PMC,a potent hydrophilic α‐tocopherol derivative,inhibits NF‐κB activation via PP2A but not IκBα‐dependent signals in vascular smooth muscle cells

The hydrophilic α‐tocopherol derivative, 2,2,5,7,8‐pentamethyl‐6‐hydroxychromane (PMC), is a promising alternative to vitamin E in clinical applications. Critical vascular inflammation leads to vascular dysfunction and vascular diseases, including atherosclerosis, hypertension and abdominal aortic aneurysms. In this study, we investigated the mechanisms of the inhibitory effects of PMC in vascular smooth muscle cells (VSMCs) exposed to pro‐inflammatory stimuli, lipopolysaccharide (LPS) combined with interferon (IFN)‐γ. Treatment of LPS/IFN‐γ‐stimulated VSMCs with PMC suppressed the expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase‐9 in a concentration‐dependent manner. A reduction in LPS/IFN‐γ‐induced nuclear factor (NF)‐κB activation was also observed in PMC‐treated VSMCs. The translocation and phosphorylation of p65, protein phosphatase 2A (PP2A) inactivation and the formation of reactive oxygen species (ROS) were significantly inhibited by PMC in LPS/IFN‐γ‐activated VSMCs. However, neither IκBα degradation nor IκB kinase (IKK) or ribosomal s6 kinase‐1 phosphorylation was affected by PMC under these conditions. Both treatments with okadaic acid, a PP2A‐selective inhibitor, and transfection with PP2A siRNA markedly reversed the PMC‐mediated inhibition of iNOS expression, NF‐κB‐promoter activity and p65 phosphorylation. Immunoprecipitation analysis of the cellular extracts of LPS/IFN‐γ‐stimulated VSMCs revealed that p65 colocalizes with PP2A. In addition, p65 phosphorylation and PP2A inactivation were induced in VSMCs by treatment with H₂O₂, but neither IκBα degradation nor IKK phosphorylation was observed. These results collectively indicate that the PMC‐mediated inhibition of NF‐κB activity in LPS/IFN‐γ‐stimulated VSMCs occurs through the ROS‐PP2A‐p65 signalling cascade, an IKK‐IκBα‐independent mechanism. Therapeutic interventions using PMC may therefore be beneficial for the treatment of vascular inflammatory diseases.     

Structural variation in human apolipoprotein E3 and E4: secondary structure, tertiary structure, and size distribution

Human apolipoprotein E (apoE) is a 299-amino-acid protein with a molecular weight of 34 kDa. The difference between the apoE3 and apoE4 isoforms is a single residue substitution involving a Cys-Arg replacement at residue 112. ApoE4 is positively associated with atherosclerosis and late-onset and sporadic Alzheimer's disease (AD). ApoE4 and its C-terminal truncated fragments have been found in the senile plaques and neurofibrillary tangles in the brain of AD patients. However, detail structural information regarding isoform and domain interaction remains poorly understood. We prepared full-length, N-, and C-terminal truncated apoE3 and apoE4 proteins and studied their structural variation. Sedimentation velocity and continuous size distribution analysis using analytical ultracentrifugation revealed apoE3(72-299) as consisting of a major species with a sedimentation coefficient of 5.9. ApoE4(72-299) showed a wider and more complicated species distribution. Both apoE3 and E4 N-terminal domain (1-191) existed with monomers as the major component together with some tetramer. The oligomerization and aggregation of apoE protein increased when the C-terminal domain (192-271) was incorporated. The structural influence of the C-terminal domain on apoE is to assist self-association with no significant isoform preference. Circular dichroism and fluorescence studies demonstrated that apoE4(72-299) possessed a more alpha-helical structure with more hydrophobic residue exposure. The structural variation of the N-terminal truncated apoE3 and apoE4 protein provides useful information that helps to explain the greater aggregation of the apoE4 isoform and thus has implication for the involvement of apoE4 in AD.     

Genetic analysis of the APC gene in taiwanese familial adenomatous polyposis

Colorectal cancer has become the third leading cause of death from cancer in Taiwan. Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease characterized by the presence of multiple adenomatous polyps in the colon and rectum. The gene responsible for FAP(APC) was cloned in 1991. Extensive analyses of the mutation spectra in FAP kindreds have been performed in different countries, but the results have been highly variable (30–80%). In this study, we used denaturing high-performance liquid chromatography (DHPLC) followed by automatic sequencing in an effort to establish the mutation spectrum of APC from DNA of peripheral blood cells. Among the 6 FAP probands analyzed, mutations were detected in 3 (50%), 2 of which were novel. The first novel mutation was at codon 2166, with a C to T transition, resulting in a stop codon. The second novel mutation was at codon 1971, with a C to G transversion, resulting in an amino acid change from serine to cysteine. The third mutation involved an A insertion in the sequence of -AAAAAA- at codons 1554–1556, which created a downstream stop codon (codon 1558). This study is the first to report mutation analysis in Taiwanese FAP probands.     

Superoxide anion burst and taxol production induced by Ce in suspension cultures of Taxus cuspidata

Generation of reactive oxygen species (ROS) induced by Ce⁴⁺ in suspension cultures of Taxus cuspidata was investigated. The burst of superoxide anions (O₂√⁻) occurred rapidly after the addition of Ce⁴⁺ and reached maximum at 4.3 h, while the total level of the cellular reactive oxygen species maintained unchanged. The intracellular superoxide dismutase (SOD) and catalase (CAT) were activated while the intra/extracellular peroxidases (PODs) were inhibited accompanying the O₂√⁻ burst. The pretreatment of the suspension cultures with diphenylene iodonium (DPI), a suicide inhibitor of the NADPH oxidase, blocked the O₂√⁻ burst, inhibiting the cell apoptosis and taxol production induced by Ce⁴⁺. These results show that NADPH oxidase played a key role in O₂√⁻ burst and O₂√⁻ served as a mediator of Ce⁴⁺ for cell apoptosis and taxol production. The pretreatments of the suspension cultures with anthracene-9-carboxylate, an ion-channel blocker, nifedipine, a Ca²⁺-channel blocker, neomycin, a phospholipase C (PLC) inhibitor, or suramin, a G-protein inhibitor, decreased O₂√⁻ burst induced by Ce⁴⁺. It is thus inferred that Ce⁴⁺-induced O₂√⁻ burst, which mediated cell apoptosis and taxol production by activating the ion-channels, PLC, G-proteins and NADPH oxidase.     

Induction of transient ion channel-like pores in a cancer cell by antibiotic peptide

The anticancer activity of anti-bacterial cecropins makes them potentially useful as peptide anti-cancer drugs. We used the cell-attached patch to study the effect of cecropin B (CB; having one hydrophobic and one amphipathic alpha-helix) and its derivative, cecropin B3 (CB3; having two hydrophobic alpha-helices) on the membrane of Ags cancer cells. Application of 10-60 microM CB onto the membrane of the cancer cell produces short outward currents. Comparative study with CB3, which induces no outward currents, shows that the amphipathic group of CB is necessary for the pore formation. The results provide a rationale to study the cell-killing activity of antimicrobial peptides at the single cancer cell level.     

The Transforming Growth Factor-β Type III Receptor Mediates Distinct Subcellular Trafficking and Downstream Signaling of Activin-like Kinase (ALK)3 and ALK6 Receptors

Bone morphogenetic proteins (BMPs) signal through the BMP type I and type II receptors to regulate cellular processes, including embryonic development. The type I BMP receptors activin-like kinase (ALK)3 and ALK6 share a high degree of homology, yet possess distinct signaling roles. Here, we report that although the transforming growth factor (TGF)-β type III receptor (TβRIII) enhanced both ALK3 and ALK6 signaling, TβRIII more potently enhanced ALK6-mediated stimulation of the BMP-responsive promoters XVent2 and 3GC2, and up-regulation of the early response gene Smad6. In contrast, TβRIII specifically enhanced ALK3-mediated up-regulation of the early response gene ID-1. TβRIII associated with ALK3 primarily through their extracellular domains, whereas its interaction with ALK6 required both the extracellular and cytoplasmic domains. TβRIII, along with its interacting scaffolding protein β-arrestin2, induced the internalization of ALK6. In contrast, TβRIII colocalized with and resulted in the cell surface retention of ALK3, independently of β-arrestin2. Although complex formation between TβRIII, ALK6, and β-arrestin2 and TβRIII/ALK6 internalization resulted in maximal BMP signaling, the TβRIII mutant unable to interact with β-arrestin2, TβRIII-T841A, was unable to do so. These studies support a novel role for TβRIII in mediating differential ALK3 and ALK6 subcellular trafficking resulting in distinct signaling downstream of ALK3 and ALK6.     

WWOX suppresses autophagy for inducing apoptosis in methotrexate-treated human squamous cell carcinoma

Squamous cell carcinoma (SCC) cells refractory to initial chemotherapy frequently develop disease relapse and distant metastasis. We show here that tumor suppressor WW domain-containing oxidoreductase (WWOX) (also named FOR or WOX1) regulates the susceptibility of SCC to methotrexate (MTX) in vitro and cure of SCC in MTX therapy. MTX increased WWOX expression, accompanied by caspase activation and apoptosis, in MTX-sensitive SCC cell lines and tumor biopsies. Suppression by a dominant-negative or small interfering RNA targeting WWOX blocked MTX-mediated cell death in sensitive SCC-15 cells that highly expressed WWOX. In stark contrast, SCC-9 cells expressed minimum amount of WWOX protein and resisted MTX-induced apoptosis. Transiently overexpressed WWOX sensitized SCC-9 cells to apoptosis by MTX. MTX significantly downregulated autophagy-related Beclin-1, Atg12–Atg5 and LC3-II protein expression and autophagosome formation in the sensitive SCC-15, whereas autophagy remained robust in the resistant SCC-9. Mechanistically, WWOX physically interacted with mammalian target of rapamycin (mTOR), which potentiated MTX-increased phosphorylation of mTOR and its downstream substrate p70 S6 kinase, along with dramatic downregulation of the aforementioned proteins in autophagy, in SCC-15. When WWOX was knocked down in SCC-15, MTX-induced mTOR signaling and autophagy inhibition were blocked. Thus, WWOX renders SCC cells susceptible to MTX-induced apoptosis by dampening autophagy, and the failure in inducing WWOX expression leads to chemotherapeutic drug resistance.     

CME‐1, a novel polysaccharide,suppresses iNOS expression in lipopolysaccharide‐stimulated macrophages through ceramide‐initiated protein phosphatase 2A activation

CME‐1, a novel water‐soluble polysaccharide purified from Ophiocordyceps sinensis mycelia, has anti‐oxidative, antithrombotic and antitumour properties. In this study, other major attributes of CME‐1, namely anti‐inflammatory and immunomodulatory properties, were investigated. Treating lipopolysaccharide (LPS)‐stimulated RAW 264.7 cells with CME‐1 concentration‐dependently suppressed nitric oxide formation and inducible nitric oxide synthase (iNOS) expression. In the CME‐1‐treated RAW 264.7 cells, LPS‐induced IκBα degradation and the phosphorylation of p65, Akt and mitogen‐activated protein kinases (MAPKs), including extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and p38, were reduced. Treatment with a protein phosphatase 2A (PP2A)‐specific inhibitor, significantly reversed the CME‐1‐suppressed iNOS expression; IκBα degradation; and p65, Akt and MAPK phosphorylation. PP2A activity up‐regulation and PP2A demethylation reduction were also observed in the cells. Moreover, CME‐1‐induced PP2A activation and its subsequent suppression of LPS‐activated RAW 264.7 cells were diminished by the inhibition of ceramide signals. LPS‐induced reactive oxygen species (ROS) and hydroxyl radical formation were eliminated by treating RAW 264.7 cells with CME‐1. Furthermore, the role of ceramide signalling pathway and anti‐oxidative property were also demonstrated in CME‐1‐mediated inhibition of LPS‐activated primary peritoneal macrophages. In conclusion, CME‐1 suppressed iNOS expression by up‐regulating ceramide‐induced PP2A activation and reducing ROS production in LPS‐stimulated macrophages. CME‐1 is a potential therapeutic agent for treating inflammatory diseases.