Upregulation of CYP1B1 by hypoxia is mediated by ERα activation in breast cancer cells.

Endocrine therapy for breast cancer often leads to drug resistance and tumor recurrence; tumor hypoxia is also associated with mortality and tumor relapse. Cytochrome P450 1B1 (CYP1B1) regulates estrogen metabolism in breast cells and is known to be overexpressed in breast cancer tissue. Although the individual association of hypoxia-induced hypoxia-inducible factor-1-alpha (HIF-1α) and CYP1B1 with tumorigenesis is well known, the association between HIF-1α and CYP1B1 leading to tumorigenesis has not been investigated. Here, we investigated the correlation between hypoxia and CYP1B1 expression in breast cancer cells for tumorigenesis-related mechanisms. Hypoxia was induced in the human breast cancer cell lines MCF-7 (Er-positive) and MDA-MB-231 (triple-negative) and the normal breast epithelial cell line MCF10A; these cell lines were then subjected to immunoblotting, transient transfection, luciferase assays, gene silencing using small interfering RNA, polymerase chain reaction analysis, chromatin immunoprecipitation, co-immunoprecipitation, and mammalian two-hybrid assays. Furthermore, immunofluorescence analysis of the tumor microarrays was performed, and the pub2015 and the Cancer Genome Atlas patient datasets were analyzed. HIF-1α expression in response to hypoxia occurred in both normal and breast cancer cells, whereas CYP1B1 was induced only in estrogen receptor α (ERα)-positive breast cancer cells under hypoxia. HIF-1α activated ERα through direct binding and in a ligand-independent manner to promote CYP1B1 expression. Therefore, we suggested the mechanism by which hypoxia and ER-positivity orchestrate breast cancer relapse.

Shinorine and porphyra-334 isolated from laver (Porphyra dentata) inhibit adipogenesis in 3T3-L1 cells.

Mycosporine-like amino acids (MAAs) such as shinorine and porphyra-334 from Porphyra spp. are bioactive compounds with strong photoprotective and antioxidant properties. In this study, the anti-adipogenic effect of shinorine and porphyra-334 was examined in vitro utilizing 3T3-L1 preadipocytes. Shinorine and porphyra-334 were extracted from laver (Porphyra dentata) 50% methanolic (MeOH) extract of and their structures were elucidated by MS and NMR spectroscopy. Both compounds had no cytotoxic effect in 3T3-L1 cells (< 200 µg/mL) and inhibited the accumulation of lipid droplets in 3T3-L1 mature adipocytes in a dose-dependent manner (0.1 and 1.0 µM). Interestingly, both compounds had also significantly reduced the expression of adipogenic-related genes such as peroxisome proliferator-activated receptor γ2 (PPARγ2), CCAAT/enhancer-binding protein α (C/EBPα), adiponectin, and leptin in 3T3-L1 cells. The findings suggest that shinorine and porphyra-334 have the potential to inhibit adipogenesis in 3T3-L1 preadipocytes.

Simplified Free Gingival Graft Around Implants Using Medical Grade Tissue Cyanoacrylate: A Case Series Report.

The free gingival graft (FGG) procedure using suturing techniques has been widely utilized to effectively increase the amount of attached keratinized gingiva. However, conventional suturing procedures are time-consuming and technique-sensitive. Simplified FGG procedure around teeth and dental implants using medical grade tissue adhesive (cyanoacrylate) is known to overcome drawbacks of traditional suture techniques. However, the clinical application of cyanoacrylate as a means of stabilizing the graft has not been a common practice. The aim of this report demonstrates simplified FGG procedures around dental implants using cyanoacrylate with follow-up results.

Effects of tobacco compound 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on the expression of epigenetically regulated genes in lung carcinogenesis.

Cigarette smoking is a major cause of lung cancer. Although tobacco smoking-induced genotoxicity has been well established, there is apparent lack of abundance functional epigenetic effects reported On cigarette smoke-induced lung carcinogenesis. The aim of this study was to determine effects of intratracheal administration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) utilizing target gene expression DNA methylation patterns in lung tissues of mice following twice weekly for 8 weeks treatment. An unbiased approach where genomic regions was undertaken to assess early methylation changes within mouse pulmonary tissues. A methylated-CpG island recovery assay (MIRA) was performed to map the DNA methylome in lung tissues, with the position of methylated DNA determined using a Genome Analyzer (MIRA-SEQ). Alterations in epigenetic-regulated target genes were confirmed with quantitative reverse transcription-PCR, which revealed 35 differentially hypermethylated genes including Cdkn1C, Hsf4, Hnf1a, Cdx1, and Hoxa5 and 30 differentially hypomethylated genes including Ddx4, Piwi1, Mdm2, and Pce1 in NNK-exposed lung tissue compared with controls. The main pathway of these genes for mediating biological information was analyzed using the Kyoto Encyclopedia of Genes and Genomes database. Among them, Rssf1 and Mdm2 were closely associated with NNK-induced lung carcinogenesis. Taken together, our data provide valuable resources for detecting cigarette smoke-induced lung carcinogenesis.

Comparison of immunohistochemical analysis on sinus augmentation using demineralized tooth graft and bovine bone.

OBJECTIVES: The purpose of this animal research was to compare bone regeneration in augmented rabbit maxillary sinuses treated with demineralized particulate human-tooth graft and anorganic bovine bone by immunohistochemical analysis. MATERIALS AND METHODS: Piezoelectric bilateral sinus augmentation was performed in eight adult rabbits. In the control group, anorganic bovine was grafted in the maxillary sinus following elevation of the sinus membrane. In the experimental group, demineralized human particulate tooth bone was grafted in the sinus. Bone regeneration in augmented sinuses was evaluated by immunohistochemical analysis using various markers of osteoprogenitor cells. RESULTS: The number of bromodeoxyuridine-labeled cells was significantly higher in the experimental group than in the control group at eight weeks. The immunoreactivity of proliferating-cell nuclear antigen was increased slightly in the experimental group relative to the control group at eight weeks. Other bone markers were expressed equally in the two groups. CONCLUSION: In the rabbit maxillary sinus, higher osteoinduction was correlated with demineralized human particulate tooth bone grafting than with anorganic bovine grafting.

Effects of Harvest Time on Phytochemical Constituents and Biological Activities of Panax ginseng Berry Extracts.

Ginseng (Panax ginseng) has long been used as a traditional medicine for the prevention and treatment of various diseases. Generally, the harvest time and age of ginseng have been regarded as important factors determining the efficacy of ginseng. However, most studies have mainly focused on the root of ginseng, while studies on other parts of ginseng such as its berry have been relatively limited. Thus, the aim of this study iss to determine effects of harvest time on yields, phenolics/ginsenosides contents, and the antioxidant/anti-elastase activities of ethanol extracts of three- and four-year-old ginseng berry. In both three- and fourfour-year-old ginseng berry extracts, antioxidant and anti-elastase activities tended to increase as berries ripen from the first week to the last week of July. Liquid chromatography-tandem mass spectrometry analysis has revealed that contents of ginsenosides except Rg1 tend to be the highest in fourfour-year-old ginseng berries harvested in early July. These results indicate that biological activities and ginsenoside profiles of ginseng berry extracts depend on their age and harvest time in July, suggesting the importance of harvest time in the development of functional foods and medicinal products containing ginseng berry extracts. To the best of our knowledge, this is the first report on the influence of harvest time on the biological activity and ginsenoside contents of ginseng berry extracts.

Suppression of PMA-induced human fibrosarcoma HT-1080 invasion and metastasis by kahweol via inhibiting Akt/JNK1/2/p38 MAPK signal pathway and NF-κB dependent transcriptional activities.

Coffee is one of the widely sales beverage worldwide and contains numerous phytochemicals that are beneficial to health. Kahweol acetate (KA), a coffee-specific diterpene, exhibits anti-tumoric properties in human tumoric cells. However, the effect of KA on the metastasis and invasion of cancer cells and the underlying mechanisms remain unclear. The objectives of this study were to estimate the anti-tumor activity of KA and reveal the possible molecular mechanisms. KA markedly inhibited the cell proliferation enhanced by phorbol 12-myristate 13-acetate (PMA) in human fibrosarcoma cells. As well as, KA attenuated PMA-induced cell migration and invasion in a concentration-dependent manner. KA suppressed PMA-enhanced activation of matrix metalloproteinase-9 (MMP-9) through suppression of nuclear factor kappa B (NF-κB) activation. KA repressed the PMA-induced phosphorylation of Akt, c-Jun N-terminal kinase (JNK) 1/2, and p38 MAPK, which are signaling molecules upstream of MMP-9 expression. In summary, we demonstrated that the anti-tumor effects of KA might occur through the inhibition of Akt/JNK1/2/p38 MAPK phosphorylation and downregulation of NF-κB activation, leading to a decrease in MMP-9 expression. Thus, KA is a useful chemotherapeutic agent that may contribute to prevent to the metastatic tumor.

Comparative Histomorphometric Analysis of Maxillary Sinus Augmentation With Deproteinized Bovine Bone and Demineralized Particulate Human Tooth Graft: An Experimental Study in Rabbits.

PURPOSE: The aim of this animal study is to evaluate, by histomorphometric analysis, new bone formation in rabbit maxillary sinuses with Bio-Oss and demineralized particulate human tooth graft. MATERIALS AND METHODS: Bilateral sinus augmentation procedures were performed in 8 adult male rabbits. After preparation of replaceable bony windows on the lateral wall of the nasal cavity with a piezoelectric surgical device, deproteinized bovine graft (Bio-Oss) was grafted in the new compartment of the maxillary sinus after elevation of the sinus membrane in the control group. In the experimental group, the demineralized human particulate tooth bone was grafted in the sinus. The replaceable bony window was repositioned over the bone graft in both groups. Animals were killed at 2 and 8 weeks after the surgical procedure. The augmented sinuses were evaluated by histomorphometric analysis using hematoxylin-eosin and Masson trichrome stains. RESULTS: Histologically, new bone was revealed along the elevated sinus membrane and both bone grafts. In the control group, the new bone area at 8 weeks was not significantly different than that at 2 weeks. In the experimental group, the new bone area at 8 weeks was significantly greater than that at 2 weeks. CONCLUSION: Significant higher new bone formation was revealed in the experimental group than in the control group.

A small-molecule inhibitor targeting the AURKC-IκBα interaction decreases transformed growth of MDA-MB-231 breast cancer cells.

The Aurora kinases, Aurora A (AURKA), Aurora B (AURKB), and Aurora C (AURKC), are serine/threonine kinases required for the control of mitosis (AURKA and AURKB) or meiosis (AURKC). Several Aurora kinase inhibitors are being investigated as novel anticancer therapeutics. Recent studies demonstrated that AURKC activation contributes to breast cancer cell transformation. Therefore, AURKC is both a promising marker and therapeutic target for breast cancer; however, its signaling network has not been fully characterized. Using translocation-based cellular assays, we identified IκBα as a binding partner of AURKC, and found that AURKC phosphorylates IκBα at Ser32, thereby activating it. In silico modeling and computational analyses revealed a small-molecule inhibitor (AKCI) that blocked the AURKC-IκBα interaction and exerted antitumor activity in MDA-MB-231 breast cancer cells. Specifically, AKCI induced G2/M cell-cycle arrest through modulation of the p53/p21/CDC2/cyclin B1 pathways. In addition, the drug significantly inhibited MDA-MB-231 cell migration and invasion, as well as decreasing colony formation and tumor growth. Via its interaction with IκBα, AURKC indirectly induced NF-κB activation; accordingly, AKCI decreased PMA-induced activation of NF-κB. Thus, the small-molecule inhibitor AKCI represents a first step towards developing targeted inhibitors of AURKC protein binding, which may lead to further advances in the treatment of breast cancer.

Leptin induces CREB-dependent aromatase activation through COX-2 expression in breast cancer cells.

Leptin plays a key role in the control of adipocyte formation, as well as in the associated regulation of energy intake and expenditure. The goal of this study was to determine if leptin-induced aromatase enhances estrogen production and induces tumor cell growth stimulation. To this end, breast cancer cells were incubated with leptin in the absence or presence of inhibitor pretreatment, and changes in aromatase and cyclooxygenase-2 (COX-2) expression were evaluated at the mRNA and protein levels. Transient transfection assays were performed to examine the aromatase and COX-2 gene promoter activities and immunoblot analysis was used to examine protein expression. Leptin induced aromatase expression, estradiol production, and promoter activity in breast cancer cells. Protein levels of phospho-STAT3, PKA, Akt, ERK, and JNK were increased by leptin. Leptin also significantly increased cAMP levels, cAMP response element (CRE) activation, and CREB phosphorylation. In addition, leptin induced COX-2 expression, promoter activity, and increased the production of prostaglandin E2. Finally, a COX-2 inhibitor and aromatase inhibitor suppressed leptin-induced cell proliferation in MCF-7 breast cancer cells. Together, our data show that leptin increased aromatase expression in breast cancer cells, which was correlated with COX-2 upregulation, mediated through CRE activation and cooperation among multiple signaling pathways.

Saponins from the roots of Platycodon grandiflorum ameliorate high fat diet-induced non-alcoholic steatohepatitis.

Platycodon grandiflorum has been healthy effects due to its various nutritious compounds and is considered as a functional food. Platycodon grandiflorum root-derived saponins (CKS) have been reported to show a variety of effects including anti-inflammatory and anti-oxidative activity. Although CKS have been studied on various bioactivities, the inhibitory effect of CKS on non-alcoholic steatohepatitis (NASH) is not examined. In this study, the inhibitory effects on HFD-induced NASH by CKS were determined. CKS suppressed HFD-induced hepatic lipid peroxidation level, collagen deposition, pro-fibrogenic and pro-inflammatory cytokines expression. CKS treatment suppressed HFD-induced COX-2 expression via inhibition of NF-κB p65 nuclear translocation and IκBα degradation. CKS treatment restored HFD-reduced Nrf2-mediated antioxidant enzymes expression. Furthermore, CKS treatment reinstated HFD-reduced peroxisomal proliferator-activated receptor alpha (PPARα)-regulated acyl-coA oxidase and carnitine-palmitoyl-coA transferase-1 expression. These findings suggest that CKS reduces HFD-induced NASH by up-regulation of Nrf2-mediated anti-oxidant enzymes and PPARα-regulated fatty acid oxidation.

Capsaicin Inhibits Dimethylnitrosamine-Induced Hepatic Fibrosis by Inhibiting the TGF-ß1/Smad Pathway via Peroxisome Proliferator-Activated Receptor Gamma Activation.

Capsaicin (CPS) exerts many pharmacological effects, but any possible influence on liver fibrosis remains unclear. Therefore, we evaluated the inhibitory effects of CPS on dimethylnitrosamine (DMN) and TGF-ß1-induced liver fibrosis in rats and hepatic stellate cells (HSCs). CPS inhibited DMN-induced hepatotoxicity, NF-κB activation, and collagen accumulation. CPS also suppressed the DMN-induced increases in α-SMA, collagen type I, MMP-2, and TNF-α. In addition, CPS inhibited DMN-induced TGF-ß1 expression (from 2.3 ± 0.1 to 1.0 ± 0.1) and Smad2/3 phosphorylation (from 1.5 ± 0.1 to 1.1 ± 0.1 and from 1.6 ± 0.1 to 1.1 ± 0.1, respectively) by activating Smad7 expression (from 0.1 ± 0.0 to 0.9 ± 0.1) via PPAR-γ induction (from 0.2 ± 0.0 to 0.8 ± 0.0) (p < 0.05). Furthermore, in HSCs, CPS inhibited the TGF-ß1-induced increases in α-SMA and collagen type I expression, via PPAR-γ activation. These results indicate that CPS can ameliorate hepatic fibrosis by inhibiting the TGF-ß1/Smad pathway via PPAR-γ activation.

Protective effect of rutaecarpine against t-BHP-induced hepatotoxicity by upregulating antioxidant enzymes via the CaMKII-Akt and Nrf2/ARE pathways.

Rutaecarpine, an indolopyridoquinazolinone alkaloid isolated from the unripe fruit of Evodia rutaecarpa, has been shown to have cytoprotective potential, but the molecular mechanism underlying this activity remains unclear. Our study was designed to investigate the cytoprotective effect of rutaecarpine against tert-butyl hydroperoxide (t-BHP) and to elucidate its action mechanism of action of rutaecarpine in a cultured HepG2 cell line and in mouse liver. Rutaecarpine decreased t-BHP-induced reactive oxygen species (ROS) production, cytotoxicity, and apoptosis in HepG2 cells. Pretreatment with rutaecarpine prior to the injection of t-BHP significantly prevented the increase in serum levels of AST, ALT, and lipid peroxidation in mice liver. It increased the transcriptional activity of NF-E2-related factor 2 (Nrf2) as well as the products of the Nrf2 target genes hemeoxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), and glutamate cysteine ligase (GCL). Moreover, rutaecarpine also enhanced the phosphorylation of Akt and Ca2+/calmodulin-dependent protein kinase-II (CaMKII). The pharmaceutical inhibitors, such as KN-93 (CaMKII inhibitor) and LY294002 (Akt inhibitor) suppressed rutaecarpine-induced HO-1 expression and cytoprotection. Our findings identify the CaMKII-PI3K/Akt-Nrf2 cascade as an antioxidant pathway mediating rutaecarpine signaling and leading to HO-1 expression in hepatocytes.

Inhibitory effects of l-theanine on airway inflammation in ovalbumin-induced allergic asthma.

l-theanine, a water-soluble amino acid isolated from green tea (Camellia sinensis), has anti-inflammatory activity, antioxidative properties, and hepatoprotective effects. However, the anti-allergic effect of l-theanine and its underlying molecular mechanisms have not been elucidated. In this study, we investigated the protective effects of l-theanine on asthmatic responses, particularly airway inflammation and oxidative stress modulation in an ovalbumin (OVA)-induced murine model of asthma. Treatment with l-theanine dramatically attenuated the extensive trafficking of inflammatory cells into bronchoalveolar lavage fluid (BALF). Histological studies revealed that l-theanine significantly inhibited OVA-induced mucus production and inflammatory cell infiltration in the respiratory tract and blood vessels. l-theanine administration also significantly decreased the production of IgE, monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-4, IL-5, IL-13, tumor necrosis factor-alpha (TNF-α), and interferon-gamma in BALF. The lung weight decreased with l-theanine administration. l-theanine also markedly attenuated the OVA-induced generation of reactive oxygen species and the activation of nuclear factor kappa B (NF-κB) and matrix metalloprotease-9 in BALF. Moreover, l-theanine reduced the TNF-α-induced NF-κB activation in A549 cells. Together, these results suggest that l-theanine alleviates airway inflammation in asthma, which likely occurs via the oxidative stress-responsive NF-κB pathway, highlighting its potential as a useful therapeutic agent for asthma management.

Ethanol extract of Lithospermum erythrorhizon Sieb. et Zucc. promotes osteoblastogenesis through the regulation of Runx2 and Osterix.

Bone remodeling and homeostasis are largely the result of the coordinated action of osteoblasts and osteoclasts. Osteoblasts are responsible for bone formation. The differentiation of osteoblasts is regulated by the transcription factors, Runx2 and Osterix. Natural products of plant origin are still a major part of traditional medicinal systems in Korea. The root of Lithospermum erythrorhizon Sieb. et Zucc. (LR), the purple gromwell, is an herbal medicine used for inflammatory and infectious diseases. LR is an anti-inflammatory and exerts anticancer effects by inducing the apoptosis of cancer cells. However, the precise molecular signaling mechanisms of osteoblastogenesis as regards LR and osteoblast transcription are not yet known. In this study, we investigated the effects of ethanol (EtOH) extract of LR (LES) on the osteoblast differentiation of C2C12 myoblasts induced by bone morphogenetic protein 4 (BMP4) and the potential involvement of Runx2 and Osterix in these effects. We found that the LES exhibited an ability to induce osteoblast differentiation. LES increased the expression of the osteoblast marker, alkaline phosphatase (ALP), as well as its activity, as shown by ALP staining and ALP activity assay. LES also increased mineralization, as shown by Alizarin Red S staining. Treatment with LES increased the protein levels (as shown by immunoblotting), as well as the transcriptional activity of Runx2 and Osterix and enhanced osteogenic activity. These results suggest that LES modulates osteoblast differentiation at least in part through Runx2 and Osterix.

Betulinic Acid Increases eNOS Phosphorylation and NO Synthesis via the Calcium-Signaling Pathway.

Betulinic acid (BA) is a naturally occurring pentacyclic triterpene that attenuates vascular diseases and atherosclerosis, but the mechanism by which it stimulates endothelial nitric oxide synthase (eNOS) is unclear. eNOS is the key regulatory enzyme in the vascular endothelium. This study examined the intracellular pathways underlying the effects of BA on eNOS activity and endothelial nitric oxide (NO) production in endothelial cells. BA treatment induced both eNOS phosphorylation at Ser1177 and NO production. It also increased the level of intracellular Ca(2+) and phosphorylation of Ca(2+)/calmodulin-dependent kinase IIα (CaMKIIα) and Ca(2+)/calmodulin-dependent protein kinase kinase ß (CaMKKß). Inhibition of the L-type Ca(2+) channel (LTCC) and the ryanodine receptor (RyR) abolished BA-induced intracellular levels of Ca(2+) and eNOS phosphorylation. Treatment with W7 (a CaM antagonist), KN-93 (a selective inhibitor of CaMKII), and STO 609 (a selective inhibitor of CaMKK) suppressed eNOS phosphorylation and NO production. Moreover, AMP-activated protein kinase (AMPK) was induced by BA, and BA-induced eNOS phosphorylation was inhibited by compound C, an AMPK inhibitor. Taken together, these results indicate that BA activates eNOS phosphorylation and NO synthesis via the Ca(2+)/CaMKII and Ca(2+)/CaMKK/AMPK pathways. These findings provide further insight into the eNOS signaling pathways involved in the antiatherosclerosis effects of BA.

Caffeic acid phenethyl ester inhibits 3-MC-induced CYP1A1 expression through induction of hypoxia-inducible factor-1α.

Caffeic acid phenethyl ester (CAPE), a natural component of propolis, is reported to have anticarcinogenic properties, although its precise chemopreventive mechanism remains unclear. In this study, we examined the effects of CAPE on 3-methylcholanthrene (3-MC)-induced CYP1A1 expression and activities. CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. Moreover, CAPE inhibited 3-MC-induced CYP1A1 activity, mRNA expression, protein level, and promoter activity. CAPE treatment also decreased 3-MC-inducible xenobiotic-response element (XRE)-linked luciferase, aryl hydrocarbons receptor (AhR) transactivation and nuclear localization. CAPE induced hypoxia inducible factor-1α (HIF-1α) protein level and HIF-1α responsible element (HRE) transcriptional activity. CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 protein expression. Taken together, CAPE decreases 3-MC-mediated CYP1A1 expression, and this inhibitory response is associated with inhibition of AhR and HIF-1α induction.

Saponins, especially platyconic acid A, from Platycodon grandiflorum reduce airway inflammation in ovalbumin-induced mice and PMA-exposed A549 cells.

We investigated the inhibitory effects of Platycodon grandiflorum root-derived saponins (Changkil saponins: CKS) on ovalbumin-induced airway inflammation in mice. CKS suppressed leukocytes number, IgE, Th1/Th2 cytokines, and MCP-1 chemokine secretion in bronchoalveolar lavage fluid. Also, ovalbumin-increased MUC5AC, MMP-2/9, and TIMP-1/-2 mRNA expression, NF-κB activation, leukocytes recruitment, and mucus secretion were inhibited by CKS treatment. Moreover, the active component of CKS, platyconic acid A (PA), suppressed PMA-induced MUC5AC mRNA expression (from 2.1 ± 0.2 to 1.1 ± 0.1) by inhibiting NF-κB activation (from 2.3 ± 0.2 to 1.2 ± 0.1) via Akt (from 3.7 ± 0.3 to 2.1 ± 0.2) (p < 0.01) in A549 cells. Therefore, we demonstrate that CKS or PA suppressed the development of respiratory inflammation, hyperresponsiveness, and remodeling by reducing allergic responses, and they may be potential herbal drugs for allergen-induced respiratory disease prevention.

Endosulfan induces COX-2 expression via NADPH oxidase and the ROS, MAPK, and Akt pathways.

Endosulfan (1,4,5,6,7,7-hexachloro-8,9,10-trinorborn-5-en-2,3-ylenebismet-hylene) is correlated with endocrine disruption, reproductive, and immune dysfunctions. Recently, endosulfan was shown to have an effect on inflammatory pathways, but its influence on cyclooxygenase-2(COX-2) expression is unclear. This study investigated the effects of COX-2 and molecular mechanisms by endosulfan in murine macrophage RAW 264.7 cells. Endosulfan significantly induced COX-2 protein and mRNA levels, as well as COX-2 promoter-driven luciferase activity and the production of prostaglandin E2, a major COX-2 metabolite. Transfection experiments with several human COX-2 promoter constructs revealed that endosulfan activated NF-κB, C/EBP, AP-1, and CREB. Moreover, Akt and mitogen-activated protein kinases (MAPK) were significantly activated by endosulfan. Moreover, endosulfan increased production of the ROS and the ROS-producing NAPDH-oxidase (NOX) family oxidases, NOX2, and NOX3. Endosulfan-induced Akt/MAPK pathways and COX-2 expression were attenuated by DPI, a specific NOX inhibitor, and the ROS scavenger N-acetylcysteine. These results demonstrate that endosulfan induces COX-2 expression via NADPH oxidase, ROS, and Akt/MAPK pathways. These findings provide further insight into the signal transduction pathways involved in the inflammatory effects of endosulfan.

Endosulfan Induces CYP1A1 Expression Mediated through Aryl Hydrocarbon Receptor Signal Transduction by Protein Kinase C.

CYP1A1 is a phase I xenobiotic-metabolizing enzyme whose expression is mainly driven by AhR. Endosulfan is an organochlorine pesticide used agriculturally for a wide range of crops. In this study, we investigated the effect of endosulfan on CYP1A1 expression and regulation. Endosulfan significantly increased CYP1A1 enzyme activity as well as mRNA and protein levels. In addition, endosulfan markedly induced XRE transcriptional activity. CH-223191, an AhR antagonist, blocked the endosulfan-induced increase in CYP1A1 mRNA and protein expression. Moreover, endosulfan did not induce CYP1A1 gene expression in AhR-deficient mutant cells. Furthermore, endosulfan enhanced the phosphorylation of calcium calmodulin (CaM)-dependent protein kinase (CaMK) and protein kinase C (PKC). In conclusion, endosulfan-induced up-regulation of CYP1A1 is associated with AhR activation, which may be mediated by PKC-dependent pathways.