The Significance of VSIG4 Expression in Ovarian Cancer.

OBJECTIVES: The protein V-set and Ig domain-containing 4 (VSIG4), a novel B7 family-related macrophage protein with the capacity to inhibit T-cell activation, has a potential role in cancer. Here we suggest its possibility as a therapeutic target and prognostic biomarker of ovarian cancer. METHODS: Between January 2011 and June 2015, tumor tissues and peripheral blood samples were obtained during surgery from 10 patients with benign ovarian tumors and 22 patients with ovarian cancers. Messenger RNA and protein expression levels of VSIG4 in benign tumor and cancer tissues were examined by the reverse transcription polymerase chain reaction and Western blot, respectively. Soluble VSIG4 concentrations were measured by an enzyme-linked immunosorbent assay. The correlation between VSIG4 expression and the prognosis of ovarian cancer was analyzed according to the patients' clinicopathologic characteristics. RESULTS: VSIG4 messenger RNA and protein expression levels in ovarian cancer tissues were higher than those in benign ovarian tumors (P = 0.0013 and 0.0001, respectively). Soluble VSIG4 concentrations were increased in patients with ovarian cancer compared with that in patients with benign ovarian tumors (P = 0.0452). Moreover, soluble VSIG4 levels were significantly increased in advanced-stage and recurrent ovarian cancer (P = 0.0244 and 0.0288, respectively). High VSIG4 expression of cancer tissue and low VSIG4 expression of plasma (soluble VSIG4) were associated with a longer disease-free interval (P = 0.0246 and 0.0398, respectively). CONCLUSIONS: VSIG4 is overexpressed in ovarian cancers compared with that in benign tumors. This finding supports VSIG4 being used as a potential therapeutic target for ovarian cancer. Furthermore, soluble VSIG4 levels are associated with the progression and recurrence of ovarian cancer, indicating that soluble VSIG4 may be used as a potential biomarker for predicting tumor prognosis.

West Nile virus T-cell ligand sequences shared with other flaviviruses: a multitude of variant sequences as potential altered peptide ligands.

Phylogenetic relatedness and cocirculation of several major human pathogen flaviviruses are recognized as a possible cause of deleterious immune responses to mixed infection or immunization and call for a greater understanding of the inter-Flavivirus protein homologies. This study focused on the identification of human leukocyte antigen (HLA)-restricted West Nile virus (WNV) T-cell ligands and characterization of their distribution in reported sequence data of WNV and other flaviviruses. H-2-deficient mice transgenic for either A2, A24, B7, DR2, DR3, or DR4 HLA alleles were immunized with overlapping peptides of the WNV proteome, and peptide-specific T-cell activation was measured by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assays. Approximately 30% (137) of the WNV proteome peptides were identified as HLA-restricted T-cell ligands. The majority of these ligands were conserved in ∼≥88% of analyzed WNV sequences. Notably, only 51 were WNV specific, and the remaining 86, chiefly of E, NS3, and NS5, shared an identity of nine or more consecutive amino acids with sequences of 64 other flaviviruses, including several major human pathogens. Many of the shared ligands had an incidence of >50% in the analyzed sequences of one or more of six major flaviviruses. The multitude of WNV sequences shared with other flaviviruses as interspecies variants highlights the possible hazard of defective T-cell activation by altered peptide ligands in the event of dual exposure to WNV and other flaviviruses, by either infection or immunization. The data suggest the possible preferred use of sequences that are pathogen specific with minimum interspecies sequence homology for the design of Flavivirus vaccines.

Dendritic cell mediated delivery of plasmid DNA encoding LAMP/HIV-1 Gag fusion immunogen enhances T cell epitope responses in HLA DR4 transgenic mice.

This report describes the identification and bioinformatics analysis of HLA-DR4-restricted HIV-1 Gag epitope peptides, and the application of dendritic cell mediated immunization of DNA plasmid constructs. BALB/c (H-2d) and HLA-DR4 (DRA1*0101, DRB1*0401) transgenic mice were immunized with immature dendritic cells transfected by a recombinant DNA plasmid encoding the lysosome-associated membrane protein-1/HIV-1 Gag (pLAMP/gag) chimera antigen. Three immunization protocols were compared: 1) primary subcutaneous immunization with 1x10(5) immature dendritic cells transfected by electroporation with the pLAMP/gag DNA plasmid, and a second subcutaneous immunization with the naked pLAMP/gag DNA plasmid; 2) primary immunization as above, and a second subcutaneous immunization with a pool of overlapping peptides spanning the HIV-1 Gag sequence; and 3) immunization twice by subcutaneous injection of the pLAMP/gag DNA plasmid. Primary immunization with pLAMP/gag-transfected dendritic cells elicited the greatest number of peptide specific T-cell responses, as measured by ex vivo IFN-gamma ELISpot assay, both in BALB/c and HLA-DR4 transgenic mice. The pLAMP/gag-transfected dendritic cells prime and naked DNA boost immunization protocol also resulted in an increased apparent avidity of peptide in the ELISpot assay. Strikingly, 20 of 25 peptide-specific T-cell responses in the HLA-DR4 transgenic mice contained sequences that corresponded, entirely or partially to 18 of the 19 human HLA-DR4 epitopes listed in the HIV molecular immunology database. Selection of the most conserved epitope peptides as vaccine targets was facilitated by analysis of their representation and variability in all reported sequences. These data provide a model system that demonstrates a) the superiority of immunization with dendritic cells transfected with LAMP/gag plasmid DNA, as compared to naked DNA, b) the value of HLA transgenic mice as a model system for the identification and evaluation of epitope-based vaccine strategies, and c) the application of variability analysis across reported sequences in public databases for selection of historically conserved HIV epitopes as vaccine targets.

Programmed death-1 receptor negatively regulates LPS-mediated IL-12 production and differentiation of murine macrophage RAW264.7 cells.

While programmed death-1 (PD-1), a co-inhibitory member of CD28 immunoglobulin superfamily plays negative roles in effector functions of T cells and B cells, little is known about the function of PD-1 expressed on innate immune cells. In this study, we demonstrate that IL-12 production was greatly suppressed in LPS-stimulated RAW264.7 cells upon PD-1 engagement with B7-H1.Fc fusion protein, and was restored in the presence of antagonistic anti-PD-1 mAb. PD-1-mediated suppression of IL-12 production in LPS-stimulated RAW264.7 cells was mediated by inhibition of Janus N-terminal-linked kinase (JNK) signaling pathway, and to a lesser extent, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway through the recruitment of SHP-2 to PD-1 cytoplasmic tail. B7-H1.Fc-mediated PD-1 engagement also downregulates the expression of co-stimulatory molecules such as CD80, CD86, MHC class I and II proteins in LPS-stimulated RAW264.7 cells. Furthermore, the endocytic activity is enhanced but the allostimulatory capacity is suppressed in LPS-treated RAW264.7 cells upon PD-1 engagement. Taken together, our results reveal a novel function of macrophage PD-1 in the negative regulation of IL-12 synthesis and differentiation into dendritic cell-like cells.

Conservation and variability of West Nile virus proteins.

West Nile virus (WNV) has emerged globally as an increasingly important pathogen for humans and domestic animals. Studies of the evolutionary diversity of the virus over its known history will help to elucidate conserved sites, and characterize their correspondence to other pathogens and their relevance to the immune system. We describe a large-scale analysis of the entire WNV proteome, aimed at identifying and characterizing evolutionarily conserved amino acid sequences. This study, which used 2,746 WNV protein sequences collected from the NCBI GenPept database, focused on analysis of peptides of length 9 amino acids or more, which are immunologically relevant as potential T-cell epitopes. Entropy-based analysis of the diversity of WNV sequences, revealed the presence of numerous evolutionarily stable nonamer positions across the proteome (entropy value of < or = 1). The representation (frequency) of nonamers variant to the predominant peptide at these stable positions was, generally, low (< or = 10% of the WNV sequences analyzed). Eighty-eight fragments of length 9-29 amino acids, representing approximately 34% of the WNV polyprotein length, were identified to be identical and evolutionarily stable in all analyzed WNV sequences. Of the 88 completely conserved sequences, 67 are also present in other flaviviruses, and several have been associated with the functional and structural properties of viral proteins. Immunoinformatic analysis revealed that the majority (78/88) of conserved sequences are potentially immunogenic, while 44 contained experimentally confirmed human T-cell epitopes. This study identified a comprehensive catalogue of completely conserved WNV sequences, many of which are shared by other flaviviruses, and majority are potential epitopes. The complete conservation of these immunologically relevant sequences through the entire recorded WNV history suggests they will be valuable as components of peptide-specific vaccines or other therapeutic applications, for sequence-specific diagnosis of a wide-range of Flavivirus infections, and for studies of homologous sequences among other flaviviruses.

Increased antimutagenic and anticlastogenic effects of doenjang (Korean fermented soybean paste) prepared with bamboo salt.

The kind of salt used in doenjang preparation is one of the most important factors that affect the chemopreventive activities of doenjang. Bamboo salt (BS) is known to exert various therapeutic effects on several diseases. In this study, antimutagenic and anticlastogenic effects of methanol extracts of doenjang made with either one-time heat-treated BS (BS1-D), nine-times heat-treated BS (BS9-D), sun-dried salt (SDS-D), or purified salt (PS-D) using the Ames test and micronucleus test. BS1-D and BS9-D showed higher inhibitory effects than PS-D and SDS-D on mutagenicity induced by aflatoxin B(1), N-methyl-N'-nitro-N-nitrosoguanidine, and 4-nitroquinoline-1-oxide. BS1-D exerted more suppressive effects on chromosome aberration than SDS-D on mitomycin C-induced micronucleus in mice. It can be concluded that the chemopreventive effect of doenjang was significantly increased by using BS instead of the more typical commercial salts, SDS and PS (P < .05). This is probably due to the changes in the chemical and physical properties of the salt, especially increased content of K and P, during the processing of BS and to differences in fermented products formed during the doenjang fermentation in the presence of BS.

Longer aging time increases the anticancer and antimetastatic properties of doenjang.

OBJECTIVE: Fermented soy foods, such as doenjang, are important components of the Korean diet. Doenjang has been shown to have greater antimutagenic and anticancer activities than other fermented soybean foods such as chungkukjang (Korean-style natto) and miso and non-fermented soybeans. This study investigated the effects of fermentation time of doenjang on its inhibitory activity against solid tumor formation and lung metastasis. METHODS: Effects of methanol extracts from doenjang prepared with long-term fermentation on tumor formation, natural killer cell activity in spleen, and glutathione S-transferase activity in liver were investigated in sarcoma-180 cell-transplanted mice. Inhibitory effects of these samples on lung metastasis of colon 26-M3.1 cells were also evaluated in Balb/c mice. To determine the effect of aging on functionality, doenjang fermented for 3, 6, and 24 mo were evaluated. RESULTS: Doenjang fermented for 24 mo exhibited a two- to three-fold increase in antitumor effects on sarcoma-180-injected mice and antimetastatic effects in colon 26-M 3.1 cells in mice compared with the 3- or 6-mo fermented doenjang. The 24-mo fermentation was the most effective in preventing cancer by decreasing tumor formation and increasing natural killer cell activity in spleens and glutathione S-transferase activity in livers of mice. CONCLUSIONS: Prolonging the fermentation period when making doenjang increases its antitumor and antimetastatic effects in vivo.

Curcumin inhibits the growth of AGS human gastric carcinoma cells in vitro and shows synergism with 5-fluorouracil.

The inhibitory effect of curcumin and its synergism with 5-fluorouracil (5-FU) on the growth of the AGS human gastric carcinoma cell line was examined. Cell cycle analysis was used to elucidate the mechanisms for the inhibition by curcumin. Curcumin significantly inhibited the growth of AGS cells in a dose- and time-dependent manner (P <.05). Curcumin caused a 34% decrease in AGS proliferation at 5 micromol/L, 51% at 10 micromol/L, and 92% at 25 micromol/L after 4 days of treatment. When curcumin (10 micromol/L) was removed after a 24-hour exposure, the growth pattern of curcumin-treated AGS cells was similar to that of control cells, suggesting reversibility of curcumin on the growth of AGS cells. Combining curcumin with 5-FU significantly increased growth inhibition of AGS cells compared with either curcumin or 5-FU alone (P <.05), suggesting synergistic actions of the two drugs. After 4 days of treatment with 10 micromol/L of curcumin, the G2/M phase fraction of cells was 60.5% compared with 22.0% of the control group, suggesting a G2/M block by curcumin treatment. Because the curcumin concentrations (5 micromol/L) used in our study were similar to steady-state concentrations (1.77 +/- 1.87 micromol/L) in human serum of subjects receiving chronic administration of a commonly recommended dose (8 g/day), curcumin may be useful for the treatment of gastric carcinoma, especially in conjunction with 5-FU.

Induction of Bax and activation of caspases during beta-sitosterol-mediated apoptosis in human colon cancer cells.

beta-sitosterol, a main dietary phytosterol found in plants, may have the potential for prevention and therapy for human cancer. The purpose of the present study was to examine the effect of beta-sitosterol on the growth of HT116 human colon cancer cells. Treatment with beta-sitosterol resulted in a dose-dependent growth inhibition coupled with the characteristic morphological features of apoptosis and with the increase of a sub-G1 cell population. Apoptosis-inducing concentrations of beta-sitosterol induced caspase-3 and caspase-9 activation accompanied by proteolytic cleavage of poly(ADP-ribose)-polymerase. In addition, beta-sitosterol-induced apoptosis in HT116 cells was associated with a decreased expression of the anti-apototic Bcl-2 protein and mRNA and a concomitant increase of the pro-apototic Bax protein and mRNA, and with release of cytochrome c from the mitochondria into the cytosol. beta-sitosterol treatment also inhibited the expression of cIAP-1 without significant changes in the level of cIAP-2. Taken together, these findings provide important new insights into the possible molecular mechanisms of the anti-cancer activity of beta-sitosterol.

Kimchi and an active component, beta-sitosterol, reduce oncogenic H-Ras(v12)-induced DNA synthesis.

The Korean fermented vegetable food, kimchi, has been demonstrated to have anticancer functional properties. This study examined the effect of kimchi samples, methanol extracts of commercially grown baechu cabbage kimchi (CK) and organically grown baechu cabbage kimchi (OK), as well as the dichloromethane fraction (DCM fr.) from CK, and the active compound (AC), which has been identified as largely beta-sitosterol, from DCM fr., on the Ras-dependent signaling pathway. CK, OK, and DCM fr. exhibited a greater inhibition against the proliferation of Rat2 fibroblasts transformed with Ras(v12) (HO6) than parental Rat2 fibroblasts. In addition, OK and DCM fr. showed a higher inhibitory effect than CK. Furthermore, we employed the single-cell microinjection technique, combined with 3-bromo-5'-deoxyuridine incorporation, to examine the effects of kimchi samples on DNA synthesis induced by microinjected oncogenic Ras(v12). When the DCM fr. and AC were used to treat Rat1 fibroblasts overexpressing human insulin receptors (HIRc-B) and microinjected with oncogenic H-Ras(v12), the DNA synthesis of injected cells was decreased, suggesting that kimchi might block the signaling pathway of oncogenic Ras(v12), thus preventing the proliferation of transformed cells. This study provides additional evidence that kimchi and its active components, including beta-sitosterol, have potential in both the prevention and treatment of cancer, and presents convincing evidence that the anticancer effects may be a result of an inhibition of Ras oncogene signaling.

Antimutagenic effects of doenjang (Korean fermented soypaste) and its active compounds.

Doenjang (Korean fermented soypaste) is one of the important fermented foods of Korea. Doenjang has been traditionally manufactured from meju, which is fermented rectangular shape molded from crushed cooked soybeans. The main microorganisms involved for meju fermentation are Bacillus subtilis and molds such as Rizopus sp., Mucor sp., and Aspergillus sp. We have already reported that doenjang is safe from mycotoxin, especially, aflatoxin contamination due to the manufacturing process of the doenjang. We have demonstrated that the doenjang extracts showed strong antimutagenic activities against various carcinogens/mutagens including aflatoxin B(1). The traditionally fermented soypaste, doenjang showed higher antimutagenic activity than the raw soybeans, cooked soybeans, meju and other fermented soybeans in the Ames test. The active compounds that were identified are genistein, linoleic acid, beta-sitosterol glucoside, soyasaponin, etc. The active compounds exhibited strong antimutagenic activities in the Ames test, SOS chromotest and Drosophila wing spot test. More genistein was formed during the doenjang fermentation from genistin in the soybeans. Genistein and linoleic acid were the most effective active compounds found in doenjang.