Suppression of intestinal polyposis in Mdr1-deficient ApcMin/+ mice

Aberrant transactivation of a certain set of target genes by the beta-catenin and T-cell factor/lymphoid enhancer factor complex has been considered crucial for the initiation of intestinal tumorigenesis. The human multidrug resistance (MDR)1 (ABCB1) gene contains multiple beta-catenin-T-cell factor4-binding elements in its promoter and is one of the immediate targets of the complex. In the current study, we have further substantiated the biological involvement of MDR1 in intestinal tumorigenesis based on the following evidence: (a) aberrant induction of the Mdr1a (Abcb1a) gene product, P-glycoprotein, associated with nuclear accumulation of the beta-catenin protein, was observed even in nascent microscopic adenomas of Min mice; (b) Mdr1-deficient Min (Apc(Min/+)Mdr1a/b(-/-)) mice developed significantly fewer intestinal polyps than did Apc(Min/+)Mdr1a/b(+/+) mice; and (c) Inhibitors of P-glycoprotein, verapamil, and cyclosporin A had a suppressive effect on the in vitro polypoid growth of IEC6 expressing stabilized (DeltaN89) beta-catenin protein. Inhibitors of P-glycoprotein may be included in a novel class of chemopreventive agents against colorectal carcinogenesis.     

Intestinal-specific PPARgamma deficiency enhances tumorigenesis in ApcMin/+ mice

Multiple investigations of the effects of peroxisome proliferator-activated receptor gamma (PPARgamma) ligands on colon cancer have produced contradictory results. While some studies demonstrated increased numbers of colonic polyps in Apc(Min/+) mice treated with various thiazolidinedione (TZD) PPARgamma ligands, others reported amelioration of tumor multiplicity and progression in both Apc(Min/+) mice and in mice with chemically-induced colon cancer. Here, we addressed the role of PPARgamma in murine intestinal tumorigenesis using gene knockout methodology. We found that either heterozygous or homozygous intestinal-specific PPARgamma deficiency enhanced the number of Apc(Min/+) tumors in both the small intestine and colon, especially in the colon, where PPARgamma deficiency also modulated tumor incidence. Gender significantly affected tumor multiplicity independent of PPARgamma genotype. Female Apc(Min/+) mice developed more tumors in the small intestine and more tumors overall, whereas male Apc(Min/+) mice developed more tumors in the colon. Nevertheless, intestinal PPARgamma deficiency enhanced tumorigenesis irrespective of gender. Our results suggest that PPARgamma functions as a tumor resistance factor in the mouse intestine and warrant further investigation of the PPARgamma-dependent and independent actions of TZDs in cancer.     

beta-Catenin-accumulated crypts in the colonic mucosa of juvenile ApcMin/+ mice

Although Apc(Min/+) mice are widely used for an animal model of human familial adenomatous polyposis (FAP), a majority of intestinal polyps locate in the small intestine. We recently reported that numerous beta-catenin-accumulated crypts (BCAC), which are reliable precursor lesions for colonic adenocarcinoma, develop in the large bowel of aged Apc(Min/+) mice. In this study, we determined the presence and location of BCAC in the large intestine of juvenile Apc(Min/+) mice (3 and 5 weeks of age). Surprisingly, BCAC were noted in the colon of even Apc(Min/+) mice of both ages, and mainly located in the distal and middle segments of the colon. Also, a few microadenomas were detected in Apc(Min/+) mice of 5-week old. Our results may indicate need of further investigation of the colorectal mucosa of Apc(Min/+) mice for examining colorectal carcinogenesis using Apc(Min/+) mice.     

Heterozygous deletion of ATG5 in ApcMin/+ mice promotes intestinal adenoma growth and enhances the antitumor efficacy of interferon-gamma

Autophagy related gene 5 (ATG5) was lost in 23% of the patients with colorectal cancer (CRC) and the role of loss of ATG5 in the pathogenesis of CRC remains unclear. Knockdown of ATG5 in cancer cells enhances the antitumor efficacy of lots of chemotherapeutic agents. However, there is still no animal model to validate these in vitro observations in vivo. In this study, we found that heterozygous deletion of ATG5 in Apc^Min/+ mice increased the number and size of adenomas as compared with those in Apc^Min/+ATG5^+/+ mice. To investigate whether ATG5 deficiency could sensitize tumors to chemotherapies, we compared the antitumor effects of Interferon-gamma (IFN-γ) between Apc^Min/+ATG5^+/+ and Apc^Min/+ATG5^+/− mice, as IFN-γ is a potential tumor suppressor for CRC and has been used clinically as an efficient adjuvant to chemotherapy of cancer. We revealed that heterozygous deletion of ATG5 significantly enhanced the antitumor efficacy of IFN-γ. Early treatment of Apc^Min/+ATG5^+/− mice with IFN-γ decreased tumor incidence rate to 16.7% and reduced the number of adenomas by 95.5% and late treatment led to regression of tumor. Moreover, IFN-γ treatment did not cause any evident toxic reaction. Mechanistic analysis revealed that heterozygous deletion of ATG5 activated EGFR/ERK1/2 and Wnt/β-catenin pathways in adenomas of Apc^Min/+ mice and enhanced the effects of IFN-γ-dependent inhibition of these 2 pathways. Our results demonstrate that ATG5 plays important roles in intestinal tumor growth and combination of IFN-γ and ATG5 deficiency or ATG5-targeted inhibition is a promising strategy for prevention and treatment of CRC.     

CD4+CD25+ regulatory lymphocytes induce regression of intestinal tumors in ApcMin/+ mice

Colorectal cancer in humans results from sequential genetic changes in intestinal epithelia commencing with inactivation of the APC tumor suppressor gene. Roles for host immunity in epithelial tumorigenesis are poorly understood. It has been previously shown that CD4+CD25+ lymphocytes inhibit colitis-associated epithelial tumors in Rag-deficient mice. Here we show that addition of CD4+CD25+ lymphocytes in ApcMin/+ mice reduces multiplicity of epithelial adenomas. Interleukin-10 was required in regulatory cells for therapeutic effect. Recipients of regulatory cells showed increased apoptosis and down-regulation of cyclooxygenase-2 within tumors coincident with tumor regression. These data suggest a role for regulatory lymphocytes in epithelial homeostasis in the ApcMin/+ mouse model of intestinal polyposis. Similarities with cancer of the breast, prostate, lung, and other sites raise the possibility of broader roles for regulatory lymphocytes in prevention and treatment of epithelial cancers in humans.     

Reduction in the resident intestinal myelomonocytic cell population occurs during ApcMin/+ mouse intestinal tumorigenesis

With its significant contribution to cancer mortality globally, advanced colorectal cancer (CRC) requires new treatment strategies. However, despite recent good results for mismatch repair (MMR)-deficient CRC and other malignancies, such as melanoma, the vast majority of MMR-proficient CRCs are resistant to checkpoint inhibitor (CKI) therapy. MMR-proficient CRCs commonly develop from precursor adenomas with enhanced Wnt-signalling due to adenomatous polyposis coli (APC) mutations. In melanomas with enhanced Wnt signalling due to stabilized β-catenin, immune anergy and resistance to CKI therapy has been observed, which is dependent on micro-environmental myelomonocytic (MM) cell depletion in melanoma models. However, MM populations of colorectal adenomas or CRC have not been studied. To characterize resident intestinal MM cell populations during the early stages of tumorigenesis, the present study utilized the Apc^Min/+ mouse as a model of MMR-proficient CRC, using enhanced green fluorescent protein (EGFP) expression in the mouse lysozyme (M-lys)^lys-EGFP/+ mouse as a pan-myelomonocytic cell marker and a panel of murine macrophage surface markers. Total intestinal lamina propria mononuclear cell (LPMNC) numbers significantly decreased with age (2.32±1.39×10⁷ [n=4] at 33 days of age vs. 1.06±0.24×10⁷ [n=8] at 109 days of age) during intestinal adenoma development in Apc^Min/+ mice (P=0.05; unpaired Student''s t-test), but not in wild-type littermates (P=0.35). Decreased total LPMNC numbers were associated with atrophy of intestinal lymphoid follicles and the absence of MM/lymphoid cell aggregates in Apc^Min/+ mouse intestine, but not spleen, compared with wild-type mice. Furthermore, during the early stage of intestinal adenoma development, there was a two-fold reduction of M-lys expressing cells (P=0.05) and four-fold reduction of ER-HR3 (macrophage sub-set) expressing cells (P=0.05; two tailed Mann-Whitney U test) in mice with reduced total intestinal LPMNCs (n=3). Further studies are necessary to determine the relevance of these findings to immune-surveillance of colorectal adenomas or MMR-proficient CRC CKI therapy resistance.     

Protease‐activated receptor‐2 accelerates intestinal tumor formation through activation of nuclear factor‐κB signaling and tumor angiogenesis in ApcMin/+ mice

Hepatocyte growth factor activator inhibitor‐1 (HAI‐1), encoded by the SPINT1 gene, is a membrane‐bound protease inhibitor expressed on the surface of epithelial cells. Hepatocyte growth factor activator inhibitor‐1 regulates type II transmembrane serine proteases that activate protease‐activated receptor‐2 (PAR‐2). We previously reported that deletion of Spint1 in Apc^Min/+ mice resulted in accelerated formation of intestinal tumors, possibly through enhanced nuclear factor‐κB signaling. In this study, we examined the role of PAR‐2 in accelerating tumor formation in the Apc^Min/+ model in the presence or absence of Spint1. We observed that knockout of the F2rl1 gene, encoding PAR‐2, not only eliminated the enhanced formation of intestinal tumors caused by Spint1 deletion, but also reduced tumor formation in the presence of Spint1. Exacerbation of anemia and weight loss associated with HAI‐1 deficiency was also normalized by compound deficiency of PAR‐2. Mechanistically, signaling triggered by deregulated protease activities increased nuclear translocation of RelA/p65, vascular endothelial growth factor expression, and vascular density in Apc^Min/+‐induced intestinal tumors. These results suggest that serine proteases promote intestinal carcinogenesis through activation of PAR‐2, and that HAI‐1 plays a critical tumor suppressor role as an inhibitor of matriptase, kallikreins, and other PAR‐2 activating proteases.     

小肠间质瘤临床诊断和治疗

目的探讨小肠间质瘤(GIST)的诊断和治疗。方法对我院1999年1月~2004年4月收治的16例小肠GIST患者的临床表现、手术处理及病理结果进行回顾性分析。结果全组平均发病年龄53岁。发病至就诊时间为半个月至5年不等。首诊主要表现为黑便、腹部不适、腹部肿块,发生部位为空肠5例,回肠8例,空、回肠均有3例。术前5例行DSA检查,诊断小肠肿瘤4例;11例行CT检查,均发现腹腔占位,诊断小肠肿瘤7例;全消化道造影检查8例,发现小肠受压2例。均行手术治疗,其中4例在腹腔镜下完成。手术标本均经病理及免疫组化证实。12例随访时间6个月至5年,平均随访27.3个月,4例失访。其中根治性切除9例,复发2例,均再次手术,仍健在;腹腔镜下小肠肠段切除术4例均无复发。结论小肠GIST缺乏特征性临床表现,DSA检查和CT检查对诊断有一定帮助,但术前确诊率较低,其确诊依赖病理结果。目前小肠GIST的治疗仍以手术切除为主,对复发或远处转移者应积极再次手术,可延长生存期。     

Inhibiting vascular endothelial growth factor receptor-2 signaling reduces tumor burden in the ApcMin/+ mouse model of early intestinal cancer

The Apc(Min/+) mouse model is a clinically relevant model of early intestinal cancer. We used AZD2171, an oral, highly potent and selective vascular endothelial growth factor (VEGF) signaling inhibitor, to investigate the role of VEGF receptor-2 (VEGFR-2) signaling in adenoma development and growth in Apc(Min/+) mice. AZD2171 (5 mg/kg body wt/day) was administered once daily for 28 days to 6-week-old (early-intervention) or 10-week-old (late intervention) mice. In the early-intervention study, AZD2171 reduced the number of macroscopic polyps in the small bowel and colon. Macropolyp diameter was lower in the small bowel, but remained unchanged in the colon. In animals receiving AZD2171, microscopic evaluation of the small intestine showed a significant reduction in the number of larger lesions. In the late-intervention study, AZD2171 treatment reduced macropolyp diameter (but not number) in the small intestine. Microscopic analysis revealed that AZD2171 significantly reduced the number of larger micropolyps in the small bowel, with no large micropolyps present in the colon. AZD2171 treatment had no effect on microvessel density or localization of beta-catenin staining in adenomas or non-tumor intestinal tissue, but significantly reduced the number of cells expressing VEGFR-2 mRNA. In conclusion, the effects of AZD2171 in the small intestine of Apc(Min/+) mice are consistent with an antiangiogenic mechanism of action, limiting growth of adenomas to < or =1 mm. These data also suggest that an early step in adenoma development may depend on VEGFR-2 signaling. Together, these results indicate that VEGFR-2 signaling may play key roles in the development and progression of intestinal adenomas.     

Cancer chemoprevention of intestinal polyposis in ApcMin/+ mice by sulforaphane, a natural product derived from cruciferous vegetable

Sulforaphane (SFN) is an isothiocyanate that is present abundantly in widely consumed cruciferous vegetables and has a particularly high content in broccoli and cauliflower. It has been shown to be an effective inhibitor of some carcinogen-induced cancers in rodents. Here, we investigated the chemopreventive efficacy of SFN in the ApcMin/+ mouse model. ApcMin/+ mice were fed with diet supplemented with two different dose levels of SFN (300 and 600 p.p.m.) for 3 weeks. Our results clearly demonstrated that ApcMin/+ mice fed with SFN-supplemented diet developed significantly less and smaller polyps with higher apoptotic and lower proliferative indices in their small intestine, in a SFN dose-dependent manner. In addition, immunohistochemical (IHC) staining of the adenomas indicated that SFN significantly suppressed the expression of phosphorylated c-Jun N-terminal kinase (p-JNK), phosphorylated extracellular signal-regulated kinases (p-ERK) and phosphorylated-Akt (p-Akt), which were found to be highly expressed in the adenomas of ApcMin/+ mice. In contrast, expression of two important biomarkers of the Wnt signaling pathway, beta-catenin and cyclin-D1 was unaffected by SFN treatment. Measurement of SFN and its metabolite SFN-GSH in the small intestine using LC-MS indicates that the concentrations between 3 and 30 nmol/g are required to prevent, or retard adenoma formation in the gastrointestinal tract of ApcMin/+ mice.     

Regulation of stromal cell cyclooxygenase-2 in the ApcMin/+ mouse model of intestinal tumorigenesis

Cyclooxygenase-2 (Cox-2) is expressed predominantly by stromal cells in intestinal adenomas from the Apc(Min/+) mouse model of familial adenomatous polyposis. We investigated the mechanistic basis of stromal cell Cox-2 expression in Apc(Min/+) mouse adenomas, as well as Cox-2 expression and activity in histologically normal (HN) Apc(Min/+) mouse intestine, in order to gain further insights into regulation of Cox-2 as a potential chemoprevention target. Upregulation of Cox-2 in intestinal tumours is not an intrinsic feature of Apc(Min/+) macrophages as bone marrow-derived Apc(Min/+) macrophages did not exhibit an abnormality in Cox-2 expression or activity. Intestinal permeability to lactulose or mannitol was similar in Apc(Min/+) mice and wild-type littermates, implying that macrophage activation by luminal antigen is unlikely to explain stromal cell Cox-2 induction. Moreover, stromal cells exhibited differential expression of Cox-2 and inducible nitric oxide synthase, suggesting 'alternative' (M2) rather than 'classical' (M1) macrophage activation. Flow cytometric sorting of isolated stromal mononuclear cells (SMNCs), on the basis of M-lysozyme and specific macrophage marker expression, demonstrated that macrophages, neutrophils and non-myelomonocytic cells all contributed to lamina propria prostaglandin (PG) E(2) synthesis. However, the majority of PGE(2) synthesis by macrophages was via a Cox-2-dependent pathway compared with predominant Cox-1-derived PGE(2) production by non-myelomonocytic cells. SMNCs from HN Apc(Min/+) intestinal mucosa exhibited similar levels of Cox-2 mRNA and protein, but produced more Cox-2-derived PGE(2) than wild-type cells at 70 days of age. There was an age-dependent decline in PGE(2) synthesis by Apc(Min/+) SMNCs, despite tumour progression. These data suggest that other Cox-2-independent factors also control PGE(2) levels during Apc(Min/+) mouse intestinal tumorigenesis. Regulation of macrophage Cox-2 expression and other steps in PGE(2) synthesis (e.g. PGE synthase) are valid targets for novel chemoprevention strategies that could minimize or avoid systemic COX-2 inhibition.     

Chemoprevention of intestinal polyps in ApcMin/+ mice fed with western or balanced diets by drinking annurca apple polyphenol extract

The Western diet (WD) is associated with a higher incidence of colorectal cancer (CRC) than the Mediterranean diet. Polyphenols extracted from Annurca apple showed chemopreventive properties in CRC cells. A multifactorial, four-arm study by using wild-type (wt) and Apc(Min/+) mice was carried out to evaluate the effect on polyp number and growth of APE treatment (60 μmol/L) ad libitum in drinking water combined with a WD or a balanced diet (BD) for 12 weeks. Compared with APE treatment, we found a significant drop in body weight (P < 0.0001), severe rectal bleeding (P = 0.0076), presence of extraintestinal tumors, and poorer activity status (P = 0.0034) in water-drinking Apc(Min/+) mice, more remarkably in the WD arm. In the BD and WD groups, APE reduced polyp number (35% and 42%, respectively, P < 0.001) and growth (60% and 52%, respectively, P < 0.0001) in both colon and small intestine. Increased antioxidant activity was found in wt animals fed both diets and in Apc(Min/+) mice fed WD and drinking APE. Reduced lipid peroxidation was found in Apc(Min/+) mice drinking APE fed both diets and in wt mice fed WD. In normal mucosa, mice drinking water had lower global levels of DNA methylation than mice drinking APE. APE treatment is highly effective in reducing polyps in Apc(Min/+) mice and supports the concept that a mixture of phytochemicals, as they are naturally present in foods, represent a plausible chemopreventive agent for CRC, particularly in populations at high risk for colorectal neoplasia.     

Exercise through Shaking Stimuli Suppresses Cancer Growth via the Wnt pathway in ApcMin/+ Mice

Objective: Exercise has been reported to suppress colorectal cancer; however, the mechanism of suppression by exercise and its effect on the Wnt pathway, which is particularly involved in the early stage of carcinogenesis, remain unclear. In this study, we subjected ApcMin/+ mice to exercise by shaking stimuli to investigate the mechanisms of suppressing colorectal cancer, and focused on the Ca2+ pathway, which is one of the β-catenin-independent Wnt signaling pathways that suppress the accumulation of β-catenin. Methods: Mice in the exercise group were subjected to exercise by shaking stimuli for 30 min/session, 6 sessions/ week, for a total of 11 weeks. The number and diameter of intestinal polyps were calculated. Expression analysis of β-catenin and Pak1 from the intestinal tract and Wnt5a-Pan and Wnt5a-Long from the gastrocnemius muscle was performed by western blotting. The expression of β-catenin and Wnt5a-Pan was observed by immunohistochemical staining. Result: The levels of expression of β-catenin and Pak1 in the small intestine were low in the exercise group, indicating that exercise suppressed the accumulation of β-catenin. In the gastrocnemius muscle, the levels of expression of Wnt5a-Pan and Wnt5a-Long were significantly higher in the exercise group (p < 0.05). Histological analysis revealed that the percentage of large polyps was significantly lower in the exercise group than in the control group (p < 0.01), revealing that exercise suppressed the growth of polyps. In addition, the villi/crypt ratio (V/C ratio) was significantly higher in the exercise group, suggesting the suppression of exercise-induced local inflammation in the small intestine. Conclusion: We believe that the mechanism of polyp growth suppression is related to the inflammatory and not the Wnt pathway. This study clarified the growth-suppressing effect of a novel exercise method on cancer. We believe that its development and clinical application might open new possibilities for the prevention treatment of colorectal cancer.     

腹腔镜小肠切除术治疗小肠肿瘤

腹腔镜技术已成为未来外科的发展方向之一,并已广泛地运用于普外科的各种手术中.但腹腔镜小肠切除术在小肠肿瘤,特别是恶性肿瘤方面的应用报道较少.我院从2003年4月至2010年3月间共施行了8例腹腔镜辅助下小肠切除术治疗小肠肿瘤,现将结果报告如下.