The use of zoledronic acid in men receiving androgen deprivation therapy for prostate cancer with severe osteopenia or osteoporosis.

OBJECTIVES: To study the effect of zoledronic acid on patients with pre-existing osteoporosis on androgen deprivation therapy (ADT), who are at highest risk for fracture. Zoledronic acid is a potent bisphosphonate that can prevent osteoporosis in patients with nonmetastatic (M0), prostate cancer (CaP) who are initiating ADT. The effect of zoledronic acid on patients with pre-existing osteoporosis on ADT, who are highest risk for fracture, has not been adequately studied. METHODS: We enrolled 28 patients with M0 CaP on ADT with severe osteopenia or osteoporosis (baseline bone-mineral density (BMD) T score < -2.0) in this open-label, single-arm trial to assess the effect of zoledronic acid on BMD. All patients also received supplemental calcium and vitamin D, and were counseled about lifestyle modifications. Patients received zoledronic acid (4 mg) intravenously every 3 months for 4 treatments. BMD was measured by dual energy X-ray absorptiometry scan at enrollment, 6 and 12 months. Primary endpoint was percent change in lumbar spine BMD. RESULTS: This was a high-risk patient population-primarily older Caucasians (mean age, 73 years), former smokers, and moderate users of alcohol. Mean duration of ADT was 2.4 years. Pre-existing osteopenia or osteoporosis was observed in a single site in 9 patients and multiple sites in 19 (68%). After 12 months of zoledronic acid, lumbar spine BMD increased 4.17% (P < .0001), and BMD increased significantly (P < .05) in both hips and the right femoral neck. Seven patients (25%) experienced improved BMD into the nonosteoporotic range (T score > -2.0). Zoledronic acid infusion was well tolerated and without substantial renal toxicity. CONCLUSIONS: Zoledronic acid improves BMD in men with M0 CaP on ADT with severe osteopenia or osteoporosis (T scores < 2.0). This novel finding identifies a high-risk patient population that can potentially benefit from bisphosphonate therapy.

Intravenous zoledronic acid to prevent osteoporosis in a veteran population with multiple risk factors for bone loss on androgen deprivation therapy.

PURPOSE: Androgen deprivation therapy for prostate cancer is associated with osteoporosis and increased fracture risk. Previous studies of zoledronic acid demonstrated bone loss prevention in patients initiating androgen deprivation therapy. There are limited data on patients on prolonged androgen deprivation therapy or in Veterans Affairs patients with multiple risk factors for osteoporosis. METHODS: We randomized 93 patients with M0 prostate cancer in this placebo controlled trial in the Veterans Affairs health care system. Preplanned strata included 50 patients on androgen deprivation therapy for less than 1 year (stratum 1) and 43 on androgen deprivation therapy for greater than 1 year (stratum 2). In each stratum patients were randomized to 4 mg zoledronic acid intravenously every 3 months for 4 treatments or intravenous placebo. The primary end point was the percent change in bone mineral density at the lumbar spine at 12 months. RESULTS: Age, race, body mass index and osteoporosis risk factors were similar for the 2 treatments. Most patients were former smokers, had moderate alcohol intake, were not on calcium/vitamin D supplements and were relatively sedentary at baseline. In stratum 1 spine bone mineral density increased 5.95% in the zoledronic acid arm and decreased 3.23% in the placebo arm (p = 0.0044). In stratum 2 spine bone mineral density increased 6.08% in the zoledronic acid arm and only increased 1.57% in the placebo arm (p = 0.0005). Treatment was well tolerated with minimal impact on renal function. CONCLUSIONS: Zoledronic acid improved bone mineral density in patients with M0 prostate cancer on androgen deprivation therapy for 1 year or less, or greater than 1 year. This finding indicates that bisphosphonate therapy remains effective when initiated later in the course of androgen deprivation therapy and is efficacious in Veterans Affairs patients with multiple risk factors for osteoporosis.

Design and activity of a murine and humanized anti-CEACAM6 single-chain variable fragment in the treatment of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease, with surgery being the only curative modality for localized disease, and gemcitabine with or without erlotinib remains the standard of therapy for unresectable or metastatic disease. CEACAM6 is overexpressed in human PDA independent of stage or grade and causes anoikis resistance when dysregulated. Because murine monoclonal antibody 13-1 possesses target-specific cytotoxicity in human PDA cell lines, we designed a humanized anti-CEACAM6 single-chain variable fragment (scFv) based on monoclonal antibody 13-1. PEGylation of the glycine-serine linker was used to enhance plasma half-life. These scFvs bound CEACAM6 with high affinity, exhibited cytotoxic activity, and induced dose-dependent poly(ADP-ribose) polymerase cleavage. Murine PDA xenograft models treated with humanized scFv alone elicited tumor growth inhibition, which was enhanced in combination with gemcitabine. Immunohistochemistry showed significant apoptosis, with inhibition of angiogenesis and proliferation, and preservation of the target. Collectively, our results have important implications for the development of novel antibody-based therapies against CEACAM6 in PDA.

Expression of bile acid transporting proteins in Barrett's esophagus and esophageal adenocarcinoma.

OBJECTIVES: Barrett's esophagus (BE) is a metaplastic lesion characterized by replacement of the normal squamous epithelium by columnar intestinal epithelium containing goblet cells. It is speculated that this process is an adaptation to protect cells from components of refluxate, such as gastric acid and bile acids. In contrast to the normal squamous epithelium, enterocytes of the distal ileum are adapted to transport bile acids from the intestinal lumen. Several bile acid transporters are utilized for effective removal of bile acids, including the apical sodium-dependent bile acid transporter (ASBT), the ileal bile acid-binding protein (IBABP), and the multidrug-resistant protein 3 (MRP3). We hypothesized that one of the possible functions of newly arising metaplastic epithelium, in the esophagus, is to transport bile acids. Our major goal was to evaluate the expression of bile acid transporters in normal squamous epithelium, BE with different grades of dysplasia, and esophageal adenocarcinoma (EAC). METHODS: A total of 101 patients were included in this study. Immunohistochemistry (IHC) and reverse transcriptase (RT)-PCR were used to detect the expression of these transporters at the mRNA and protein levels. RESULTS: Our immunohistochemical studies showed that all three bile acid transporters are expressed in BE glands, but not in squamous epithelium. ASBT was found in the apical border in BE biopsies. The highest frequency of ASBT expression was in patients with nondysplastic BE (9 of 15, 60%), and a progressive loss of ASBT was observed through the stages of dysplasia. ASBT was not detected in EAC (0 of 15). IBABP staining was observed in the cytoplasm of BE epithelial surface cells. Expression of IBABP was found in 100% of nondysplastic BE (14 of 14), in 93% of low-grade dysplasia (LGD, 15 of 16), in 73% of high-grade dysplasia (HGD, 10 of 14), and in 33% of EAC (5 of 15). MRP3 was expressed in the basolateral membrane in 93% of nondysplastic BE (13 of 14), in 60% of LGD (10 of 16), and in 86% of HGD (11 of 13). Only weak MRP3 staining was detected in EAC biopsies (5 of 15, 33%). In addition, RT-PCR studies showed increased expression of mRNA coding for ASBT (6.1x), IBABP (9.1x), and MRP3 (2.4x) in BE (N=13) compared with normal squamous epithelium (N=15). Significantly increased mRNA levels of IBABP (10.1x) and MRP3 (2.5x) were also detected in EAC (N=21) compared with normal squamous epithelium. CONCLUSIONS: We found that bile acid transporters expression is increased in BE tissue at the mRNA and protein levels and that expression of bile acid transporter proteins decreased with progression to cancer.

Deoxycholate, an endogenous cytotoxin/genotoxin, induces the autophagic stress-survival pathway: implications for colon carcinogenesis.

We report that deoxycholate (DOC), a hydrophobic bile acid associated with a high-fat diet, activates the autophagic pathway in non-cancer colon epithelial cells (NCM-460), and that this activation contributes to cell survival. The DOC-induced increase in autophagy was documented by an increase in autophagic vacuoles (detected using transmission electron microscopy, increased levels of LC3-I and LC3-II (western blotting), an increase in acidic vesicles (fluorescence spectroscopy of monodansycadaverine and lysotracker red probes), and increased expression of the autophagic protein, beclin-1 (immunohistochemistry/western blotting). The DOC-induced increase in beclin-1 expression was ROS-dependent. Rapamycin (activator of autophagy) pre-treatment of NCM-460 cells significantly (P < .05) decreased, and 3-MA (inhibitor of autophagy) significantly (P < .05) increased the cell loss caused by DOC treatment, alone. Rapamycin pre-treatment of the apoptosis-resistant colon cancer cell line, HCT-116RC (developed in our laboratory), resulted in a significant decrease in DOC-induced cell death. Bafilomycin A(1) and hydroxychloroquine (inhibitors of the autophagic process) increased the DOC-induced percentage of apoptotic cells in HCT-116RC cells. It was concluded that the activation of autophagy by DOC has important implications for colon carcinogenesis and for the treatment of colon cancer in conjunction with commonly used chemotherapeutic agents.

Gene Expression and Serum Cytokine Profiling of Low Stage CLL Identify WNT/PCP, Flt-3L/Flt-3 and CXCL9/CXCR3 as Regulators of Cell Proliferation, Survival and Migration.

Gene expression profiling (GEP) of 8 stage 0/I untreated Chronic Lymphocytic Leukemia (CLL) patients showed over-expression of Frizzled 3 (FZD3)/ROR-1 receptor tyrosine kinase (RTK), FLT-3 RTK and CXCR3 G-protein coupled receptor (GPCR). RT-PCR of 24 genes in 21 patients of the WNT pathway corroborated the GEP. Transforming growth factorß, fibromodulin, TGFßRIII and SMAD2 are also over-expressed by GEP. Serum cytokine profiling of 26 low stage patients showed elevation of IFNγ, CSF3, Flt-3L and insulin-like growth factor binding protein 4. In order to ascertain why CLL cells grow poorly in culture, a GEP of 4 CLL patients cells at 0 hr and 24 hr in culture demonstrated over expression of CXCL5, CCL2 and CXCL3, that may recruit immune cells for survival. Treatment with thalidomide, an immunomodulatory agent, showed elevation of CCL5 by GEP but was not cytotoxic to CLL cells. Our data suggest an interplay of several oncogenic pathways, cytokines and immune cells that promote a survival program in CLL.

Field defects in progression to gastrointestinal tract cancers.

A field of defective tissue may represent a pre-malignant stage in progression to many cancers. However, field defects are often overlooked in studies of cancer progression through assuming tissue at some distance from the cancer is normal. We indicate, however, the generality of field defects in gastrointestinal cancers, including cancers of the oropharynx, esophagus, stomach, bile duct, pancreas, small intestine and colon/rectum. Common features of these field defects are reduced apoptosis competence, aberrant proliferation and genomic instability. These features are often associated with high bile acid exposure and may explain the association of dietary-related factors with cancer progression.

A novel dietary-related model of esophagitis and Barrett's esophagus, a premalignant lesion.

Barrett's esophagus (BE) is a premalignant lesion in which columnar epithelium (containing goblet cells) replaces esophageal squamous cells. Previous evidence suggested that hydrophobic bile acids and zinc deficiency each play a role in BE development. We fed wild-type C57BL/6 mice a zinc-deficient diet containing the hydrophobic bile acid, deoxycholic acid for various times up to 152 days. All mice fed this diet developed esophagitis by 69 days on the diet and 63% of the mice on this diet for 88 to 152 days also developed a BE-like lesion. Esophageal tissues showed thickened mucosa, increased proliferation, and increased expression of markers associated with oxidative and nitrosative stress. The newly formed BE-like lesions expressed Mucin-2, a marker of columnar differentiation. They also showed translocation of the p65 subunit of nuclear factor-kappaB and beta -catenin to the nucleus and typical histological changes associated with BE lesions. This mouse model of esophagitis and BE is expected to contribute to a deeper understanding of BE pathogenesis and to strategies for prevention of BE progression to cancer.

Activation of the interleukin-6/STAT3 antiapoptotic pathway in esophageal cells by bile acids and low pH: relevance to barrett's esophagus.

OBJECTIVES: The molecular factors contributing to the development of Barrett's esophagus (BE) are unclear. Our previous studies showed that BE tissues secrete interleukin-6 (IL-6) and express proteins associated with IL-6 signaling, including IL-6 receptor, activated signal transducer and activators of transcription 3 (STAT3), and antiapoptotic proteins Bcl-x(L) and Mcl-1. Here, we test the hypothesis that bile acids and gastric acids, two components of refluxate associated with gastresophageal reflux disease, activate the IL-6/STAT3 pathway. MATERIALS AND METHODS: Immunohistochemistry was used to assess levels of phosphorylated STAT3 in esophageal tissue samples from BE patients with different grades of dysplasia. Seg-1 esophageal adenocarcinoma cells were evaluated for STAT3 activation and IL-6 and Bcl-x(L) expression by molecular biology techniques, including Western blot, reverse transcription-PCR, and ELISA after exposure to control media (pH 7.4), media supplemented with a 0.1 mmol/L bile acid cocktail with media at pH 4 or media at pH 4 with bile acid cocktail. RESULTS: Immunohistochemical analysis showed that activated, phosphorylated STAT3 is expressed in nuclei of dysplastic BE and cancer tissues. Treatment of Seg-1 cells with media containing bile acid cocktail and acidified to pH 4 resulted in increased activation of STAT3, IL-6 secretion, and increased expression of Bcl-x(L). Inhibition of the STAT3 pathway using STAT3 small interfering RNA or Janus-activated kinase inhibitor resulted in increased apoptosis. CONCLUSIONS: The IL-6/STAT3 antiapoptotic pathway is induced by short exposure to bile acid cocktail and low pH. This alteration, if persistent in vivo, may underlie the development of dysplastic BE and tumor progression.

Five-year colon surveillance after screening colonoscopy.

BACKGROUND & AIMS: Outcomes of colon surveillance after colorectal cancer screening with colonoscopy are uncertain. We conducted a prospective study to measure incidence of advanced neoplasia in patients within 5.5 years of screening colonoscopy. METHODS: Three thousand one hundred twenty-one asymptomatic subjects, age 50 to 75 years, had screening colonoscopy between 1994 and 1997 in the Department of Veterans Affairs. One thousand one hundred seventy-one subjects with neoplasia and 501 neoplasia-free controls were assigned to colonoscopic surveillance over 5 years. Cohorts were defined by baseline findings. Relative risks for advanced neoplasia within 5.5 years were calculated. Advanced neoplasia was defined as tubular adenoma greater than > or =10 mm, adenoma with villous histology, adenoma with high-grade dysplasia, or invasive cancer. RESULTS: Eight hundred ninety-five (76.4%) patients with neoplasia and 298 subjects (59.5%) without neoplasia at baseline had colonoscopy within 5.5 years; 2.4% of patients with no neoplasia had interval advanced neoplasia. The relative risk in patients with baseline neoplasia was 1.92 (95% CI: 0.83-4.42) with 1 or 2 tubular adenomas <10 mm, 5.01 (95% CI: 2.10-11.96) with 3 or more tubular adenomas <10 mm, 6.40 (95% CI: 2.74-14.94) with tubular adenoma > or =10 mm, 6.05 (95% CI: 2.48-14.71) for villous adenoma, and 6.87 (95% CI: 2.61-18.07) for adenoma with high-grade dysplasia. CONCLUSIONS: There is a strong association between results of baseline screening colonoscopy and rate of serious incident lesions during 5.5 years of surveillance. Patients with 1 or 2 tubular adenomas less than 10 mm represent a low-risk group compared with other patients with colon neoplasia.

Deoxycholate-induced colitis is markedly attenuated in Nos2 knockout mice in association with modulation of gene expression profiles.

Nos2 knockout mice were compared to wild-type mice for susceptibility to colitis in response to a diet supplemented with deoxycholate, a bile acid increased in the colon of individuals on a high-fat diet. Wild-type mice fed a fat-related diet, supplemented with 0.2% DOC, develop colonic inflammation associated with increases in nitrosative stress, proliferation, oxidative DNA/RNA damage, and angiogenesis, as well as altered expression of numerous genes. However, Nos2 knockout mice fed a diet supplemented with deoxycholate were resistant to these alterations. In particular, 35 genes were identified whose expression was significantly altered at the mRNA level in deoxycholate-fed Nos2(+/+) mice but not in deoxycholate-fed Nos2(-/-) mice. Some of these alterations in NOS2-dependent gene expression correspond to those reported in human inflammatory bowel disease. Overall, our results indicate that NOS2 expression is necessary for the development of deoxycholate-induced colitis in mice, a unique dietary-related model of colitis.

Bile acids in combination with low pH induce oxidative stress and oxidative DNA damage: relevance to the pathogenesis of Barrett's oesophagus.

BACKGROUND: Barrett's oesophagus is a premalignant condition associated with an increased risk for the development of oesophageal adenocarcinoma (ADCA). Previous studies indicated that oxidative damage contributes to the development of ADCA. OBJECTIVE: To test the hypothesis that bile acids and gastric acid, two components of refluxate, can induce oxidative stress and oxidative DNA damage. METHODS: Oxidative stress was evaluated by staining Barrett's oesophagus tissues with different degrees of dysplasia with 8-hydroxy-deoxyguanosine (8-OH-dG) antibody. The levels of 8-OH-dG were also evaluated ex vivo in Barrett's oesophagus tissues incubated for 10 min with control medium and medium acidified to pH 4 and supplemented with 0.5 mM bile acid cocktail. Furthermore, three oesophageal cell lines (Seg-1 cells, Barrett's oesophagus cells and HET-1A cells) were exposed to control media, media containing 0.1 mM bile acid cocktail, media acidified to pH 4, and media at pH 4 supplemented with 0.1 mM bile acid cocktail, and evaluated for induction of reactive oxygen species (ROS). RESULTS: Immunohistochemical analysis showed that 8-OH-dG is formed mainly in the epithelial cells in dysplastic Barrett's oesophagus. Importantly, incubation of Barrett's oesophagus tissues with the combination of bile acid cocktail and acid leads to increased formation of 8-OH-dG. An increase in ROS in oesophageal cells was detected after exposure to pH 4 and bile acid cocktail. CONCLUSIONS: Oxidative stress and oxidative DNA damage can be induced in oesophageal tissues and cells by short exposures to bile acids and low pH. These alterations may underlie the development of Barrett's oesophagus and tumour progression.

Abnormal expression of biomarkers in incompletely ablated Barrett's esophagus.

OBJECTIVE: The aim of this study was to evaluate expression of cancer risk-associated biomarkers in columnar epithelium at squamocolumnar junctions produced by an ablation procedure and proton pump inhibitors in incompletely ablated Barrett's esophagus (BE) patients that were nondysplastic prior to ablation. SUMMARY BACKGROUND DATA: Ablation of BE to squamous epithelium is achievable by combining a re-injury method with acid suppression. We previously reported that, when there is complete ablation, the neo-squamous epithelium is normal histologically and in biomarker expression. However, squamous islands observed after prolonged use of PPIs were associated with abnormalities in p53 expression and Ki-67 labeling. METHODS: Twenty-one nondysplastic BE cases with incomplete ablation were evaluated for the expression of Ki-67 (proliferation), cyclooxygenase-2 (COX-2), and p53 by immunohistochemistry. RESULTS: Pre-ablation biopsies showed the normal staining patterns in columnar epithelium, ie, normal Ki-67 labeling, rare positive COX-2 staining of interstitial cells, and negative or mild staining for p53 in the majority of patients' biopsies. However, post-ablation biopsies demonstrated abnormal staining patterns in the glandular area at the new squamocolumnar junctions. In 13 of 21 post-ablation cases (62%), increased Ki-67 staining was seen in BE glands. In 8 of 21 patients (38%), increased COX-2 expression was seen in columnar epithelium. Similarly, in 8 of 21 post-ablation junctions (38%), there was increased p53 staining. CONCLUSIONS: Our findings of increased expression of cancer-associated biomarkers in incompletely ablated BE patients raise a cautionary note regarding this procedure. We hypothesize that newly formed junctions contain cells undergoing replication, differentiation, etc, and are thus more susceptible to genomic damage.

Pathogenesis of diarrhea in the adult: diagnostic challenges and life-threatening conditions.

The mechanisms that regulate ionic balance, fluid absorption and secretion in the gastrointestinal tract are complex. Disturbance of this homeostatic state by bile acids, bacterial enterotoxins and neoplasm-derived secretagogues, for example, can lead to diarrhea. Determining the causes of chronic diarrhea in individual patients may, therefore, represent a diagnostic challenge. The gastrointestinal tract has finely tuned mechanisms to maintain ionic balance, fluid absorption and secretion. To achieve this homeostasis, various ionic transport proteins/complexes are targeted to the apical, basal or basolateral membranes of epithelial cells. Na, K and Cl ion transport proteins are regulated by endogenous activators (e.g. cAMP, PGE2, Ca), dietary-related factors (e.g. bile acids) and through post-translational modifications (e.g. phosphorylation). In certain pathophysiologic states, this homeostatic ionic/fluid exchange becomes dysfunctional as a result of failure of compensatory pro-absorptive/anti-secretory mechanisms. The excessive secretion of Na and Cl ions followed by the release of a large amount of H2O into the colonic lumen results in diarrhea, which may be acute or chronic, and can be life-threatening if not ameliorated. Diverse infectious organisms (e.g. bacteria, protozoa, viruses) utilize different mechanisms to cause intestinal disease and diarrhea. These pathophysiologic mechanisms include alterations in ion transport, disruption of tight junctions and elicitation of inflammatory responses. Studies of patients infected with certain pathogens, those with ulcerative colitis and those harboring extra-colonic or colonic neoplasms have elucidated some of these pathophysiologic mechanisms of diarrhea. Determining the etiology of chronic diarrhea in specific cases may be a diagnostic challenge for both gastroenterologists and primary care physicians.

Reduced Pms2 expression in non-neoplastic flat mucosa from patients with colon cancer correlates with reduced apoptosis competence.

Pms2 protein is a component of the DNA mismatch repair complex responsible both for post-replication correction of DNA nucleotide mispairs and for early steps in apoptosis. Germline mutations in DNA mismatch repair genes give rise to hereditary non-polyposis colon cancer, which accounts for about 4% of colon cancers. However, little is known about the expression of mismatch repair proteins in relation to sporadic colon cancer, which accounts for the great majority of colon cancers. Multiple samples were taken from the non-neoplastic flat mucosa of colon resections from patients with no colonic neoplasia, a tubulovillous adenoma, or an adenocarcinoma. Expression of Pms2 was assessed using semiquantitative immunohistochemistry. Apoptosis was assessed in polychrome-stained epoxy sections using morphologic criteria. Samples from patients without colonic neoplasia had moderate to strong staining for Pms2 in cell nuclei at the base of crypts, while samples from 2 of the 3 colons with a tubulovillous adenoma, and from 6 of the 10 colons with adenocarcinomas, showed reduced Pms2 expression. Samples from patients with an adenocarcinoma that had reduced Pms2 expression also exhibited reduced apoptosis capability in nearby tissue samples, evidenced when this paired tissue was stressed ex vivo with bile acid. Reduced Pms2 expression in the colonic mucosa may be an early step in progression to colon cancer. This reduction may cause decreased mismatch repair, increased genetic instability, and/or reduced apoptotic capability. Immunohistochemical determination of reduced Pms2 expression, upon further testing, may prove to be a promising early biomarker of risk of progression to malignancy.

Unique dietary-related mouse model of colitis.

BACKGROUND: A high-fat diet is a risk factor for the development of inflammatory bowel disease (IBD) in humans. Deoxycholate (DOC) is increased in the colonic contents in response to a high-fat diet. Thus, an elevated level of DOC in the colonic lumen may play a role in the natural course of development of IBD. METHODS: Wild-type B6.129 mice were fed an AIN-93G diet, either supplemented with 0.2% DOC or unsupplemented and sacrificed at 1 week, 1 month, 3 months, 4 months, and 8 months. Colon samples were assessed by histopathological, immunohistochemical, and cDNA microarray analyses. RESULTS: Mice fed the DOC-supplemented diet developed focal areas of colonic inflammation associated with increases in angiogenesis, nitrosative stress, DNA/RNA damage, and proliferation. Genes that play a central role in inflammation and angiogenesis and other related processes such as epithelial barrier function, oxidative stress, apoptosis, cell proliferation/cell cycle/DNA repair, membrane transport, and the ubiquitin-proteasome pathway showed altered expression in the DOC-fed mice compared with the control mice. Changes in expression of individual genes (increases or reductions) correlated over time. These changes were greatest 1 month after the start of DOC feeding. CONCLUSIONS: The results suggest that exposure of the colonic mucosa to DOC may be a key etiologic factor in IBD. The DOC-fed mouse model may reflect the natural course of development of colitis/IBD in humans, and thus may be useful for determining new preventive strategies and lifestyle changes in affected individuals.

Identification of S-nitrosylated proteins after chronic exposure of colon epithelial cells to deoxycholate.

Apoptosis resistance, a condition favoring genomic instability, is associated with higher risk of colorectal cancer. Deoxycholate (DOC) is a hydrophobic bile salt found in high concentrations in colon cancer patients, and induces apoptosis in cultured colonic cells and ex vivo in colonic biopsies. We showed previously that the chronic exposure of colon cancer cells to increasing concentrations of DOC leads to apoptosis resistance, and the suggested mechanism involves oxidative/nitrosative stress. Nitric oxide (NO) is a key signaling molecule that regulates cell function in a variety of physiologic and pathophysiologic states. In part, NO exerts its actions by S-nitrosylation of target thiols, and several proteins are regulated through this PTM, including the caspases, the main effectors of apoptosis. Here, we performed a proteomics study in the DOC-induced apoptosis-resistant colon cell line, HCT-116RC. Its profile of S-nitrosylated proteins was compared to a control cell line not exposed to DOC. Eighteen differentially S-nitrosylated proteins were identified in the HCT-116RC cell line, 14 of these are novel targets of S-nitrosylation not previously reported. These proteins include cytoskeletal and signaling proteins, metabolic enzymes, chaperones, and redox- and differentiation-related proteins. These results broaden our knowledge of potential signal transduction pathways that may lead to the development of new biomarkers and therapy targets.

Clinical predictors of Barrett's esophagus length.

BACKGROUND: Assessment of clinical factors associated with Barrett's esophagus (BE) length remained within the realm of anecdotal reports or one center's experience. The aim of this multicenter study was to determine which clinical factors are highly correlated with the length of BE. METHODS: Patients diagnosed with BE were recruited into the study from 5 academic centers in the United States. All patients had an upper endoscopy that documented BE by the presence of intestinal metaplasia in biopsy specimens. All patients were evaluated by a validated demographic questionnaire and the GERD Symptom Checklist. RESULTS: A total of 263 patients with BE were recruited into the study. Mean BE length length for the entire sample was 4 +/- 3.3 cm. A longer hiatal hernia (r = 0.22, p < 0.01), any dysplasia (t = -2.3, p < 0.05), H2 receptor antagonist (H2-RA) consumption (t = 1.98, p < 0.05), and nonsmoking (t = -2.5, p < 0.05) were correlated with a longer segment of BE. Proton pump inhibitors (PPI) (t = 1.96, p < 0.05) were correlated with a shorter segment of BE. CONCLUSIONS: PPIs were correlated with shorter lengths of BE. In contrast, a longer hiatal hernia, any dysplasia, nonsmoking, or use of H2-RAs were correlated with a longer BE segment.

Crypt-restricted loss and decreased protein expression of cytochrome C oxidase subunit I as potential hypothesis-driven biomarkers of colon cancer risk.

There is an increasing demand for the development of intermediate biomarkers to assess colon cancer risk. We previously determined that a live cell bioassay, which assesses apoptosis resistance in the nonneoplastic colonic mucosa, detects approximately 50% of patients with colon cancer. A hypothesis-driven biomarker that reflects apoptosis resistance in routine formalin-fixed, paraffin-embedded tissue would be easier to use. Cytochrome c oxidase is a critical enzyme that controls mitochondrial respiration and is central to apoptosis. We did an immunohistochemical study of cytochrome c oxidase subunit I expression in 46 colonic mucosal samples from 16 patients who had undergone a colonic resection. These included five patients without evidence of colonic neoplasia (three normal and two diverticulitis), three patients with tubulovillous adenomas, and eight patients with colonic adenocarcinomas. Analysis of aberrancies in expression of cytochrome c oxidase subunit I showed that, compared with nonneoplasia, the patients with neoplasia had a higher mean incidence of crypts having decreased expression (1.7 versus 22.8, P = 0.03) and a higher mean incidence having crypt-restricted loss (0.6 versus 3.2, P = 0.06). The percentage with segmented loss was low and was similar in the two groups. Combining these results, the mean % normal (i.e., with none of the three types of abnormality) was 96.7 in nonneoplasia versus only 73.2 in patients with neoplasia (P = 0.02). It should be noted that a defect in cytochrome c oxidase subunit I immunostaining was not detected in all biopsy samples from each patient for whom some abnormality was found, indicating a "patchiness" in the cytochrome c oxidase subunit I field defect. As a result of this "patchiness," the increased variability in the incidence of crypt-restricted loss of cytochrome c oxidase subunit I expression was a statistically significant feature of the neoplasia group. Crypt-restricted loss of cytochrome c oxidase subunit I has not been previously reported in colonic mucosa and is presumably the result of a crypt-restricted stem cell mutation. Decreased cytochrome c oxidase subunit I expression also significantly correlated with apoptosis resistance, a factor known to contribute to carcinogenesis. The results suggest, however, that aberrant cytochrome c oxidase subunit I expression may be a better biomarker than loss of apoptosis competence for increased colon cancer risk.

Assessment of apoptosis by immunohistochemical markers compared to cellular morphology in ex vivo-stressed colonic mucosa.

Apoptosis competence is central to the prevention of cancer. Frequency of apoptotic cells, after a sample of colonic tissue is stressed, can be used to gauge apoptosis competence and, thus, possible susceptibility to colon cancer. The gold standard for assessment of apoptosis is morphological evaluation, but this requires an experienced microscopist. Easier-to-use immunohistochemical markers of apoptosis, applicable in archived paraffin-embedded tissue, have been commercially developed. Potentially useful apoptosis markers include cleaved cytokeratin-18 (c-CK18), cleaved caspase-3 (c-cas-3), cleaved lamin A (c-lam-A), phosphorylated histone H2AX (gammaH2AX), cleaved poly(ADP ribose) polymerase (c-PARP), and translocation of apoptosis-inducing factor (AIF). When tissue samples from freshly resected colon segments were challenged ex vivo with the bile acid deoxycholate, approximately 50% of goblet cells became apoptotic by morphologic criteria. This high level of morphologic apoptosis allowed quantitative comparison with the usefulness and specificity of immunohistochemical markers of apoptosis. The antibody to c-CK18 was almost as useful and about as specific as morphology for identifying apoptotic colonic epithelial cells. Antibodies to c-cas-3, c-lam-A, and gammaH2AX, though specific for apoptotic cells, were less useful. The antibody to c-PARP, though specific for apoptotic cells, had low usefulness, and the antibody to AIF was relatively nonspecific, under our conditions.