Antiproliferative activity of IL-27 on melanoma.

IL-27 is a member of the IL-6/IL-12 family and activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130. We previously demonstrated that IL-27 has potent antitumor activities, which are mediated through CD8(+) T cells, NK cells, or its own antiangiogenic activity. In this study, we demonstrate that IL-27 also possesses a direct antiproliferative activity on melanoma. Although WSX-1 expression was hardly detected in parental mouse melanoma B16F10 cells, IL-27 activated STAT1 and STAT3 and up-regulated MHC class I in B16F10 transfectants expressing wild-type WSX-1. In contrast, IL-27 failed to activate STAT1 and up-regulate MHC class I in those expressing mutant WSX-1, in which the putative STAT1-binding Tyr-609 of the cytoplasmic region was replaced by Phe. IL-27 inhibited the tumor growth of transfectants expressing wild-type WSX-1 in a dose-dependent manner. IL-27 augmented the expression of IFN regulatory factor (IRF)-1 and IRF-8, which possess tumor suppressor activities, in B16F10 transfectants expressing wild-type WSX-1. Down-regulation of IRF-1 but not IRF-8 with small interfering RNA partially blocked the IL-27-induced growth inhibition. A small, but significant, direct antiproliferative effect of IL-27 was also observed in vivo. Moreover, several human melanoma cells were revealed to express both IL-27 receptor subunits, and activation of STAT1 and STAT3 and growth inhibition by IL-27 were detected. These results suggest that IL-27 has an antiproliferative activity on melanomas through WSX-1/STAT1 signaling. Thus, IL-27 may be an attractive candidate as an antitumor agent applicable to cancer immunotherapy.     

Structure and the evolutionary implication of the triplicated complement factor B genes of a urochordate ascidian, Ciona intestinalis

To elucidate the evolution of the complement system and MHC class III region, we analyzed the complement factor B (Bf) genes of a urochordate ascidian, Ciona intestinalis. Three different cDNA species, termed CiBf-1, CiBf-2 and CiBf-3, were identified. The deduced amino-acid sequences all contained the usual domains of vertebrate Bf and, in addition, three extra domains at the N-terminus. Furthermore, the serine protease domain of these CiBfs shared unique features with vertebrate complement components C1r/s and mannose-binding lectin-associated serine protease (MASP)-2/3, the absence of the disulfide bond designated histidine loop, and the usage of the AGY codon for the catalytic serine residue. These results indicate that complement genes have evolved through extensive exon shuffling events in the early stage of chordate evolution. Overall deduced amino-acid identity between CiBf-1 and -2 was 88%, whereas CiBf-3 showed 49% identity to both CiBf-1 and CiBf-2. These three CiBf genes were located within an approximately 50-kb genomic region, and exons 3 and 5 of all the three Bf genes showed an extremely high degree of nucleotide identity, indicating that the CiBf genes experienced extensive reorganization, such as duplication and gene conversion, since its divergence from the vertebrate Bf/C2 gene. Fluorescent in situ hybridization (FISH) to the chromosomes showed that genetic loci for the CiBfs, CiC3-1 and CiC3-2 genes are present on three different chromosomes, suggesting the possibility that the linkage among the MHC class III complement genes was established in the vertebrate lineage after its divergence from urochordates.The sequences reported in this paper have been deposited in the DDBJ database (accession nos. AB180044–AB180051).     

Proteomic analysis of sera from hepatocellular carcinoma patients after radiofrequency ablation treatment

Comparative proteomic analysis was used to search for characteristic alterations in the sera of hepatocellular carcinoma (HCC) patients who had undergone curative radiofrequency ablation treatment. Serum samples collected from eight patients before and after treatment were subjected to 2-DE. Eighty-eight protein spots differentially expressed with the treatment were selected by clustering analysis, and the proteins were identified by MS based on MALDI-TOF/TOF analysis and public database searches. The statistical analysis suggested that four proteins decreased after treatment (pro-apolipoprotein, alpha2-HS glycoprotein, apolipoprotein A-IV precursor, and PRO1708/PRO2044, which is the carboxy terminal fragment of albumin) and that seven proteins were increased after treatment, including leucine-rich alpha2-glycoprotein and alpha1-antitrypsin. These data facilitate the identification of differentially expressed proteins that are involved in HCC carcinogenesis and provide candidate biomarkers for the development of diagnostic and therapeutic tools.     

Overexpression of thioredoxin reductase 1 regulates NF-kappa B activation

Thioredoxin reductase (TrxR) is a flavoprotein that contains a C-terminal penultimate selenocysteine (Sec) and has an ability to reduce thioredoxin (Trx), which regulates the activity of NF-kappa B. To date, three TrxR isozymes, TrxR1, TrxR2, and TrxR3, have been identified. In the present study, we found that among these isozymes only TrxR1 was induced by tumor necrosis factor-alpha (TNF alpha) in vascular endothelial cells. Furthermore, the overexpression of TrxR1 enhanced TNF alpha-induced DNA-binding activity of NF-kappa B and NF-kappa B-dependent gene expression. The catalytic Sec residue of TrxR1, which is essential for reducing Trx, was required for this NF-kappa B activation, and aurothiomalate, an inhibitor of TrxR, suppressed TNF alpha-induced activation of NF-kappa B and the expression of NF-kappa B-targeted proinflammatory genes such as E-selectin and cyclooxygenase-2. These results suggest that TrxR1 may act as a positive regulator of NF-kappa B and may play an important role in the cellular inflammatory response.     

Phosphodiesterase 5 inhibitor acts as a potent agent sensitizing acute myeloid leukemia cells to 67-kDa laminin receptor-dependent apoptosis

(−)-Epigallocatechin-3-O-gallate (EGCG), a polyphenol in green tea, induces apoptosis in acute myeloid leukemia (AML) cells without affecting normal cells. In this study, we observed that cGMP acts as a cell death mediator of the EGCG-induced anti-AML effect through acid sphingomyelinase activation. EGCG activated the Akt/eNOS axis, a well-known mechanism in vascular cGMP upregulation. We also observed that a major cGMP negative regulator, phosphodiesterase 5, was overexpressed in AML cells, and PDE5 inhibitor, an anti-erectile dysfunction drug, synergistically enhanced the anti-AML effect of EGCG. This combination regimen killed AML cells via overexpressed 67-kDa laminin receptors.     

Crucial Role of Perilipin-3 (TIP47) in Formation of Lipid Droplets and PGE2 Production in HL-60-Derived Neutrophils

Cytosolic lipid droplets (LDs), which are now recognized as multifunctional organelles, accumulate in leukocytes under various inflammatory conditions. However, little is known about the characteristic features of LDs in neutrophils. In this study, we show that perilipin-3 (PLIN3; formerly called TIP47) is involved in LD formation and the inflammatory response in HL-60-derived neutrophils. HL-60, a promyelocytic cell line, was differentiated into neutrophils via treatment with all-trans retinoic acid. After differentiation, cells were stimulated with Porphyromonas gingivalis lipopolysaccharide (P.g-LPS), a major pathogen in adult periodontitis. When HL-60-derived neutrophils were stimulated with P.g-LPS, LDs increased in both number and size. In the differentiated cells, PLIN3 was induced while PLIN1, PLIN2 and PLIN5 were not detected. PGE₂ production and the PLIN3 protein level were increased by the P.g-LPS treatment of the cells in a dose-dependent manner. When PLIN3 was down-regulated with siRNA treatment, LDs essentially disappeared and the level of PGE₂ secreted in the cell culture medium decreased by 65%. In addition, the suppression of PLIN3 repressed the PGE₂ producing enzymes; i.e., microsomal PGE synthase-1, -2 and cyclooxygenase-2. These findings indicate that PLIN3 has a pivotal role in LD-biogenesis in HL-60-derived neutrophils, and that PLIN3 is associated with the synthesis and secretion of PGE₂.