Min-Chul Cho

Both E6 and E7 Oncoproteins of Human Papillomavirus 16 Inhibit IL-18-Induced IFN-γ Production in Human Peripheral Blood Mononuclear and NK Cells

Cervical carcinoma is the predominant cancer among malignancies in women throughout the world, and human papillomavirus (HPV) 16 is the most common agent linked to human cervical carcinoma. The present study was performed to investigate the mechanisms of immune escape in HPV-induced cervical cancer cells. The presence of HPV oncoproteins E6 and E7 in the extracellular fluids of HPV-containing cervical cancer cell lines SiHa and CaSki was demonstrated by ELISA. The effect of HPV 16 oncoproteins E6 and E7 on the production of IFN-γ by IL-18 was assessed. E6 and E7 proteins reduced IL-18-induced IFN-γ production in both primary PBMCs and the NK0 cell line. FACS analysis revealed that the viral oncoproteins reduced the binding of IL-18 to its cellular surface receptors on NK0 cells, whereas there was no effect of oncoproteins on IL-1 binding to its surface IL-1 receptors on D10S, a subclone of the murine Th cell D10.G4.1. In vitro pull-down assays also revealed that the viral oncoproteins and IL-18 bound to IL-18R α-chain competitively. These results suggest that the extracellular HPV 16 E6 and E7 proteins may inhibit IL-18-induced IFN-γ production locally in HPV lesions through inhibition of IL-18 binding to its α-chain receptor. Down-modulation of IL-18-induced immune responses by HPV oncoproteins may contribute to viral pathogenesis or carcinogenesis.

Down modulation of IL-18 expression by human papillomavirus type 16 E6 oncogene via binding to IL-18.

To understand modulation of a novel immune‐related cytokine, interleukin‐18, by human papillomavirus type (HPV) 16 oncogenes, HaCaT, normal keratinocyte cell line, and C‐33A, HPV‐negative cervical cancer cell line, were prepared to establish stable cell lines expressing E6, E6 mutant (E6m), E6E7, or E7 constitutively. Expressions of various HPV oncogene transcripts were identified by RT‐PCR. Expression of HPV oncogene E6 was reversely correlated to the expression of interleukin‐18, a novel pro‐inflammatory cytokine. The expression of E6 in C‐33A, independent of E6 splicing, resulted in decreased IL‐18 expression and that of IL‐18 was also significantly reduced in HaCaT cells expressing E6. The level of p53 was reduced in C‐33A cells expressing E6 whereas not altered in HaCaT cells expressing E6, suggesting that E6 downregulated IL‐18 expression via an independent pathway of p53 degradation in HaCaT cells which have a mutated p53 form. However, E7 did not affect IL‐18 expression significantly in both C‐33A and HaCaT cells. Cotransfection experiments showed that E6 oncogene did not inhibit the activities of IL‐18 promoter P1 and P2, suggesting that E6 oncogene indirectly inhibited IL‐18 expression. Taken together, E6, E6m and E6/E7 inhibited IL‐18 expression with some variation, assuming that cells expressing E6 oncogene can evade immune surveillance by downregulating the expression of immune stimulating cytokine gene, IL‐18, and inhibiting the cascade of downstream effects that follow activation of the IL‐18 receptor.

Peroxisome Proliferators-Activated Receptor (PPAR) Modulators and Metabolic Disorders

Overweight and obesity lead to an increased risk for metabolic disorders such as impaired glucose regulation/insulin resistance, dyslipidemia, and hypertension. Several molecular drug targets with potential to prevent or treat metabolic disorders have been revealed. Interestingly, the activation of peroxisome proliferator-activated receptor (PPAR), which belongs to the nuclear receptor superfamily, has many beneficial clinical effects. PPAR directly modulates gene expression by binding to a specific ligand. All PPAR subtypes (α, γ, and σ) are involved in glucose metabolism, lipid metabolism, and energy balance. PPAR agonists play an important role in therapeutic aspects of metabolic disorders. However, undesired effects of the existing PPAR agonists have been reported. A great deal of recent research has focused on the discovery of new PPAR modulators with more beneficial effects and more safety without producing undesired side effects. Herein, we briefly review the roles of PPAR in metabolic disorders, the effects of PPAR modulators in metabolic disorders, and the technologies with which to discover new PPAR modulators.

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.

A proinflammatory cytokine interleukin-32β promotes the production of an anti-inflammatory cytokine interleukin-10

A new proinflammatory cytokine interleukin‐32 (IL‐32) has six isoforms. Although IL‐32 can be detected in sera from patients suffering from Crohn’s disease and rheumatoid arthritis, it is unclear which isoforms are involved. To this end, we investigated the functions of the most abundant IL‐32β by generating K562‐IL‐32β stable cell lines. This report confirms, using IL‐32 small interfering RNA, that IL‐32β induces an anti‐inflammatory cytokine IL‐10 in K562‐IL‐32β cells and U937 promonocytic cells, which express endogenous IL‐32β upon phorbol 12‐myristate 13‐acetate (PMA) treatment, and monocyte‐derived dendritic cells (DC) upon lipopolysaccharide (LPS) treatment. Interleukin‐32β was induced in monocyte‐derived macrophages by LPS and in monocyte‐derived DC by LPS, poly(I:C), or anti‐CD40 antibody, but was not induced by PMA. We showed that IL‐32β expression was increased in a time‐dependent manner in monocyte‐derived DC upon LPS treatment and peaked at 24 hr. Production of IL‐10 was exactly coincident with IL‐32β expression, but IL‐1β and tumour necrosis factor‐α production peaked at 6 hr after LPS treatment, then steeply declined. Interleukin‐12 p40 was induced at 9 hr and gradually increased until 48 hr, at which time IL‐32β and IL‐10 were no longer increased. Knock‐down of IL‐32β by IL‐32 small interfering RNA led to the decrease of IL‐10, but the increase of IL‐12 in monocyte‐derived DC, which means that IL‐32β promotes IL‐10 production, but limits IL‐12 production. We also showed that IL‐10 neutralization increases IL‐12, IL‐1β and tumour necrosis factor‐α production, which implies that IL‐10 suppresses such proinflammatory cytokines. Taken together, our results suggest that IL‐32β upregulates the production of an anti‐inflammatory cytokine IL‐10, and then IL‐10 suppresses proinflammatory cytokines.

Indole-3-carbinol induces apoptosis through p53 and activation of caspase-8 pathway in lung cancer A549 cells.

Indole-3-carbinol (I3C) has anti-tumor effects in various cancer cell lines. However, the anti-tumor effect of I3C on human lung cancers has been rarely reported. We investigated the anti-tumor effects and its mechanism of I3C on human lung carcinoma A549 cell line. Treatment of the A549 cells with I3C significantly reduced cell proliferation, increased formations of fragmented DNA and apoptotic body, and induced cell cycle arrest at G0/G1 phase. I3C increased not only the protein levels of cyclin D1, phosphorylated p53, and p21 but also the expression of Fas mRNA. Cleavage of caspase-9, -8, -3 and PARP also was increased by I3C. Treatment with wortmannin significantly suppressed both I3C-induced Ser15 phosphorylation and accumulation of p53 protein. The inhibition of caspase-8 by z-IETD-FMK significantly decreased cleavage of procaspase-8,-3 and PARP in I3C-treated A549 cells. Taken together, these results demonstrate that I3C induces cell cycle arrest at G0/G1 through the activation of p-p53 at Ser 15 and induces caspase-8 mediated apoptosis via the Fas death receptor. This molecular mechanism for apoptotic effect of I3C on A549 lung carcinoma cells may be a first report and suggest that I3C may be a preventive and therapeutic agent against lung cancer.

Signaling pathways implicated in α‐melanocyte stimulating hormone‐induced lipolysis in 3T3‐L1 adipocytes

Melanocortins, besides their central roles, have also recently been reported to regulate adipocyte metabolism. In this study, we attempted to characterize the mechanism underlying α‐melanocyte‐stimulating hormone (MSH)‐induced lipolysis, and compared it with that of the adrenocorticotrophin hormone (ACTH) in 3T3‐L1 adipocytes. Similar to ACTH, MSH treatment resulted in the release of glycerol into the cell supernatant. The activity of hormone‐sensitive lipase, a rate‐limiting enzyme, which is involved in lipolysis, was significantly increased by MSH treatment. In addition, a variety of kinases, including protein kinase A (PKA) and extracellular signal‐regulated kinase (ERK) were also phosphorylated as the result of MSH treatment, and their specific inhibitors caused a reduction in MSH‐induced glycerol release and HSL activity, indicating that MSH‐induced lipolysis was mediated by these kinases. These results suggest that PKA and ERK constitute the principal signaling pathways implicated in the MSH‐induced lipolytic process via the regulation of HSL in 3T3‐L1 adipocytes. J. Cell. Biochem.

Cytotoxic Flavonoids as Agonists of Peroxisome Proliferator-Activated Receptor γ on Human Cervical and Prostate Cancer Cells

We conducted in silico screening for human peroxisome proliferator-activated receptor gamma (hPPARgamma) by performing an automated docking study with 450 flavonoids. Among the eight flavonoids as possible agonists of hPPARgamma, only 3,6-dihydroxyflavone (4) increased the binding between PPARgamma and steroid receptor coactivator-1 (SRC-1), approximately 5-fold, and showed one order higher binding affinity for PPARgamma than a reference compound, indomethacin. The 6-hydroxy group of the A-ring of 3,6-dihydroxyflavone (4) participated in hydrogen-bonding interactions with the side chain of Tyr327, His449, and Tyr473. The B-ring formed a hydrophobic interaction with Leu330, Leu333, Val339, Ile341, and Met364. Therefore, 3,6-dihydroxyflavone is a potent agonist of hPPAR with cytotoxic effects on human cervical and prostate cancer cells.

Novel PPARγ partial agonists with weak activity and no cytotoxicity; identified by a simple PPARγ ligand screening system.

Peroxisome proliferator-activated receptors (PPARs) are the transcriptional factor that regulate glucose and lipid homeostasis and widely well-known as molecular targets for improvement of metabolic disorder. Because major transcriptional activity of PPARs depends on their proper ligands, the studies for PPAR ligands have been continuously developed. We previously reported the simple enzyme-linked immunosorbent assay (ELISA) systems to screen PPAR ligands and a chemical library including flavonoid derivatives have applied to these systems. In this study, we introduce two compounds (KU16476 and KU28843) identified as PPARγ partial agonists by a screening ELISA for PPARγ ligand. KU16476 and KU28843 significantly increased binding between PPARγ and SRC-1 in a simple ELISA system. Co-activator recruiting-induced abilities of two compounds were less than that of indomethacin, a well-known PPARγ agonist. To determine whether these compounds would be PPARγ partial agonists, each candidate with indomethacin were applied to a simple ELISA based on binding between PPARγ and SRC-1. Cotreatment with indomethacin significantly increased binding between PPARγ and SRC-1 than treatment of indomethacin or candidate alone. Two compounds had no considerable cytotoxicities, induced partial adipogenesis, and accumulated lipid droplets in 3T3-L1 fibroblast. Also, these two compounds enhanced expression of PPARγ-mediated genes such as aP2 and UCP-2. By docking study, we confirmed that two compounds bound well to the active site of PPARγ with hydrophobic interactions. We suggest that two compounds identified by a simple ELISA system can be PPARγ partial agonists. These PPARγ partial agonists and these studies to find out novel PPARγ agonists may contribute to drug development against metabolic disorders.

Epiregulin expression by Ets-1 and ERK signaling pathway in Ki-ras-transformed cells.

Epiregulin belongs to the epidermal growth factor family, binds to the epidermal growth factor receptor, and its expression is upregulated in various cancer cells, but the regulatory mechanism is unclear. We investigated the regulatory mechanism of epiregulin expression in Ki-ras-transformed cancer cells. In 267B1/Ki-ras cells, the RAF/MEK/ERK pathway was constitutively activated, epiregulin was up-regulated, and the expression and phosphorylation of Ets-1 were augmented. The inhibition of ERK by PD98059 decreased epiregulin and Ets-1 expression and suppressed the growth of 267B1/Ki-ras cells. A chromatin immunoprecipitation assay demonstrated that Ets-1 was bound to human epiregulin promoter, and this binding was abolished by PD98059. Silencing of Ets-1 by RNA interference decreased cellular epiregulin transcript expression. We suggest that the Ki-ras mutation in 267B1 prostate cells constitutively activates the RAF/MEK/ERK pathway and induces the activation of the Ets-1 transcription factor, ultimately leading to the increased expression of epiregulin.