MLH1 Promotor Hypermethylation in Colorectal and Endometrial Carcinomas from Patients with Lynch Syndrome.

Screening for Lynch syndrome (LS) in colorectal cancer (CRC) and endometrial cancer patients generally involves immunohistochemical staining of the mismatch repair (MMR) proteins. In case of MLH1 protein loss, MLH1 promotor hypermethylation (MLH1-PM) testing is performed to indirectly distinguish the constitutional MLH1 variants from somatic epimutations. Recently, multiple studies have reported that MLH1-PM and pathogenic constitutional MMR variants are not mutually exclusive. This study describes 6 new and 86 previously reported MLH1-PM CRCs or endometrial cancers in LS patients. Of these, methylation of the MLH1 gene promotor C region was reported in 30 MLH1, 6 MSH2, 6 MSH6, and 3 PMS2 variant carriers at a median age at diagnosis of 48.5 years [interquartile range (IQR), 39-56.75 years], 39 years (IQR, 29-51 years), 58 years (IQR, 53.5-67 years), and 68 years (IQR, 65.6-68.5 years), respectively. For 31 MLH1-PM CRCs in LS patients from the literature, only the B region of the MLH1 gene promotor was tested, whereas for 13 cases in the literature the tested region was not specified. Collectively, these data indicate that a diagnosis of LS should not be excluded when MLH1-PM is detected. Clinicians should carefully consider whether follow-up genetic MMR gene testing should be offered, with age <60 to 70 years and/or a positive family history among other factors being suggestive for a potential constitutional MMR gene defect.

Role of epoxide hydrolase, NAD(P)H:quinone oxidoreductase, cytochrome P450 2E1 or alcohol dehydrogenase genotypes in susceptibility to colorectal cancer.

Colorectal cancer (CRC) is one of the most common forms of cancer in Western countries. CRC has been associated with genetic and lifestyle factors. Individual susceptibility to CRC may be due partly to variations in detoxification capacity in the gastrointestinal tract. Genetic polymorphisms in detoxification enzymes may result in variations in detoxification activities, which subsequently might influence the levels of toxic/carcinogenic compounds, and this may influence the risk for CRC. Therefore, we determined whether polymorphisms in the genes coding for microsomal epoxide hydrolase (EPHX1), NAD(P)H:quinone oxidoreductase (NQO1), cytochrome P450 2E1 (CYP2E1) and alcohol dehydrogenase (ADH3) predispose to the development of CRC. DNA samples were obtained from 371 patients with sporadic CRC and 415 healthy controls. Patients and controls were all of Caucasian origin. All genetic polymorphisms were determined by polymerase chain reaction, eventually followed by restriction-fragment-length-polymorphism analyses, except for the EPHX1 codon 113 polymorphism, which was genotyped by an allele-specific discrimination assay. Calculation of crude Odds Ratios (ORs) revealed an increased risk for CRC associated with variant NQO1 (OR 1.5, 95% CI 1.1-2.0) and CYP2E1 intron 6 genotypes (OR 2.2, 95% CI 1.3-3.8). However, after adjustment for age and gender, logistic regression analyses only showed a statistically significant risk for CRC associated with variant NQO1 genotypes (OR 1.6, 95% CI 1.03-2.4). No associations were found between CRC and the other polymorphic genes as mentioned above. In conclusion, these data suggest that the presence of variant NQO1 genotypes, with expected reduced enzyme activities might enhance susceptibility to CRC.

High oxygen radical production in patients with sporadic colorectal cancer.

It is hypothesized that excessive generation of reactive oxygen species (ROS) by phagocytes or leakage from mitochondria may harm key genes or proteins responsible for intestinal cell homeostasis. This may initiate the multistage process of colon cancer development. The present study investigates whether ROS production by whole blood may contribute to the etiology of colorectal cancer (CRC). Whole-blood oxygen radical production was measured by luminol-enhanced chemiluminescence and performed in fourfold with and without the stimuli phorbol 12-myristate 13-acetate (PMA) and serum-treated zymosan (STZ). We evaluated patients (i) with a history of sporadic CRC at least 3 months after surgery, (ii) who were hereditary nonpolyposis colorectal cancer (HNPCC) gene carriers, and (iii) with familial adenomatous polyposis (FAP). For each patient group (n = 20) an age- and gender-matched healthy control group was measured. Unstimulated and PMA-stimulated values for maximal oxygen radical production were significantly higher in patients with sporadic CRC in comparison to controls (p = 0.01, p = 0.04, respectively). Furthermore, trends toward higher unstimulated and PMA-stimulated area under the curve chemiluminescence were seen in CRC patients compared with controls (p = 0.08, p = 0.09, respectively). In patients with HNPCC or FAP, unstimulated or PMA- or STZ-stimulated chemiluminescence did not differ compared to their control groups. In conclusion, whole-blood oxygen radical production was higher in patients with a history of sporadic CRC, in comparison with age- and gender-matched controls, which indicates that ROS may play a role in the etiology of sporadic CRC.

Glibenclamide depletes ATP in renal proximal tubular cells by interfering with mitochondrial metabolism.

Sulfonylurea drugs, like glibenclamide, stimulate insulin secretion by blocking ATP-sensitive potassium channels on pancreatic beta cells. Renal tubular epithelial cells possess a different class of K(ATP) channels with much lower affinities for sulfonylurea drugs, necessitating the use of micromolar glibenclamide concentrations to study these channels. Here, we describe the toxic effects of these concentrations on mitochondrial energy metabolism in freshly isolated renal proximal tubular cells. Glibenclamide, at concentrations of 50 and 100 microM, reduced intracellular ATP levels by 28+/-4 and 53+/-5%, respectively (P<0.01). This decline in ATP could be attributed to a dose-dependent inhibition of mitochondrial respiration. Glibenclamide (10-500 microM) inhibited ADP-stimulated mitochondrial oxygen consumption. In addition to this toxic effect, specific association of radiolabeled and fluorescent glibenclamide to renal mitochondria was found. Association of [(3)H]glibenclamide with renal mitochondria revealed a low-affinity site with a K(D) of 16+/-6 microM and a B(max) of 167+/-29 pmol mg(-1). We conclude that micromolar concentrations of glibenclamide interfere with mitochondrial bioenergetics and, therefore, should be used with care for inhibition of epithelial K(ATP) channels.

No association between genetic polymorphisms in NAD(P)H oxidase p22phox and paraoxonase 1 and colorectal cancer risk.

BACKGROUND: Impaired metabolism of ingested toxic or carcinogenic compounds is a postulated mechanism underlying colorectal cancer (CRC). Furthermore, it is suggested that reactive oxygen species (ROS) may play a role in human cancer development. Polymorphic variations in NAD(P)H oxidase p22phox and paraoxonase 1 (PON1) enzyme activities may alter superoxide production or the rate of chemical metabolism, respectively, and this may influence the risk for CRC. Therefore, this study was designed to determine whether the distribution of polymorphisms in NAD(P)H oxidase p22phox and PON1 genes was different in sporadic CRCpatients versus healthy controls. MATERIALS AND METHODS: The study participants (365 cases and 354 controls) were all of Caucasian origin. NAD(P)H oxidase p22phox H72Y, and PON1 L55M and Q192R polymorphisms were genotyped by polymerase chain reaction, eventually followed by restriction-fragment-length-polymorphism analyses. RESULTS: Comparison of CRC patients and controls revealed no significant differences in genotype distributions or allele frequencies for polymorphisms in the NAD(P)H oxidase p22phox and PON1 genes. Investigation of potential associations between the variant NAD(P)H oxidase p22phox or PON1 alleles and the clinical characteristics, tumour location or tumour stage, also did not reveal statistically significant associations. CONCLUSION: Variant genotypes of NAD(P)H oxidase p22phox and PON1 do not contribute to the susceptibility to CRC.

Fully automated fluorescent in situ hybridization (FISH) staining and digital analysis of HER2 in breast cancer: a validation study.

HER2 assessment is routinely used to select patients with invasive breast cancer that might benefit from HER2-targeted therapy. The aim of this study was to validate a fully automated in situ hybridization (ISH) procedure that combines the automated Leica HER2 fluorescent ISH system for Bond with supervised automated analysis with the Visia imaging D-Sight digital imaging platform. HER2 assessment was performed on 328 formalin-fixed/paraffin-embedded invasive breast cancer tumors on tissue microarrays (TMA) and 100 (50 selected IHC 2+ and 50 random IHC scores) full-sized slides of resections/biopsies obtained for diagnostic purposes previously. For digital analysis slides were pre-screened at 20x and 100x magnification for all fluorescent signals and supervised-automated scoring was performed on at least two pictures (in total at least 20 nuclei were counted) with the D-Sight HER2 FISH analysis module by two observers independently. Results were compared to data obtained previously with the manual Abbott FISH test. The overall agreement with Abbott FISH data among TMA samples and 50 selected IHC 2+ cases was 98.8% (κ = 0.94) and 93.8% (κ = 0.88), respectively. The results of 50 additionally tested unselected IHC cases were concordant with previously obtained IHC and/or FISH data. The combination of the Leica FISH system with the D-Sight digital imaging platform is a feasible method for HER2 assessment in routine clinical practice for patients with invasive breast cancer.

Effects of dietary anticarcinogens and nonsteroidal anti-inflammatory drugs on rat gastrointestinal UDP-glucuronosyltransferases.

BACKGROUND: Dietary compounds or nonsteroidal anti-inflammatory drugs (NSAIDs) may reduce cancer rates. Elevation of phase II detoxification enzymes might be one of the mechanisms leading to cancer prevention. We investigated the effects of dietary anticarcinogens and NSAIDs on rat gastrointestinal UDP-glucuronosyltransferases (UGT). MATERIALS AND METHODS: Diets of Wistar rats were supplemented with oltipraz, alpha-tocopherol, beta-carotene, phenethylisothiocyanate (PEITC), sulforaphane analogue compound-30, indole-3-carbinol, D-limonene, relafen, indomethacin, ibuprofen, piroxicam, acetyl salicylic acid or sulindac. Hepatic and intestinal UGT enzyme activities were quantified by using 4-nitrophenol and 4-methylumbelliferone as substrates. RESULTS: Compound-30, D-limonene, indomethacin, ibuprofen or sulindac enhanced proximal small intestinal UGT activities. Only compound-30 was able to induce mid- and distal small intestinal UGT activities. Large intestinal UGT activities were increased by ibuprofen and sulindac, whereas oltipraz, PEITC and D-limonene gave enhanced hepatic UGT activities. CONCLUSION: Mainly rat proximal small intestinal and hepatic UGT enzyme activities were induced by dietary anticarcinogens or NSAIDs. Enhanced UGT activities might lead to a more efficient detoxification of carcinogenic compounds and thus could contribute to the prevention of gastrointestinal cancer.

Westernized high-fat diet accelerates weight loss in dextran sulfate sodium-induced colitis in mice, which is further aggravated by supplementation of heme.

The Western diet, rich in fat and red meat, predisposes for inflammatory bowel disease (IBD); however, little is known about mechanisms involved. Red meat contains high levels of heme, a well-known inducer of the cytoprotective enzyme heme oxygenase-1 (HO-1). Pharmacological induction of HO-1 ameliorates experimental colitis. We analyzed the effect of a westernized high-fat (HF) diet supplemented with heme on intestinal HO-1 expression and dextran sulfate sodium (DSS)-induced colitis. Mice were fed chow or HF diets for 2 weeks. In the second week, the HF diet was supplemented with or without 0.5 µmol/g heme. Subsequently, the 3 diet groups were given drinking water with or without 4% DSS to induce colitis. Significant body weight reduction was first observed after 4 days in the chow/DSS mice (-5±3%), whereas this was evident already after 2 days (-6±2%) in HF/DSS mice, showing increased weight loss compared to chow/DSS mice in the following days. Heme supplementation further aggravated DSS-induced weight loss in HF mice (-18±4% vs. -7±5% for HF+heme/DSS vs. HF/DSS, P<.01). Heme increased HO-1 expression in the colon epithelium but decreased villin messenger RNA levels, indicating epithelial damage. In contrast, heme did not affect DSS-induced colon shortening and histological scores of epithelial damage and inflammation. A westernized diet accelerates DSS-induced weight loss in mice, which is further aggravated by heme, despite the induction of HO-1 in the colon epithelium. Our data warrant a detailed analysis of the association of (red) meat-containing diets and the development of IBD.

COX-2 polymorphisms -765G-->C and -1195A-->G and colorectal cancer risk.

AIM: To determine the possible modulating effect of the COX-2 polymorphisms, -765G-->C and -1195A-->G, on the risk of colorectal cancer (CRC) in a Dutch population. METHODS: This case-control study includes 326 patients with CRC and 369 age- and gender-matched controls. Genotypes of the COX-2 polymorphisms -765G-->C and -1195A-->G were determined by polymerase chain reaction-based restriction fragment length polymorphism. COX-2 genotypes and haplotypes were analyzed and odds ratios with 95% confidence intervals were estimated by logistic regression. RESULTS: The -765GG genotype was associated with an increased risk of developing CRC (OR, 1.45; 95% CI, 1.03-2.04). No significant difference was observed in the genotype distribution of the -1195A-->G polymorphism between patients and controls. The GG/AC haplotype was present significantly less often in patients than in controls (OR 0.44; 95% CI, 0.22-0.85). When the AC, AG and GG haplotypes were investigated separately, the AC haplotype showed a tendency to be less frequent in patients than in controls (OR((AG/AC)) 0.78; 95% CI, 0.57-1.06). CONCLUSION: The -765GG genotype is associated with an increased risk of developing CRC and the GG/AC haplotype seems to protect against CRC. These findings suggest a modulating role for the COX-2 polymorphisms -765G-->C and -1195A-->G in the development of CRC in a Dutch population.

Effects of active and passive smoking on disease course of Crohn's disease and ulcerative colitis.

BACKGROUND: Smoking is a remarkable risk factor for inflammatory bowel disease (IBD), aggravating Crohn's disease (CD) while having beneficial effects on ulcerative colitis (UC). We studied the effects of active and passive smoking in Dutch IBD patients. METHODS: A questionnaire focusing on cigarette smoke exposure was sent to 820 IBD patients. Returned questionnaires were incorporated into a retrospective chart review, containing details about disease behavior and received therapy. RESULTS: In all, 675 IBD patients (380 [56%] CD and 295 [44%] UC) responded. At diagnosis there were 52% smokers in CD, 41% in the general population, and 28% in UC. The number of present smokers in CD is lower than in the general population (26% versus 35%). No detrimental effects of active smoking on CD were observed, but passive smokers needed immunosuppressants and infliximab more frequently than nonpassive smokers. Active smoking had beneficial effects on UC, indicated by reduced rates of colectomy, primary sclerosing cholangitis, and backwash-ileitis in active smokers compared to never smokers, and higher daily cigarette dose correlated with less extensive colitis and a lower need for therapy. Furthermore, smoking cessation after diagnosis was detrimental for UC patients, indicated by increased needs for steroids and hospitalizations for patients that stopped smoking after compared to before the diagnosis. CONCLUSIONS: Active smoking is a risk factor for CD, but does not affect the outcome; passive smoking is detrimental for the outcome of CD patients. In UC, active smoking shows dose-dependent beneficial effects. Our data suggest that passive smoking is a novel risk factor for CD.