MicroRNAs: biogenesis, roles for carcinogenesis and as potential biomarkers for cancer diagnosis and prognosis

MicroRNAs (miRNAs) are short non-coding RNAs of 20-24 nucleotides that play important roles in carcinogenesis. Accordingly, miRNAs control numerous cancer-relevant biological events such as cell proliferation, cell cycle control, metabolism and apoptosis. In this review, we summarize the current knowledge and concepts concerning the biogenesis of miRNAs, miRNA roles in cancer and their potential as biomarkers for cancer diagnosis and prognosis including the regulation of key cancer-related pathways, such as cell cycle control and miRNA dysregulation. Moreover, microRNA molecules are already receiving the attention of world researchers as therapeutic targets and agents. Therefore, in-depth knowledge of microRNAs has the potential not only to identify their roles in cancer, but also to exploit them as potential biomarkers for cancer diagnosis and identify therapeutic targets for new drug discovery.

REVERSIBILITY OF ADENOMATOUS HYPERPLASIA IN THE GASTRIC STUMP AFTER DIVERSION OF BILE REFLUX IN RATS

Morphological and phenotypical patterns of proliferative epithelial lesions induced in the gastric stump mucosa by duodenal content reflux after Billroth II partial gastrectomy (BII) were evaluated in rats. Control animals were either sham-operated or submitted at different times after BII to Roux-en-Y (RY) surgical procedure which prevents duodenal reflux. The lesions were analysed using routine haematoxylin and eosin staining, immunohistochemical staining for pepsinogen isoenzyme 1 and histochemical procedures for mucins (paradoxical Concanavalin A, galactose oxidase Schiff and sialidase galactose oxidase Schiff reactions). Mucosal hyperplasia (H) was observed in the group submitted to BII procedure 6 weeks after surgery. Adenomatous hyperplasia (AH) also appeared 6 weeks after induction of the reflux and its incidence and size increased until the 54th week of the experiment. RY procedure performed in the normal animals at the beginning of the experiment or at the 24th week after BII gastrectomy led to a significantly lower incidence of AH which was related to the moment of surgery. Most of H was due to pyloric mucosal hyperplasia. AH consisted mainly of gastric type glands but in some animals glands of the intestinal type Were present probably originating from the intestinal mucosa. Six mucinous adenocarcinomas were observed, all of them of intestinal type. This study demonstrates that AH induced by BII procedure is a reversible lesion and that the anomalous epithelial proliferation in the stoma may lead to adenocarcinomas.

REDUCTION OF CELL PROLIFERATIVE ACTIVITIES OF GASTRIC STUMP ADENOMATOUS HYPERPLASIAS AFTER BILE REFLUX DIVERSION IN RATS

Previously we reported the majority of lesions induced hy bile reflux, in the absence of chemical carcinogens, in the rat remnant stomach to consist primarily of gastric type and secondarily of intestinal type cells, and that they are reversible after diversion of bile reflux. The present study was designed to evaluate changes in proliferative activities in cells of each type under these conditions. The frequency of adenomatous hyperplasia (AH) induced in the gastric stump mucosa by duodenal content reflux after Billroth II partial gastrectomy (BII) increased until the 54th week of the experiment. Roux-en-Y (RY) surgical procedure which prevents duodenal reflux performed at the 24th or 36th week after BII led to a decrease in AH. Cell content of the lesions was analyzed using routine H&E staining, immunohistochemical staining for pepsinogen isoenzyme 1 and histochemical procedures for mucins (paradoxical concanavalin A, galactose oxidase Schiff and sialidase galactose oxidase Schiff reactions) and proliferation in each compartment evaluated by an immunohistochemical method using bromodeoxyuridine (BrdU) and a monoclonal antibody against BrdU. At the 54th week the number of BrdU-labeled cells per normal pyloric column was significantly (P < 0.05) increased to 10.63/pit after the BII operation, while it diminished to 5.23/pit after RY diversion, this being the same level as with the RY procedure alone. AH maintained a high rate of BrdU incorporation at 12.7% after BII operation, which was also significantly reduced (P < 0.01) to 7.0% by the RY surgery. The intestinal type cell showed highest (22.2%), the surface mucous type cell showed the next (16.5%) and the pyloric gland type cell showed lowest (5.2%) BrdU labeling indices after BII operation. All the cell types in AH showed similar proportional decreases in BrdU incorporation after RY diversion. Thus surgical intervention reverses the cell proliferation caused by bile reflux in the gastric stump.

Apoptosis in different gastric lesions and gastric cancer: Relationship with Helicobacter pylori, overexpression of p53 and aneuploidy

Apoptosis has an essential function in maintaining the integrity of the gastrointestinal mucosa. Its deregulation is associated with the occurrence of lesions such as in atrophic gastritis, peptic ulcers, intestinal metaplasia, and stomach tumorigenesis. Thus, the aim of the present study was to investigate the frequency of apoptotic cells (apoptotic index, AI) by using two different immunohistochemical techniques, TUNEL and anti-activated caspase-3 antibody (CPP32), in gastric dyspepsia [chronic gastritis (CG, N = 34), chronic atrophic gastritis (CAG, N = 11), gastric ulcer (GU, N = 17), and intestinal metaplasia (IM, N = 15)], normal gastric mucosae (NM, N = 8), and gastric adenocarcinoma (GC, N = 12). The relationship was investigated between the AI and Helicobacter pylori infection, diagnosed by PCR, overexpression of p53 protein determined by immunohistochemistry, and aneuploidy by fluorescence in situ hybridization, as performed by our laboratory in previous studies. No significant differences were observed in AI between the different groups, whether by the TUNEL technique (F = 1.60; P = 0.1670) or by CPP32 antibody (F = 1.70; P = 0.1420). Nonetheless, CAG and CG groups had AI statistically higher than those of normal mucosae. These two groups (CAG and CG) also showed a higher frequency of apoptosis-positive cases (TUNEL+ or CPP32+). Generally, there was no correlation between the AI detected by the TUNEL and CPP32 techniques in the groups studied, except in the GC group (r = 0.70). Moreover, there was no significant association between apoptosis and H. pylori infection, overexpression of p53 protein and aneuploidy, but the H. pylori-positive cases only of GU (P = 0.0233) and IM (P = 0.0253) groups displayed a statistically higher AI compared to H. pylori-negative NM, when the CPP32 antibody technique was used. Thus, CG and CAG have increased apoptosis, which may occur independent of an association with H. pylori infection, aneuploidy and overexpression of p53 protein. ©FUNPEC-RP.

Expression of annexin-A1 and galectin-1 anti-inflammatory proteins and mRNA in chronic gastritis and gastric cancer

Objective. The anti-inflammatory proteins annexin-A1 and galectin-1 have been associated with tumor progression. This scenario prompted us to investigate the relationship between the gene and protein expression of annexin-A1 (ANXA1/AnxA1) and galectin-1 (LGALS1/Gal-1) in an inflammatory gastric lesion as chronic gastritis (CG) and gastric adenocarcinoma (GA) and its association with H. pylori infection. Methods. We analyzed 40 samples of CG, 20 of GA, and 10 of normal mucosa (C) by the quantitative real-time PCR (qPCR) technique and the immunohistochemistry assay. Results. High ANXA1 mRNA expression levels were observed in 90% (36/40) of CG cases (mean relative quantification RQ = 4.26 ± 2.03) and in 80% (16/20) of GA cases (mean RQ = 4.38 ± 4.77). However, LGALS1 mRNA levels were high (mean RQ = 2.44 ± 3.26) in 60% (12/20) of the GA cases, while low expression was found in CG (mean RQ = 0.43 ± 3.13; P < 0.01). Normal mucosa showed modest immunoreactivity in stroma but not in epithelium, while stroma and epithelium displayed an intense immunostaining in CG and GA for both proteins. Conclusion. These results have provided evidence that galectin-1 and mainly annexin-A1 are overexpressed in both gastritis and gastric cancer, suggesting a strong association of these proteins with chronic gastric inflammation and carcinogenesis. © 2013 Yvana Cristina Jorge et al.

Digit ratio (2D:4D) is associated with gastric cancer

Background: Sex steroid exposure during early human development may influence disease susceptibility. Digit ratio (2D:4D) is a putative marker for prenatal hormone exposure and sensitivity, as well as the action of genes closely related to carcinogenesis. Digit ratio could act as a possible marker for cancer predisposition. Aims: The aim of this study is to investigate the possible correlations between right hand, left hand and right minus left (R. - L) 2D:4D and gastric cancer (GCA) in men and women and assess the correlations with tumor staging and histological diagnosis. Methods: Digital images of the right and left hand palms of patients diagnosed with GCA (n = 57, 42 males, 15 females) and age and sex-matched controls (n = 59, 41 males, 18 females) were obtained. Means for 2D:4D were compared. Data were analyzed by repeated-measures one-way ANOVA and Student's t-test for finger measurements and group comparisons and Pearson's and Spearman's tests for correlations with tumor staging (α = 0.05). Results: GCA group presented significantly higher left 2D:4D, but significantly lower R. - L in comparison to healthy controls, particularly so for males. Digit ratio did not correlate to clinical staging or TNM staging. However, low R. - L was significantly related to adenocarcinomas. Conclusions: Early developmental conditions, including prenatal testosterone seem to play a role on the malignant transformation of gastric lesions. The 2D:4D pattern found for gastric cancer parallels that earlier described for breast cancer. The findings suggest that 2D:4D could add to the list of etiological factors and be a putative marker for the screening of patients' susceptibility to develop gastric cancer. © 2012 Elsevier Ltd.

Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice

Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (NQO1). Constitutive hepatic and gastric activities of GST and NQO1 were reduced by 50–80% in nrf2-deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice by the chemoprotective agent oltipraz, which is currently in clinical trials, was almost completely abrogated in the nrf2-deficient mice. In parallel with the enzymatic changes, nrf2-deficient mice had a significantly higher burden of gastric neoplasia after treatment with benzo[a]pyrene than did wild-type mice. Oltipraz significantly reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no effect on tumor burden in nrf2-deficient mice. Thus, Nrf2 plays a central role in the regulation of constitutive and inducible expression of phase 2 enzymes in vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the total loss of anticarcinogenic efficacy of oltipraz in the nrf2-disrupted mice highlights the prime importance of elevated phase 2 gene expression in chemoprotection by this and similar enzyme inducers.

Transcutaneous immunization with novel lipid-based adjuvants induces protection against gastric Helicobacter pylori infection

The development of vaccines to combat pathogens that infect across mucosal surfaces has been a major goal of vaccine research. Successful mucosal vaccination requires the co-administration of adjuvants that can overcome the state of immune tolerance normally associated with mucosal application of proteins. In the case of oral immunization, delivery systems are also required to protect vaccine antigens against destruction by gastric pH and digestive enzymes. Furthermore, adjuvants used for mucosal delivery must be free of neurotoxic effects like those induced by the commonly used experimental mucosal adjuvant cholera toxin. Maintenance of the "cold chain" is also essential for the effectiveness of any vaccine and adjuvants/delivery systems that enhance the stability of a vaccine would offer a significant advantage. Needle-free methods of vaccination that induce protective immunity at multiple mucosal surfaces are also desirable for rapid vaccination of large populations. In the present study we show that transcutaneous immunization (TCI) using Lipid C, a novel lipid-based matrix originally developed for oral immunization, containing soluble Helicobacter sonicate significantly reduces the gastric bacterial burden in mice following gastric challenge with live Helicobacter pylori. Protection is associated with the production of splenic gamma interferon and gastric IgA and was achieved without the co-administration of potent and potentially toxic adjuvants, although protection was further enhanced by inclusion of CpG-ODN and cholera toxin in the lipid delivery system.

The effects of weight loss strategies on gastric emptying and appetite control

The gastrointestinal tract plays an important role in the improved appetite control and weight loss in response to bariatric surgery. Other strategies which similarly alter gastrointestinal responses to food intake could contribute to successful weight management. The aim of this review is to discuss the effects of surgical, pharmacological and behavioural weight loss interventions on gastrointestinal targets of appetite control, including gastric emptying. Gastrointestinal peptides are also discussed because of their integrative relationship in appetite control. This review shows that different strategies exert diverse effects and there is no consensus on the optimal strategy for manipulating gastric emptying to improve appetite control. Emerging evidence from surgical procedures (e.g., sleeve gastrectomy and Roux en-Y gastric bypass) suggests a faster emptying rate and earlier delivery of nutrients to the distal small intestine may improve appetite control. Energy restriction slows gastric emptying, while the effect of exercise-induced weight loss on gastric emptying remains to be established. The limited evidence suggests that chronic exercise is associated with faster gastric emptying which we hypothesise will impact on appetite control and energy balance. Understanding how behavioural weight loss interventions (e.g., diet and exercise) alter gastrointestinal targets of appetite control may be important to improve their success in weight management.

Involvement of KLF4 in sulforaphane- and iberin-mediated induction of p21waf1/cip1

Sulforaphane (SF; 4-methylsulfinylbutyl isothiocyanate), a dietary compound derived from broccoli, may exhibit chemopreventive properties by inducing cell cycle arrest via induction of cyclin-dependent kinase inhibitor 1A (p21(waf1/cip1)), but the exact molecular mechanism has not been determined. Here we evaluate the role of the transcription factor Kruppel-like factor 4 (KLF4) in mediating the induction of p21(waf1/cip1) and cellular differentiation by SF and iberin (IB; 3-methylsulphinyl propyl isothiocyanate), also derived from broccoli. Exposure of Caco-2 and Caco-2/TC7 cells to SF and IB increased expression of both KLF4 and p21(waf1/cip1), whereas exposure of HT29 cells resulted only in induction of p21(waf1/cip1). In Caco-2 cells, small interfering RNA knock down of KLF4 expression attenuated induction of p21(waf1/cip1) in response to either SF or IB treatment. Contrary to expectation, prolonged exposure to SF reduced sucrase isomaltase activity, a marker of small intestinal differentiation in Caco-2 cells. Additional support for the SF-mediated induction of p21(waf1/cip1) by KLF4 was obtained from analyses of gastric tissue of Apc(Min/+) mice following acute intervention with SF but not from the analyses of other tissue of the intestinal tract. These results suggest that induction of p21(waf1/cip1) by SF or IB may be partly mediated by KLF4 in some colon cancer cells and tissues.

Nutritional analysis of gastric contents and body condition score at a single time point in feral horses in Australia

Objective: To determine the impact of a free-choice diet on nutritional intake and body condition of feral horses. Animals: Cadavers of 41 feral horses from 5 Australian locations. Procedures: Body condition score (BCS) was determined (scale of 1 to 9), and the stomach was removed from horses during postmortem examination. Stomach contents were analyzed for nutritional variables and macroelement and microelement concentrations. Data were compared among the locations and also compared with recommended daily intakes for horses. Results: Mean BCS varied by location; all horses were judged to be moderately thin. The BCS for males was 1 to 3 points higher than that of females. Amount of protein in the stomach contents varied from 4.3% to 14.9% and was significantly associated with BCS. Amounts of water-soluble carbohydrate and ethanol-soluble carbohydrate in stomach contents of feral horses from all 5 locations were higher than those expected for horses eating high-quality forage. Some macroelement and microelement concentrations were grossly excessive, whereas others were grossly deficient. There was no evidence of ill health among the horses. Conclusions and Clinical Relevance: Results suggested that the diet for several populations of feral horses in Australia appeared less than optimal. However, neither low BCS nor trace mineral deficiency appeared to affect survival of the horses. Additional studies on food sources in these regions, including analysis of water-soluble carbohydrate, ethanol-soluble carbohydrate, and mineral concentrations, are warranted to determine the provenance of such rich sources of nutrients. Determination of the optimal diet for horses may need revision.

Reproducibility of gastric emptying in overweight and obese males

Background & aim To understand whether any change in gastric emptying (GE) is physiologically relevant, it is important to identify its variability. Information regarding the variability of GE in overweight and obese individuals is lacking. The aim of this study was to determine the reproducibility of GE in overweight and obese males. Methods Fifteen overweight and obese males [body mass index 30.3 (4.9) kg/m2] completed two identical GE tests 7 days apart. GE of a standard pancake breakfast was assessed by 13C-octanoic acid breath test. Data are presented as mean (±SD). Results There were no significant differences in GE between test days (half time (t1/2): 179 (15) and 176 (19 min), p = 0.56; lag time (tlag): 108 (14) and 104 (8) min, p = 0.26). Mean intra-individual coefficient of variation for t1/2 was 7.9% and tlag 7.5%. Based on these findings, to detect a treatment effect in a paired design with a power of 80% and α = 0.05, minimum mean effect sizes for t1/2 would need to be ≥14.4 min and tlag ≥ 8.1 min. Conclusions These data show that GE is reproducible in overweight and obese males and provide minimum mean effect sizes required to detect a hypothetical treatment effect in this population.

The relationship between angiogenesis and the immune response in carcinogenesis and the progression of malignant disease

Recent studies have demonstrated that angiogenesis and suppressed cell- mediated immunity (CMI) play a central role in the pathogenesis of malignant disease facilitating tumour growth, invasion and metastasis. In the majority of tumours, the malignant process is preceded by a pathological condition or exposure to an irritant which itself is associated with the induction of angiogenesis and/or suppressed CMI. These include: cigarette smoking, chronic bronchitis and lung cancer; chronic oesophagitis and oesophageal cancer; chronic viral infections such as human papilloma virus and ano-genital cancers, chronic hepatitis B and C and hepatocellular carcinoma, and Epstein- Barr virus (EBV) and lymphomas; chronic inflammatory conditions such as Crohn's disease and ulcerative colitis and colorectal cancer; asbestos exposure and mesothelioma and excessive sunlight exposure/sunburn and malignant melanoma. Chronic exposure to growth factors (insulin-like growth factor-I in acromegaly), mutations in tumour suppressor genes (TP53 in Li Fraumeni syndrome) and long-term exposure to immunosuppressive agents (cyclosporin A) may also give rise to similar environments and are associated with the development of a range of solid tumours. The increased blood supply would facilitate the development and proliferation of an abnormal clone or clones of cells arising as the result of: (a) an inherited genetic abnormality; and/or (b) acquired somatic mutations, the latter due to local production and/or enhanced delivery of carcinogens and mutagenic growth factors. With progressive detrimental mutations and growth-induced tumour hypoxia, the transformed cell, to a lesser or greater extent, may amplify the angiogenic process and CMI suppression, thereby facilitating further tumour growth and metastasis. There is accumulating evidence that long-term treatment with cyclo-oxygenase inhibitors (aspirin and indomethacin), cytokines such as interferon-α, anti-oestrogens (tamoxifen and raloxifene) and captopril significantly reduces the incidence of solid tumours such as breast and colorectal cancer. These agents are anti-angiogenic and, in the case of aspirin, indomethacin and interferon-α have proven immunomodulatory effects. Collectively these observations indicate that angiogenesis and suppressed CMI play a central role in the development and progression of malignant disease. (C) 2000 Elsevier Science Ltd.