Search

Abstract: Ceramide synthase 5 is involved in the de novo synthesis of ceramide, a sphingolipid involved in cell death and proliferation. In this study, we investigated the role of ceramide synthase 5 in colorectal cancer by examining ceramide synthase 5 expression, clinico‐pathological parameters and association with survival/death signalling pathways in cancer. Immunohistochemical analysis of CerS5 was performed on 102 colorectal cancer samples using tissue microarrays constructed from formalin‐fixed and paraffin‐embedded tissues. We found strong membranous ceramide synthase 5 staining in 57 of 102 (56%) colorectal cancers. A multivariate Cox regression analysis of ceramide synthase 5 expression adjusted for disease stage, differentiation and lymphovascular invasion revealed reduced 5‐year overall survival (p = 0.001) and 5‐year recurrence‐free survival (p = 0.002), with hazard ratios of 4.712 and 4.322, respectively. The effect of ceramide synthase 5 expression on tumourigenic processes was further characterised by reverse phase protein array analysis. Reverse phase protein arrays were generated from laser capture microdissection‐enriched carcinoma cells from 19 fresh‐frozen colorectal cancer tissues. Measurements of phosphorylation and total levels of signalling proteins involved in apoptosis, autophagy and other cancer‐related pathways revealed two distinct signalling networks; weak membranous ceramide synthase 5 intensity was associated with a proteomic network dominated by signalling proteins linked to apoptosis, whereas strong ceramide synthase 5 intensity was associated with a proteomic sub‐network mostly composed of proteins linked to autophagy. In conclusion, high ceramide synthase 5 expression was found in colorectal cancer tissue and was associated with poorer patient outcomes. Our findings suggest that this may be mediated by a transition from apoptotic to autophagy signalling pathways in ceramide synthase 5 High expressing tumours, thus implicating ceramide synthase 5 in the progression of colorectal cancer. [ABSTRACT FROM AUTHOR]

Background and Aim TRIM28 is a multi-domain nuclear protein with pleotropic effects in both normal and tumor cells. In this study, TRIM28 expression in epithelial and stromal tumor microenvironment and its prognostic role in colorectal cancer were investigated. Methods Immunohistological staining of TRIM28 was evaluated in tissue microarrays constructed from 137 colorectal cancer patients. The correlations of TRIM28 expression with clinicopathological features and p53 expression were studied. Kaplan- Meier analysis and Cox proportional hazard modeling were used to assess overall survival ( OS) and recurrence-free survival ( RFS). Results Strong epithelial TRIM28 expression was found in 42% of colorectal cancer tissues. TRIM28 expression correlated significantly with p53 expression in matched cases ( P = 0.0168, Spearman rank test). A high epithelial to stromal TRIM28 expression ratio was associated with shorter OS ( P = 0.033; log-rank test) and RFS ( P = 0.043; log-rank test). Multivariate analysis showed that the epithelial to stromal TRIM28 expression ratio was an independent predictor of OS (hazard ratio = 2.136; 95% confidence interval 1.015-4.498, P = 0.046) and RFS (hazard ratio = 2.100; confidence interval 1.052-4.191, P = 0.035). Conclusion A high TRIM28 expression ratio between stromal and epithelial compartments in colorectal cancer tissue is an independent predictor of poor prognosis. The pathophysiological role of TRIM28 in carcinogenesis may be dependent on expression levels and cell type within the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinases (MAPK) are considered to play significant roles in colonic carcinogenesis and kinase inhibitor therapy has been proposed as a potential tool in the treatment of this disease. Reverse-phase microarray assays using phospho-specific antibodies can directly measure levels of phosphorylated protein isoforms. In the current study, samples from 35 cases of untreated colorectal cancer colectomies were laser capture-microdissected to isolate epithelium and stroma from cancer as well as normal (i.e. uninvolved) mucosa. Lysates generated from these four tissue types were spotted onto reverse-phase protein microarrays and probed with a panel of antibodies to ERK, p-ERK, p38, p-p38, p-JNK, MEK and p-MEK. Whereas total protein levels were unchanged, or slightly elevated (p38, p = 0.0025) in cancers, activated isoforms, including p-ERK, p-p38 and p-JNK, were decreased two- to four-fold in cancers compared with uninvolved mucosa ( p < 0.0023 in all cases except for p-JNK in epithelium, where decrement was non-significant). This was backed up by western blotting. Dukes' stage B and C cancers displayed lower p-ERK and p-p38 expression than Dukes' stage A cancers, although this was not statistically significant. It is concluded that MAPK activity may be down-regulated in colorectal cancer and that further exploration of inhibitory therapy in this system should be carefully evaluated if this finding is confirmed in larger series. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background and Aim: Cyclooxygenase-2 (COX-2), a target of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs), is upregulated in chronic hepatitis B and may have a role in hepatocellular carcinoma. Little is known about the expression of COX-2 in chronic hepatitis C (HCV) infection. The aim the present study was to evaluate the extent of COX-2 expression in liver biopsies in patients with HCV infection and to determine the effect of treatment with interferon α (IFN). Methods: Percutaneous liver biopsy specimens were retrieved. Following formalin fixation and paraffin embedding, the biopsies were histologically evaluated for inflammation and fibrosis. The extent of COX-2 expression was measured by the avidin biotin immunohistochemical technique using a monoclonal COX-2 antibody. The extent of COX-2 expression was graded according to the number of hepatocytes expressing COX-2. Data were analyzed using Student's t-test. Results: Liver biopsies from 10 patients before and after treatment with IFN were obtained and compared with nine normal liver biopsies. All of the liver biopsies showed some COX-2 expression. COX-2 expression was confined to hepatocytes and bile duct epithelium and was not detected in vascular endothelium or inflammatory cells. The extent of COX-2 expression was greater in hepatitis C infected liver biopsies than in normal biopsies. Following treatment with IFN, there was a greater than threefold reduction in COX-2 expression ( P < 0.01). This result was independent of the sustained virological response. Conclusion: In this small pilot study we have shown that COX-2 is overexpressed in liver biopsies infected with HCV and COX-2 expression is reduced following treatment with IFN. [ABSTRACT FROM AUTHOR]

Calcium sparks result from the concerted opening of a small number of Ca2+ release channels (ryanodine receptors, RyRs) organized in clusters in the membrane of the sarcoplasmic reticulum (SR). Calcium sparks represent the elementary events of SR Ca2+ release in cardiac myocytes, and their spatial and temporal summation results in whole-cell [Ca2+]i transients observed during excitation–contraction coupling (ECC). Atrial myocytes generally lack transverse tubules; however, during ECC Ca2+ release is initiated from junctional SR (j-SR) in the cell periphery from where activation propagates inwardly through Ca2+-induced Ca2+ release (CICR) from non-junctional SR (nj-SR). Despite the structural differences in the microdomains of RyRs of j-SR and nj-SR, spontaneous Ca2+ sparks are observed from both types of SR, albeit at different frequencies. In cells that showed spontaneous Ca2+ sparks from j-SR and nj-SR, subsarcolemmal (SS) Ca2+ sparks from the j-SR were 3–4 times more frequent than central (CTR) Ca2+ sparks occurring from nj-SR. Subsarcolemmal Ca2+ sparks had a slightly higher amplitude, but were essentially identical in their spatial spread and duration when compared to CTR Ca2+ sparks. Sensitization of RyRs with a low concentration (0.1 mm) of caffeine led to a 107% increase in the frequency of CTR Ca2+ sparks, whereas the SS Ca2+ spark frequency increased by only 58%, suggesting that the nj-SR is capable of much higher Ca2+ spark activity than observed normally in unstimulated cells. The L-type Ca2+ channel blocker verapamil reduced SS Ca2+ spark frequency to 38% of control values, whereas Ca2+ spark activity from nj-SR was reduced by only 19%, suggesting that SS Ca2+ sparks are under the control of Ca2+ influx from the extracellular space. Removal of extracellular Ca2+ eliminated SS Ca2+ sparks completely, whereas Ca2+ sparks from the nj-SR continued, albeit at a lower frequency. In membrane-permeabilized (saponin-treated) atrial myocytes, where [Ca2+] can be experimentally controlled throughout the entire myocyte, j-SR and nj-SR Ca2+ spark frequencies were identical, and Ca2+ sparks could be observed spaced at sarcomeric distances throughout the entire cell, suggesting that all release sites of the nj-SR can become active. Measurement of SR Ca2+ load (10 mm caffeine) revealed no difference between j-SR and nj-SR. The data suggest that in atrial myocytes, which lack a t-tubular system, the nj-SR is fully equipped with a three-dimensional array of functional SR Ca2+ release sites; however, in intact cells under resting conditions, peripheral RyR clusters have a higher probability of activation owing to their association with surface membrane Ca2+ channels, leading to higher spontaneous Ca2+ spark activity. In conclusion, Ca2+ sparks originating from both j-SR and nj-SR are rather stereotypical and show little differences in their spatiotemporal properties. In intact cells, however, the higher frequency of spontaneous SS Ca2+ sparks arises from the structural arrangement of sarcolemma and j-SR membrane and thus from the difference in the trigger mechanism. [ABSTRACT FROM AUTHOR]