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_II32, 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_II32 in the cytoplasm and suppresses p53-mediated transcriptional transactivation and apoptosis during HCV infection, which may contribute to the hepatocarcinogenesis of HCV infection.

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. Glucose-induced activation of PI3K resulted in the downstream effector Akt phosphorylation. PI3K inhibitors effectively attenuated the intracellular reactive oxygen species (ROS) generation and nuclear factor κB (NF-κB) activation. Blocking the PI3K and Akt activities with the dominant-negative vectors greatly diminished the HG-triggered NF-κB activation and COX-2 expression and apoptosis. Conclusions— These results suggest that HG, via PI3K/Akt signaling, induces NF-κB–related upregulation of COX-2, which in turn triggers the caspase-3 activity that facilitates HUVEC apoptosis. Also, HG may cause ROS generation in HUVECs through a PI3K/Akt–dependent pathway.

Purpose: Persistent interleukin-8 (IL-8) production contributes to chronic inflammation of the stomach. The proinflammatory IL-1β polymorphisms, which enhance the cytokine production, are associated with increased risk of gastric cancer. The −251A/T polymorphism of the IL-8 promoter is involved in several human diseases. Particularly, the −251A is associated with decreased risk of colorectal cancer. We aimed to determine whether the −251 allele resulting in high IL-8 expression was associated with increased risk of gastric carcinoma.Experimental Design: The −251A/T promoters were cloned and analyzed by luciferase assay. Binding of nuclear proteins to the −251A/T promoters was analyzed by electrophoretic mobility shift assay. The −251A/T promoters were differentiated by PCR-RFLP. Comparison of gastric cancer risk between the −251A/T promoters was done by a case-control study.Results: The −251T allele possessed transcriptional activity 2- to 5-fold stronger than the −251A counterpart. Electrophoretic mobility shift assay showed that the −251A promoter had strong ability to bind to an unknown protein or multiprotein complex. The −251T allele was associated with increased risk of noncardia (Ptrend = 0.012) and cardia (Ptrend = 0.029) carcinomas. Gastric carcinoma patients with the low-risk AA genotype had a tendency to sustain intestinal-type carcinomas (χ2 = 6.816; P = 0.033); however, the high-risk −251T allele 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 −251T allele is significantly associated with increased risk of gastric carcinoma, particularly the diffuse and mixed types in Chinese population.

Overexpression of Her2/neu is implicated in the development of resistance to the antiestrogen tamoxifen (TAM) that exerts its inhibitory effect through interaction with estrogen receptor (ER). Whereas Her2/neu and ER are believed to be important cell survival/death factors in human breast cancer cells, if and how they interact to confer resistance to hormone therapy is not known. This prompted us to investigate whether modulation of the effect of TAM occurs via the Her2/neu pathway and whether targeting the interaction between the Her2/neu pathway and the ER pathway is beneficial. There are 2 forms of ER that are localized to the cell membrane and to the nucleus. For the first time, we found that Her2/neu directly interacts with ER at the cell membrane. We then investigated the role of Her2/neu overexpression in the regulation of the cell membrane ER pathway in TAM‐resistant breast cancer cells and the nature of this interaction in apoptotic signaling. Relief of TAM resistance was associated with Her2/neu downregulation and ER upregulation. TAM‐induced apoptosis occurred immediately after dissociation of Her2/neu from cell membrane ER. These results demonstrate a novel mechanism by which Her2/neu regulates the cell membrane ER‐coupled apoptosis and the possible involvement of the Her2/neu in TAM resistance of breast cancer cells. Moreover, the antiproliferative activity of TAM should rely on the integration between the signal transduction from the cell membrane ER and the gene regulation by the nuclear ER. Coordinated modulation on the cell membrane ER/Her2/neu pathway and the nuclear ER/RAR pathway may provide a new approach for treatment of ER‐positive, Her2/neu overexpressing breast cancer.

Accumulation of advanced glycation end products (AGEs) in joints is important in the development of cartilage destruction and damage in age-related osteoarthritis (OA). The aim of this study was to investigate the roles of peroxisome proliferator-activated receptor γ (PPARγ), toll-like receptor 4 (TLR4), and receptor for AGEs (RAGE) in AGEs-induced inflammatory signalings in human OA chondrocytes. Human articular chondrocytes were isolated and cultured. The productions of metalloproteinase-13 and interleukin-6 were quantified using the specific ELISA kits. The expressions of related signaling proteins were determined by Western blotting. Our results showed that AGEs enhanced the productions of interleukin-6 and metalloproteinase-13 and the expressions of cyclooxygenase-2 and high-mobility group protein B1 and resulted in the reduction of collagen II expression in human OA chondrocytes. AGEs could also activate nuclear factor (NF)-κB activation. Stimulation of human OA chondrocytes with AGEs significantly induced the up-regulation of TLR4 and RAGE expressions and the down-regulation of PPARγ expression in a time- and concentration-dependent manner. Neutralizing antibodies of TLR4 and RAGE effectively reversed the AGEs-induced inflammatory signalings and PPARγ down-regulation. PPARγ agonist pioglitazone could also reverse the AGEs-increased inflammatory signalings. Specific inhibitors for p38 mitogen-activated protein kinases, c-Jun N-terminal kinase and NF-κB suppressed AGEs-induced PPARγ down-regulation and reduction of collagen II expression. Taken together, these findings suggest that AGEs induce PPARγ down-regulation-mediated inflammatory signalings and reduction of collagen II expression in human OA chondrocytes via TLR4 and RAGE, which may play a crucial role in the development of osteoarthritis pathogenesis induced by AGEs accumulation.