Identification of potential small-molecule protein-protein inhibitors of cancer metastasis by 3D epitope-based computational screening.

In cancer cells exposed to extracellular pressure or shear stress, AKT1-FAK interaction drives focal adhesion kinase (FAK) phosphorylation, leading to force-activated cancer cell adhesion and metastasis. Blocking the AKT1-FAK interaction is therefore an attractive target for cancer therapy, avoiding the side effects of global FAK inhibition. Starting with our previous identification of a short FAK peptide that binds AKT1, we identified a series of small-molecule inhibitor candidates using a novel approach for inhibiting protein-protein interactions. Using a 3D structural fragment of the FAK peptide as the query, millions of drug-like, commercially available molecules were screened to identify a subset mimicking the volume and chemistry of the FAK fragment to test for their ability to block pressure-sensitive FAK phosphorylation by AKT1. Two compounds reduced the stimulation of FAK phosphorylation in response to extracellular pressure in human SW620 colon cancer cells without affecting basal FAK phosphorylation. Thus, using a 3D protein interaction epitope as a novel query for ligand-based virtual screening can successfully identify small-molecules that show promise in modulating cancer cell adhesion and metastasis.

The C-terminal region of the focal adhesion kinase F1 domain binds Akt1 and inhibits pressure-induced cell adhesion.

Increased extracellular pressure or shear stress activate a complex signal pathway that stimulates integrin binding affinity and potentiates metastatic cell adhesion. Inhibiting either focal adhesion kinase (FAK) and Akt1 can block this pathway, but risks interfering with the diverse other functions of each kinase. However, the mechanotransduced signal pathway involves a novel Akt1-FAK interaction not required for most FAK or Akt1 function, so modeling and blocking this interaction seems a desirable target. Building upon previous work suggesting that FAK-Akt1 binding is mediated by the FAK F1 lobe, we demonstrated that independently expressing the F1 domain in human Caco-2 or murine CT-26 colon cancer cells by transient or stable inducible plasmid expression respectively prevents the stimulation of cancer cell adhesion by increased extracellular pressure. Serial further truncation of the FAK F1 lobe identified shorter regions capable of pulling down Akt1 on a glutathione S-transferase (GST) - conjugated column. Ultimately, we identified a 33 residue segment (residues 94-126) at the C-terminal of the F1 lobe as sufficient to pull down Akt1. These findings raise the possibility of developing a treatment modality around the disruption of the FAK-Akt1 interaction using peptides modeled from FAK.

Hierarchies of healing in gut mucosal injury.

The mucosal response to injury can best be understood as a complex layering of different responses that may be invoked depending upon the size and nature of the injury, the persistence of the injurious stimulus, and the availability of various trophic host factors that stimulate repair. There is a hierarchy of responses that ranges from small rapid resealing of acute apical membrane or single cell wounds through the long term attempt to heal a giant peptic ulcer or respond to inflammatory bowel disease. Although much previous attention has been paid to the local factors that regulate these pathways and responses, there is increasing interest in systemic factors that may act either by the production of cytokines and trophic factors or by the direct migration of bone marrow derived stem cells into the mucosal wound.

Akt1 binds focal adhesion kinase via the Akt1 kinase domain independently of the pleckstrin homology domain.

Akt1 and focal adhesion kinase (FAK) are protein kinases that play key roles in normal cell signaling. Individually, aberrant expression of these kinases has been linked to a variety of cancers. Together, Akt1/FAK interactions facilitate cancer metastasis by increasing cell adhesion under conditions of increased extracellular pressure. Pathological and iatrogenic sources of pressure arise from tumor growth against constraining stroma or direct perioperative manipulation. We previously reported that 15 mmHg increased extracellular pressure causes Akt1 to both directly interact with FAK and to phosphorylate and activate it. We investigated the nature of the Akt1/FAK binding by creating truncations of recombinant FAK, conjugated to glutathione S-transferase (GST), to pull down full-length Akt1. Western blots probing for Akt1 showed that FAK/Akt1 binding persisted in FAK truncations consisting of only amino acids 1-126, FAK(NT1), which contains the F1 subdomain of its band 4.1, ezrin, radixin, and moesin (FERM) domain. Using FAK(NT1) as bait, we then pulled down truncated versions of recombinant Akt1 conjugated to HA (human influenza hemagglutinin). Probes for GST-FAK(NT1) showed Akt1-FAK binding to occur in the absence of the both the Akt1 (N)-terminal pleckstrin homology (PH) domain and its adjacent hinge region. The Akt1 (C)-terminal regulatory domain was equally unnecessary for Akt1/FAK co-immunoprecipitation. Truncations involving the Akt1 catalytic domain showed that the domain by itself was enough to pull down FAK. Additionally, a fragment spanning from the PH domain to half way through the catalytic domain demonstrated increased FAK binding compared to full length Akt1. These results begin to delineate the Akt1/FAK interaction and can be used to manipulate their force-activated signal interactions. Furthermore, the finding that the N-terminal half of the Akt1 catalytic domain binds so strongly to FAK when cleaved from the rest of the protein may suggest a means for developing novel inhibitors that target this specific Akt1/FAK interaction.

Spontaneous hernia through the posterior rectus abdominis sheath: case report and review of the published literature 1937-2008.

Spontaneous ventral hernia through the rectus abdominis sheath is perhaps the rarest hernia, with eight previously reported cases since 1937. The condition has not been reflected in the popular treatises on hernia. An 83-year-old male patient underwent surgery for intestinal obstruction. A spontaneous Richter's hernia of the small intestine through the posterior rectus abdominis sheath was successfully treated with bowel resection and tissue repair. Hernias of this variety are not well known and thus are rarely suspected. Their successful management is based on accurate emergency diagnosis and surgery, which we recommend as the best chance for a successful outcome.

Pressure activates colon cancer cell adhesion via paxillin phosphorylation, Crk, Cas, and Rac1.

Physical forces can activate colon cancer cell adhesion, critical for metastasis. Paxillin is phosphorylated by FAK and required for pressure-stimulated adhesion. However, whether paxillin acts as an inert scaffolding protein or whether paxillin phosphorylation is required is unknown. Transfection with paxillin point-phosphorylation mutants demonstrated that phosphorylation at tyrosines 31 and 118 together is necessary for pressure-stimulated adhesion. We further evaluated potential paxillin partners. Reducing the adaptor protein Crk or the focal adhesion protein p130Cas blocked pressure-stimulated adhesion. Furthermore, Crk and p130Cas both displayed increased co-immunoprecipitation with paxillin in response to increased pressure, except in cells transfected with a Y31Y118 paxillin mutant. Inhibiting the small GTPase Rac1 also abolished pressure-stimulated adhesion, and reducing paxillin by siRNA blocked Rac1 phosphorylation by pressure. Thus, paxillin phosphorylation at tyrosines 31 and 118 together is necessary for pressure-induced adhesion. Paxillin, Crk and Cas form a trimeric complex that activates Rac1 and mediates this effect.

Mondor's disease mimicking a Spigelian hernia.

Superficial thrombophlebitis of the thoracoepigastric veins (also known as Mondor's disease) is an uncommon disorder that typically affects middle-aged women and classically involves the chest wall including the breasts. Only one previously published, non-operative case of the disease, describes how the condition can resemble a strangulated Spigelian hernia. Herein we describe another similar case in which the diagnosis was made intra-operatively. The extremely unusual and similar clinical findings we observed demonstrate that Mondor's disease can occur in the Spigelian hernia belt and cause diagnostic confusion.

Subxiphoid incisional hernias after median sternotomy.

BACKGROUND: Subxiphoid incisional hernias are notoriously difficult to repair and are prone to recurrence. The few reports on subxiphoid hernia published over the last two decades have not fully addressed the etiology, pathology, treatment, and outcome of this problem. This review was performed to analyze the published experience and increase the understanding of these difficult hernias. METHODS: We reviewed the extensive literature, including the Medline and Current Contents computerized database searches, and searched the available bibliographies. RESULTS: Seven retrospective studies of a total of 113 patients who had clinical subxiphoid hernias after median sternotomy were found. An additional surgical technique describing a modified median sternotomy preventing the hernia, and a single review article on selected technical considerations of subxiphoid ventral repair were also found. CONCLUSIONS: The incidence of subxiphoid hernia after median sternotomy can be possibly reduced by paraxiphoid extension of the sternotomy, reinforcement near the xiphoid end of the incision, or by optimizing closure of the distal sternotomy and the linea alba. Abdominal wall reinforcement by open-mesh closure or laparoscopic transperitoneal prosthetic repair can effectively deal with the defect. Long-term outcome analyses are not yet available.

Effects of repetitive deformation on intestinal epithelial cells.

The intestinal mucosa is subject to a variety of physical forces in a complex manner during normal gut function and in disease. Intestinal mucosal atrophy during some disease states is a troubling phenomenon that is only partially explained by nutritional parameters. Increasing evidence suggests the possibility that repetitive deformation engendered by peristalsis, villous motility, and interaction with luminal chyme may be trophic for the gut mucosa in normal function, but that the intestinal epithelial response to repetitive deformation may be altered by inflammatory or other states in which plasma or tissue fibronectin levels are increased.

Extracellular pressure stimulates tumor cell adhesion in vitro by paxillin activation.

Metastasizing colon cancer cells bind target tissues primarily via integrins. Extracellular pressure or shear stress stimulates integrin-mediated adhesion to matrix proteins or endothelial cells by activating the focal adhesion proteins FAK and Src. Because this effect is blocked by cytoskeletal perturbation and paxillin may link the cytoskeleton to the focal adhesion complex, we evaluated the role of paxillin in pressure-induced malignant colonocyte adhesion. We studied SW620 colon cancer cells and confirmed key results in Caco-2 colon cancer cells, primary human colon cancer cells, and a murine colonic adenocarcinoma. We evaluated adhesion to collagen at ambient and 15 mmHg increased pressure after 30 minutes, and paxillin, FAK, and Src phosphorylation in suspended cells prior to adhesion. Some cells were treated with siRNA to paxillin or FAK, or the Src inhibitor PP2. We also compared pressure-induced signals in suspended cells with adhesion-induced signals after adhesion to collagen. Pressure stimulated adhesion and paxillin phosphorylation in SW620 and Caco-2 cells and human primary colon cancer cells. Pressure also increased paxillin phosphorylation in murine carcinoma cells. SiRNA to paxillin decreased SW620 and Caco-2 paxillin without altering basal levels of phosphorylated paxillin. Paxillin reduction decreased basal adhesion to collagen, and inhibited pressure-stimulated adhesion, as well as paxillin, FAK397, FAK576, and Src476 phosphorylation. Neither PP2 nor siRNA to FAK inhibited induction of paxillin phosphorylation by pressure. In contrast, adhesion stimulated FAK, Src, and paxillin phosphorylation regardless of paxillin reduction. In summary, pressure induced paxillin phosphorylation in colon cancer cells. Paxillin reduction inhibited basal adhesion and blocked the pressure-mediated increase in adhesion, as well as pressure-induced FAK and Src signals, while adhesion-induced signals were preserved. Paxillin may be an upstream mediator of pressure-stimulated adhesion, important in metastasis.

Extracellular pressure stimulates colon cancer cell proliferation via a mechanism requiring PKC and tyrosine kinase signals.

UNLABELLED: Pressure in colonic tumours may increase during constipation, obstruction or peri-operatively. Pressure enhances colonocyte adhesion by a c-Src- and actin-cytoskeleton-dependent PKC-independent pathway. We hypothesized that pressure activates mitogenic signals. METHODS: Malignant colonocytes on a collagen I matrix were subjected to 15 mmHg pressure. ERK, p38, c-Src and Akt phosphorylation and PKCalpha redistribution were assessed by western blot after 30 min and PKC activation by ELISA. Cells were counted after 24 h and after inhibition of each signal, tyrosine phosphorylation or actin depolymerization. RESULTS: Pressure time-dependently increased SW620 and HCT-116 cell counts on collagen or fibronectin (P < 0.01). Pressure increased the SW620 S-phase fraction from 28 +/- 1 to 47 +/- 1% (P = 0.0002). Pressure activated p38, ERK, and c-Src (P < 0.05 each) but not Akt/PKB. Pressure decreased cytosolic PKC activity, and translocated PKCalpha to a membrane fraction. Blockade of p38, ERK, c-Src or PI-3-K or actin depolymerization did not inhibit pressure-stimulated proliferation. However, global tyrosine kinase blockade (genistein) and PKC blockade (calphostin C) negated pressure-induced proliferation. CONCLUSIONS: Extracellular pressure stimulates cell proliferation and activates several signals. However, the mitogenic effect of pressure requires only tyrosine kinase and PKCalpha activation. Pressure may modulate colon cancer growth and implantation by two distinct pathways, one stimulating proliferation and the other promoting adhesion.

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.

Differential dietary effects on colonic and small bowel neoplasia in C57BL/6J Apc Min/+ mice.

The effects of fiber on colon cancer are controversial. Twenty 5-week old C57BL/6J Apc Min/+ mice were fed for 60 days with a commercial mouse diet (Teklad LM-485) and eight semidefined diets containing 5-10% various fibers and 20% soybean oil. Ten additional C57BL/6J congenic litter-mates were fed each diet to assay colonic SCFA. SCFA, stool bulk, and colonic tumor incidence differed only slightly among the semidefined diets despite variations in fiber content and source. However, food consumption, caloric intake, stool bulk, and SCFA were substantially increased by the Teklad diet compared with all other groups. The Teklad diet significantly increased the number of mice with colonic tumors, average number of tumors/mouse, total tumor burden, colonic atypical hyperplasia, and small bowel tumors. Mice fed high-fat, no-fiber diets had more small bowel tumors (29.8 +/- 3.1) than mice fed diets with fiber (8.2 +/- 2.1) or with low fat and no fiber (18.1 +/- 3.4) (P < 0.05 for each group). These studies suggest that fat predisposes to and fiber protects against small bowel tumors but not colon tumors in these mice. Thus, diets high in fiber or yielding high colonic luminal SCFA may not necessarily protect against colonic cancer. Furthermore, the effects of dietary fiber in Teklad appear overshadowed by some other biologically active factors in this animal model.

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.

Environmental factors of temperature, humidity, serum accumulation, and cell seeding increase colon cancer cell adhesion in vitro, with partial characterization of the serum component responsible for pressure-stimulated adhesion.

Physical characteristics of surgical wounds and viable tumor cells shed may differ between open and laparoscopic procedures. Because environmental factors may vary between the laparoscopic milieu and that of open surgical procedures, we sought to characterize the effect of these factors on tumor cell adhesion, an early step in the process of wound implantation. Human SW620 colon cancer cells were placed in matrix-precoated dishes for 30 min at concentrations of 90,000-540,000 cells/well, at 25-37 degrees C, in the native state of the matrix proteins and after drying for 60 min, and in 0-10% serum. As increased pressure has previously been reported to stimulate colon cancer cell adhesion synergistically with serum, we then further partially characterized the serum components responsible for this potentiating effect. The number of adherent cells varied linearly with cells seeded. Adhesion was temperature-dependent, and also was dependent on the matrix conformation. Less adhesion occurred to dry matrix proteins. Serum dose-dependently potentiated SW620 pressure-stimulated adhesion, with a maximal increase in adhesion compared with ambient pressure conditions at 5% serum concentration. Heat inactivating the serum at 60 degrees C for 30 min ablated the effect. Filtration to remove molecules over 10 kDa produced no change in adhesion relative to ambient conditions, but filtration to 100 kDa preserved the serum effect. When the serum was passed over a gelatin-Sepharose column, which binds numerous proteins including fibronectin, the serum effect was lost. Addition of fibronectin to serum-free media did not reconstitute the effect. The environmental factors of warm temperature, moisture, and serum accumulation may contribute to increased colon cancer cell adhesion. However, the most important determinant of malignant adhesion to surgical wounds, laparoscopic or open, is likely to be the size of the tumor cell inoculum. Pressure stimulation of colon cancer cell adhesion is potentiated by heat-labile serum components of molecular weight 10-100 kDa which bind gelatin-Sepharose, and is not fibronectin alone. Irrigating serum from surgical wounds may decrease tumor implantation.

Effect of nabumetone and aspirin on colonic mucosal bleeding time.

BACKGROUND: The management of patients taking aspirin or non-steroidal anti-inflammatory drugs (NSAIDs) who require colonoscopy remains controversial because of concerns over bleeding after biopsy or polypectomy. AIM: To determine whether patients using the NSAID nabumetone, a non-acidic prodrug with mixed activity against cyclooxygenase-1 (COX-1) and COX-2, exhibited prolonged mucosal bleeding times and how this might compare with mucosal bleeding times in patients using aspirin. METHODS: We assessed triplicate mucosal bleeding times in patients undergoing screening flexible sigmoidoscopy. We compared 90 patients who had taken no aspirin or NSAIDs within the previous 2 weeks, to 60 patients who had received nabumetone 1 g b.d. by mouth for the previous 2 weeks, and 30 patients who had taken 325 mg aspirin daily for the previous 2 weeks. In each case, the investigator performing the study was blinded to the patient's medication. RESULTS: Mucosal bleeding times did not differ significantly among control or nabumetone-using patients. However, the patients receiving aspirin exhibited significant prolongation. Mucosal bleeding time correlated statistically significantly, but weakly, with skin bleeding time. CONCLUSIONS: Nabumetone does not appear to prolong mucosal bleeding time after mucosal pinch biopsy, and skin bleeding time does not reliably screen for prolonged mucosal bleeding time.