Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis and hepatocellular carcinoma. HCV NS5A, one of non-structural proteins of HCV, was suggested to play a role in oncogenic transformation. Since the tumor suppressor p53 is important for preventing neoplastic transformation, we investigated the functional effects of HCV NS5A on p53. In vitro and in vivo coimmunoprecipitation and confocal microscopy were used to determine the interaction of NS5A and p53. HCV NS5A binds directly to p53 and colocalizes p53 in the perinuclear region. NS5A inhibits transcriptional transactivation by p53 in a dose-dependent manner by use of a reporter assay. Down regulation of endogenous p21/waf1 expression, which is activated by p53 in Hep3B cells, by NS5A was demonstrated by using FLAG-and FLAG-NS5A Hep3B stable cell lines. The effect of NS5A on p53-mediated apoptosis was determined by flow cytometry in both NS5A permanently and transiently transfected Hep3B cells with exogenous p53. The p53-induced apoptosis was abrogated by NS5A and the inhibition effect correlates well with the binding ability of NS5A to p53. In addition, HCV NS5A protein interacts with and colocalizes hTAF II 32, a component of TFIID and an essential coactivator of p53, in vivo. These results suggest that HCV NS5A interacts with and partially sequestrates p53 and hTAF II 32 in the cytoplasm and suppresses p53-mediated transcriptional transactivation and apoptosis during HCV infection, which may contribute to the hepatocarcinogenesis of HCV infection.

Diabetic myopathy, a less studied complication of diabetes, exhibits the clinical observations characterized by a less muscle mass, muscle weakness and a reduced physical functional capacity. Accumulation of advanced glycation end-products (AGEs), known to play a role in diabetic complications, has been identified in ageing human skeletal muscles. However, the role of AGEs in diabetic myopathy remains unclear. Here, we investigated the effects of AGEs on myogenic differentiation and muscle atrophy in vivo and in vitro. We also evaluated the therapeutic potential of alagebrium chloride (Ala-Cl), an inhibitor of AGEs. Muscle fibre atrophy and immunoreactivity for AGEs, Atrogin-1 (a muscle atrophy marker) and phosphorylated AMP-activated protein kinase (AMPK) expressions were markedly increased in human skeletal muscles from patients with diabetes as compared with control subjects. Moreover, in diabetic mice we found increased blood AGEs, less muscle mass, lower muscular endurance, atrophic muscle size and poor regenerative capacity, and increased levels of muscle AGE and receptor for AGE (RAGE), Atrogin-1 and phosphorylated AMPK, which could be significantly ameliorated by Ala-Cl. Furthermore, in vitro, AGEs (in a dose-dependent manner) reduced myotube diameters (myotube atrophy) and induced Atrogin-1 protein expression in myotubes differentiated from both mouse myoblasts and primary human skeletal muscle-derived progenitor cells. AGEs exerted a negative regulation of myogenesis of mouse and human myoblasts. Ala-Cl significantly inhibited the effects of AGEs on myotube atrophy and myogenesis. We further demonstrated that AGEs induced muscle atrophy/myogenesis impairment via a RAGE-mediated AMPK-down-regulation of the Akt signalling pathway. Our findings support that AGEs play an important role in diabetic myopathy, and that an inhibitor of AGEs may offer a therapeutic strategy for managing the dysfunction of muscle due to diabetes or ageing.

Objectives-Diabetes mellitus causes endothelial dysfunction. The precise molecular mechanisms by which hyperglycemia causes apoptosis in endothelial cells are not yet well understood. The aim of this study was to explore the role of cyclooxygenase-2 (COX-2) and the possible involvement of phosphoinositide 3-kinase (PI3K) signaling in high glucose (HG)-induced apoptosis in human umbilical vein endothelial cells (HUVECs). Methods and Results-For detection of apoptosis, the morphological Hoechst staining and Annexin V/propidium iodide staining were used. Glucose upregulated COX-2 protein expression, which was associated with the induction of prostaglandin (PG) E 2 (PGE 2 ), caspase-3 activity, and apoptosis. Unexpectedly, we found that PI3K inhibitors could suppress COX-2 expression, PGE 2 production, caspase-3 activity, and the subsequent apoptosis under HG condition.

Purpose: Persistent interleukin-8 (IL-8) production contributes to chronic inflammation of the stomach. The proinflammatory IL-1h polymorphisms, which enhance the cytokine production, are associated with increased risk of gastric cancer. The À251A/T polymorphism of the IL-8 promoter is involved in several human diseases. Particularly, the À251A is associated with decreased risk of colorectal cancer. We aimed to determine whether the À251 allele resulting in high IL-8 expression was associated with increased risk of gastric carcinoma. Experimental Design: The À251A/T promoters were cloned and analyzed by luciferase assay. Binding of nuclear proteins to the À251A/T promoters was analyzed by electrophoretic mobility shift assay.The À251A/T promoters were differentiated by PCR-RFLP. Comparison of gastric cancer risk between the À251A/T promoters was done by a case-control study. Results:The À251Tallele possessed transcriptional activity 2-to 5-fold stronger than the À251A counterpart. Electrophoretic mobility shift assay showed that the À251A promoter had strong ability to bind to an unknown protein or multiprotein complex. The À251T allele was associated with increased risk of noncardia (P trend = 0.012) and cardia (P trend = 0.029) carcinomas. Gastric carcinoma patients with the low-risk AA genotype had a tendency to sustain intestinal-type carcinomas (m 2 = 6.816; P = 0.033); however, the high-risk À251Tallele was associated with >2-fold increased risk of diffuse-type (AA versus AT + TT: odds ratio, 2.52; 95% confidence interval,1.16-5.49; P = 0.017) and mixed-type (AA versus AT + TT: odds ratio, 2.22; 95% confidence interval, 1.12-4.40; P = 0.019) carcinomas. Conclusions:The IL-8 À251Tallele is significantly associated with increased risk of gastric carcinoma, particularly the diffuse and mixed types in Chinese population.