Inhibition Effect of Adipogenesis and Lipogenesis via Activation of AMPK in Preadipocytes Treated with Canavalia gladiata Extract.

The aim of this study was to investigate the effect of Canavalia gladiata extract (CGE) on the regulation of AMP-activated protein kinase (AMPK) in 3T3-L1 preadipocytes and evaluate the adipogenesis and lipogenesis mechanisms. In 3T3-L1 preadipocytes, lipid accumulation and differentiation were suppressed by 1.1, 1.3, and 1.4 times under the CGE treatment at 0.25, 0.5, and 1.0 mg/mL, respectively. The expression of the main genes involved in the inhibition of adipogenesis was evaluated at the mRNA level via a transcription-polymerase chain reaction. The extract at 1.0 mg/mL increased the mRNA expressions of AMPK and carnitine palmitoyl transferase-1 (CPT-1) by 1.9 and 1.2 times, respectively, while it decreased the expression of sterol regulatory element binding proteins-1c (SREBP-1c), peroxisome proliferator activated receptor-γ (PPAR-γ), CCAAT enhancer binding protein-α (C/EBP-α), and fatty acid synthase (FAS) by 1.1, 1.2, 1.8, and 1.5 times, respectively, indicating inhibition of the adipogenesis and lipogenesis potential of CGE. Gallic acid (4.02 mg/g) was identified as the main component of the CGE via LC-MS/MS and HPLC analysis. The results of this study suggested that CGE can be utilized as an anti-obesity food additive or medication by activating the AMPK-induced regulation and suppressing adipogenesis transcription factors.

Platelet-activating factor enhances tumour metastasis via the reactive oxygen species-dependent protein kinase casein kinase 2-mediated nuclear factor-κB activation.

Platelet-activating factor (PAF) promotes tumour metastasis via activation of the transcription factor nuclear factor-κB (NF-κB). We here investigated the role of the protein kinase CK2 (formerly Casein Kinase 2 or II) in PAF-induced NF-κB activation and tumour metastasis, given that PAF has been reported to increase CK2 activity, and that CK2 plays a key role in NF-κB activation. PAF increased CK2 activity, phosphorylation and protein expression in vivo as well as in vitro. CK2 inhibitors inhibited the PAF-mediated NF-κB activation and expression of NF-κB-dependent pro-inflammatory cytokines and anti-apoptotic factors. Pre-treatment with the antioxidant N-Acetyl-L-Cysteine (NAC) resulted in a significant inhibition in PAF-induced enhancement of CK2 activity, phosphorylation and protein expression in vivo as well as in vitro. H2 O2 and known reactive oxygen species inducers, lipopolysaccharide (LPS) and tumour necrosis factor-α (TNF-α) enhanced CK2 activity, phosphorylation and protein expression, which was again inhibited by antioxidant. PAF, LPS and TNF-α induced increased CK2 activity, phosphorylationand protein expression, which were inhibited by p38 inhibitor. PAF, LPS or TNF-α increased pulmonary metastasis of B16F10, which was inhibited by antioxidants, CK2 inhibitor and p38 inhibitor. Our data suggest that (i) reactive oxygen species activate CK2 via p38, which, in turn, induces NF-κB activation, and (ii) PAF, LPS and TNF-α increase pulmonary tumour metastasis via the induction of the reactive oxygen species (ROS)/p38/CK2/NF-κB pathway.

Cholera toxin breakdowns oral tolerance via activation of canonical NF-κB.

The mechanisms of mucosal immunogenicity and adjuvanticity of bacterial exotoxins remains unknown. In this study, we investigated the role of the transcription factor nuclear factor-κB (NF-κB) in cholera toxin (CT)-induced alteration of oral tolerance. Feeding CT abrogated ovalbumin (OVA)-induced oral tolerance, as evaluated by OVA-specific serum antibody responses, and CD4(+) T cell proliferation. CT feeding activated canonical NF-κB (one heterodimer type, p50-p65) and mRNA expression of NF-κB-dependent proinflammatory cytokines in mesenteric lymph node (MLN) and Peyer's patch (PP) cells. CT no longer showed abrogation of oral tolerance in mice pretreated with p50 small interfering RNAs (siRNAs). ADP-ribosylation inhibitors inhibited CT-induced NF-κB activation. These data suggest that CT induces canonical NF-κB activation in intestinal lymphoid cells, which plays a key role in mucosal immunogenicity and adjuvanticity.

Protein kinase CK2/PTEN pathway plays a key role in platelet-activating factor-mediated murine anaphylactic shock.

Platelet-activating factor (PAF) is a major mediator in the induction of fatal hypovolemic shock in murine anaphylaxis. This PAF-mediated effect has been reported to be associated with PI3K/Akt-dependent eNOS-derived NO. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is phosphatidylinositol phosphate phosphatase, which negatively controls PI3K by dephosphorylating the signaling lipid, phosphatidylinositol 3,4,5-triphosphate. In this study, we examined the possible involvement of PTEN in PAF-mediated anaphylactic shock. Induction of anaphylaxis or PAF injection resulted in a rapid decrease in PTEN activity, followed by increases in PI3K activity and phosphorylation of Akt and eNOS. Systemic administration of adenoviruses carrying PTEN cDNA (adenoviral PTEN), but not the control AdLacZ, not only attenuated anaphylactic symptoms, but also reversed anaphylaxis- or PAF-induced changes in PTEN and PI3K activities, as well as phosphorylation of Akt and eNOS. We found that the decreased PTEN activity was associated with PTEN phosphorylation, the latter effect being prevented by the protein kinase CK2 inhibitor, DMAT. DMAT also inhibited anaphylactic symptoms as well as the anaphylaxis- or PAF-mediated PTEN/PI3K/Akt/eNOS signaling cascade. CK2 activity was increased by PAF. The present data provide, as the key mechanism underlying anaphylactic shock, PAF triggers the upstream pathway CK2/PTEN, which ultimately leads to the activation of PI3K/Akt/eNOS. Therefore, CK2/PTEN may be a potent target in the control of anaphylaxis and other many PAF-mediated pathologic conditions.

Mechanisms of platelet-activating factor-induced enhancement of VEGF expression.

It has been previously reported that platelet-activating factor (PAF) induces the expression of vascular endothelial growth factor (VEGF) via the downregulation of p53 activity. In this study, we attempted to characterize the mechanism by which p53 activity negatively regulates PAF-induced VEGF expression. PAF increased luciferase activity as well as VEGF mRNA expression in human non-small cell lung cancer cell line H1299 transfected with VEGF luciferase reporter plasmid (VEGF-Luc). Cotransfection of the cells with wt p53, but not mutant p53, effected a blockage of PAF-induced VEGF mRNA expression. The ChIP assay revealed that p53 did not bind to the VEGF promoter. Transfection of Egr-1 or Sp-1 expression vector increased VEGF luciferase activity in VEGF-Luc-transfected cells, and this was inhibited by transfection with wt p53. The results of the Immunoprecipitation and immunoblot analysis showed that p53 binds to Egr-1 and Sp-1. Additionally, our electrophoretic mobility shift assay demonstrated that PAF induced the mobilization of Egr-1 and Sp-1 to the nucleus, and this activity was inhibited by transfection with wt p53. These data indicate that PAF inhibits protein complexes between p53 and Egr-1/Sp-1 via the downregulation of p53 levels, thus increasing the free form levels of Egr-1 and Sp-1, ultimately resulting in the transcriptional activation of VEGF.

Hierarchically Structured Core-Shell Design of a Lithium Transition-Metal Oxide Cathode Material for Excellent Electrochemical Performance.

Tuning geometrical parameters of lithium-mixed transition-metal oxide (LiTM) cathode materials is a promising strategy for resource-efficient design of high-performance Li-ion batteries. In this paper, we demonstrate that simple and facile geometrical tailoring of the secondary microstructure of LiTM cathode materials without complex chemical modification or heterostructure engineering can significantly improve overall electrochemical performance of the active cathode materials. An optimized LiTM with a bimodal size distribution of primary particles inside the secondary particles exhibits a 53.8% increase in capacity at a high discharge rate (10 C) compared to a commercially available reference and comparable rate capability after 100 charge/discharge cycles. The key concept of this approach is to maximize the beneficial effects arising from the controlled sizes of primary particles. Multimodal/multiscale microscopic characterizations based on electron tomography and scanning transmission electron microscopy, combined with electron energy-loss spectroscopy and energy-dispersive X-ray spectroscopy from the atomic level to the microscale level, were employed to elucidate structural origins of enhanced battery performance. This study paves the way for the resource-efficient microstructure design of LiTM cathode materials to maximize capacity and stability via simple adjustment of processing conditions, which is advantageous for mass-production applications.

Nitric oxide plays a key role in the platelet-activating factor-induced enhancement of resistance against systemic candidiasis.

Platelet-activating factor (PAF) has been demonstrated to augment resistance against Candida albicans infection. In this study, the role of nitric oxide (NO) in PAF-induced resistance in the kidneys was investigated. Pretreatment of the C. albicans-infected mice with PAF resulted in strong expression of messenger RNA (mRNA) and the protein synthesis of inducible nitric oxide synthase (iNOS). These PAF effects were inhibited to a significant degree by pretreatment with the nuclear factor-kappaB inhibitor, pyrrolidinedithiocarbamate. Pretreatment with PAF protected the mice from death caused by C. albicans infection and reduced the growth of fungus in the kidneys. The protective activity of PAF was abrogated by pretreatment with the iNOS inhibitor, aminoguanidine, and in the iNOS(-/-) mice. The PAF markedly increased the infiltration of neutrophils, but not macrophages, and also enhanced the mRNA expression levels of the CXC chemokine, keratinocyte-derived chemokine, in C. albicans-infected kidneys. These effects of PAF were attenuated in the aminoguanidine-treated mice and the iNOS(-/-) mice. These data show that NO plays an important role in PAF-induced protection against C. albicans.

Platelet-activating factor induces up-regulation of antiapoptotic factors in a melanoma cell line through nuclear factor-kappaB activation.

In this study, we investigated the influence of platelet-activating factor (PAF) on the induction of apoptosis-regulating factors in B16F10 melanoma cells. PAF increased the expression of mRNA and the protein synthesis of antiapoptotic factors, such as Bcl-2 and Bcl-xL, but did not increase the expression of the proapoptotic factor, Bax. A selective nuclear factor-kappaB (NF-kappaB) inhibitor, parthenolide, inhibited the effects of PAF. Furthermore, PAF inhibited etoposide-induced increases in caspase-3, caspase-8, and caspase-9 activities, as well as cell death. p50/p65 heterodimer increased the mRNA expression of Bcl-2 and Bcl-xL and decreased etoposide-induced caspase activities and cell death. In an in vivo model in which Matrigel was injected s.c., PAF augmented the growth of B16F10 cells and attenuated etoposide-induced inhibition of B16F10 cells growth. These data indicate that PAF induces up-regulation of antiapoptotic factors in a NF-kappaB-dependent manner in a melanoma cell line, therefore suggesting that PAF may diminish the cytotoxic effect of chemotherapeutic agents.

Platelet-activating factor-induced NF-kappaB activation enhances VEGF expression through a decrease in p53 activity.

We investigated the role of p53 in nuclear factor (NF)-kappaB dependent, platelet-activating factor (PAF)-induced vascular endothelial growth factor (VEGF) expression. Transfected NF-kappaB subunits in ECV304 cells increased the tumor necrosis factor-alpha promoter activity, which was completely inhibited by p53. Transfected p53 increased p53RE promoter activity, which was completely inhibited by NF-kappaB subunits, indicating that cross-regulation occurs between NF-kappaB and p53. PAF-induced increase in VEGF expression was correlated with decreased p53 activity. These data suggest that NF-kappaB-dependency of the PAF-induced increase in VEGF expression is due to decreased p53 activity, which is reciprocally regulated by increased NF-kappaB activity.

Involvement of matrix metalloproteinase-9 in platelet-activating factor-induced angiogenesis.

Platelet-activating factor (PAF) augments angiogenesis by promoting the synthesis of various angiogenic factors, via the activation of NF-kappaB. In this study, we investigated the role of the matrix metalloproteinase (MMP)-9, in PAF-induced angiogenesis. PAF increased mRNA expression, protein synthesis, and MMP-9 activity in ECV304 cells, in a NF-kappaB-dependent manner. PAF increased MMP-9 promoter activity in ECV304, which was inhibited by WEB2107, and NF-kappaB inhibitors. Transfected NF-kappaB subunits, p65 or/and p50, increased luciferase activity in the reporter plasmid MMP-9, resulting in an increase not only of MMP-9 luciferase activity, but also of mRNA expression in MMP-9. MMP-9 or NF-kappaB inhibitors significantly inhibited PAF-induced angiogenesis, in a dose-dependent manner, in an in vivo mouse Matrigel implantation model. In a parallel to the Matrigel implantation study, MMP-9 or NF-kappaB inhibitors inhibited PAF-induced sprouting of porcine pulmonary arterial endothelial cells. These data indicate that NF-kappaB-dependent MMP-9 plays a key role in PAF-induced angiogenesis.

PTEN/MAPK pathways play a key role in platelet-activating factor-induced experimental pulmonary tumor metastasis.

In this study, we investigated the role of PTEN (phosphatase and tensin homolog deleted on chromosome 10) in a platelet-activating factor (PAF)-induced experimental pulmonary tumor metastasis model. An adenovirus carrying PTEN cDNA (Ad-PTEN) reversed PAF-induced increase in phosphorylation of AKT as well as pulmonary metastasis of B16F10. PAF-induced pulmonary metastasis was inhibited by MAPK inhibitors, but not by PI3K inhibitor. Ad-PTEN abrogated PAF-induced phosphorylation of MAPKs. These data indicate PTEN/MAPK pathways play a key role in PAF-induced tumor metastasis.

Glutamine inhibits platelet-activating factor-mediated pulmonary tumour metastasis.

Inflammation has been increasingly recognised as an important component of tumourigenesis. Platelet-activating factor (PAF), a potent inflammatory mediator, has the ability to enhance tumour growth and metastasis. In this study, we have investigated (i) the role of mitogen-activated protein kinases (MAPKs) and (ii) the therapeutic efficacy of the non-essential amino acid, l-glutamine (Gln), which evidences MAPKs inhibition activity in PAF-mediated B16F10 melanoma metastasis to the lungs. Mice were given intraperitoneal injection of PAF. ERK, JNK, and p38 MAPKs were activated rapidly by PAF in the lungs, and the PAF-induced metastasis of B16F10 was inhibited in a dose-dependent manner by pretreatment with either U0126 (ERK inhibitor), SP600125 (JNK inhibitor), or SB202190 (p38 inhibitor). Intraperitoneal administration of Gln after, but not before, PAF injection deactivated ERK, JNK, and p38 by dephosphorylating them. Gln inhibited PAF-induced metastasis when Gln was administered either intraperitoneally or orally. PAF induced pronounced angiogenic activity in an in vivo mouse Matrigel implantation model. MAPK inhibitors as well as Gln significantly inhibited PAF-induced angiogenesis. These data indicate that Gln exerts a beneficial effect against inflammation-associated enhanced tumour metastasis via the deactivation of MAPKs.

CpG-ODN-based immunotherapy is effective in controlling the growth of metastasized tumor cells.

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG-ODN) act as potent immune stimulators by activating innate immunity through toll-like receptor 9. These immunomodulatory effects of CpG-ODN have been reported to be associated with anti-tumor immunity. In this study, we used a murine B16F10 melanoma model and a CT26 colon cancer model to assess whether CpG-ODN-based immunotherapy was effective in inhibiting tumor cells that have already metastasized to distant organs. Systemic administration of CpG-ODN after melanoma cell injection resulted in a significant inhibition of pulmonary colonization. When CpG-ODN was administered after tumor cell injection, it also inhibited pulmonary metastasis of the tumor cells, albeit to a lesser degree in the latter case. Systemic administration of CpG-ODN after subcutaneous inoculation of CT26 colon cancer cells diminished pulmonary metastasis from the primary tumor sites. Additionally, CpG-ODN also inhibited the growth of pulmonary colonization of the colon tumor cells when CpG-ODN was administered after the primary tumors had been surgically removed. These data indicate that CpG-ODN was effective in inhibiting pulmonary metastasis of the B16F10 melanoma and CT26 colon cancer cells, as well as the growth of metastasized tumor cells. Our results suggest that CpG-ODN-based immunotherapy may be beneficial in controlling micrometastasis after surgery in clinical settings.

Critical role for matrix metalloproteinase-9 in platelet-activating factor-induced experimental tumor metastasis.

In this study, the roles of matrix metalloproteinase (MMP)-2 and MMP-9 in platelet-activating factor (PAF)-induced experimental pulmonary metastasis of the murine melanoma cell, B16F10, were investigated. An injection of PAF resulted in increases in mRNA expression, protein levels and the activities of both MMP-2 and MMP-9 in the lungs. The overall expression of MMP-9 was stronger than that of MMP-2. The increased MMP-9 expression was inhibited by both NF-kappaB and AP-1 inhibitors, whereas the increased MMP-2 expression was inhibited by only AP-1 inhibitors. Immunohistochemical analysis revealed that MMP-9 was expressed in bronchial epithelial cells as well as in the walls of blood vessels, whereas MMP-2 expression was observed only in bronchial epithelial cells. PAF significantly enhanced the pulmonary metastasis of B16F10, which was inhibited by both NF-kappaB and c-jun inhibitors. MMP-9 inhibitor, but not that of MMP-2, completely inhibited PAF-induced B16F10 metastasis. These data indicate that MMP-9, the expression of which was regulated by NF-kappaB and AP-1, plays a critical role in PAF-induced enhancement of pulmonary melanoma metastasis.