Recipient Organization
TRUSTEES OF TUFTS COLLEGE
136 HARRISON AVE
Boston,MA 021111817
Performing Department
Vitamins & Carcinogenesis
Non Technical Summary
Colorectal cancer (CRC) remains a major public health problem in the US - with approximately 135,000 new cases being diagnosed and 50,000 deaths from this disease each year. Although several agents are reported to reduce cancer formation in laboratory rodents, aspirin remains the only agent with robust evidence in human populations - however its adverse side effects may outweigh its benefits. Thus, there is a clear need to identify new, effective and safe strategies to prevent CRC.Diet is a major determinant of CRC risk - recent estimates indicate that up to 38% of all CRC cases can be attributed to poor diet. The consensus of several human population studies is that consumption of a Mediterranean diet (MD) is associated with a significantly reduced risk for CRC. The MD is characterized by a high intake of vegetables, legumes, fruits, nuts, grains, fish, seafood, olive oil, and moderate intake of red wine.Changes in the composition of the gut bacteria play a role in CRC formation. Moreover, the composition of the gut bacteria is also sensitive to diet. The goal of our current studies is to understand the involvement of gut bacteria in CRC prevention by MD and whether the CRC protective effect of MD can be enhanced by giving specific bacteria with known anti-cancer propertiesWe will achieve this goal by conducting two experiments utilizing a mouse model of CRC. Mice will be fed standard diet or a Mediterranean and undergo various bacteria treatments including depletion, depletion with adding back inflammatory bacteria components and supplementation with protective bacteria.Successful completion of these studies will enhance our understanding of how MD protects against CRC and may outline a novel approach to further enhance the protective effect of MD.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Colorectal cancer (CRC) remains a major public health burden in the US - with approximately 135,000 new cases being diagnosed and 50,000 deaths from this disease each year. Our long-term goal is to create knowledge that will contribute to the development of strategies to reduce the burden of this disease on our society.The Mediterranean diet (MD) is one of the dietary patterns most strongly associated with a reduced risk for CRC. Disturbances in the composition of gut microbial communities are also etiologically involved in CRC. Given that gut microbial populations are sensitive to diet, our lack of understanding of how the MD affects the gut microbiome, and the physiological significance of these effects, is surprising.The major goal of this project is to understand the role of the gut microbiome in the chemopreventive effect of the MDThe specific objectives of this proposal are to:To understand the contribution of gut microbiota to the chemopreventive effect of MD.To determine if the chemopreventive effect of MD is enhanced by Pd.To determine if MD increases the abundance of Pd in the gut.
Project Methods
The objectives of this proposal will be addressed by conducting two mouse experiments, each with a 2 x 3 factorial design. The first experiment will address Aim 1 and will involve randomizing wildtype C57BL6/J mice to receive either of two diets (control, or Mediterranean Diet) and either of three gut microbiota manipulations (control, microbiota depletion or microbiota depletion with restored inflammatory capacity). The second experiment will address Aims 2 and 3 and will involve randomizing wildtype C57BL6/J mice to receive either of two diets (control, or Mediterranean Diet) and either of three gut microbiota manipulations (control, freeze dried Pd or live Pd).Mice will be kept on a 12hr light/dark cycle and food and water provided ad libitum.Experiment 1: Mice will be randomized into one of six experimental groups 1) control diet; 2) control diet with microbiota depletion; 3) control diet with microbiota depletion and restored inflammatory capacity; 4) Mediterranean diet; 5) Mediterranean diet with microbiota depletion; 6) Mediterranean diet with microbiota depletion and restored inflammatory capacity. The groups undergoing gut microbiota depletion will be provided an antibiotic (Abx) cocktail of streptomycin (2 g/l), gentamicin (0.5 g/l), bacitracin (1 g/l) and ciprofloxacin (0.125 g/l) in autoclaved water ad libitum. LPS will be added in drinking water to at a concentration of 60 mg/ml to restore the inflammatory milieu of Abx treat mice in groups 3 and 6.Experiment 2: Mice will be randomized into one of six experimental groups 1) control diet; 2) control diet supplemented with freeze dried Parabacteroides distasonis, 3) control diet and weekly gavage with live Parabacteroides distasonis; 4) Mediterranean diet; 5) Mediterranean diet supplemented with freeze dried Parabacteroides distasonis; 6) Mediterranean diet and weekly gavage with live Parabacteroides distasonis.Four weeks after beginning diets, mice will be given one dose of the colorectal carcinogen azoxymethane (AOM. 10mg/kg IP) to initiate tumorigenesis. On the fifth (days 35-42) and eighth (days 56-63) weeks mice will be given dextran sulfate sodium (DSS. MWt 36-50 kDa) in drinking water (3%) to promote colitis.Mice will be euthanized after 12 weeks, which is 8 weeks after the AOM injection and 3 weeks after the second DSS cycle. To assess gut "leakiness" all mice will be administered 0.6 mg/g FITC-dextran four hours prior to euthanasia by oral gavage. Mice will be weighed weekly throughout the protocol. We will assess body composition by MRI in the 11th week of the protocol. Stool will be collected weekly and stored frozen. Microbiota depletion in Abx treated mice will be confirmed by routine stool culture and PCR with universal primers.Mice will be euthanized by isoflurane anesthesia combined with exsanguination by cardiac puncture (EDTA tubes) and finally cervical dislocation. The abdomen will be opened and liver and spleen weighed, frozen in liquid N2 and stored at -80°C. The large intestine and cecum will be removed, measured and contents removed and stored. The intestines will then be flushed, opened longitudinally and rinsed thoroughly in cold PBS with protease inhibitors.Colons will be scrutinized under a dissecting microscope for the presence of tumors by an observer unaware to the groups. Tumors will be measured, excised and fixed for later confirmation and grading by a rodent histopathologist.A 0.5cm section will be cut from the center of the colon and fixed in formalin for later histologic analyses. The remaining colon will be scraped with glass slides to collect the mucosa which will then be stored frozen for later analysis of inflammatory markers.Total protein will be isolated from colonic mucosal scrapings with RIPA buffer and quantified using the BCA assay. Cytokines (IL-1b, Tnfa, IL-2, IL-6, IL-8, IL-10, Il-17, Tgfb, Ifng) will be measured in mucosa by ELISA. Mediators of pro-inflammatory signaling MyD88 and IkB phosphorylation (ser32) will be measured in mucosa by western blotting with GAPDH as loading control. Tight junction proteins ZO-1 and occludin will be measured in mucosa by western blotting. Active (non-phospho) b-catenin will be also be measured by western blot.Multiple microscope slides will be prepared from the fixed and embedded colon tissue section: H&E stained slides will be evaluated for histologic inflammation and Disease Activity Index calculated according to established criteria. To assess inflammatory signaling nuclear NF-kB (p65) will be measured by immunohistochemistry. Proliferation and apoptosis will be measured by IHC for Ki-67 and cleaved caspase 3.To assess gut leakiness we will measure blood FITC concentrations (from gavage) in serum with a microplate reader at an excitation/emission wavelength of 485/528nm. In addition, serum endotoxin (LPS) will be measured by the Limulus Amebocyte Lysate assay. Fecal calprotectin will also be measured at 0, 4, 9 and 12 weeks as an additional measure of colonic inflammation.Gut microbial composition will be assessed in stool by 16S rRNA sequencing.