Han-A Kim

Platelet-Activating Factor Induces Up-regulation of Antiapoptotic Factors in a Melanoma Cell Line through Nuclear Factor-κB 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-κB (NF-κB) 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-κB-dependent manner in a melanoma cell line, therefore suggesting that PAF may diminish the cytotoxic effect of chemotherapeutic agents. (Cancer Res 2006; 66(9): 4681-6)

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‐κB 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‐κB 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‐κB and AP‐1, plays a critical role in PAF‐induced enhancement of pulmonary melanoma metastasis.

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.

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.

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. H2O2 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.

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.

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 Peyers 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.

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.

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‐κB 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.