM) and dextran sulphate (DSS). Mutyh^−/− mice were more sensitive than wild-type (WT) animals to AOM/DSS toxicity and accumulated DNA 8-oxoG in their gastrointestinal tract. AOM/DSS-induced colonic adenomas were significantly more numerous in Mutyh^−/− than in WT animals, and frequently showed a tubulo-villous feature along with high-grade dysplasia and larger size lesions. This condition resulted in a greater propensity to develop adenocarcinomas. The colon of untreated Mutyh^−/− mice expressed higher basal levels of pro-inflammatory cytokines GM-CSF and IFNγ, and treatment with AOM/DSS induced an early decrease in circulating CD4+ and CD8+ T lymphocytes and an increase in myeloid-derived suppressor cells (MDSCs). Adenomas from Mutyh^−/− mice had a greater infiltrate of Foxp3+ T regulatory cells, granulocytes, macrophages, MDSCs and strong expression of TGF-β-latency-associated peptide and IL6. Our findings indicate that MUTYH loss is associated with an increase in CRC risk, which involves immunosuppression and altered inflammatory response. We propose that the AOM/DSS initiation/promotion protocol in Mutyh^−/− mice provides a good model for MAP.     

TiaochangXiaoliu decoction inhibits azomethane (AOM)/dextran sulfate sodium (DSS)-induced colorectal cancer by regulating immune response

BackgroundTiaochangXiaoliu decoction (TXD) has an anti-tumor effect in clinical practice. We further investigated the role of TXD in colorectal cancer (CRC).MethodsMouse models of CRC were induced by azomethane (AOM)/dextran sulfate sodium (DSS), with sixty male C57BL/6 mice randomly divided into six groups (10 mice/group): a control group, AOM/DSS group, TXD at low dose (L-dose) group, middle dose (M-dose) group, high dose (H-dose) group, and Celecoxib (Cel) group. The colorectum, serum, and plasma of mice in each group was collected following sacrifice to record the number of tumors. HE staining was utilized for observing pathological damage to colorectal tissues, ELISA used for detecting INF-γ, IL-2, and TNF-α expression in serum, and flow cytometry used for measuring the proportion of CD4⁺, CD8⁺, CD4⁺/CD8⁺, and NK cells in plasma.ResultsCompared with the control group, the AOM/DSS group showed tumor masses in the colorectum and different degrees of pathological damage in the intestine. AOM/DSS induction also resulted in an increase in INF-γ, IL-2, and TNF-α expression in serum, and a decrease in the percentages of CD4⁺, CD8⁺, CD4⁺/CD8⁺, and NK cells(P<0.05). In comparison with the AOM/DSS group, with the increase of TXD dose, the number of tumors decreased significantly, and intestinal structure and mucosal inflammatory cell infiltration also improved. Further, in comparison with the AOM/DSS group, all three doses of TXD and celecoxib caused an increase in the contents of CD4⁺, CD8⁺, CD4⁺/CD8⁺, and NK cells in plasma. In addition, in the M-dose, H-dose, and Cel groups, INF-γ, IL-2, and TNF-α expression showed a marked decrease, and the reduction in these two groups treated with TXD was dose-dependent.ConclusionsTXD leads to a marked reduction in the number of tumors and inflammatory cell infiltration in CRC mice. This decoction significantly decreased the levels of INF-γ, IL-2, and TNF-α in serum, and increased the contents of CD4⁺, CD8⁺, CD4⁺/CD8⁺, and NK cell in the plasma of mice with AOM/DSS-induced CRC.     

Dvl2 facilitates the coordination of NF‐κB and Wnt signaling to promote colitis‐associated colorectal progression

Colitis‐associated colorectal cancer (CAC) arises due to prolonged inflammation and has distinct molecular events compared with sporadic colorectal cancer (CRC). Although inflammatory NF‐κB signaling was activated by pro‐inflammatory cytokines (such as TNFα) in early stages of CAC, Wnt/β‐catenin signaling later appears to function as a key regulator of CAC progression. However, the exact mechanism responsible for the cross‐regulation between these 2 pathways remains unclear. Here, we found reciprocal inhibition between NF‐κB and Wnt/β‐catenin signaling in CAC samples, and the Dvl2, an adaptor protein of Wnt/β‐catenin signaling, is responsible for NF‐κB inhibition. Mechanistically, Dvl2 interacts with the C‐terminus of tumor necrosis factor receptor 1 (TNFRI) and mediates TNFRI endocytosis, leading to NF‐κB signal inhibition. In addition, increased infiltration of the pro‐inflammatory cytokine interleukin‐13 (IL‐13) is responsible for upregulating Dvl2 expression through STAT6. Targeting STAT6 effectively decreases Dvl2 levels and restrains colony formation of cancer cells. These findings demonstrate a unique role for Dvl2 in TNFRI endocytosis, which facilitates the coordination of NF‐κB and Wnt to promote CAC progression.     

IL-32α suppresses colorectal cancer development via TNFR1-mediated death signaling

Inflammation is associated with cancer-prone microenvironment, leading to cancer. IL-32 is expressed in chronic inflammation-linked human cancers. To investigate IL-32α in inflammation-linked colorectal carcinogenesis, we generated a strain of mice, expressing IL-32 (IL-32α-Tg). In IL-32α-Tg mice, azoxymethane (AOM)-induced colon cancer incidence was decreased, whereas expression of TNFR1 and TNFR1-medicated apoptosis was increased. Also, IL-32α increased ROS production to induce prolonged JNK activation. In colon cancer patients, IL-32α and TNFR1 were increased. These findings indicate that IL-32α suppressed colon cancer development by promoting the death signaling of TNFR1.     

阿克曼菌对AOM/DSS炎症相关性结直肠癌发生的作用

目的 探讨阿克曼菌（AKK）对氧化偶氮甲烷（AOM）/葡聚糖硫酸钠（DSS）诱导的炎症相关性结直肠癌小鼠模型及其肠道干细胞的影响。方法 AOM/DSS诱导小鼠炎症相关性结直肠癌模型随机分为三组,通过灌胃方式给予三组不同的药物分别为模型组（Model）、AKK组及阿司匹林组（Aspirin）。干预10周后观察小鼠的肿瘤数目、肿瘤大小、肿瘤分布及分析肿瘤负荷情况。免疫组织化学分析表征肿瘤恶性化的蛋白Ki67和表征干细胞的特异性蛋白Lgr5的表达变化。qRT-PCR检测干细胞分化特性的基因Lgr5、CD133、Nanog和ALDH1的mRNA表达。结果 与模型组相比,AKK组的肿瘤数目、肿瘤大小及肿瘤负荷明显减小（P<0.01）;相较于模型组小鼠,AKK组的肿瘤组织中Ki67和Lgr5表达明显下降（P<0.05）;CD133、Nanog和ALDH1的mRNA表达明显下调。结论 AKK对AOM/DSS诱导的结肠炎相关性结直肠癌小鼠具有防治作用,其作用机制可能与结直肠干细胞活性密切相关。     

薏苡附子败酱散联合电针治疗对AOM/DSS小鼠炎症相关性结直肠癌的防治作用及机制

目的:探究薏苡附子败酱散(Yiyi Fuzi Baijiang San,YYFZBJS)联合电针(electroacupuncture,EA)对AOM/DSS模型小鼠炎症相关性结直肠癌（colitis-associated colorectal cancer,CAC）的防治作用及对结直肠癌上皮间质转化（epithelial-mesenchymal transition,EMT）相关蛋白表达情况的影响。方法:建立AOM/DSS诱导的CAC小鼠模型,将40只小鼠随机分为模型组、针药结合组、薏苡附子败酱散组、电针组和阿司匹林组,于实验第2周开始干预治疗,治疗过程中记录各组小鼠一般情况,统计体质量变化、疾病活动指数（disease activity index,DAI）评分及生存情况,于实验末比较各组小鼠结直肠长度、成瘤数量及肿瘤大小,通过HE染色观察结直肠组织及肿瘤组织的病理变化,免疫组织化学染色观察结直肠肿瘤Ki-67、PCNA、p-NF-κB、NF-κB、E-cadherin、ZO-1、Occludin、Vimentin和N-cadherin蛋白的表达。结果:与模型组相比,针药结合组小鼠体质量显著升高(P＜0.05),DAI分值降低(P＜0.05),生存率为87.5%,结直肠长度显著增加（P＜0.01）,直径小于2 mm的肿瘤数量减少(P＜0.05),直径介于2~4 mm之间和大于4 mm的肿瘤数量显著减少（P＜0.01）,总成瘤数量明显下降（P＜0.01）,结直肠形态明显改善;肿瘤组织病理评分降低（P＜0.01）,肿瘤组织中Ki-67、PCNA、p-NF-κB、NF-κB、Vimentin和N-cadherin蛋白表达量降低（P＜0.01）,E-cadherin、ZO-1和Occludin蛋白表达量升高(P＜0.05)。结论:薏苡附子败酱散联合电针对AOM/DSS模型小鼠的CAC发生发展有防治作用,其机制可能与下调NF-κB信号通路,减缓EMT进程有关。     

Hepatocyte growth factor ameliorates mucosal injuries leading to inhibition of colon cancer development in mice

Hepatocyte growth factor (HGF), which facilitates the repair of injured mucosa, has the potential to be a new therapeutic agent for inflammatory bowel disease (IBD). However, given that the incidence of colorectal cancer increases continuously with disease duration in patients with IBD, the fact that HGF is a potent mitogen for intestinal epithelial cells may further heighten the risk of bowel cancer in this patient population. In this study, we examined the effects of recombinant HGF on colorectal cancer development in mice with or without experimentally induced colitis. Although HGF stimulated proliferation of colonic epithelial cells in normal mucosa, the development of colorectal cancer induced by repeated injection of azoxymethane (AOM) was significantly inhibited by HGF treatment. In a mouse model of colitis-associated cancer, colorectal cancer frequently developed despite only a single injection of AOM prior to three cycles of dextran sulfate sodium administration. However, HGF treatment significantly facilitated the repair of injured mucosa, leading to inhibition of colorectal cancer development in a dose-dependent manner. Thus, HGF-induced repair of injured mucosa inhibits rather than accelerates the development of colorectal cancer, and these results also suggest the importance of blocking the cycles of mucosal injury and repair to prevent colitis-associated colorectal cancer.     

Increased nicotinamide adenine dinucleotide pool promotes colon cancer progression by suppressing reactive oxygen species level

Nicotinamide adenine dinucleotide (NAD) exists in an oxidized form (NAD⁺) and a reduced form (NADH). NAD⁺ plays crucial roles in cancer metabolism, including in cellular signaling, energy production and redox regulation. However, it remains unclear whether NAD(H) pool size (NAD⁺ and NADH) could be used as biomarker for colon cancer progression. Here, we showed that the NAD(H) pool size and NAD⁺/NADH ratio both increased during colorectal cancer (CRC) progression due to activation of the NAD⁺ salvage pathway mediated by nicotinamide phosphoribosyltransferase (NAMPT). The NAMPT expression was upregulated in adenoma and adenocarcinoma tissues from CRC patients. The NADH fluorescence intensity measured by two‐photon excitation fluorescence (TPEF) microscopy was consistently increased in CRC cell lines, azoxymethane/dextran sodium sulfate (AOM/DSS)‐induced CRC tissues and tumor tissues from CRC patients. The increases in the NAD(H) pool inhibited the accumulation of excessive reactive oxygen species (ROS) levels and FK866, a specific inhibitor of NAMPT, treatment decreased the CRC nodule size by increasing ROS levels in AOM/DSS mice. Collectively, our results suggest that NAMPT‐mediated upregulation of the NAD(H) pool protects cancer cells against detrimental oxidative stress and that detecting NADH fluorescence by TPEF microscopy could be a potential method for monitoring CRC progression.     

Ebselen abrogates TNFα induced pro‐inflammatory response in glioblastoma

We investigated the pro‐inflammatory response mediated by TNFα in glioblastoma and whether treatment with organoselenium Ebselen (2‐phenyl‐1,2‐benzisoselenazol‐3[2H]one) can affect TNFα induced inflammatory response. Exposure to TNFα increased the expression of pro‐inflammatory mediator interleukin IL‐6, IL‐8, monocyte chemoattractant protein‐1 (MCP‐1) and cyclooxygenase (COX‐2). Treatment with Ebselen abrogated TNFα induced increase in pro‐inflammatory mediators. Ebselen not only abrogated TNFα induced enhanced invasiveness of glioma cells by down‐regulating matrix metallo proteinase (MMP‐9) and urokinase plasminogen (uPa) activity, but also inhibited glioma cell migration. Treatment with Ebselen also down‐regulated the enhanced ROS production of TNFα treated glioma cells. In addition, Ebselen induced DNA damage repair signaling response in glioma cells both in the presence and absence of TNFα. These studies indicate that together with its known ability to sensitize glioma cell to TNFα induced apoptosis, Ebselen can overcome TNFα induced pro‐inflammatory mediators to prevent a build up of a deleterious pro‐inflammatory tumor microenvironment.     

Suppressor of cytokine signaling 3 (SOCS3) limits damage-induced crypt hyper-proliferation and inflammation-associated tumorigenesis in the colon

Intestinal injury or chronic inflammation induce cytokines that promote crypt regeneration and mucosal repair. If excessive or prolonged, such mechanisms may increase colon cancer risk. Factors that terminate or limit cytokine action in intestinal epithelial cells (IEC) may protect against crypt hyperplasia and neoplasia. We hypothesized that suppressor of cytokine signaling-3 (SOCS3) is such a factor. Mice with Vilin-promoter/Cre-recombinase (VC)-mediated IEC-specific SOCS3 gene disruption (VC/HO), WT/HO littermates with floxed but intact SOCS3 genes and VC/WT mice were studied. Colon was examined after acute dextran sodium sulfate (DSS)-induced mucosal injury or after azoxymethane (AOM) and chronic DSS. Signaling pathways were examined in colon, cultured IEC or colon cancer cell lines. VC/HO mice showed no basal phenotype. After acute DSS, VC/HO exhibited enhanced crypt proliferation and crypt hyperplasia and reduced transforming growth factor (TGF) beta expression in colon. Inflammation and mucosal damage were similar across genotypes. Following AOM/DSS, VC/HO mice had increased size, number and load of colonic tumors and increased STAT3 and nuclear factor-kappa B (NF-kappaB) activation in colon. In vitro, SOCS3 overexpression reduced proliferation, IL-6-mediated STAT3 activation and tumor necrosis factor (TNF) alpha-mediated NF-kappaB activation. We conclude that cytokine induction of SOCS3 normally provides an intrinsic mechanism to limit injury-induced crypt hyperproliferation and inflammation-associated colon cancer by regulating both STAT3 and NF-kappaB pathways.     

Troglitazone, a ligand for peroxisome proliferator-activated receptor gamma, inhibits chemically-induced aberrant crypt foci in rats.

The biological roles of peroxisome proliferator-activated receptors (PPARs) in various diseases, including inflammation and cancer, have been highlighted recently. Although PPARgamma ligand is suspected to play an important role in carcinogenesis, its effects on colon tumorigenesis remain undetermined. The present time-course study was conducted to investigate possible modifying effects of a PPARgamma ligand, troglitazone, on the development and growth of aberrant crypt foci (ACF), putative precursor lesions for colon carcinoma, induced by azoxymethane (AOM) or dextran sodium sulfate (DSS) in male F344 rats. Oral troglitazone (10 or 30 mg / kg body weight (b.w.)) significantly reduced AOM (two weekly subcutaneous injections, 20 mg / kg b.w.)-induced ACF. Treatment with troglitazone increased apoptosis and decreased polyamine content and ornithine decarboxylase (ODC) activity in the colonic mucosa of rats treated with AOM. Gastric gavage of troglitazone also inhibited colitis and ACF induced by DSS (1% in drinking water), in conjunction with increased apoptosis and reduced colonic mucosal polyamine level and ODC activity. Our results suggest that troglitazone, a synthetic PPARgamma ligand, can inhibit the early stage of colon tumorigenesis with or without colitis.     

Troglitazone, a Ligand for Peroxisome Proliferator-activated Receptor γ Inhibits Chemically-induced Aberrant Crypt Foci in Rats

The biological roles of peroxisome proliferator-activated receptors (PPARs) in various diseases, including inflammation and cancer, have been highlighted recently. Although PPARγ ligand is suspected to play an important role in carcinogenesis, its effects on colon tumorigenesis remain undetermined. The present tune-course study was conducted to investigate possible modifying effects of a PPARγ ligand, troglitazone, on the development and growth of aberrant crypt foci (ACF), putative precursor lesions for colon carcinoma, induced by azoxymethane (AOM) or dextran sodium sulfate (DSS) in male F344 rats. Oral troglitazone (10 or 30 mg/kg body weight (b.w.)) significantly reduced AOM (two weekly subcutaneous injections, 20 mg/kg b.w.)-induced ACF. Treatment with troglitazone increased apoptosis and decreased polyamine content and ornithine decarboxylase (ODC) activity in the colonic mucosa of rats treated with AOM. Gastric gavage of troglitazone also inhibited colitis and ACF induced by DSS (1% in drinking water), in conjunction with increased apoptosis and reduced colonic mucosal polyamine level and ODC activity. Our results suggest that troglitazone, a synthetic PPARγ ligand, can inhibit the early stage of colon tumorigenesis with or without colitis.     

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.      

Mucin‐depleted foci show strong activation of inflammatory markers in 1,2‐dimethylhydrazine‐induced carcinogenesis and are promoted by the inflammatory agent sodium dextran sulfate

Mucin‐depleted foci (MDF), formed by dysplastic crypts devoid of mucins, have been identified in the colon of carcinogen‐treated rodents and in humans at high risk for colon cancer. The lack of the protective layer of mucus may cause inflammation which has been linked to colon carcinogenesis, therefore, the expression of markers such as cyclooxygenase‐2 (COX‐2), inducible nitric oxide synthase (i‐NOS) and macrophage infiltration was studied with immunohistochemistry (IH) in MDF harvested from F344 rats treated with the colon carcinogen 1,2‐dimethylhydrazine (DMH). The same determinations were performed in aberrant crypt foci (ACF) and, at a later time point, in tumours. A dramatic increase in COX‐2, i‐NOS and macrophage infiltration was observed in MDF but ACF showed a moderate increase compared with the paired normal mucosa. Tumours were positive for all the markers. RT‐PCR experiments demonstrated that i‐NOS RNA expression was increased in a set of MDF confirming the results obtained with immunohistochemistry. In an inflammation‐cancer experimental model [mice treated with azoxymethane (AOM) and dextran sodium sulphate (DSS)], we observed that DSS‐induced inflammation promoted MDF in a dose‐dependent manner, whereas ACF were not affected. In conclusion, we report here for the first time a strong activation of the inflammatory process in MDF, which may contribute to the further progression of MDF to tumours. © 2009 UICC