Marc D. Basson

Human enterocyte (Caco-2) migration is modulated in vitro by extracellular matrix composition and epidermal growth factor.

The modulation of enterocyte sheet migration was studied using Caco-2 cells, a well-differentiated human colonic cell line. Although Caco-2 cells attached and spread equivalently over collagen types I, III, IV, and V and laminin, migration over laminin was significantly slower than migration over the collagen types. Fibronectin was a poor substrate for attachment, spreading, and migration. Epidermal growth factor (EGF) stimulated migration over laminin but did not alter Caco-2 migration over collagen or fibronectin. This effect was independent of cell proliferation, which was stimulated equivalently on both laminin and collagen I. Expression and organization of cell surface receptors for matrix (integrins) were studied using antibodies specific for beta and alpha integrin subunits. Integrin surface expression was assessed by immunoprecipitation of surface 125iodinated control and EGF-treated cells. Beta 1 surface pools did not change substantially in any condition studied. Alpha 1 subunit pools were decreased after EGF treatment on collagen I but alpha 1 pools increased after EGF treatment on laminin. Surface pools of alpha 2 subunits were increased following EGF treatment whether cells were cultured on laminin or collagen I. However, traditional immunofluorescent and laser confocal imaging demonstrated substantial differences in the character of alpha 2 subunit organization between collagen and laminin in the migrating cell front. Furthermore, a functional antibody to the alpha 2 subunit inhibited EGF stimulation of migration over laminin without substantial effects on basal migration over laminin or collagen I. Thus, EGF appears to exert a matrix-specific effect on enterocyte migration by modulation of integrin expression and organization.

Complications of endoscopy

BACKGROUND Although most gastrointestinal endoscopic procedures are performed by gastroenterologists, surgeons often assist in the management of patients with complications. This review provides an introduction to the incidence, prevention, and treatment of complications that may occur after upper endoscopy, colonoscopy, percutaneous endoscopic gastrostomy, and endoscopic retrograde cholangiopancreatography. METHODS Systematic review of the literature. RESULTS Preprocedural complications include medication effects and adverse effects of bowel preparation. Major procedural complications consist primarily of perforation and hemorrhage. Percutaneous endoscopic gastrostomy tube placement may be complicated by fistula and obstruction. There is also a risk of infectious disease transmission, both to and from the patient. CONCLUSIONS Endoscopy, like all invasive procedures, carries significant potential risks for the patient. In practiced hands, and with awareness of the problems that may arise, many complications may be avoided and others successfully managed.

FAK association with multiple signal proteins mediates pressure-induced colon cancer cell adhesion via a Src-dependent PI3K/Akt pathway

Cancer cell adhesion is traditionally viewed as random, occurring if the cells receptors match the substrate. Cancer cells are subjected to pressure and shear during growth against a constraining stroma, surgical manipulation, and passage through the venous and lymphatic system. Cells shed into a cavity such as the abdomen postoperatively also experience increased pressure from postoperative edema. Increased extracellular pressure stimulates integrin‐medi‐ated cancer cell adhesion via FAK and Src. PI 3‐kinase (PI3K) inhibitors (LY294002 or wortmannin), Akt inhibitors, or Aktl siRNA blocked adhesion stimulated by 15 mmHg pressure in SW620 or primary human malignant colonocytes. Pressure activated PI3K, tyrosine‐phosphorylated and membrane‐translocated the p85 subunit, and phosphorylated Akt. PI3K inhibitor (LY294002) prevented pressure‐stimulated Akt Ser473 and FAK Tyr397, but not FAK576 or Src416 phosphor‐ylation. PP2 inhibited PI3K activity and Akt phosphor‐ylation. FAK siRNA did not affect pressure‐induced PI3K activation but blocked Akt phosphorylation. Pressure also stimulated FAK or FAKY397F mutant translocation to the membrane. Akt inhibitor IV blocked pressure‐induced Akt and FAK translocation. Pressure activated Src‐ and PI3K‐dependently induced p85 interaction with FAK, and FAK with βl integrin. These results delineate a novel force‐activated inside‐out Src/ PI3K/FAK/Akt pathway by which cancer cells regulate their own adhesion. These signals may be potential targets for inhibition of metastatic adhesion.—Thamil‐selvan V., Craig D. H., Basson M. D. FAK association with multiple signal proteins mediates pressure‐induced colon cancer cell adhesion via a Src‐dependent PI3K/Akt pathway. FASEB J. 21, 1730–1741 (2007)

Deoxycholate is an important releaser of peptide YY and enteroglucagon from the human colon.

Peptide YY (PYY) and enteroglucagon are hormonal peptides found in endocrine cells of the distal intestinal mucosa. Although it is known that plasma concentrations of both peptides increase in response to feeding, the mechanism by which ingested food causes release of colonic hormones is not understood. The release of PYY and enteroglucagon was measured in response to intraluminal stimuli in 176 patients having investigative colonoscopy. Introduction of air, saline (isotonic and hypertonic), glucose (isotonic and hypertonic), oleic acid (without bile salts), and casein hydrolysate all failed to release PYY but glucose caused a small but significant increase in enteroglucagon concentrations. In contrast with the lack of effect of nutrients, infusion of deoxycholic acid produced a rapid and marked dose responsive increase in plasma PYY concentrations when introduced into the sigmoid colon. PYY release was statistically significant at doses between 3.3 mM to 30 mM; for example 10 mM deoxycholate caused a sixfold increase in plasma PYY concentrations. Infusion of 10 mM deoxycholate into the transverse colon or caecum produced an increase of PYY that was similar to the responses in the sigmoid colon. There was also a significant release of enteroglucagon in response to infusion of this bile salt into the sigmoid colon at doses between 3.3 mM and 30 mM. The enteroglucagon response to 10 mM deoxycholate was similar in all three colonic regions. When oleic acid was added to deoxycholate as an emulsion, the release of PYY and enteroglucagon was similar to that seen with the bile salt alone. These findings suggest that bile salts may play an important part in the control of colonic endocrine function and may explain the increased circulating concentrations of colonic regulatory peptides that are seen in malabsorption states and after small bowel resection in humans.

Short-Chain Fatty Acids Inhibit Invasive Human Colon Cancer by Modulating uPA, TIMP-1, TIMP-2, Mutant p53, Bcl-2, Bax, p21 and PCNA Protein Expression in an In Vitro Cell Culture Model

High intakes of dietary fiber or resistant starches have been associated with a lower incidence of colon cancers. Because short-chain fatty acids (SCFA) such as butyrate are produced in the colonic lumen by the bacterial fermentation of dietary fibers and resistant starches, we hypothesized that SCFA may inhibit the development of invasive human colon cancers. To test this hypothesis, primary human invasive colonocytes were isolated from fresh surgical specimens and treated with 0.01 mol/L acetate, propionate or butyrate; cell invasion, cell adhesion, F-actin polymerization, urokinase plasminogen activator (uPA), tissue inhibitor matrix metalloproteinase (TIMP)-1, TIMP-2 and mutant p53, Bcl-2, Bax, p21 and proliferating cell nuclear antigen (PCNA) protein expression levels were examined. Although each of the SCFA tested significantly reduced primary cell invasion, butyrate was the most potent, inhibiting primary invasive human colon cancer invasion by 54% (P < 0.0001). The effects of SCFA on primary cell invasion appeared to be independent of cell adhesion and F-actin polymerization but dependent on the inhibition of uPA (P < 0.05) and the stimulation of TIMP-1 and TIMP-2 activities (P < 0.05). Protein expression levels of mutant p53, p21, Bax, Bcl-2 and PCNA were significantly altered by each of the SCFA tested (P < 0.05). These data indicate that SCFA inhibit invasive human colon cancer by modulating proteolytic uPA and antiproteolytic TIMP-1 and TIMP-2 activities, but their mechanisms of action on tumor suppression, apoptosis and growth arrest may differ.

Repetitive Deformation Activates Focal Adhesion Kinase and ERK Mitogenic Signals in Human Caco-2 Intestinal Epithelial Cells through Src and Rac1

Intestinal epithelial cells are subject to repetitive deformation during peristalsis and villous motility, whereas the mucosa atrophies during sepsis or ileus when such stimuli are abnormal. Such repetitive deformation stimulates intestinal epithelial proliferation via focal adhesion kinase (FAK) and extracellular signal-regulated kinases (ERK). However, the upstream mediators of these effects are unknown. We investigated whether Src and Rac1 mediate deformation-induced FAK and ERK phosphorylation and proliferation in human Caco-2 and rat IEC-6 intestinal epithelial cells. Cells cultured on collagen-I were subjected to an average 10% cyclic strain at 10 cycles/min. Cyclic strain activated Rac1 and induced Rac1 translocation to cell membranes. Mechanical strain also induced rapid sustained phosphorylation of c-Src at Tyr418, Rac1 at Ser71, FAK at Tyr397 and Tyr576, and ERK1/2 at Thr202/Tyr204. The mitogenic effect of cyclic strain was blocked by inhibition of Src (PP2 or short interfering RNA) or Rac1 (NSC23766). Src or Rac1 inhibition also prevented strain-induced FAK phosphorylation at Tyr576 and ERK phosphorylation but not FAK phosphorylation at Tyr397. Reducing FAK using short interfering RNA blocked strain-induced mitogenicity and attenuated ERK phosphorylation but not Src or Rac1 phosphorylation. Src inhibition blocked strain-induced Rac1 phosphorylation, but Rac inhibition did not alter Src phosphorylation. Transfection of a two-tyrosine phosphorylation-deficient FAK mutant Y576F/Y577F prevented activation of cotransfected myc-ERK2 by cyclic strain. Repetitive deformation induced by peristalsis or villus motility may support the gut mucosa by a pathway involving Src, Rac1, FAK, and ERK. This pathway may present important targets for interventions to prevent mucosal atrophy during prolonged ileus or fasting.

Effects of increased ambient pressure on colon cancer cell adhesion

Forces such as strain modulate intestinal epithelial biology. Shear and pressure influence other cells. The effects of pressure on human colon cancer cells are poorly understood. Increasing ambient pressure for 30 min by 15 mm Hg over atmospheric stimulated adhesion to matrix proteins of four human colon cancer cell lines and primary cells from three human colon cancers, but not bovine aortic smooth‐muscle cells. This effect was energy dependent and cation dependent (blocked by azide and chelation), accompanied by tyrosine phosphorylation of intracellular proteins including focal adhesion kinase, and blocked by tyrosine kinase inhibition (genistein, tyrphostin, and erbstatin) and a functional antibody to the β1 integrin subunit. Although pressure stimulated adhesion even in a balanced salt solution, baseline and pressure‐stimulated adhesion were each substantially diminished in the absence of serum. These data suggest that relatively low levels of increased pressure may stimulate malignant colonocyte adhesion by a cation‐dependent β1‐integrin‐mediated mechanism, perhaps via focal adhesion kinase–related tyrosine phosphorylation. In addition to elucidating another aspect of physical force regulation of colonocyte biology, these findings may be relevant to the effects of increased pressure engendered by colonic peristalsis, surgical manipulation, or laparoscopic surgery on colon cancer cell adhesion. J. Cell. Biochem. 78:47–61, 2000.

Association Between 6-n-Propylthiouracil (PROP) Bitterness and Colonic Neoplasms

Inadequate vegetable intake appears to increase colon cancer risk. Since genetic variation in taste influences vegetable preference, we tested associations between bitterness of 6-n-propylthiouracil (PROP), a measure of taste genetics, and number of colonic polyps, a measure of colon cancer risk, in 251 men who underwent screening lower endoscopy. Patients used the general Labeled Magnitude Scale to rate bitterness of 1.6 mg PROP delivered via filter paper. A subset of 86 patients reported weekly vegetable intakes, excluding salad or potatoes. PROP bitterness correlated significantly with polyp number, an effect separate from age-associated increases in polyp number. The PROP–polyp relationship was strongest in men over 66 years, and older men with polyps were most likely to be overweight or obese. In the subset reporting vegetable intake, men who tasted PROP as more bitter consumed fewer vegetables. These preliminary findings suggest that taste genetics may influence colon cancer risk, possibly through intake of vegetables.

Autoantibody Approach for Serum-Based Detection of Head and Neck Cancer

Currently, no effective tool exists for screening or early diagnosis of head and neck squamous cell carcinoma (HNSCC). Here, we describe an approach for cancer detection based on analysis of patterns of serum immunoreactivity against a panel of biomarkers selected using microarray-based serologic profiling and specialized bioinformatics. We biopanned phage display libraries derived from three different HNSCC tissues to generate 5,133 selectively cloned tumor antigens. Based on their differential immunoreactivity on protein microarrays against serum immunoglobulins from 39 cancer and 41 control patients, we reduced the number of clones to 1,021. The performance of a neural network model (Multilayer Perceptron) for cancer classification on a data set of 80 HNSCC and 78 control samples was assessed using 10-fold cross-validation repeated 100 times. A panel of 130 clones was found to be adequate for building a classifier with sufficient sensitivity and specificity. Using these 130 markers on a completely new and independent set of 80 samples, an accuracy of 84.9% with sensitivity of 79.8% and specificity of 90.1% was achieved. Similar performance was achieved by reshuffling of the data set and by using other classification models. The performance of this classification approach represents a significant improvement over current diagnostic accuracy (sensitivity of 37% to 46% and specificity of 24%) in the primary care setting. The results shown here are promising and show the potential use of this approach toward eventual development of diagnostic assay with sufficient sensitivity and specificity suitable for detection of early-stage HNSCC in high-risk populations. (Cancer Epidemiol Biomarkers Prev 2007;16(11):2396–405)

Regulation of the intestinal epithelial response to cyclic strain by extracellular matrix proteins.

Repetitive mechanical deformation may stimulate intestinal epithelial proliferation. Because the extracellular matrix modulates static intestinal epithelial biology, we examined whether matrix proteins influence intestinal epithelial responses to deformation. Human Caco‐2bbe cells and nontransformed human enterocytes (HIPEC) were subjected to 10% average cyclic strain at 10 cycles/min on flexible membranes precoated with matrix proteins without or with plasma fibronectin or functional anti‐integrin antibodies in the medium. Strain stimulated proliferation, focal adhesion kinase, extracellular signal‐regulated protein kinase (ERK), p38, and Jun N‐terminal kinase similarly on collagen I or IV, and more weakly on laminin, but had no effect on fibronectin. MEK blockade (PD98059) prevented strain‐stimulated proliferation on collagen but did not affect proliferation on fibronectin. Adding tissue fibronectin to a collagen substrate or plasma fibronectin to the media suppressed strains mitogenic and signal effects, but not those of epidermal growth factor. Functional antibodies to the α5 or αv integrin subunit blocked strains effects on Caco‐2 proliferation and ERK activation, although ligation of the α2 or α6 subunit did not. Repetitive strain also stimulated, and fibronectin inhibited, human intestinal primary epithelial cell proliferation. Repetitive deformation stimulates transformed and nontransformed human intestinal epithelial proliferation in a matrix‐dependent manner. Tissue or plasma fibronectin may regulate the intestinal epithelial response to strain via integrins containing α5 or av.