David C. Montrose

Genetic Deletion of mPGES-1 Suppresses Intestinal Tumorigenesis

Elevated levels of prostaglandin E(2) (PGE(2)) are often found in colorectal cancers. Thus, nonsteroidal anti-inflammatory drugs, including selective cyclooxygenase-2 (COX-2) inhibitors, are among the most promising chemopreventive agents for colorectal cancer. However, their long-term use is restricted by the occurrence of adverse events believed to be associated with a global reduction in prostaglandin production. In the present study, we evaluated the chemopreventive efficacy of targeting the terminal synthase microsomal PGE(2) synthase 1 (mPGES-1), which is responsible for generating PGE(2), in two murine models of intestinal cancer. We report for the first time that genetic deletion of mPGES-1 in Apc-mutant mice results in marked and persistent suppression of intestinal cancer growth by 66%, whereas suppression of large adenomas (>3 mm) was almost 95%. This effect occurred despite loss of Apc heterozygosity and beta-catenin activation. However, we found that mPGES-1 deficiency was associated with a disorganized vascular pattern within primary adenomas as determined by CD31 immunostaining. We also examined the effect of mPGES-1 deletion on carcinogen-induced colon cancer. The absence of mPGES-1 reduced the size and number of preneoplastic aberrant crypt foci (ACF). Importantly, mPGES-1 deletion also blocked the nuclear accumulation of beta-catenin in ACF, confirming that beta-catenin is a critical target of PGE(2) procarcinogenic signaling in the colon. Our data show the feasibility of targeting mPGES-1 for cancer chemoprevention with the potential for improved tolerability over traditional nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors.

Anti-inflammatory effects of freeze-dried black raspberry powder in ulcerative colitis

Ulcerative colitis (UC) is a chronic inflammatory disease of the colonic mucosa that can dramatically increase the risk of colon cancers. In the present study, we evaluated the effects of a dietary intervention of freeze-dried black raspberries (BRB), a natural food product with antioxidant and anti-inflammatory bioactivities, on disease severity in an experimental mouse model of UC using 3% dextran sodium sulfate (DSS). C57BL/6J mice were fed either a control diet or a diet containing BRB (5 or 10%) for 7-14 days and then the extent of colonic injury was assessed. Dietary BRB markedly reduced DSS-induced acute injury to the colonic epithelium. This protection included better maintenance of body mass and reductions in colonic shortening and ulceration. BRB treatment, however, did not affect the levels of either plasma nitric oxide or colon malondialdehyde, biomarkers of oxidative stress that are otherwise increased by DSS-induced colonic injury. BRB treatment for up to 7 days suppressed tissue levels of several key pro-inflammatory cytokines, including tumor necrosis factor α and interleukin 1β. Further examination of the inflammatory response by western blot analysis revealed that 7 day BRB treatment reduced the levels of phospho-IκBα within the colonic tissue. Colonic cyclooxygenase 2 levels were also dramatically suppressed by BRB treatment, with a concomitant decrease in the plasma prostaglandin E₂ (276 versus 34 ng/ml). These findings demonstrate a potent anti-inflammatory effect of BRB during DSS-induced colonic injury, supporting its possible therapeutic or preventive role in the pathogenesis of UC and related neoplastic events.

CD13 is dispensable for normal hematopoiesis and myeloid cell functions in the mouse

The robust and consistent expression of the CD13 cell surface marker on very early as well as differentiated myeloid hematopoietic cells has prompted numerous investigations seeking to define roles for CD13 in myeloid cells. To address the function of myeloid CD13 directly, we created a CD13 null mouse and assessed the responses of purified primary macrophages or DCs from WT and CD13 null animals in cell assays and inflammatory disease models, where CD13 has been implicated previously. We find that mice lacking CD13 develop normally with normal hematopoietic profiles except for an increase in thymic but not peripheral T cell numbers. Moreover, in in vitro assays, CD13 appears to be largely dispensable for the aspects of phagocytosis, proliferation, and antigen presentation that we tested, although we observed a slight decrease in actin‐independent erythrocyte uptake. However, in agreement with our published studies, we show that lack of monocytic CD13 completely ablates anti‐CD13‐dependent monocyte adhesion to WT endothelial cells. In vivo assessment of four inflammatory disease models showed that lack of CD13 has little effect on disease onset or progression. Nominal alterations in gene expression levels between CD13 WT and null macrophages argue against compensatory mechanisms. Therefore, although CD13 is highly expressed on myeloid cells and is a reliable marker of the myeloid lineage of normal and leukemic cells, it is not a critical regulator of hematopoietic development, hemostasis, or myeloid cell function.

Planar spindle orientation and asymmetric cytokinesis in the mouse small intestine.

A major feature of epithelial cell polarity is regulated positioning of the mitotic spindle within the cell. Spindles in cells of symmetrically expanding tissues are predicted to align parallel to the tissue plane. Direct measurement of this alignment has been difficult in mammalian tissues. Here, we analyzed the position of spindles in intact mouse intestinal epithelium using microtubule immunofluorescence and three-dimensional confocal imaging. Mitotic cells were identified in the proliferative zone of intestinal crypts. Spindle angle relative to the apical cell surface was determined either by direct measurement from confocal images or with a computational algorithm. Angles averaged within 10° of parallel to the apical surface in metaphase and anaphase cells, consistent with robust planar spindle positioning, whereas spindles in prometaphase cells showed much greater angle variability. Interestingly, cytokinetic furrows appeared to extend from the basal cell surface toward the apical surface. This type of image analysis may be useful for studying the regulation of spindle position during tissue remodeling and tumor formation.

Selective PGE2 suppression impairs colon carcinogenesis and modifies local mucosal immunity

Prostaglandin E2 (PGE2) is a bioactive lipid that mediates a wide range of physiologic effects and plays a central role in inflammation and cancer. PGE2 is generated from arachidonic acid by the sequential actions of the COX and terminal synthases (PGES). Increased levels of COX-2, with a concomitant elevation of PGE2, are often found in colorectal cancers (CRC), providing the rationale for the use of COX-2 inhibitors for chemoprevention. Despite their proven efficacy in cancer prevention, however, COX-2 inhibitors exhibit dose-dependent toxicities that are mediated in part by their nonspecific reduction of essential prostanoids, thus limiting their chemopreventive benefit. To achieve enhanced specificity, recent efforts have been directed toward targeting the inducible terminal synthase in the production of PGE2, microsomal PGES (mPGES-1). In the present study, we show that genetic deletion of mPGES-1 affords significant protection against carcinogen-induced colon cancer. mPGES-1 gene deletion results in an about 80% decrease in tumor multiplicity and up to a 90% reduction in tumor load in the distal colon of azoxymethane (AOM)-treated mice. Associated with the striking cancer suppression, we have identified a critical role for PGE2 in the control of immunoregulatory cell expansion (FoxP3-positive regulatory T cells) within the colon-draining mesenteric lymph nodes, providing a potential mechanism by which suppression of PGE2 may protect against CRC. These results provide new insights into how PGE2 controls antitumor immunity. Cancer Prev Res; 4(8); 1198–208. ©2011 AACR.

Cytoplasmic Phospholipase A2 Levels Correlate with Apoptosis in Human Colon Tumorigenesis

Colon cancers often display perturbations in arachidonic acid metabolism, with elevated levels of cyclooxygenase (COX)-2 expression and prostaglandin E2 (PGE2) production frequently observed. Whereas COX-2 and PGE2 are associated with cancer cell survival and tumor angiogenesis, arachidonic acid itself is a strong apoptotic signal that may facilitate cancer cell death. To further explore how cancer cells exploit the progrowth actions of prostaglandins while suppressing the proapoptotic actions of intracellular arachidonic acid, we determined the cytoplasmic phospholipase A2 (cPLA2) and COX-2 expression levels in a panel of human colon tumors by immunohistochemistry. Although high levels of cPLA2 and COX-2 expression are predicted to facilitate maximal prostaglandin production, tumors frequently displayed a high-COX-2/low-cPLA2 phenotype. The least represented phenotype was the high expression of cPLA2, a characteristic predicted to generate the highest levels of intracellular arachidonic acid. The potential proapoptotic role of cPLA2 was supported by a higher frequency of terminal deoxynucleotidyl transferase–mediated nick end labeling staining in cPLA2-positive tumors. Moreover, analysis of preneoplastic aberrant crypt foci from high-risk patients suggests that acquisition of the high-COX-2/low-cPLA2 phenotype may arise at an early stage of colon carcinogenesis. We additionally inhibited cPLA2 in HT-29 cells using antisense oligonucleotides. Our results indicate that cPLA2 plays an important role in tumor necrosis factor α–induced apoptosis in human colon cancer cells. Our data further support the model in which colon cancer growth is favored when intracellular arachidonic acid levels are suppressed by inhibition of cPLA2 or by a high-COX-2/low-cPLA2 phenotype.

Probiotics Used in Human Studies

Goal: To investigate the literature from 1980 to 2004 for the types of studies and organisms used as probiotics in human studies and diseases. Methods: PubMed search for probiotic organisms used in studies from 1980 to August 2004. The data from those papers were evaluated for organisms used, dose, vehicle, frequency, and investigators report of positive or negative results. Results: A total of 185 manuscripts were identified. Investigators reported results of both single and multiple organisms in the same manuscript. A single organism was used in 125 reports, and multiple organisms, ranging from 2 to 9, were used in 60 reports. Positive results were reported in 239 clinical situations and negative results reported in 49 in a total of 288 clinical conclusions drawn by the respective investigators. The studies are tabulated in this review. Conclusion: There is a large literature available in credible journals that report benefit of probiotic administration using a wide range of doses, organisms, and clinical situations. Most of the studies are simple reports of a clinical situation and require additional evaluation.

Metabolic Profiling, a Noninvasive Approach for the Detection of Experimental Colorectal Neoplasia

Colorectal cancer is the second leading cause of cancer-related deaths in the United States. Although noninvasive stool-based screening tests are used for the early detection of colorectal neoplasia, concerns have been raised about their sensitivity and specificity. A metabolomics-based approach provides a potential noninvasive strategy to identify biomarkers of colorectal carcinogenesis including premalignant adenomas. Our primary objective was to determine whether a distinct metabolic profile could be found in both feces and plasma during experimental colorectal carcinogenesis. Feces, plasma as well as tumor tissue and normal colorectal mucosa were obtained from A/J mice at several time points following administration of azoxymethane or saline. Ultra-performance liquid chromatography tandem mass spectroscopy and gas chromatography mass spectroscopy were used to quantify metabolites in each of these matrices. Here, we show that colorectal carcinogenesis was associated with significant metabolic alterations in both the feces and plasma, some of which overlap with metabolic changes in the tumor tissue. These consisted of 33 shared changes between feces and tumor, 14 shared changes between plasma and tumor, and 3 shared changes across all 3 matrices. For example, elevated levels of sarcosine were found in both tumor and feces whereas increased levels of 2-hydroxyglutarate were found in both tumor and plasma. Collectively, these results provide evidence that metabolomics can be used to detect changes in feces and plasma during azoxymethane-induced colorectal carcinogenesis and thus provide a strong rationale for future studies in humans. Cancer Prev Res; 5(12); 1358–67. ©2012 AACR.

Deoxycholic acid promotes the growth of colonic aberrant crypt foci.

AKR/J mice are resistant to the tumorigenic properties of the colon carcinogen, azoxymethane (AOM). Following AOM exposure, limited numbers of preneoplastic lesions, referred to as aberrant crypt foci (ACF), are formed in the colon, and their progression to tumors rarely occurs. To determine whether genetic resistance can be overcome by exposure to a dietary tumor promoter, AOM‐exposed AKR/J mice were fed a diet containing 0.25% deoxycholic acid (DCA). DCA exposure was begun 1 wk prior to or 1 wk after tumor initiation with AOM. Mice placed on the DCA diet prior to AOM treatment developed a significantly higher multiplicity of ACF compared to AOM‐exposed mice fed a control diet (15.50 ± 0.96 vs. 6.17 ± 0.48, respectively; P < 0.05). When DCA exposure was begun after AOM treatment (post‐initiation), ACF formation was further enhanced (34.00 ± 1.22). Interestingly, increased numbers of ACF were associated with the presence of nuclear β‐catenin, assessed by immunohistochemistry. While ∼33% of ACF from mice exposed to DCA prior to AOM treatment contained positive nuclear β‐catenin staining, ∼77% of ACF from mice fed DCA after AOM were positive. Accumulation of nuclear β‐catenin was not associated with a loss of E‐cadherin from the plasma membrane, although loss of APC staining was a consistent feature of most AOM‐induced ACF, regardless of DCA exposure. These results demonstrate that exposure to DCA, an important digestive component, is sufficient to sensitize the resistant AKR/J colon to formation of high‐grade dysplasia, and that nuclear translocation of β‐catenin may play an important role in this process.

S1P1 localizes to the colonic vasculature in ulcerative colitis and maintains blood vessel integrity

Signaling through sphingosine-1-phosphate receptor1 (S1P1) promotes blood vessel barrier function. Degradation of S1P1 results in increased vascular permeability in the lung and may explain side effects associated with administration of FTY720, a functional antagonist of the S1P1 receptor that is currently used to treat multiple sclerosis. Ulcerative colitis (UC) is characterized by an increased density of abnormal vessels. The expression or role of S1P1 in blood vessels in the colon has not been investigated. In the present study, we show that S1P1 is overexpressed in the colonic mucosa of UC patients. This increase in S1P1 levels reflects increased vascular density in the inflamed mucosa. Genetic deletion of S1pr1 in mice increases colonic vascular permeability under basal conditions and increases bleeding in experimental colitis. In contrast, neither FTY720 nor AUY954, two S1P receptor-targeting agents, increases bleeding in experimental colitis. Taken together, our findings demonstrate that S1P1 is critical to maintaining colonic vascular integrity and may play a role in UC pathogenesis.