Profiling of transcripts and proteins modulated by K-ras oncogene in the lung tissues of K-ras transgenic mice by omics approaches.

The mutated K-ras gene is involved in approximately 30% of human cancers. In order to search for K-ras oncogene-induced modulators in lung tissues of K-ras transgenic mice, we performed microarray and proteomics (LC/ESI-MS/MS) analysis. Genes (RAB27b RAS family, IL-1RA, IL-33, chemokine ligand 6, epiregulin, EGF-like domain and cathepsin) related to cancer development (Wnt signaling pathway) and inflammation (chemokine/cytokine signaling pathway, Toll receptor signaling) were up-regulated while genes (troponin, tropomodulin 2, endothelial lipase, FGFR4, integrin alpha8 and adenylate cyclase 8) related to the tumor suppression such as p53 pathway, TGF-beta signaling pathway and cadherin signaling pathway were down-regulated by K-ras oncogene. Proteomics approach revealed that up-regulated proteins in lung adenomas of K-ras mice were classified as follows: proteins related to the metabolism/catabolism (increased from 7 to 22% by K-ras gene), proteins related to translation/transcription and nucleotide (from 4 to 6%), proteins related to signal transduction (from 3 to 5%), proteins related to phosphorylation (from 1 to 2%). ATP synthase, Ras oncogene family, cytochrome c oxidase, flavoprotein, TEF 1, adipoprotein A-1 BP, glutathione oxidase, fatty acid BP 4, diaphorase 1, MAPK4 and transgelin were up-regulated by K-ras oncogene. However, integrin alpha1, Ras-interacting protein (Rain), endothelin-converting enzyme-1d and splicing factor 3b were down-regulated. These studies suggest that genes related to cancer development and inflammation were up-regulated while genes related to the tumor suppression were down-regulated by K-ras, resulting in the tumor growth. Putative biomarkers such as cell cycle related genes (Cdc37), cancer cell adhesion (Glycam 1, integrin alpha8, integrin alphaX and Clec4n), signal transduction (Tlr2, IL-33, and Ccbp2), migration (Ccr1, Ccl6, and diaphorase 1 (Cyb5r3) and cancer development (epiregulin) can be useful for diagnosis and as prognosis markers and some of the target molecules can be applied for prevention of cancer.

Profiling of transcripts and proteins modulated by the E7 oncogene in the lung tissue of E7-Tg mice by the omics approach.

The E6 and E7 oncoproteins of human papilloma virus (HPV) type 16 have been known to cooperatively induce the immortalization and transformation of primary keratinocytes. We established an E7 transgenic mouse model to screen HPV-related biomakers using the omics approach. The methods used to identify HPV-modulated factors were genomics analysis by microarray using the Affymetrix 430 2.0 array to screen E7-modulated genes, and proteomics analysis using nano-LC-ESI-MS/MS to screen E7-modulated proteins with the lung tissue of E7 transgenic mice. According to omics data, cyclin B1, cyclin E2, topoisomerase IIα, calnexin, activated leukocyte cell adhesion molecule CD166, actinin α1, diaphorase 1, gelsolin, platelet glycoprotein, and annexin A2 and A4 were up-regulated in the E7-Tg mice, while proteoglycan 4, sarcolipin, titin, vimentin, drep 1, troponin and cofilin-1 were down-regulated. We further confirmed the significance of differences between the expression levels of the selected factors in E7-Tg and non-Tg mice by real-time PCR. Genes related to cancer cell adhesion, cell cycle and migration, proliferation and apoptosis, as well as to the intermediate filament network and to endoplasmic reticulum proteins, were selected. Taken together, the results suggest that the E7 oncogene modulates the expression levels of cell cycle-related (cyclin B1, cyclin E2) and cell adhesion- and migration-related (actinin α1, CD166) factors, which may play important roles in cellular transformation in cancer. In addition, the solubilization of the rigid intermediate filament network by specific proteolysis mediated via up-regulating gelsolin and down-regulating cofilin-1, as well as increased levels of endoplasmic reticulum protein calnexin with chaperone functions, might also be involved in E7-lung epithelial cells.

Detection of expressed IL-32 in human stomach cancer using ELISA and immunostaining.

Interleukin (IL)-32 is a recently identified proinflammatory cytokine that is one of the IL-18 inducible genes, and plays an important role in autoimmune and inflammatory diseases. We produced antibodies against IL-32 and studied the expression of IL-32 in human stomach cancer. We detected IL-32 secreted from K-562 cells that werw stably transfected with IL-32 and in the sera of stomach cancer patients, by a sandwich ELISA using a monoclonal antibody KU32-52 and a polyclonal antibody. In order to optimize a sandwich immunoassay, recombinant IL-32alpha was added, followed by the addition of a biotinylated KU32-52 into microtiter plate wells precoated with a goat anti-IL-32 antibody. The bound biotinylated KU32-52 was probed with a streptavidin conjugated to HRP. This sandwich ELISA was highly specific and had a minimal detection limit of 80 pg/ml (mean+/-SD of zero calibrator) and measuring up to 3,000 pg/ml. This ELISA showed no cross-reaction with other cytokines such as hIL-1alpha, hIL-1beta, hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-alpha. Intra-assay coefficients of variation were 18.5% to 4.6% (n=10), and inter-assay coefficients were 23% to 9% (n=10). The average IL-32 level in the sera of 16 stomach cancer patients (189 pg/ml) was higher than that of 12 healthy control men (109 pg/ml). Our results indicate that serum IL-32 level can be detected by using an established ELISA, and that this immunoassay and mAb KU32-09 specific for immunohistochemistry can be used in the detection of expressed and secreted IL- 32 in stomach cancer patients.

KR-31831, benzopyran derivative, inhibits VEGF-induced angiogenesis of HUVECs through suppressing KDR expression.

Angiogenesis is important in the development and progression of cancer, therefore the therapeutic approach based on anti-angiogenesis may represent a promising therapeutic option. KR-31831 is a novel anti-ischemic agent. Previously, we reported the anti-angiogenic activity of KR-31831. In the present study we investigated the molecular mechanisms underlying anti-angiogenic activity of KR-31831. We show that KR-31831 inhibits vascular endothelial growth factor (VEGF)-induced proliferation and tube formation via release of intracellular Ca2+ and phosphorylation of extra-cellular regulated kinase 1/2 (Erk 1/2) in human umbilical vein endothelial cells (HUVECs). Moreover, the expression of VEGF receptor 2 (VEGFR2, known as Flk-1 or KDR) was reduced by the treatment of KR-31831. These results suggest that KR-31831 may have inhibitory effects on tumor angiogenesis through down-regulation of KDR expression.

Effects of a tetramethoxyhydroxyflavone p7f on the expression of inflammatory mediators in LPS-treated human synovial fibroblast and macrophage cells.

The inhibitory effects of 5,6,3',5'-tetramethoxy 7,4'-hydroxyflavone (labeled as p7F) were elucidated on the productions of proinflammatory cytokines as well as inflammatory mediators in human synovial fibroblasts and macrophage cells. p7F inhibited IL-1beta or TNF-alpha induced expressions of inflammatory mediators (ICAM-1, COX-2, and iNOS). p7F also inhibited LPS-induced productions of nitric oxide and prostaglandin E2 in RAW 264.7 cells. In order to investigate whether p7F would inhibit IL-1 signaling, p7F was added to the D10S Th2 cell line (which is responsive to only IL-1beta and thus proliferates), revealing that p7F inhibited IL-1beta-induced proliferation of D10S Th2 cells in a doseresponse manner. A flow cytometric analysis revealed that p7F reduced the intracellular level of free radical oxygen species in RAW 264.7 cells treated with hydrogen peroxide. p7F inhibited IkappaB degradation and NF-kappaB activation in macrophage cells treated with LPS, supporting that p7F could inhibit signaling mediated via toll-like receptor. Taken together, p7F has inhibitory effects on LPS-induced productions of inflammatory mediators on human synovial fibroblasts and macrophage cells and thus has the potential to be an antiinflammatory agent for inhibiting inflammatory responses.

Interleukin-32 monoclonal antibodies for immunohistochemistry, Western blotting, and ELISA.

The members of the IL-1 family play important roles in the development and pathogenesis of autoimmune and inflammatory diseases. Especially, IL-1 and IL-18 belong to the IL-1 family because they share structural similarity and require caspase-1 for processing. Currently, IL-18 has been studied for its biological effects in the broad spectrum of Th1- or Th2- related autoimmune diseases. IL-18 also uses a similar signaling pathway as that of IL-1 family members. Taken together these results, IL-18-inducible genes might also contribute to autoimmune and inflammatory diseases. It has recently been reported that an inducer of TNF-alpha was identified as one of IL-18 inducible genes in IL-18 responsible cells and named as a new cytokine IL-32. We have produced novel monoclonal anti IL-32 antibodies in order to help study IL-32 function and to develop improved diagnosis of IL-32-expressing tumors. Several mAbs reactive to IL-32 isoforms were prepared and characterized by the epitope analysis and Western blotting performed using various deletion mutants and IL-32 isoforms (IL-32alpha, beta, gamma, and delta). In order to optimize the sandwich ELISA for IL-32, recombinant IL-32alpha was added, followed by the addition of a biotinylated mAb KU32-52 into the microtiter plate wells pre-coated with a mAb KU32-07 or mAb KU32-56. The bound mAb was probed with a streptavidin conjugated to HRP. The epitope analysis and Western blot analysis revealed that mAb KU32-07 could detect only IL-32alpha and KU32-52 was bound to all isoforms, whereas KU32-56 were reactive to IL-32 alpha, beta, delta isoforms but not gamma isoform. These sandwich ELISAs were highly specific and had a minimal detection limit of 80 pg/ml (mean+3 SD of zero calibrator) and measuring range of up to 3000 pg/ml. An ELISA using a coating mAb KU32-07 and a capturing biotinylated mAb KU32-52 had no cross-reaction with other cytokines such as IL-32beta, IL-32gamma, IL-32delta, hIL-1alpha , IL-1beta , hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-alpha. Intra-assay coefficients of variation were 11 to 6% (n=16) and inter-assay coefficients were 10 to 5% (n=9). Another ELISA using a coating mAb KU32-56 and a capturing biotinylated mAb KU32-52 detected both IL-32alpha and IL-32beta isoforms but not gamma and delta isoforms and had no cross-reaction with other cytokines such as hIL-1alpha , IL-1beta , hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-alpha. One mAb KU32-09 was shown to react strongly on immunohistochemistry. Our newly established mAbs, KU32-07, KU32-09, KU32-52, KU32-56, have different and useful properties for the detection of IL-32 by immunohistochemistry, ELISA, and Western blotting.