Nathaniel S. Rial

Activated K-RAS Increases Polyamine Uptake in Human Colon Cancer Cells Through Modulation of Caveolar Endocytosis

Endocytic pathways have been implicated in polyamine transport in mammalian cells, but specific mechanisms have not been described. We have shown that expression of a dominant negative (DN) form of the GTPase Dynamin, but not Eps15, diminished polyamine uptake in colon cancer cells indicating a caveolar and nonclathrin uptake mode. Polyamines co‐sediment with lipid raft/caveolin‐1 rich fractions, of the plasma membrane in a sucrose density gradient. Knock down of caveolin‐1 significantly increased polyamine uptake. Conversely, ectopic expression of this protein resulted in diminished polyamine uptake. We also found that presence of an activated K‐RAS oncogene significantly increased polyamine uptake by colon cancer cells. This effect is through an increase in caveolin‐1 phosphorylation at tyrosine residue 14. Caveolin‐1 is a negative regulator of caveolar endocytosis and phosphorylation in a K‐RAS dependent manner leads to an increase in caveolar endocytosis. In cells expressing wild type K‐RAS, addition of exogenous uPA was sufficient to stimulate caveolar endocytosis of polyamines. This effect was abrogated by the addition of a SRC kinase inhibitor. These data indicate that polyamine transport follows a dynamin‐dependent and clathrin‐independent endocytic uptake route, and this route is positively regulated by the oncogenic expression of K‐RAS in a caveolin‐1 dependent manner.

Nuclear factor kappa B (NF-κB): A novel cause for diabetes, coronary artery disease and cancer initiation and promotion?

Obesity is a growing epidemic in the United States (US). Obesity has been recognized as a modifiable risk factor for many diverse diseases including diabetes, cardiovascular disease and cancer burden. Common contributors to obesity include a high fat diet, smoking and physical inactivity. Systemic effects of obesity include increased micro-inflammatory molecules such as nuclear factor kappa B (NF-κB) that influence the both endothelial and epithelial layers as well as the supportive stroma. An emerging risk factor for micro-inflammation also includes periodontal disease. These pro-inflammatory states are hypothesized to contribute to diabetes as well as cardiovascular disease and cancer through the direct activation of NF-κB. Therefore, a comprehensive health care strategy would include reduction of diabetes, cardiovascular and cancer risk through the decrease in micro-inflammation.

Polyamines as mediators of APC-dependent intestinal carcinogenesis and cancer chemoprevention

Combination chemoprevention for cancer was proposed a quarter of a century ago, but has not been implemented in standard medical practice owing to limited efficacy and toxicity. Recent trials have targeted inflammation and polyamine biosynthesis, both of which are increased in carcinogenesis. Preclinical studies have demonstrated that DFMO (difluoromethylornithine), an irreversible inhibitor of ODC (ornithine decarboxylase) which is the first enzyme in polyamine biosynthesis, combined with NSAIDs (non-steroidal anti-inflammatory drugs) suppresses colorectal carcinogenesis in murine models. The preclinical rationale for combination chemoprevention with DFMO and the NSAID sulindac, was strengthened by the observation that a SNP (single nucleotide polymorphism) in the ODC promoter was prognostic for adenoma recurrence in patients with prior sporadic colon polyps and predicted reduced risk of adenoma in those patients taking aspirin. Recent results from a phase III clinical trial showed a dramatic reduction in metachronous adenoma number, size and grade. Combination chemoprevention with DFMO and sulindac was not associated with any serious toxicity. A non-significant trend in subclinical ototoxicity was detected by quantitative audiology in a subset of patients identified by a genetic marker. These preclinical, translational and clinical data provide compelling evidence for the efficacy of combination chemoprevention. DFMO and sulindac is a rational strategy for the prevention of metachronous adenomas, especially in patients with significant risk for colorectal cancer. Toxicities from this combination may be limited to subsets of patients identified by either past medical history or clinical tests.

Ratio images and ultraviolet C excitation in autofluorescence imaging of neoplasms of the human colon

Abstract. The accepted screening technique for colon cancer is white light endoscopy. While most abnormal growths (lesions) are detected by this method, a significant number are missed during colonoscopy, potentially resulting in advanced disease. Missed lesions are often flat and inconspicuous in color. A prototype ultraviolet spectral imager measuring autofluorescence (AF) and reflectance has been developed and applied in a study of 21 fresh human colon surgical specimens. Six excitation wavelengths from 280 to 440 nm and formulaic ratio imaging were utilized to increase lesion contrast and cause neoplasms to appear bright compared to normal tissue. It was found that in the subset of lesions which were most difficult to visualize in standard color photographs [low contrast lesions, (LCLs)] a ratio image (F340/F440) of AF images excited at 340 and 440 nm produced extraordinary images and was effective in about 70% of these difficult cases. Contrast may be due to increased levels of reduced nicotinamide adenine dinucleotide, increased hemoglobin absorption, and reduced signal from submucosal collagen. A second successful ratio image (R480/R555) combined two reflectance images to produce exceptional images especially in particular LCLs where F340/F440 was ineffective. The newly discovered ratio images can potentially improve detection rate in screening with a novel AF colonoscope.

Regulation of deoxycholate induction of CXCL8 by the adenomatous polyposis coli gene in colorectal cancer.

Elevated deoxycholic acid (DCA), mutations in the adenomatous polyposis coli (APC) gene and chronic inflammation are associated with increased risk of colorectal cancer. APC status was manipulated to determine whether DCA mediates inflammatory molecules in normal or initiated colonic mucosa. DCA increased steady state mRNA and protein levels of CXCL8 in cells which do not express wild‐type APC. Steady‐state CXCL8 mRNA and protein were suppressed when cells with conditional expression of wild‐type APC were exposed to DCA. Immunostaining did not detect CXCL8 in normal human colonic mucosa. CXCL8 was expressed in adenomatous polyps and adenocarcinomas. CXCL8 expression correlated with nuclear β‐catenin localization in epithelial cells of adenomas, but was associated with endothelial cells and neutrophils in the adenocarcinomas. DCA‐mediated CXCL8 promoter–reporter activity was elevated in a mutant APC background. Wild‐type APC suppressed this effect. Mutation of activator protein‐1 (AP‐1) or nuclear factor kappa B (NF‐κB) sites suppressed the activation of the CXCL8 promoter–reporter by DCA. Chromatin immunoprecipitation revealed that AP‐1 and NF‐κB binding to the 5′‐promoter of CXCL8 was induced by DCA. The β‐catenin transcription factor was bound to the 5′‐promoter of CXCL8 in the absence or presence of DCA. Phenotypic assays determined that DCA‐mediated invasion was blocked by antibody‐directed against CXCL8 or wild‐type APC. CXCL8 exposure led to matrix metalloproteinase‐2 production and increased invasion on laminin‐coated filters. These data suggest that DCA‐mediated CXCL8 occurs in initiated colonic epithelium and neutralizing CXCL8 could reduce the invasive potential of tumors.

Clinical end points for developing pharmaceuticals to manage patients with a sporadic or genetic risk of colorectal cancer

To reduce the morbidity and mortality from colorectal cancer (CRC), current clinical practice focuses on screening for early detection and polypectomy as a form of secondary prevention, complemented with surgical interventions when appropriate. No pharmaceutical agent is currently approved for use in clinical practice for the management of patients at risk for CRC. This article will review earlier attempts to develop pharmaceuticals for use in managing patients with a sporadic or genetic risk of CRC. It will also discuss therapeutic end points under evaluation in current efforts to develop drugs for treating CRC risk factors.

Use of endoscopic ultrasound for diagnosis of cholangiocarcinoma in auto-immune hepatitis

In this report, a patient was exposed to an herbal remedy for hypercholesterolemia. She became acutely jaundiced while taking the remedy and presented for medical care. Endoscopic ultrasound was utilized, and found a distal common bile duct mass. Endoscopic retrograde cholangiopancreatography guided bile duct biopsies revealed that the mass was cholangiocarcinoma (CCA). This case highlights a unique association between autoimmune hepatitis and CCA. It also highlights that EUS can be safely used in patients with cirrhosis to spare invasive evaluation such as exploratory laporotomy for diagnosis and staging of cholangiocarcinoma.

Enhanced visibility of colonic neoplasms using formulaic ratio imaging of native fluorescence

Colonoscopy is the preferred method for colon cancer screening, but can miss polyps and flat neoplasms with low color contrast. The objective was to develop a new autofluorescence method that improves image contrast of colonic neoplasms.

Endoscopic ultrasound with biopsy of omental mass for cholangiocarcinoma diagnosis in cirrhosis

In this report, a patient had a previous diagnosis of cholangiocarcinoma with an extended cholecystectomy. Three years later, he was evaluated for recurrent ascites. The patient had several large volume paracentesis, without evidence of malignant cells. Subsequently, endoscopic ultrasound (EUS) with fine needle aspiration (FNA) of both lymph and omental nodules was utilized. While the lymph nodes were negative for malignancy, the omental nodule was interrogated with multiple antibodies and was found to be positive for neoplasia. EUS with FNA can safely be used in patients with cirrhosis to spare the patient invasive evaluation such as exploratory laparotomy (ex-lap) for diagnosis and staging of cholangiocarcinoma.

Sa1839 Spectral Imaging of Neoplasms of the Colon by Targeting Tryptophan, FAD and Collagen

G A A b st ra ct s vs. 60.6% (p=0.0003), respectively. Also, the area under the ROC curve (AUC) was greater for endoscopists than pathologist (0.83 vs. 0.55, p=0.0001) (figure). While the overall agreement between endoscopists and pathologist was moderate for all GI lesions (kappa coefficient [k] = 0.43; 95% CI, 0.26 0.61), luminal lesions (k = 0.40; 95% CI, 0.20 0.60) and those of dysplastic/neoplastic pathology (k = 0.55; 95% CI, 0.37 0.72), the agreement was poor for benign (k = 0.13; 95% CI, -0.097 0.36) and pancreaticobiliary lesions (k = 0.19; 95% CI, -0.26 0.63). Conclusion: There is a wide discrepancy in the interpretation of p-CLE findings between endoscopists and pathologist, particularly for benign and pancreaticobiliary lesions. Given these findings, further studies are needed to identify the cause of this poor agreement.