RESUMO
Modulation of regional growth within specific segments of the bowel may have clinical value for several gastrointestinal conditions. We therefore examined the effects of different dietary protein sources on regional gut growth and luminal growth factor bioactivity as potential therapies. Rats were fed for 14 days on isonitrogenous and isocaloric diets comprising elemental diet (ED) alone (which is known to cause gut atrophy), ED supplemented with casein or whey or a soya protein-rich feed. Effects on regional gut growth and intraluminal growth factor activity were then determined. Despite calorie intake being similar in all groups, soya rich feed caused 20% extra total body weight gain. Stomach weight was highest on soya and casein diets. Soya enhanced diet caused greatest increase in small intestinal weight and preserved luminal growth factor activity at levels sufficient to increase proliferation in vitro. Regional small intestinal proliferation was highest in proximal segment in ED fed animals whereas distal small intestine proliferation was greater in soya fed animals. Colonic weight and proliferation throughout the colon was higher in animals receiving soya or whey supplemented feeds. We conclude that specific protein supplementation with either soya, casein or whey may be beneficial to rest or increase growth in different regions of the bowel through mechanisms that include differentially affecting luminal growth factor bioactivity. These results have implications for targeting specific regions of the bowel for conditions such as Crohn's disease and chemotherapy.
Assuntos
Caseínas/metabolismo , Colo/crescimento & desenvolvimento , Colo/metabolismo , Intestino Delgado/crescimento & desenvolvimento , Intestino Delgado/metabolismo , Proteínas de Soja/metabolismo , Animais , Colo/lesões , Colo/fisiopatologia , Proteínas Alimentares , Alimentos Formulados/efeitos adversos , Intestino Delgado/lesões , Intestino Delgado/fisiopatologia , Masculino , Ratos , Ratos Sprague-Dawley , Regeneração , Glycine max/metabolismo , Soro do Leite/metabolismoRESUMO
The measurement of cell proliferation in vivo is usually carried out by the examination of static measures. These comprise the mitotic index or labeling indices using incorporation of DNA synthesis markers such as bromodeoxyuridine or tritiated thymidine, or intrinsic markers, such as Ki67 and proliferative cell nuclear antigen (PCNA). But static measures only provide a 'snapshot' of cell proliferation. Rate measures, including double labeling methods and the metaphase arrest method, can actually measure cell production rates but they are far less utilized at present. Transit times and migration rates can also be measured using pulse and chase labeling or by following the transit of labeled cells through the tissue. Simple indices of cell division can easily be confounded by concomitant changes in the compartment size and many alleged markers of proliferation have serious shortcomings, as the markers may be involved in multiple aspects of cell regulation. The complexities of studying proliferation in vivo are illustrated here with a focus on the gastrointestinal tract. Some of these methods can help elucidate the role of the stem cells and their relationship to label retaining cells. WIREs Dev Biol 2017, 6:e274. doi: 10.1002/wdev.274 For further resources related to this article, please visit the WIREs website.
Assuntos
Ciclo Celular/fisiologia , Proliferação de Células/fisiologia , Células Epiteliais/citologia , Animais , Células Epiteliais/metabolismo , Humanos , Cinética , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and 'roll' mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card, tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.
Assuntos
Colo/anatomia & histologia , Intestino Delgado/anatomia & histologia , Técnicas de Cultura de Órgãos/métodos , Focos de Criptas Aberrantes/patologia , Animais , Colite/patologia , Colo/citologia , Colo/patologia , Pólipos do Colo/patologia , Intestino Delgado/citologia , Intestino Delgado/patologia , Camundongos , Técnicas de Cultura de Órgãos/instrumentaçãoRESUMO
To assess effects of epidermal growth factor (EGF) and pegylated granulocyte colony-stimulating factor (P-GCSF; pegfilgrastim) administration on the cellular origin of renal tubular epithelium regenerating after acute kidney injury initiated by mercuric chloride (HgCl2 ). Female mice were irradiated and male whole bone marrow (BM) was transplanted into them. Six weeks later recipient mice were assigned to one of eight groups: control, P-GCSF+, EGF+, P-GCSF+EGF+, HgCl2 , HgCl2 +P-GCSF+, HgCl2 +EGF+ and HgCl2 +P-GCSF+EGF+. Following HgCl2 , injection tubular injury scores increased and serum urea nitrogen levels reached uraemia after 3 days, but EGF-treated groups were resistant to this acute kidney injury. A four-in-one analytical technique for identification of cellular origin, tubular phenotype, basement membrane and S-phase status revealed that BM contributed 1% of proximal tubular epithelium in undamaged kidneys and 3% after HgCl2 damage, with no effects of exogenous EGF or P-GCSF. Only 0.5% proximal tubular cells were seen in S-phase in the undamaged group kidneys; this increased to 7-8% after HgCl2 damage and to 15% after addition of EGF. Most of the regenerating tubular epithelium originated from the indigenous pool. BM contributed up to 6.6% of the proximal tubular cells in S-phase after HgCl2 damage, but only to 3.3% after additional EGF. EGF administration attenuated tubular necrosis following HgCl2 damage, and the major cause of this protective effect was division of indigenous cells, whereas BM-derived cells were less responsive. P-GCSF did not influence damage or regeneration.
Assuntos
Células da Medula Óssea/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Necrose do Córtex Renal/induzido quimicamente , Necrose do Córtex Renal/metabolismo , Cloreto de Mercúrio/efeitos adversos , Regeneração/fisiologia , Animais , Feminino , Humanos , Túbulos Renais/metabolismo , Masculino , CamundongosRESUMO
The Aurora family of kinases, play a fundamental role in cell division and are overexpressed in several cancers including colon. The activity of barasertib-hQPA, a selective inhibitor of Aurora-B kinase (ABK) was investigated in a range of preclinical models of gastrointestinal cancer. Treatment with barasertib-hQPA produced anti-proliferative and cytotoxic effects across a panel of human colorectal cancer (CRC) cell lines in vitro. Prodrug, barasertib [48-h subcutaneous (s.c.) infusion; 150 mg/kg/day] inhibited the growth of SW620, Colo205, HCT116 human colorectal tumor xenografts in nude mice significantly (Student's t-test, P<0.05, n=10-12 per group). Flow cytometric analysis of single cells from disaggregated barasertib-treated SW620 tumors revealed a decrease in phosphorylated histone H3 (phH3) and an increase in tumor cells with ≥4N DNA content P<0.05). The activity of barasertib was then examined in ApcMin/+ mice, a spontaneous model of early intestinal neoplasia. Macroscopic evaluation of the small intestine revealed that barasertib treatment [25 mg/kg intra-peritoneal (i.p.) Q1Dx4 each week for 3 weeks] of 8-week old ApcMin/+ mice produced a 39% reduction in macroadenoma number (P=0.02) and a 43% reduction in overall adenoma burden (P=0.02) compared with vehicle-treated controls. Quantification of microscopic adenomas revealed a >64% reduction in the number of adenomas spanning more than one villus. Histological analysis of these adenomas revealed a number of distinct changes in barasertib-treated ApcMin/+ mice, including a 94% reduction in the proportion of phospho-histone H3-positive cells (P<0.001) and a 53% reduction in the number of cells per adenoma (P=0.001). These results provide a scientific rationale for investigating ABK inhibitors as a treatment for intestinal cancer.
Assuntos
Aurora Quinase B/biossíntese , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Gastrointestinais/genética , Animais , Aurora Quinase B/antagonistas & inibidores , Aurora Quinase B/genética , Neoplasias Colorretais/patologia , Neoplasias Gastrointestinais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Histonas/genética , Humanos , Camundongos , Fosforilação , Quinazolinas/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The Apc(MIN/+) mouse is a well-characterised model of intestinal tumourigenesis in which animals develop macroscopically detectable adenomas. However, most of the adenomas are formed in the small intestine and resolution of events in the colon, the most relevant site for human disease, is limited. Inducing colitis with dextran sodium sulphate (DSS) can selectively enhance the development of lesions in the colon. We demonstrated that a DSS pre-treatment is well tolerated and effective at inducing colon adenomas in an Apc(MIN/+) mouse model. We then investigated the effect of inhibiting vascular endothelial growth factor (VEGFR)- and epidermal growth factor receptor (EGFR)-dependent signalling pathways on the development of adenomas induced in DSS-pretreated (DSS/Apc(MIN/+)) or non-DSS-pretreated (Apc(MIN/+)) mice using vandetanib (ZD6474), a potent and selective inhibitor of VEGFR and EGFR tyrosine kinase activity. Eight-week old Apc(MIN/+) mice were given either drinking water or 1.8% DSS and then vandetanib (ZD6474) (50 mg/kg/day) or vehicle by oral gavage for 28 days and sacrificed 24 h after the last dose and assessed for adenoma formation in the intestines. DSS pre-treatment was well tolerated and significantly enhanced formation of adenomas in the colon of control Apc(MIN/+) mice. Vandetanib treatment significantly reduced adenoma formation in the small intestine by 68% (P=0.001) and the colon by 77% (from 13.8 to 3.1, P=0.01) of DSS-pretreated Apc(MIN/+) mice. In the Apc(MIN/+) group, vandetanib also reduced the mean number of adenomas in the small intestine by 76% (P<0.001) and in the colon by 60% (from 3.9 to 1.5, P=0.1). DSS-pre-treatment increased the resolution of the model, allowing us to confirm statistically significant effects of vandetanib on the development and growth of colon adenomas in the Apc(MIN/+) mouse. Moreover these preclinical data provide a rationale for studying the effects of vandetanib in early stages of intestinal cancer in the clinic.
Assuntos
Adenoma/prevenção & controle , Antineoplásicos/farmacologia , Colite/induzido quimicamente , Neoplasias do Colo/prevenção & controle , Sulfato de Dextrana , Genes APC , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Adenoma/induzido quimicamente , Adenoma/enzimologia , Adenoma/genética , Adenoma/patologia , Animais , Colite/complicações , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/enzimologia , Intestino Delgado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , beta Catenina/metabolismoRESUMO
OBJECTIVES: To evaluate the role of GH in colon carcinogenesis, we examined the formation of aberrant crypt foci (ACFs) and tumor development in wild type (WT) and GH-deficient, spontaneous dwarf rats (SDRs) exposed to the carcinogen azoxymethane (AOM). DESIGN: ACF were quantified by stereomicroscopy and tumor number and weights were recorded for each animal. Cell proliferation was measured by vincristine metaphase arrest, flow cytometry, and bromodeoxyuridine (BrdU) incorporation. Apoptosis was measured by TUNEL staining and cleaved caspase-3 immunohistochemistry. IGF-I was measured by radioimmunoassay (RIA). Hexokinase activity was measured by spectrophotometric assay. PARP cleavage, and IGF-IR, and p27(kip/cip) expression were measured by Western blotting. RESULTS: ACFs detected by stereomicroscopy were markedly reduced ( approximately 85%) in SDRs vs. WT rats at 10, 25, and 28 weeks after AOM. Tumor incidence, number, and weight also were reduced in SDR vs. WT animals. AOM treatment increased cell proliferation in the distal colon (where tumors occur) of WT rats but not SDRs, and these changes corresponded to increased ACF and tumor formation. Apoptosis rates were similar in AOM-treated WT and SDRs. Alterations in serum IGF-I levels may contribute to differences in the proliferative response to AOM and decreased ACF formation in SDR vs. WT rats. CONCLUSIONS: We conclude that early neoplastic lesions (ACFs) were reduced in GH-deficient animals. This effect corresponds with differences in AOM-induced proliferation, but not apoptosis. These data indicate that GH is required for the full effect of AOM on colon ACF and tumor development, and that the SDR rat is a promising model for studies regarding the role of GH/IGF system in the initiation and promotion of colon cancer.
Assuntos
Azoximetano/toxicidade , Carcinógenos/toxicidade , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Hormônio do Crescimento/genética , Animais , Apoptose , Neoplasias do Colo/patologia , Suscetibilidade a Doenças , Hormônio do Crescimento/metabolismo , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
In this study, we have used metabolic profiling (metabolomics/metabonomics) via high resolution magic angle spinning (HRMAS) and solution state (1)H NMR spectroscopy to characterize small bowel and colon tissue from the Apc(Min/+) mouse model of early gastrointestinal (GI) tumorigenesis. Multivariate analysis indicated the presence of metabolic differences between the morphologically normal/non-tumor tissue from approximately 10 week-old Apc(Min/+) mice and their wild-type litter mates. The metabolic profile of isolated lamina propria and epithelial cells from the same groups could also be discriminated on the basis of genotype. Accounting for systematic variation in individual metabolite levels across different anatomical regions of the lower GI tract, the metabolic phenotype of Apc(Min/+) lamina propria tissue was defined by significant increases in the phosphocholine/glycerophosphocholine ratio (PC/GPC, +21%) and decreases in GPC (-25%) and the gut-microbial cometabolite dimethylamine (DMA, -40%) relative to wild type. In the whole tissue, elevated lactate (+15%) and myo-inositol (+19%) levels were detected. As the metabolic changes occurred in non-tumor tissue from animals of very low tumor burden (<2 polyps/animal), they are likely to represent the specific consequence of reduced Apc function and very early events in tumorigenesis. The observed increase in PC/GPC ratio has been previously reported with immortalisation and malignant transformation of cells and is consistent with the role of Apc as a tumor suppressor. Phospholipase A2, which hydrolyses phosphatidylcholine to Acyl-GPC, is a known modifier gene of the model phenotype (Mom1), and altered expression of choline phospholipid enzymes has been reported in gut tissue from Apc(Min/+) mice. These results indicate the presence of a metabolic phenotype associated with "field cancerization", highlighting potential biomarkers for monitoring disease progression, for early evaluation of response to chemoprevention, and for predicting the severity of the polyposis phenotype in the Apc(Min/+) model.
Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Transformação Celular Neoplásica/metabolismo , Neoplasias Gastrointestinais/metabolismo , Mucosa/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Colo/metabolismo , Colo/patologia , Dimetilaminas/metabolismo , Células Epiteliais/metabolismo , Inositol/metabolismo , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Ácido Láctico/metabolismo , Espectroscopia de Ressonância Magnética , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Fosforilcolina/metabolismo , Lesões Pré-Cancerosas/metabolismoRESUMO
Sulforaphane (SF; 4-methylsulfinylbutyl isothiocyanate), a dietary compound derived from broccoli, may exhibit chemopreventive properties by inducing cell cycle arrest via induction of cyclin-dependent kinase inhibitor 1A (p21(waf1/cip1)), but the exact molecular mechanism has not been determined. Here we evaluate the role of the transcription factor Kruppel-like factor 4 (KLF4) in mediating the induction of p21(waf1/cip1) and cellular differentiation by SF and iberin (IB; 3-methylsulphinyl propyl isothiocyanate), also derived from broccoli. Exposure of Caco-2 and Caco-2/TC7 cells to SF and IB increased expression of both KLF4 and p21(waf1/cip1), whereas exposure of HT29 cells resulted only in induction of p21(waf1/cip1). In Caco-2 cells, small interfering RNA knock down of KLF4 expression attenuated induction of p21(waf1/cip1) in response to either SF or IB treatment. Contrary to expectation, prolonged exposure to SF reduced sucrase isomaltase activity, a marker of small intestinal differentiation in Caco-2 cells. Additional support for the SF-mediated induction of p21(waf1/cip1) by KLF4 was obtained from analyses of gastric tissue of Apc(Min/+) mice following acute intervention with SF but not from the analyses of other tissue of the intestinal tract. These results suggest that induction of p21(waf1/cip1) by SF or IB may be partly mediated by KLF4 in some colon cancer cells and tissues.
Assuntos
Ciclo Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Isotiocianatos/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , Tiocianatos/farmacologia , Animais , Brassica/química , Células CACO-2 , Diferenciação Celular , Linhagem Celular , Genes APC , Inibidores do Crescimento/metabolismo , Células HT29 , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Complexo Sacarase-Isomaltase/metabolismo , SulfóxidosRESUMO
P-cadherin is normally expressed in the basal layer of squamous epithelia and absent from the healthy intestine and colon. We have previously shown it to be expressed in all inflamed, hyperplastic, and dysplastic intestinal and colonic mucosa. This study aimed to better understand the mechanisms controlling the expression of P-cadherin and the biological effects of its ectopic presence in the intestine and colon. We investigated the CpG methylation status of the P-cadherin (CDH3) promoter and P-cadherin mRNA and protein expression in cases of familial and sporadic colorectal cancer (CRC). The CDH3 promoter was hypomethylated in colonic aberrant crypt foci, in CRC, and, occasionally, in the normal epithelium adjacent to cancer, demonstrating a potential "field effect" of cancerization. The hypomethylation was also associated with induction of P-cadherin expression in the neoplastic colon (P < 0.0001). We then created transgenic mice that overexpressed P-cadherin specifically in the intestinal and colonic epithelium under the liver fatty acid binding protein promoter. Forced ectopic expression of P-cadherin accompanied by indomethacin-induced inflammation resulted in a 3-fold higher crypt fission rate within the small and large intestines in the homozygous mice compared with the wild-type animals (P < 0.02). We conclude that epigenetic demethylation of the P-cadherin promoter in the human intestine permits its ectopic expression very early in the colorectal adenoma-carcinoma sequence and persists during invasive cancer. Induced P-cadherin expression, especially in mucosal damage, leads to an increased rate of crypt fission, a common feature of clonal expansion in gastrointestinal dysplasia.
Assuntos
Adenoma/genética , Caderinas/genética , Proliferação de Células , Neoplasias Colorretais/genética , Metilação de DNA , Mucosa Intestinal/patologia , Regiões Promotoras Genéticas , Adenoma/metabolismo , Adenoma/patologia , Animais , Caderinas/metabolismo , Divisão Celular/genética , Divisão Celular/fisiologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Fatores de TempoRESUMO
BACKGROUND AND AIMS: The relationship between obesity, weight reduction, and future risk of colorectal cancer is not well understood. Therefore, we compared mucosal biomarkers in normal weight individuals [body mass index (BMI), 18.5-24.9 kg/m(2)] with those in morbidly obese patients (BMI >40 kg/m(2)) before and 6 months after Roux-en-Y gastric bypass (RYGB). METHODS: Rectal epithelial cell mitosis, crypt area, and crypt branching were measured following whole crypt microdissection. Apoptosis was measured by immunohistochemistry for neo-cytokeratin 18 on fixed tissue sections. Serum levels of C-reactive protein and cytokines were assayed in combination with quantification of mucosal proinflammatory gene expression by real-time RT-PCR. RESULTS: Twenty-six morbidly obese patients (mean BMI, 54.4 kg/m(2)) had significantly increased mitosis, crypt area, and crypt branching (all P < 0.01) compared with 21 age- and sex-matched normal weight individuals (mean BMI, 22.5 kg/m(2)). Morbidly obese patients underwent a mean excess weight loss of 41.7% at a mean of 26 weeks after RYGB. Surprisingly, this was associated with a further increase in mitosis and decreased apoptosis of epithelial cells. At the same time, lower levels of serum C-reactive protein and interleukin-6 following RYGB were accompanied by a reduction in mucosal IL-6 protein content but elevated mucosal expression of other proinflammatory genes such as cyclooxygenase-1 and cyclooxygenase-2. CONCLUSIONS: Mucosal biomarkers, accepted as indicators of future colorectal cancer risk, are increased in morbidly obese patients compared with normal weight controls. The hyperproliferative state that exists 6 months after RYGB may have important implications for long-term colorectal cancer risk in bariatric surgery patients.
Assuntos
Biomarcadores Tumorais/análise , Colo/citologia , Derivação Gástrica , Obesidade Mórbida/cirurgia , Reto/citologia , Adulto , Apoptose , Proteína C-Reativa/análise , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Neoplasias Colorretais/patologia , Citocinas/análise , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Mitose , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não ParamétricasRESUMO
Both the epidermal growth factor (EGF) and the vascular endothelial growth factor (VEGF) pathways are associated with intestinal cancer, and therapeutic approaches targeting either EGF receptor (EGFR) or VEGF receptor (VEGFR) signaling have recently been approved for patients with advanced colorectal cancer. The Apc(Min/+) mouse is a well-characterized in vivo model of intestinal tumorigenesis, and animals with this genetic mutation develop macroscopically detectable adenomas from approximately 6 weeks of age. Previous work in the Apc(Min/+) mouse has shown that therapeutic approaches targeting either VEGFR or EGFR signaling affect predominantly the size or number of adenomas, respectively. In this study, we have assessed the effect of inhibiting both these key pathways simultaneously using ZD6474 (Vandetanib, ZACTIMA), a selective inhibitor of VEGFR and EGFR tyrosine kinases. To assess the effects of ZD6474 on early- and later-stage disease, treatment was initiated in 6- and 10-week-old Apc(Min/+) mice for 28 days. ZD6474 markedly reduced both the number and the size of polyps when administered at either an early or a later stage of polyp development. This reduction in both adenoma number and size resulted in a total reduction in tumor burden in the small intestine of nearly 75% in both studies (P < 0.01). The current data build on the concept that EGFR-dependent tumor cell proliferation and VEGF/VEGFR2-dependent angiogenesis and survival are distinct key mechanisms in polyp development. Pharmacologic inhibition of both signaling pathways has significant antitumor effects at both early and late stages of polyp development. Therefore, targeting both VEGFR- and EGFR-dependent signaling may be a beneficial strategy in early intestinal cancer.
Assuntos
Adenoma/patologia , Receptores ErbB/metabolismo , Genes APC , Neoplasias Intestinais/patologia , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transdução de Sinais , Adenoma/genética , Adenoma/metabolismo , Animais , Feminino , Neoplasias Intestinais/genética , Neoplasias Intestinais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Fermentation of carbohydrates in the colon can stimulate cell proliferation and could thus be a cancer risk. The effects of resistant carbohydrates, i.e. those not digested and absorbed in the small intestine, on cell proliferation, crypt fission and polyp development were investigated in wild-type and adenomatous polyposis coli multiple intestinal neoplasia (Apc(Min/+)) mice. Fifteen 4-week-old female wild-type and fifteen Apc(Min/+) mice were used for each group and fed a chow diet, a semi-synthetic diet or the semi-synthetic supplemented with wheat bran or an apple pomace preparation, both high in resistant carbohydrates, for 8 weeks. Tissue from all mice was used to measure cell proliferation and crypt fission and tissue from the Apc(Min/+) mice was scored for polyp number and tumour burden. There were slight reductions in intestinal mass in the mice fed the semi-synthetic diets and this was increased by the inclusion of resistant carbohydrates. The Apc(Min/+) mice had elevated cell proliferation and crypt fission in the distal small intestine and colon and these were increased by the resistant carbohydrates. Bran or apple pomace significantly increased polyp number in the proximal third of the small intestine. Apple pulp more than doubled polyp number throughout the small bowel (99.2 (SEM 11.1) v. 40.0 (SEM 8.2), P<0.004). Bran and apple pomace increased polyp diameter and hence burden in the colon by 243 and 150 %, respectively (P<0.05). In conclusion, both types of resistant carbohydrates increased polyp number and tumour burden and this was associated with elevated epithelial cell proliferation and crypt fission.
Assuntos
Polipose Adenomatosa do Colo/patologia , Colo , Fibras na Dieta/administração & dosagem , Mucosa Intestinal/patologia , Pólipos Intestinais/patologia , Intestino Delgado , Animais , Proliferação de Células , Feminino , Fermentação , Genes APC , Malus , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Modelos Animais , Tamanho do Órgão , Baço/patologiaRESUMO
Pancreatic secretory trypsin inhibitor (PSTI) is a serine protease inhibitor, expressed in gut mucosa, whose function is unclear. We, therefore, examined the effects of PSTI on gut stability and repair. Transgenic mice overexpressing human PSTI within the jejunum (FABPi(-1178 to +28) hPSTI construct) showed no change in baseline morphology or morphometry but reduced indomethacin-induced injury in overexpressing hPSTI region by 42% (P < 0.01). Systemic recombinant hPSTI did not affect baseline morphology or morphometry but truncated injurious effects in prevention and recovery rat models of dextran-sodium-sulfate-induced colitis. In vitro studies showed PSTI stimulated cell migration but not proliferation of human colonic carcinoma HT29 or immortalized mouse colonic YAMC cells. PSTI also induced changes in vectorial ion transport (short-circuit current) when added to basolateral but not apical surfaces of polarized monolayers of Colony-29 cells. Restitution and vectorial ion transport effects of PSTI were dependent on the presence of a functioning epidermal growth factor (EGF) receptor because cells with a disrupted (EGFR(-/-) immortalized cells) or neutralized (EGFR blocking antibodies or tyrosine kinase inhibitor) receptor prevented these effects. PSTI also reduced the cytokine release of lipopolysaccharide-stimulated dendritic cells. We conclude that administration of PSTI may provide a novel method of stabilizing intestinal mucosa against noxious agents and stimulating repair after injury.
Assuntos
Proteínas de Transporte/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Animais , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Linhagem Celular , Movimento Celular , Proliferação de Células , Colite/induzido quimicamente , Colite/patologia , Colite/prevenção & controle , Células Dendríticas/fisiologia , Sulfato de Dextrana , Receptores ErbB/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Humanos , Indometacina , Mucosa Intestinal/fisiologia , Transporte de Íons , Doenças do Jejuno/induzido quimicamente , Doenças do Jejuno/metabolismo , Doenças do Jejuno/patologia , Masculino , Camundongos , Camundongos Transgênicos , Fosforilação , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Inibidor da Tripsina Pancreática de KazalAssuntos
Dieta , Fibras na Dieta/administração & dosagem , Intestinos/efeitos dos fármacos , Dieta/normas , Fibras na Dieta/metabolismo , Fermentação , Aditivos Alimentares/administração & dosagem , Aditivos Alimentares/efeitos adversos , Aditivos Alimentares/metabolismo , Frutas , Humanos , Intestinos/fisiologia , Pectinas/administração & dosagem , Pectinas/efeitos adversos , Pectinas/metabolismo , Verduras , Aumento de PesoRESUMO
BACKGROUND AND AIMS: Neonatal necrotizing enterocolitis (NEC) is a common and serious acquired gastrointestinal tract condition. This clinical study assessed the potential clinical efficacy and microscopic effects of recombinant human epidermal growth factor 1-48 (EGF(1-48)) in neonates with NEC. METHODS: This prospective, double-blind, randomized controlled study included 8 neonates with NEC. The study compared the effects of a 6-day continuous intravenous infusion of EGF(1-48) at 100 ng kg(-1) h(-1) against placebo. Clinical outcomes and morphological evaluation of serial rectal mucosal biopsies were assessed at baseline and 4, 7, and 14 days after starting EGF infusions. RESULTS: There was no difference between the clinical safety outcomes recorded for EGF(1-48) or placebo patients. Quantitative morphologic differences in the rectal mucosa biopsies were noted with EGF(1-48) treatment compared with baseline or placebo and included a statistically significant increase in the number of mitoses per mucosal crypt on study day 4, significantly increased thickness of rectal mucosa from baseline on study days 4 and 7, and increased crypt surface area of rectal mucosa in parallel with increased mucosa thickness on day 14. CONCLUSION: This study of EGF(1-48) in neonates with severe NEC showed that growth factor treatment was well tolerated and produced positive and measurable remodeling trophic effects on the gastrointestinal mucosa.
Assuntos
Enterocolite Necrosante/tratamento farmacológico , Fator de Crescimento Epidérmico/farmacologia , Fármacos Gastrointestinais/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Método Duplo-Cego , Enterocolite Necrosante/fisiopatologia , Fator de Crescimento Epidérmico/uso terapêutico , Feminino , Fármacos Gastrointestinais/uso terapêutico , Humanos , Lactente , Recém-Nascido , Mucosa Intestinal/fisiopatologia , Masculino , Fragmentos de Peptídeos/uso terapêutico , Estudos ProspectivosRESUMO
Novel therapies for the treatment of MOF (multiple organ failure) are required. In the present study, we examined the effect of synthetic GHRP-6 (growth hormone-releasing peptide-6) on cell migration and proliferation using rat intestinal epithelial (IEC-6) and human colonic cancer (HT29) cells as in vitro models of injury. In addition, we examined its efficacy when given alone and in combination with the potent protective factor EGF (epidermal growth factor) in an in vivo model of MOF (using two hepatic vessel ischaemia/reperfusion protocols; 45 min of ischaemia and 45 min of reperfusion or 90 min of ischaemia and 120 min of reperfusion). In vitro studies showed that GHRP-6 directly influenced gut epithelial function as its addition caused a 3-fold increase in the rate of cell migration of IEC-6 and HT29 cells (P<0.01), but did not increase proliferation ([3H]thymidine incorporation). In vivo studies showed that, compared with baseline values, ischaemia/reperfusion caused marked hepatic and intestinal damage (histological scoring), neutrophilic infiltration (myeloperoxidase assay; 5-fold increase) and lipid peroxidation (malondialdehyde assay; 4-fold increase). Pre-treatment with GHRP-6 (120 microg/kg of body weight, intraperitoneally) alone truncated these effects by 50-85% (all P<0.05) and an additional benefit was seen when GHRP-6 was used in combination with EGF (1 mg/kg of body weight, intraperitoneally). Lung and renal injuries were also reduced by these pre-treatments. In conclusion, administration of GHRP-6, given alone or in combination with EGF to enhance its effects, may provide a novel simple approach for the prevention and treatment of MOF and other injuries of the gastrointestinal tract. In view of these findings, further studies appear justified.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/uso terapêutico , Insuficiência de Múltiplos Órgãos/prevenção & controle , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/farmacologia , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Insuficiência de Múltiplos Órgãos/metabolismo , Insuficiência de Múltiplos Órgãos/patologia , Infiltração de Neutrófilos/efeitos dos fármacos , Oligopeptídeos , Ratos , Ratos Wistar , Proteínas Recombinantes/farmacologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Células Tumorais CultivadasRESUMO
BACKGROUND AND AIMS: The gastrointestinal trefoil factor family (TFF1, TFF2, TFF3) peptides are considered to play an important role in maintaining the integrity of the mucosa. The physiological role of TFF2 in the protection of the GI tract was investigated in TFF2 deficiency. METHODS: TFF2-/- mice were generated and differential expression of various genes was assessed by using a mouse expression microarray, quantitative real time PCR, Northern blots or immunohistochemistry. RESULTS: On an mRNA level we found 128 differentially expressed genes. We observed modulation of a number of crucial genes involved in innate and adaptive immunity in the TFF2-/- mice. Expression of proteasomal subunits genes (LMP2, LMP7 and PSMB5) involved in the MHC class I presentation pathway were modulated indicating the formation of immunoproteasomes improving antigen presentation. Expression of one subunit of a transporter (TAP1) responsible for importing degraded antigens into ER was increased, similarly to the BAG2 gene that modulates chaperone activity in ER helping proper loading on MHC class I molecules. Several mouse defensin (cryptdin) genes coding important intestinal microbicidal proteins were up-regulated as a consequence of TFF2 deficiency. Normally moderate expression of TFF3 was highly increased in stomach.
Assuntos
Sistema Digestório/imunologia , Sistema Digestório/metabolismo , Mucinas/deficiência , Proteínas Musculares/deficiência , Peptídeos/deficiência , Animais , Apresentação de Antígeno , Sequência de Bases , Primers do DNA/genética , Perfilação da Expressão Gênica , Marcação de Genes , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucinas/genética , Mucinas/imunologia , Proteínas Musculares/genética , Proteínas Musculares/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/genética , Peptídeos/imunologia , Complexo de Endopeptidases do Proteassoma/genética , Precursores de Proteínas/genética , Fator Trefoil-2RESUMO
BACKGROUND: Parenteral nutrition and the absence of luminal feeding result in impaired intestinal growth and differentiation of enterocytes. Glucagon-like peptide 2 (GLP-2) and epidermal growth factor (EGF) have each been shown to have trophic effects on the intestine, and thus have the potential to benefit patients fed parenterally, such as those with intestinal failure from short bowel syndrome. We report studies aimed to determine whether there may be synergistic effects of these 2 peptides. METHODS: Rats were established on parenteral nutrition (PN) and infused for 6 days with GLP-2 (20 microg/d), EGF (20 microg/d), or GLP-2 + EGF (20 microg/d of each). These groups were compared with untreated PN-fed and orally-fed controls. Tissue was obtained from small intestine and colon to determine growth, proliferation, and representative gene expression. RESULTS: Small intestinal weight was increased by 75%, 43%, and 116% in the GLP-2, EGF, and GLP-2 + EGF groups, respectively, compared with PN controls (all p < .001). Cell proliferation increased with GLP-2, EGF, and GLP-2 + EGF in proximal small intestine by factors of 2.3, 1.7, and 3.4 respectively (p < .001). A synergistic effect on villous and crypt area was observed in the proximal small intestine when GLP-2 and EGF were combined (p < .05). GLP-2 had no effect in the colon, unlike EGF. Further studies showed GLP-2 + EGF significantly increased expression in distal small intestine of transcripts for the bile acid transport protein IBABP (p < .05) and showed a significant correlation between the expression of IBABP and the transcription factor HNF-4. CONCLUSIONS: Both GLP-2 and EGF upregulate growth of the small intestine, and this is augmented when GLP-2 and EGF are combined. These findings may lead to improved treatment of patients receiving PN.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Intestino Delgado/efeitos dos fármacos , Nutrição Parenteral , Peptídeos/farmacologia , Síndrome do Intestino Curto/terapia , Adaptação Fisiológica , Animais , Divisão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Sinergismo Farmacológico , Peptídeo 2 Semelhante ao Glucagon , Peptídeos Semelhantes ao Glucagon , Intestino Delgado/crescimento & desenvolvimento , Intestino Delgado/patologia , Intestino Delgado/cirurgia , Masculino , Tamanho do Órgão , Distribuição Aleatória , Ratos , Ratos WistarRESUMO
The increase in gastrointestinal epithelial tissue mass and the development of the gut can occur through three main mechanisms, namely elevated cell production from the intestinal crypts, by raised crypt number, which occurs through the process of crypt fission or by altered apoptosis. The small bowel and the colon have various rates of these, which were studied in rats of various ages. Wistar rats were fed ad libitum, and were killed at 3, 4, 6, 9, 12, 18, 26 and 48 weeks of age. Tissue was later stained and microdissected and the number of native mitoses and apoptotic figures per crypt and the percentage of crypts in fission were determined. There was an almost linear increase in body weight from 3 to 9 weeks, followed by a more gradual rise until 18 weeks. The weight of the stomach and the small intestine reached maximum values at 9 weeks, whereas the caecum and the colon approached this at 12 weeks. Mitotic activity per crypt in the small intestine increased from 3.8 +/- 0.1 at 3 weeks to 7.8 +/- 0.4 mitoses per crypt (P < 0.001) at 9 weeks and then decreased slightly; crypt fission increased from 4.6% +/- 0.8 at 3 weeks to 8.4 +/- 0.9% at 6 weeks and then decreased gradually reaching a value of 1.5 +/- 0.4% at 48 weeks. Apoptosis also peaked at 6 weeks and was then very low. In the colon, the proliferation decreased from 4.2 +/- 0.2 mitoses per crypt in the young (3 weeks) rat and reached a plateau by 9 weeks (2.5 +/- 0.1 mitoses per crypt, P < 0.001). Crypt fission also declined rapidly in the first 9 weeks (from 67.6 +/- 4.2 to 23.1 +/- 4.6%, P < 0.01) and then continued to decline, although at a lower rate. The crypt fission index at 48 weeks was 9.8 +/- 1.0. Apoptosis in the colon persisted throughout the duration of the study, 0.19 +/- 0.06 apoptotic bodies per crypt were seen at week 48. The development of the small intestine is more dependent on cell proliferation, whereas in the colon crypt fission is far more predominant, with the colon having fission indices approximately six times greater than those of the small intestine. Proliferative activity in the colon was approximately half that of the small intestine.