Epithelial to mesenchymal transitions are vital for tumor growth and metastasis. Several inducers of epithelial to mesenchymal transition are transcription factors that repress E-cadherin expression, ...such as Snail, Slug, and Twist. In this study, we aimed to examine the expression of these transcription factors in pancreatic cancer.
The expression of Snail, Slug, and Twist was detected by immunohistochemistry in tissue samples from patients with pancreatic ductal adenocarcinoma. Five human pancreatic cancer cell lines (AsPC-1, Capan-1, HPAF-2, MiaPaCa-2, and Panc-1) were analyzed by reverse transcription-PCR, real-time PCR, and Western blotting. An orthotopic nude mouse model of pancreatic cancer was applied for in vivo experiments.
Seventy-eight percent of human pancreatic cancer tissues showed an expression of Snail, and 50% of the patients displayed positive expression of Slug. Twist showed no or only weak expression. Snail expression was higher in undifferentiated cancer cell lines (MiaPaCa-2 and Panc-1) than in more differentiated cell lines (Capan-1, HPAF-2, AsPC-1). Expression of Slug was detected in all cell lines with different intensities. Twist was not expressed. After exposure to hypoxia, the Twist gene was activated in all five pancreatic cancer cell lines.
The transcription factors Snail and Slug are expressed in pancreatic cancer but not in normal tissue, suggesting a role in the progression of human pancreatic tumors. Twist, activated by hypoxia, may play an important role in the invasive behavior of pancreatic tumors.
Muscarinic acetylcholine receptor M3 (M3R) has repeatedly been shown to be prominently expressed in human colorectal cancer (CRC), playing roles in proliferation and cell invasion. Its therapeutic ...targetability has been suggested in vitro and in animal models. We aimed to investigate the clinical role of MR3 expression in CRC for human survival. Surgical tissue samples from 754 CRC patients were analyzed for high or low immunohistochemical M3R expression on a clinically annotated tissue microarray (TMA). Immunohistochemical analysis was performed for established immune cell markers (CD8, TIA-1, FOXP3, IL 17, CD16 and OX 40). We used Kaplan-Meier curves to evaluate patients' survival and multivariate Cox regression analysis to evaluate prognostic significance. High M3R expression was associated with increased survival in multivariate (hazard ratio (HR) = 0.52; 95% CI = 0.35-0.78;
= 0.001) analysis, as was TIA-1 expression (HR = 0.99; 95% CI = 0.94-0.99;
= 0.014). Tumors with high M3R expression were significantly more likely to be grade 2 compared to tumors with low M3R expression (85.7% vs. 67.1%,
= 0.002). The 5-year survival analysis showed a trend of a higher survival rate in patients with high M3R expression (46%) than patients with low M3R expression CRC (42%) (
= 0.073). In contrast to previous in vitro and animal model findings, this study demonstrates an increased survival for CRC patients with high M3R expression. This evidence is highly relevant for translation of basic research findings into clinically efficient treatments.
Cholinergic signaling via the muscarinic M3 acetylcholine receptor (M3R) is involved in the development and progression of colorectal cancer (CRC). The present study aimed to analyze the blocking of ...M3R signaling in CRC using darifenacin, a selective M3R antagonist. Darifenacin effects were studied on HT-29 and SW480 CRC cells using MTT and BrdU assays, Western blotting and real time RT-PCR. In vivo, blocking of M3R was assessed in an orthotopic CRC xenograft BALB/cnu/nu mouse model. M3R expression in clinical tumor specimens was studied by immunohistochemistry on a tissue microarray of 585 CRC patients. In vitro, darifenacin decreased tumor cell survival and proliferation in a dose-dependent manner. Acetylcholine-induced p38, ERK1/2 and Akt signaling, and MMP-1 mRNA expression were decreased by darifenacin, as well as matrigel invasion of tumor cells. In mice, darifenacin reduced primary tumor volume and weight (p < 0.05), as well as liver metastases, compared to controls. High expression scores of M3R were found on 89.2% of clinical CRC samples and correlated with infiltrative tumor border and non-mucinous histology (p < 0.05). In conclusion, darifenacin inhibited components of tumor growth and progression in vitro and reduced tumor growth in vivo. Its target, M3R, was expressed on the majority of CRC. Thus, repurposing darifenacin may be an attractive addition to systemic tumor therapy in CRC patients expressing M3R.
The TNF-superfamily member TRAIL is known to mediate selective apoptosis in tumor cells suggesting this protein as a potential antitumor drug target. However, initial successful pr-clinical results ...could not be translated into the clinic. Reasons for the ineffectiveness of TRAIL-targeting in tumor therapies could include acquired TRAIL resistance. A tumor cell acquires TRAIL resistance, for example, by upregulation of antiapoptotic proteins. In addition, TRAIL can also influence the immune system and thus, tumor growth. We were able to show in our previous work that TRAIL
mice show improved survival in a mouse model of pancreatic carcinoma. Therefore, in this study we aimed to immunologically characterize the TRAIL
mice. We observed no significant differences in the distribution of CD3
, CD4
, CD8
T-cells, Tregs, and central memory CD4
and CD8
cells. However, we provide evidence for relevant differences in the distribution of effector memory T-cells and CD8
CD122
cells but also in dendritic cells. Our findings suggest that T-lymphocytes of TRAIL
mice proliferate at a lower rate, and that the administration of recombinant TRAIL significantly increases their proliferation, while regulatory T-cells (Tregs) from TRAIL
mice are less suppressive. Regarding the dendritic cells, we found more type-2 conventional dendritic cells (DC2s) in the TRAIL
mice. For the first time (to the best of our knowledge), we provide a comprehensive characterization of the immunological landscape of TRAIL-deficient mice. This will establish an experimental basis for future investigations of TRAIL-mediated immunology.
OBJECTIVES
The purpose of this study was to determine whether atrial expression of the extracellular signal-regulated kinases Erk1/Erk2 and of the angiotensin-converting enzyme (ACE) is altered in ...patients with atrial fibrillation (AF).
BACKGROUND
Recent studies have demonstrated that atrial fibrosis can provide a pathophysiologic substrate for AF. However, the molecular mechanisms responsible for the development of atrial fibrosis are unclear.
METHODS
Atrial tissue samples of 43 patients undergoing open heart surgery were examined. Seventeen patients had chronic persistent AF (≥6 months; CAF), 8 patients had paroxysmal AF (PAF) and 18 patients had no history of AF. Erk expression was analyzed at the mRNA (quantitative reverse transcription polymerase chain reaction), the protein (immunoblot techniques) and atrial tissue (immunohistochemistry) levels. Erk-activating kinases (MEK1/2) and ACE were analyzed by immunoblot techniques.
RESULTS
Increased amounts of Erk2-mRNA were found in patients with CAF (75 ± 20 U vs. sinus rhythm: 31 ± 25 U; p < 0.05). Activated Erk1/Erk2 and MEK1/2 were increased to more than 150% in patients with AF compared to patients with sinus rhythm. No differences between CAF and PAF were found. The expression of ACE was three-fold increased during CAF. Amounts of activated Erk1/Erk2 were reduced in patients treated with ACE inhibitors. Patients with AF showed an increased expression of Erk1/Erk2 in interstitial cells and marked atrial fibrosis.
CONCLUSIONS
An ACE-dependent increase in the amounts of activated Erk1/Erk2 in atrial interstitial cells may contribute as a molecular mechanism for the development of atrial fibrosis in patients with AF. These findings may have important impact on the treatment of AF.
ADAMs (A Disintegrin and Metalloprotease) are multifunctional, membrane-bound cell surface glycoproteins, which have numerous functions in cell growth, differentiation, and motility. We wished to ...investigate the expression of ADAM 9, 10, 12, 15, and in human breast cancer.
Expression of ADAMs was determined in breast cancer specimens and the corresponding non-neoplastic breast tissue from 24 patients, and in the MCF-7 and MDA-MB 453 breast cancer cell lines via quantitative RT-PCR and immunohistochemistry. The effects of anti-ADAM antibodies on cell proliferation were assessed by measuring DNA-synthesis.
Breast cancer tissue samples showed increased mRNA expression of ADAM 9, 12, and 17, whereas ADAM 10 and 15 were not differently expressed. Protein expression was studied by immunohistochemistry. All ADAMs were expressed in MCF-7 and MDA-MB453 cell lines, with the highest expression levels being observed for ADAM 9, 12, and 17. Application of anti-ADAM 15 and anti-ADAM 17 antibodies significantly inhibited the proliferation of both MCF-7 and MDA-MB453 breast cancer cell lines. In contrast, the growth of MCF-7 cells appeared to be stimulated by the administration of anti-ADAM 12 antibody.
The results of this study suggest that ADAMs are differentially expressed in human breast cancer and are capable of modulating tumour cell growth.
Response Surface Methodology (RSM) models may encounter limitations when incorporated with non-orthogonalities in the design matrix. This holds especially when performing End-of-Life (EoL) tests or ...demonstrating multivariate reliability. Exemplarily applied to Central-Composite Designs (CCDs) - as a popular type of test design for RSM and nonlinear dependencies - unexpectedly inexecutable test runs, variations in test point settings, and further factor level shifts can lead to impairments in the design model and prediction capability. This issue is addressed within this work. The consequences of common orthogonality offsets and their implications for predictive modeling relevant to multivariate reliability estimation with confidence intervals are discussed here. By analyzing selected orthogonality disparities, trends in variance shaping in the parameter space and beyond are captured and described. For this purpose, visualizations like contours and variance dispersions of the test designs are interpreted. Considering the marginal range of predicted values, this approach thus serves to evaluate effects relevant for extrapolation from accelerated parameter ranges with respect to an estimate of their confidence intervals. In this way, the results show that experimental design manipulations justified from practical application exemplarily in a k=2 factors CCD lead to a more manageable handling of variances in the prediction of model values - which is lastly illustrated in an exemplary layout.
Previous studies have suggested that atrial fibrillation (AF) is associated with the activation of the atrial angiotensin system. However, it is not known whether the expression of angiotensin II ...receptors changes during AF. The purpose of this study was to determine the atrial expression of angiotensin II type 1 and type 2 receptors (AT(1)-R and AT(2)-R) in patients with AF.
Atrial tissue samples from 30 patients undergoing open heart surgery were examined. Eleven patients had chronic persistent AF (> or =6 months; cAF), 8 patients had paroxysmal AF (pAF), and 11 patients were in sinus rhythm. AT(1)-R and AT(2)-R were localized in the atrial tissue by immunohistochemistry and quantified at the protein and mRNA level by Western blotting and quantitative polymerase chain reaction. Both types of AT-R were predominantly expressed in atrial myocytes in all groups. The amount of AT(1)-R was reduced to 34.9% during cAF (P<0.01) and to 51.7% during pAF (P<0.05) compared with patients in sinus rhythm. In contrast, AT(2)-R was increased during cAF (246%; P=NS) and pAF (505%; P<0.01). AT(1)-R/AT(2)-R mRNA content was similar in all groups.
AF is associated with the down-regulation of atrial AT(1)-R and the up-regulation of AT(2)-R proteins. These findings may help define the pathophysiological role of the angiotensin system in the structural remodeling of the fibrillating atria.
In this paper, a decision methodology for reliability-based stress-strength design optimization of machine components under economic aspects is described. For this purpose, pertinent influencing ...parameters were exemplarily identified, conditioned by a case study with the aid of statistical toleration. The obtained parameters were transferred into a stress-strength relationship through a feature chain. Whereas in standardized design procedures and guidelines, machine components are compulsorily dimensioned in an iterative process to the next most stable component modification in case of overstressing, here the probabilistic reliability analysis with subsequent tolerance synthesis and design optimization is used. Within the methodology, the statistically quantified probability of failure is deployed as the target value. Derived from the presented method, recommendations for customized design parameter values towards a cost-efficient, reliable construction layout can be made.
Proinflammatory cytokines play an important role in abdominal surgery and are often associated with the development of postoperative ileus, especially in Crohn's disease. The aim of this study was to ...investigate proinflammatory cytokine levels in mesenteric fat in Crohn's disease and patients without Crohn's disease.
Human mesenteric tissue specimen were divided into 3 patient groups (n = 10 each): minor surgery (laparoscopic cholecystectomy), major surgery (colectomy) in patients without Crohn's disease, and major surgery (colectomy) in patients with Crohn's disease. Levels of interleukin 6, interleukin 1-β, and tumor necrosis factor α were determined by cytometric bead array, enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction. The Kruskal-Wallis and the Mann-Whitney U test were used to compare continuous variables. For categorical variables, the χ2 test or Fisher exact test was used.
In minor surgery, cytokines levels of interleukin 6, interleukin 1-β and Tumor necrosis factor α were low (ie, interleukin 6: 1 pg/mL 0–36, interleukin 1-β: 0 fg/mL 0–18, tumor necrosis factor α: 157 fg/mL 91–237) compared with major surgery in patients with and without Crohn's disease. Cytokines were significantly higher in major surgery (ie, interleukin 6: 147 pg/mL 29–347, interleukin 1-β: 660 fg/mL 0–2580, tumor necrosis factor α: 532 fg/mL 289–1647; P = .02 and major surgery with CD (cytometric bead array: interleukin 6: 94 pg/mL 24–627, interleukin 1-β: 708 fg/mL 0–1664, tumor necrosis factor α: 733 fg/mL 209–1,354; P < .05). Cytokine levels in major surgery with Crohn's disease showed a further increase of interleukin 6 in polymerase chain reaction in comparison to major surgery in patients without Crohn's disease (1.2 vs 4, P = .04).
Proinflammatory cytokines are increased in the mesenteric fat in major operations compared to minor operations, which indicates local mesenteric inflammation. In Crohn's disease, levels of proinflammatory cytokines are even higher, which may put the patients at risk for postoperative ileus.
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