DNA Methylation of Estrogen Receptor α Gene by PhthalatesChan Kang, Se; Mu Lee, Byung
doi: 10.1080/15287390491008913pmid: 16326419
The phthalates are ubiquitous industrial plasticizers and include agents such as di(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), and butyl benzyl phthalate (BBP), which are classified as endocrine disruptors because of their anti-androgenic or pro-estrogenic effects. A recent study suggested that DBP produced the hypomethylation of c-myc protooncogene in mouse liver to activate c-myc. In the present study, DNA hypomethylation at the promoter region of the estrogen receptor α (ERα) gene was investigated by methylation-specific polymerase chain reaction (MSP) in a human breast cancer (MCF7) and in a normal (MCF10A) cell line after DBP treatment. Yeast-based estrogen receptor transcription assays showed that hERα gene expression was induced by BBP but not DBP. Moreover, MCF7 cells treated with BBP or DBP at 10−5M led to the demethylation of ERα promoter-associated CpG islands. These data suggest that an altered ER mRNA expression by BBP might be related to aberrant DNA methylation in the promoter region of ERα.
β-Carboline Alkaloid Suppresses NF-κB Transcriptional Activity Through Inhibition of IKK Signaling PathwayWoo Yoon, Jong; Ku Kang, Jae; Ro Lee, Kang; Woo Lee, Hyang; Whan Han, Jeung; Wan Seo, Dong; Kee Kim, Yong
doi: 10.1080/15287390491009110pmid: 16326420
Nuclear factor (NF)-κB transcription factors play an evolutionarily conserved and critical role in the triggering and coordination of both innate and adaptive immune responses. Therefore, there is intense interest in understanding the regulation of this transcription factor in the context of various diseases. Studies investigated the suppression mechanism of NF-κB signaling pathways by a β-carboline alkaloid (C-1) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. β-Carboline alkaloid decreased the level of inducible nitric oxide sythase (iNOS) protein and NOS promoter activities in a concentration-dependent manner. This effect was accompanied by the reduction of NF-κB DNA binding activity as well as NF-κB nuclear translocation. In addition, β-carboline alkaloid reduced the degradation and phosphorylation of IκB, and attenuated IKK activity in LPS-stimulated RAW 264.7 cells. Taken together, these results indicate that β-carboline alkaloid has the capability to suppress NF-κB signaling pathway through inhibition of IKK activity in LPS-stimulated RAW 264.7 cells.
Mechanism of NF-κB Activation Induced by γ-irradiation in B Lymphoma Cells : Role of RasRho, Hyun-Sook; Kim, Seol-Hee; Lee, Choong-Eun
doi: 10.1080/15287390491009129pmid: 16326421
Nuclear factor (NF)-κB is a ubiquitous transcription factor involved in diverse cellular responses to various stimuli, including growth factors and radiation stress. Recently it was reported that γ‐irradiation (γ-IR) upregulates allergy-associated adhesion molecule CD23 on B cells and monocytes via NF-κB activation. In the present study, the mechanism of NF-κB activation by γ-IR was investigated to understand the signaling pathways involved in IR-induced, NF-κB-mediated enhancement of CD23 expression. In human B-cell line Ramos, γ-IR induced a dose-dependent increase of nuclear translocation and transcriptional activity of NF-κB. The γ-IR-induced NF-κB activation in these cells was sensitive to a proteosome inhibitor MG132 and an antioxidant, pyrollidine dithiocarbamate (PDTC), which suggests that γ-IR-induced NF-κB activation proceeds via IκB gradation and redox regulation. Since Ras was shown to play a role in NF-κB-mediated survival and inflammation of cancer cells against radiation, the role of Ras signaling in the γ-IR-induced NF-κB activation in these transformed B cells was examined. Transfection and overexpression of dominant active Ras produced an increase in NF-κB activity as shown by DNA binding and transcriptional activities of the κB-dependent reporter gene. γ-IR, however, did not induce Erk activation, nor the γ-IR-induced κB activity that was suppressed by inhibitors of Ras/Raf interaction or MEK/Erk. Importantly, it was noted that Ras significantly augmented both the γ-IR-induced NF-κB activity and the γ-IR-induced CD23 expression. Together these results suggest that while γ-IR and Ras both contribute to the upregulation of CD23 expression via NF-κB Raf or Erk is not involved in γ-IR-induced NF-κB activation.
Effect of Alpha-Tocopherol on the Expression of Hepatic Vascular Stress Genes in Response to SepsisKim, Joo-Young; Lee, Sun-Mee
doi: 10.1080/15287390491009327pmid: 16326423
Sepsis is the leading cause of death in critically ill surgical patients. Septic hepatic dysfunction, an important determinant of outcome, although poorly understood, includes inappropriate expression of vasoregulatory genes. In this study the effect of alpha-tocopherol was determined on the expression of hepatic vascular stress genes in response to sepsis. Rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). Rats received either vehicle or alpha-tocopherol (AT, 15 mg/kg), intraperitoneally injected for 3 d prior to CLP procedure. Serum aminotransferase activities and hepatic lipid peroxides levels markedly increased 24 h after CLP, and this rise was attenuated by AT treatment. The hepatic concentrations of reduced glutathione decreased in CLP animals, which was inhibited by AT. CLP significantly increased mRNA levels of endothelin (ET)-1 and ETB receptor in livers, which was not prevented by AT treatment. There were no significant changes in ETA mRNA expression among any of the experimental groups. There were significant increases in the mRNA expression of inducible nitric oxide synthase and heme oxygenase-1 in livers of CLP animals, and this was prevented by AT treatment. The expression of tumor necrosis factor-α and cyclooxygenase-2 mRNA increased 4.9-fold and 4.4-fold, respectively, in livers of CLP animals. This increase was attenuated by AT treatment. Our data suggest that sepsis induces an imbalance in hepatic vasoregulatory gene expression and that AT ameliorates altered expression of vasodilators through its free radical scavenging activity.
Eupatilin Blocks Mediator Release Via Tyrosine Kinase Inhibition in Activated Guinea Pig Lung Mast CellsYoung Kim, Ji; Young Kwon, Eun; Song Lee, Yun; Bae Kim, Won; Youl Ro, Jai
doi: 10.1080/15287390500177024pmid: 16326424
Eupatilin, an extract from Artemisia asiatica Nakai, is known to exert anti-gastric ulcer, anticancer, and anti-inflammatory effects. The aim of this study was to elucidate whether eupatilin has antiallergic reactions in activated guinea pig lung mast cells compared to apigenin and genistein. Mast cells were purified from guinea pig lung tissues by using enzyme digestion and rough and discontinuous density Percoll gradient. The purified mast cells were sensitized with immunoglobulin (Ig) G1 (anti-OVA antibody) and challenged with ovalbumin (OVA). Histamine was assayed using an automated fluorometric analyzer, leukotrienes by radioimmunoassay, and tyrosine phosphorylation by immunoblotting. Intracellular Ca2+ was analyzed by confocal laser scanning microscopy, protein kinase C (PKC) activity using protein phosphorylated with [γ-32P]ATP, and phopholipase D activity (PLD) and phosphatidic acid by using labeled phosphatidyl alcohol. Eupatilin, apigenin, or genistein reduced histamine release and leukotriene synthesis in a does-dependent manner. Eupatilin inhibited mediators to a greater extent than apigenin or genistein. Eupatilin, apigenin, and genistein initially blocked phosphorylation of Syk tyrosine and Ca2+ influx, PLD activity, phosphatidic acid, and Ca2+-dependent PKC α/βII activities during mast cell activation in a dose-dependent manner. Our data suggest that eupatilin initially inhibits Syk kinase, and then blocks downstream multisignal pathways and Ca2+ influx during mast cell activation triggered by a specific antigen–antibody reaction. Thus, eupatilin may have use clinically as a treatment for inflammatory disorders associated with allergic diseases including asthma.
Effects of Chemical Carcinogens and Physicochemical Factors on the UV Spectrophotometric Determination of DNASik Kim, Hyung; Hyun Byun, Soo; Mu Lee, Byung
doi: 10.1080/15287390500182503pmid: 16326425
The ultraviolet (UV) absorbance ratio of 260/280 nm has been used as an indicator of DNA purity. However, the A260/A280 ratio may be beyond the normal range (1.8–1.9) due to physicochemical alterations produced by pH and temperature, and carcinogenic chemical modification. When the pH of the DNA solution buffer increased from 3 to 11, the A260/A280 ratio changed significantly from 1.5 to 2.2 in mixtures of DNA bases [A:T:C:G = 28.5:28.5: 21.5:21.5, i.e., (A + T)/(all four bases) = 57%, expressed as mole percent], of deoxyribonucleosides (adenosine:thymidine:cytidine:guanosine= 28.5:28.5:21.5:21.5, as mole percent), or of deoxyribonucleotides (dAMP:dTMP:dGMP:dCMP = 28.5:28.5:21.5:21.5, as mole percent) examined. The A260/A280 ratio increased with RNA contamination and exceeded 1.9 when RNA concentration was >30%, as mole percent. In contrast, the A260/A280 ratio was linearly reduced by increasing the protein concentration. Phenol (>0.02%) contamination also reduced the A260/A280 ratio to below 1.8. Benzo[a]pyrene diol epoxide (BPDE), a reactive carcinogen metabolite of benzo[a]pyrene (BaP), decreased the A260/A280 ratio correlated with the degree to which it modified the DNA. These results suggest that the UV A260/A280 ratio is significantly affected by pH and the presence of contaminating species of macromolecules and chemicals.
Comparative Mutagenicity of Apicidin and Apicidin Derivatives (SD-0203 and SD-2007), Histone Deacetylase InhibitorsJeong Yoo, Eun; Mu Lee, Byung
doi: 10.1080/15287390500182511pmid: 16326426
The fungal metabolite apicidin [cyclo(N-O-methyl-l-tryptophanyl-l-isoleucinyl-d-pipecolinyl-l-2-amino-8-oxodecanoyl)] is known to inhibit histone deacetylase (HDAC). In this study, the genotoxicity of apicidin and its derivatives were tested using three tests: a bacterial reverse mutation assay (Ames test), an in vitro chromosome aberration (CA) test, and an in vivo micronucleus (MN) test. Apicidin was negative in the Ames test in the presence and absence of the microsomal metabolizing enzyme system. Apicidin induced a significant increase in the total chromosome aberrations in Chinese hamster ovary (CHO) cells. In the MN test, apicidin induced mutagenic activity at the highest dose (1000 μM/kg). The apicidin derivatives SD-0203 and SD-2007 did not induce mutagenic activity in the Ames test and no significant mutagenic potency was observed in the CA test. However, these compounds significantly and dose-dependently increased the number of micronucleated polychromatic erythrocytes (MNPCEs) as well as the PCE/(PCE + NCE) ratio in the MN test. These results suggest that apicidin and its derivatives preferentially induce CA and MN but are not effective in the Ames test.
Suppression of Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Expression in Raw 264.7 Macrophages by Sesquiterpene LactonesGyu Shin, Sang; Ku Kang, Jae; Ro Lee, Kang; Woo Lee, Hyang; Whan Han, Jeung; Soo Choi, Wahn
doi: 10.1080/15287390591009506pmid: 16326428
The molecular mechanism underlying the suppression of lipopolysaccharide (LPS)/interferon-γ (IFN-γ)-induced nitric oxide (NO) and prostaglandin (PG) E2 production was investigated in RAW 264.7 macrophages treated with sesquiterpene lactones, zaluzanin-C and estafiatone, isolated from Ainsliaea. Zaluzanin-C and estafiatone decreased NO production in LPS/IFN-γ-stimulated RAW 264.7 macrophages with an IC50 of about 6.61 μM and 3.80 μM, respectively. In addition, these compounds inhibited the synthesis of PGE2 in LPS/IFN-γ-treated RAW 264.7 macrophages. Furthermore, treatment with zaluzanin-C and estafiatone resulted in a decrease in inducible No Synthase (iNOS) and Cyclooxygenase-2 (COX-2) protein and mRNA expression levels. Zaluzanin-C and estafiatone inhibited nuclear factor-κB (NF-κB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-γ. This effect was accompanied by parallel reduction of phosphorylation and degradation of inhibitor of κB (IkB). In addition, these effects were completely blocked by treatment with cysteine, indicating that the inhibitory effect of zaluzanin-C and estafiatone might be mediated by alkylation of either NF-κB itself or an upstream molecule of NF-κB. These results demonstrate that the suppression of NF-κB activation by zaluzanin-C and estafiatone might be attributed to inhibition of nuclear translocation of NF-κB resulting from blockade of the degradation of IκB, leading to suppression of the expression of iNOS and COX-2, which play important roles in inflammatory signaling pathways.