Inhibited Phorbol 12-myristate 13-acetate Research Articles

Piperlonguminine Downregulates Endothelial Protein C Receptor Shedding In Vitro and In Vivo

Endothelial cell protein C receptor (EPCR) plays an important role in coagulation and inflammation. EPCR can be shed from the cell surface, and this is mediated by tumor necrosis factor-α-converting enzyme (TACE). Piperlonguminine (PL), an important component of Piper longum fruits, is known to exhibit antihyperlipidemic, antiplatelet, and antimelanogenesis activities. However, little is known about the effects of PL on EPCR shedding. Here, we investigated this issue by monitoring the effects of PL on phorbol-12-myristate 13-acetate (PMA) and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. PL induced potent inhibition of PMA, and CLP induced EPCR shedding through suppression of TACE expression. And treatment with PL resulted in reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). Given these results, PL might have potential as an anti-sEPCR shedding reagent against PMA- and CLP-mediated EPCR shedding.

Inhibitory effects of purpurogallin on the endothelial protein C receptor shedding in vitro and in vivo

Endothelial cell protein C receptor (EPCR) plays important roles in the regulation of blood coagulation and inflammation. Activity of EPCR is markedly changed by ectodomain cleavage and released as soluble protein (sEPCR). EPCR can be shed from the cell surface, and this is mediated by tumor necrosis factor-α converting enzyme (TACE). Purpurogallin (PPG) plays an important role in inhibiting glutathione S-transferase and xanthine oxidase as well as effective in the cell protection of several cell types. Here, we investigated the effects of PPG on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. Human umbilical vein endothelial cells pretreated with PPG (0, 5, 10, 20 or 50 μg/mL) for 6 h and exposed to PMA (1 μM) for 1 h, and CLP-operated mice were administrated with PPG. Data showed that treatment with PPG resulted in potent inhibition of PMA, TNF-α, IL-1β, and CLP-induced EPCR shedding by suppression of TACE expression. In addition, PPG reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases 1/2, and c-Jun N-terminal kinase. These results suggest the potential for use of PPG as an anti-sEPCR shedding reagent against PMA and CLP-mediated EPCR shedding.

Inhibitory effects of epi-sesamin on endothelial protein C receptor shedding in vitro and in vivo

Endothelial protein C receptor (EPCR) plays a pivotal role in augmenting Protein C activation by the thrombin-thrombomodulin complex. The activity of EPCR is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). The EPCR shedding is mediated by the tumor necrosis factor-α converting enzyme (TACE). Epi-sesamin (ESM), from the roots of Asarum siebodlii, is known to exhibit anti-allergic and anti-fungal activities. However, little is known about the effects of ESM on EPCR shedding. We investigated this issue by monitoring the effects of ESM on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and cecal ligation and puncture (CLP)-mediated EPCR shedding. Data showed that ESM induced potent inhibition of PMA, TNF-α, IL-1β, and CLP-induced EPCR shedding, likely through suppression of TACE expression. In addition, treatment with ESM resulted in a reduction of PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). Given these results, ESM should be viewed as a candidate therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of EPCR shedding.

Rosmarinic acid down-regulates endothelial protein C receptor shedding in vitro and in vivo

The endothelial protein C receptor (EPCR) plays pivotal roles in coagulation and inflammation, however, its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). According to previous studies, there are approximately 100ng/ml sEPCR in human plasma and the levels increase in inflammatory diseases. EPCR can be shed from the cell surface, and this is mediated by tumor necrosis factor-α converting enzyme (TACE). We recently reported on the anti-inflammatory and barrier protective activities of rosmarinic acid (RA), an important component of the leaves of Perilla frutescens. However, little is known about the effects of RA on EPCR shedding. Here, we investigated this issue by monitoring the effects of RA on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β, and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. Data showed that treatment with RA resulted in potent inhibition of PMA, TNF-α, IL-induced EPCR shedding by suppression of TACE expression. In addition, RA reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). These results suggest the potential for use of RA as an anti-sEPCR shedding reagent against PMA, TNF-α, IL-1β and CLP-mediated EPCR shedding.

Down-regulation of endothelial protein C receptor shedding by persicarin and isorhamnetin-3-O-galactoside

Increasing evidence has shown that beyond its role in coagulation, endothelial protein C receptor (EPCR) plays an important role in the cytoprotective pathway. Previous reports have shown that EPCR can be shed from the cell surface, and that this is mediated by tumor necrosis factor-α converting enzyme (TACE) and that sEPCR levels are increased in patients with systemic inflammatory diseases. Persicarin and isorhamnetin-3-O-galactoside (I3G) are active compounds from Oenanthe javanica, which has been widely studied for its neuroprotective, antioxidant, and barrier protective activities. However, little is known of the effects of persicarin on EPCR shedding. Here, we investigated this issue by monitoring the effects of persicarin and I3G on phorbol-12-myristate 13-acetate (PMA) and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. According to the results, persicarin and I3G induced potent inhibition of PMA and CLP-induced EPCR shedding by suppressing expression of TACE. In addition, persicarin and I3G reduced PMA-stimulated phosphorylation of p38MAPK, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). Given these results, persicarin and I3G could be used as a candidate therapeutic for treatment of severe vascular inflammatory diseases.

PKCε‐mediated P‐Rex1 downregulation suppresses breast cancer cell proliferation

P‐Rex1, a Rac1 selective activator, is upregulated and promotes tumorgenesis and metastasis of various cancers. We recently found that phorbol‐12‐myristate‐13‐acetate (PMA) downregulates P‐Rex1 in breast cancer cells. The present study was to investigate the importance and mechanism underlying PMA downregulation of P‐Rex1. P‐Rex1 mRNA and protein expression were determined by real‐time RT‐PCR and Western blot analysis, respectively. Cell proliferation was analyzed by counting cell numbers. We found that P‐Rex1 expression is elevated in breast cancer MCF‐7, T47D and BT‐474 cell lines compared to normal breast epithelial cells. PMA treatment decreased P‐Rex1 expression and proliferation of breast cancer cells by over 70%. Restoring P‐Rex1 expression attenuated the PMA inhibition of cell proliferation. Go6983, a protein kinase C (PKC) inhibitor, blocked the PMA inhibition of P‐Rex1 expression and breast cancer cell proliferation. Breast cancer cells express PMA‐sensitive PKCδ, PKCε and PKCη. Overexpression of constitutively active PKCε, but not PKCδ or PKCη, significantly suppressed P‐Rex1 expression and breast cancer cell proliferation. In conclusion, PMA downregulates P‐Rex1 to suppress breast cancer cell proliferation via a PKCε signaling pathway, which could be a new therapeutic target for development of a novel breast cancer treatment. Supported by NIH CA125661 and Nebraska State LB595.

EGCG inhibits recepteur d’origine nantais expression by suppressing Egr-1 in gastric cancer cells

Abnormal accumulation and activation of the recepteur d'origine nantais (RON) has been implicated in epithelial tumor carcinogenesis. In the present study, we examined the effect of epigallocatechin-3-gallate (EGCG), the major green tea catechin, on the induction of RON and tumor growth in human gastric cancer. EGCG inhibited phorbol 12-myristate 13-acetate (PMA)-induced RON expression and reduced RON transcriptional activity. However, (-)-epigalloca-techin (EGC), (-)-epicatechin gallate (ECG) and (-)‑epicatechin (EC) did not affect RON expression. Experiments with deleted and site-directed mutagenesis of the RON promoter indicated that Egr-1 binding sites in the RON promoter may be the EGCG‑response element acting as a cis-element in gastric cancer cells. EGCG also inhibited PMA-induced Egr-1 expression and DNA binding in a dose-dependent manner. Furthermore, gastric cancer cells pretreated with PMA showed markedly enhanced invasiveness, which was partially abrogated by EGCG and siRNA-targeted RON and Egr-1. EGCG significantly reduced tumor growth in an in vivo tumor model, whereas RON expression was downregulated. These results suggest that EGCG may exert at least part of its anticancer effect by controlling RON expression through suppression of Egr-1 activation.

Opposition between PKC isoforms regulates histone deimination and neutrophil extracellular chromatin release

In response to inflammation, neutrophils deiminate histones and externalize chromatin. Neutrophil extracellular traps (NETs) are an innate immune defense mechanism, yet NETs also may aggravate chronic inflammatory and autoimmune disorders. Activation of peptidylarginine deiminase 4 (PAD4) is associated with NET release (NETosis) but the precise mechanisms of PAD4 regulation are unknown. We observed that, in human neutrophils, calcium ionophore induced histone deimination, whereas phorbol myristate acetate (PMA), an activator of protein kinase C (PKC), suppressed ionophore-induced deimination. Conversely, low doses of chelerythrine and sanguinarine, two inhibitors of PKC, reversed PMA inhibition and enhanced ionophore-stimulated deimination. In addition, a peptide inhibitor of PKCα superinduced ionophore activation of PAD4, thus identifying PKCα as the PMA-induced inhibitor of PAD4. At higher doses, chelerythrine, sanguinarine, and structurally unrelated PKC inhibitors blocked histone deimination, suggesting that a different PKC isoform activates histone deimination. We identify PKCζ as activator of PAD4 because a specific peptide inhibitor of this PKC isoform suppressed histone deimination. Confocal microscopy confirmed that, in the presence of PMA, NETosis proceeds without detectable histone deimination, and that ionophore cooperates with PMA to induce more extensive NET release. Broad inhibition of PKC by chelerythrine or specific inhibition of PKCζ suppressed NETosis. Our observations thus reveal an intricate antagonism between PKC isoforms in the regulation of histone deimination, identify a dominant role for PKCα in the repression of histone deimination, and assign essential functions to PKCζ in the activation of PAD4 and the execution of NETosis. The precise balance between opposing PKC isoforms in the regulation of NETosis affirms the idea that NET release underlies specific and vitally important evolutionary selection pressures.

Anti-allergic activity of crystallinity controlled N-acetyl glucosamine

Context: Chitin is the polysaccharide and is found in insects, parasites and fungi. Chitin has shown various immunological effects in in vivo and in vitro models. In this study, crystallinity controlled N-acetyl glucosamine (CCG) which has a high solubility was prepared from the low molecular weight chitosan. However, the effect of CCG in an allergic response is not clear.Objective: To investigate the effect and regulatory mechanism of CCG on allergic responses.Methods: To demonstrate the effect of CCG, we induced systemic anaphylactic shock, ear swelling response, passive cutaneous anaphylaxis (PCA), and inflammatory reaction.Results: CCG inhibited the compound 48/80-induced systemic anaphylactic shock and ear swelling responses. IgE-mediated PCA was inhibited by the oral administration or topical application of CCG. Histamine and β-hexosaminidase release from mast cells was decreased with the treatment of CCG. CCG also inhibited phorbol 12-myristate 13-acetate and calcium ionophore A23187-induced interleukin-1β production and mRNA expression by suppressing NF-κB activation and IκBα phosphorylation. Furthermore, CCG suppressed the activation of caspase-1.Conclusion: These results suggest that CCG may have beneficial applicability as a candidate for an anti-allergic agent.

Anti-Allergic Asthma Properties of Brazilin through Inhibition of TH2 Responses in T Cells and in a Murine Model of Asthma (175.8)

Abstract The aim of the study was to determine whether brazilin exhibits any anti-inflammatory effects that inhibit T helper cell type II (TH2) responses and whether it suppresses allergic inflammation reactions in a murine model of asthma. We found that brazilin inhibited phorbol myristate acetate (PMA) + cyclic adenosine monophosphate (cAMP)-induced IL-4 and IL-5 expression in EL-4 T cells with respect to both RNA and protein levels in a dose-dependent manner. After intratracheal instillation of brazilin in ovalbumin (OVA)-immunized mice, we found that brazilin-treated mice exhibited decreases in the release of IL-4, IL-5, IL-13, eotaxin, and TNF-α in bronchoalveolar lavage fluid (BALF), inhibited TH2 functioning by a decrease in IL-4 production, and the attenuation of the OVA-induced lung eosinophilia, and airway hyperresponsiveness and remodeling. These results suggest that brazilin exhibits an anti-TH2 reaction both in vitro and in vivo. Brazilin as demonstrates therapeutic potential for allergic diseases.

Scopoletin attenuates allergy by inhibiting Th2 cytokines production in EL-4 T cells

Scopoletin is an antioxidant found in certain weedy plants. It exerts anti-inflammatory, anti-allergic, and anti-diabetic activities. It remains unknown whether scopoletin regulates T helper (Th) cells, including Th 1 and Th 2 cells. We found that scopoletin significantly inhibited phorbol myristate acetate (PMA)/ionomycin-induced interleukin-4 (IL-4), IL-5, and IL-10 production in EL-4 T cells. In addition, scopoletin significantly enhanced the inhibitory action of PMA/ionomycin on interferon-γ (IFN-γ) expression. In EL-4 T cells, PMA/ionomycin treatment markedly increased the expression of nuclear factor of activated T cells (NFAT) and GATA-3; in contrast, scopoletin significantly down-regulated expressions of these transcription factors. Furthermore, this downregulation depended on protein kinase C (PKC) attenuation. This leads us to suggest that the anti-allergic properties of scopoletin might be mediated by the downregulation of cytokine expression in Th 2 cells.

Inactivation of Cystein-Aspartic Acid Protease (Caspase)-1 by Saikosaponin A

This work investigates the anti-inflammatory mechanism of saikosaponin A (SA), a major component of Bupleurum falcatum LINNE. SA significantly inhibited phorbol myristate acetate (PMA) plus A23187-induced the production and expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in human mast cell (HMC)-1 cells. SA suppressed PMA plus A23187-induced phosphorylation of extracellular signal-regulated kinase and p38. When HMC-1 cells were treated with SA, translocation of nuclear factor (NF)-κB/Rel A into nucleus and degradation of inhibitor of NF-κB (IκB) in cytoplasm were inhibited. SA decreased PMA plus A23187-induced cysteine-aspartic acid protease (caspase)-1 activity. IL-1β production was also inhibited by SA. Finally, SA significantly decreased the number of nasal rubs and serum TNF-α level in the ovalbumin-sensitized allergic rhinitis mouse model. The underlying mechanism involves, at least in part, inactivation of caspase-1, which provides new evidence for therapeutic application of SA to target inflammatory processes.

Suppression of COX-2, IL-1β and TNF-α expression and leukocyte infiltration in inflamed skin by bioactive compounds from Rosmarinus officinalis L.

In the present study, we evaluated the effects of extracts and purified compounds from fresh leaves of Rosmarinus officinalis L. Pretreatment with the major anti-inflammatory compounds, carnosic acid (CA) and carnosol (CS), inhibited phorbol 12-myristate 13-acetate (PMA)-induced ear inflammation in mice with an EC 50 of 10.20 μg/cm 2 and 10.70 μg/cm 2, respectively. To further understand the anti-inflammatory mechanism of these compounds, we analyzed the in vivo expression of several inflammation-associated genes in mouse skin by reverse transcriptase-polymerase chain reaction (RT-PCR). Our data showed that CA and CS reduced the expression of IL-1β and TNF-α but had less effect on fibronectin and ICAM-1 expression. Interestingly, both compounds selectively inhibited COX-2 but not COX-1. Histopathological analysis of hematoxylin and eosin (H&E)-stained tissue revealed a marked reduction in leukocyte infiltration and epidermal ulceration of PMA-treated ears when ears were pretreated with ethanolic extracts or pure CA. In vitro, we showed that ethanolic extract, carnosic acid and carnosol significantly inhibited the overproduction of nitric oxide (NO) in a dose-dependent manner in the RAW 264.7 murine macrophage cell line. For the first time in vivo, we showed that CA and CS differentially regulate the expression of inflammation-associated genes, thus demonstrating the pharmacological basis for the anti-inflammatory properties reported for CA and CS.

CADPE Inhibits PMA-Stimulated Gastric Carcinoma Cell Invasion and Matrix Metalloproteinase-9 Expression by FAK/MEK/ERK–Mediated AP-1 Activation

Metastasis is one of the main causes of death for patients with malignant tumors. Aberrant expression of matrix metalloproteinase-9 (MMP-9) has been implicated in the invasion and metastasis of various cancer cells. Here, we found that caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE) could inhibit the migration and invasion of human gastric carcinoma cells in Transwell migration assays. To understand the underlying mechanism, we showed that CADPE significantly inhibited phorbol 12-myristate 13-acetate (PMA)-induced increases in MMP-9 expression and activity in a dose-dependent manner. The inhibitory effect of CADPE on MMP-9 expression correlated well with the suppression of MMP-9 promoter activity and the reduction of MMP-9 mRNA. Reporter gene assay and electrophoretic mobility shift assay showed that CADPE inhibited MMP-9 expression by suppressing the activation of the nuclear transcription factor activator protein-1 (AP-1) and c-Fos, but not NF-κB. Moreover, CADPE inhibited PMA-induced phosphorylation of protein kinases involved in AP-1 activation, such as focal adhesion kinase (FAK), mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK), and ERK1/2, whereas CADPE had little effect on the phosphorylation of p38 and c-jun NH(2)-terminal kinase. Taken together, our findings indicate that CADPE could be a unique antitumor agent that specifically inhibits MMP-9 activity by targeting the activation of FAK/MEK/ERK protein kinases and AP-1 transcription factor.

The dietary flavonoid apigenin blocks phorbol 12-myristate 13-acetate-induced COX-2 transcriptional activity in breast cell lines

Cyclooxygenase (COX)-2 is the inducible isozyme catalyzing the conversion of arachidonic acid to prostanoids. Its expression has been linked to the process of carcinogenesis, including tumorigenesis of the breast. Apigenin (APG) is a flavone commonly found in fruit and vegetables, and is shown to be a potential modulator in inflammatory diseases. This study examined the potential suppressive effect of the flavone on phorbol ester-induced COX-2 expression in the breast cell lines MCF-10A and MCF-7. Real-time PCR and/or Western blotting indicated that APG in micromolar range significantly inhibited phorbol 12-myristate 13-acetate (PMA)-induced COX-2 expression in these breast cells. APG treatment reduced the amount of phospho-mitogen-activated protein kinase (MAPK) ERK-1/2, but it did not alter the activity of PKC. Activated ERKs might trigger the transactivation of AP-1 or CRE, which can be located at COX-2 promoter region (−72/−53). Reporter gene assay as well as electrophoretic mobility shift assays (EMSA) illustrated that APG inhibited transcription factor binding at this region in a dose-dependent manner. This study showed that APG down-regulated PMA-induced COX-2 expression in breast cells without affecting PKC activity. These findings could provide a scientific basis for developing APG nutraceutical against breast carcinogenesis.

The nitric oxide-donating pravastatin, NCX 6550, inhibits cytokine release and NF-κB activation while enhancing PPARγ expression in human monocyte/macrophages

Previous studies have shown that NCX 6550 (NCX), a nitric oxide (NO)-donating pravastatin, induces anti-inflammatory effects in murine macrophage cell lines. Here, we have studied its activity in human monocyte/macrophages, by investigating cytokine release, NF-κB translocation and peroxisome proliferator-activated receptor γ (PPARγ) expression and function. For comparison, pravastatin, isosorbide-5-mononitrate (ISMN), sodium nitroprusside (SNP) and the PPARγ ligand 15-deoxy-Δ 12,14-prostaglandin J 2 (PGJ) were also tested. Monocytes and macrophages (MDM: monocyte-derived macrophages) were isolated from healthy donors; cytokine release was measured by ELISA, NF-κB by electrophoretic mobility shift assay and PPARγ by Western blot and Real-Time PCR. NCX (1 nM–50 μM) dose-dependently inhibited phorbol 12-myristate 13-acetate (PMA)-induced TNF-α release from monocytes (IC 50 = 240 nM) and MDM (IC 50 = 52 nM). At 50 μM, it was more effective than pravastatin, ISMN and SNP ( P < 0.05), but less efficient than PGJ. Similar results were obtained for IL-6. Likewise, NCX was more effective than pravastatin and the other NO donors in inhibiting PMA-induced NF-κB translocation in both cell types, and, at the highest concentration, significantly ( P < 0.05) enhanced PPARγ protein expression in monocytes. We conclude that NCX 6550 exerts a significant anti-inflammatory activity in human monocyte/macrophages, that is also contributed by its NO donating properties, as the effects exerted by NCX are significantly higher than those evoked by pravastatin in many experimental assays. These data further indicate that the incorporation of a NO-donating moiety into a statin structure confers pharmacological properties which may translate into useful therapeutic benefits.

Selectivity of PKC isoforms in the regulation of Na+‐K+‐2Cl− cotransporter (NKCC1) in secretory epithelia

NKCC1 drives basolateral Cl− entry in secretory epithelia. Activation of PKC by phorbol 12‐myristate 13‐acetate (PMA) reduces colonic epithelial Cl− secretory response to cAMP and surface NKCC1. The study was to define selectivity of PKCs in regulating epithelial NKCC1.T84 monolayers uninfected or infected with adenoviruses expressing human PKC (α, δ, ε)‐specific shRNAs or non‐target LacZ‐shRNA were treated with PMA or vehicle, followed by measurement of Cl− secretion (short‐circuit current, Isc). NKCC1 signal in the cells was assessed by immunofluorescence. NKCC1 surface expression was examined by basolateral surface biotinylation assay.We found proteins of PKC (α, δ, ε) in shPKCs cells were decreased by ~85% after 72h infection. PMA reduced forskolin (FSK)‐stimulated Cl− secretion much less in shPKCδ or shPKCε cells than shLacZ or uninfected cells. There was no significant difference in PMA‐reduced Cl− secretion between shPKCα and shLacZ. In shLacZ and shPKCα cells, PMA induced a redistribution of NKCC1 signal to intracellular vesicles; the effect was not observed in shPKCδ or shPKCε cells. PMA caused a dramatic decrease in basolateral biotinylated NKCC1 in shLacZ or shPKCα, but not in shPKCδ or shPKCε cells.In conclusion, depletion of PKC δ or ε, but not PKCα, prevents PMA inhibition of T84 Cl− secretion via a mechanism involving stabilization of the basolateral membrane NKCC1.Received NIH grant DK48010.

The Licorice Flavonoid Isoliquiritigenin Suppresses Phorbol Ester-induced Cyclooxygenase-2 Expression in the Non-tumorigenic MCF-10A Breast Cell Line

Cyclooxygenase (COX) is the rate-limiting enzyme for the conversion of prostaglandins from arachidonic acid. Upregulation of COX-2 has been well documented during tumorigenesis and metastasis of breast cancer. Isoliquiritigenin (ILN), a flavonoid isolated from licorice (the rhizomes of GLYCYRRHIZA GLABRA, a member of the bean plant family), is known to be a potential suppressor of COX-2 expression. This study focuses on phorbol ester-induced COX-2 expression in the non-tumorigenic MCF-10A cells. Real-time PCR and Western blotting indicated that ILN at 5 microM or above significantly inhibited phorbol 12-myristate 13-acetate (PMA)-induced COX-2 expression in the breast cells. The activated PKC alpha appeared to be not affected, whereas its downstream mitogen-activated protein kinase (MAPK) ERK-1/2 was deactivated. ERK can activate the transcriptional factor binding of AP-1 or CRE, which can be located at the COX-2 promoter region (- 72/- 53). Electrophoretic mobility shift assays illustrated that ILN suppressed DNA binding at this region. The shifted bands could be competed off with consensus sequences of AP-1 and CRE, and the supershift assay demonstrated that CREB-1 instead of c-Jun was responsible for the binding. This study showed that ILN downregulated PMA-induced COX-2 expression by modulating ERK-1/2 signaling, a finding that may be relevant to the disease prevention properties of licorice.

Free radical scavenging activity and immunomodulatory effect of Stachytarpheta angustifolia leaf extract

Plant extracts with antioxidant activity could also have immunomodulatory ability. The free radical scavenging activity of an ethanol extract of the leaves of Stachytarpheta angustifolia was assessed by measuring its capability for scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide anion radical ( O 2 - ), hydroxyl radical ( OH), nitric oxide radicals (NO ), as well as its ability to inhibit lipid peroxidation, using appropriate assay systems. The extract was also assessed for its ability to decrease the phenotypic expression of the immune activation markers CD38 and CD69. This extract showed a potent antioxidant activity in the DPPH radical-scavenging assay (EC 50 = 9.65 μg/ml), significantly inhibited OH radical (IC50 = 99.43 μg/ml), O 2 - anion radical (IC50 = 64.68 μg/ml), non-enzymatic lipid peroxidation (IC50 = 282.91 μg/ml) and accumulation of nitrite in vitro. Ex vivo the extract inhibited phorbol myristate acetate (PMA)-induced production of superoxide anion ( O 2 - ), and also exhibited a dose-dependent reduction in the levels of the immune activation marker CD38 and CD69 on phytohemagglutinin A (PHA)-stimulated human peripheral blood mononuclear cells (PBMC). The observed antioxidant activity and immunomodulatory potentials of the extract suggest that it could impart health benefits when consumed. However, further investigation to verify its effect in vivo is warranted.

Losartan Metabolite EXP3179 Blocks NADPH Oxidase-Mediated Superoxide Production by Inhibiting Protein Kinase C

Oxidative stress plays a critical role in the pathogenesis of hypertension. The NADPH oxidase constitutes a major source of superoxide anion in phagocytic cells, and its activation is associated with matrix metalloproteinase (MMP)-9 secretion by these cells. We investigated the effects of the angiotensin II type 1 receptor antagonist losartan and its metabolites (EXP3174 and EXP3179) on NADPH oxidase activity and MMP-9 secretion in human phagocytic cells. EXP3179, but not losartan and EXP3174, dose-dependently inhibited (P<0.05) phorbol myristate acetate and insulin-stimulated NADPH oxidase activity. EXP3179 also inhibited phorbol myristate acetate-induced NADPH oxidase in endothelial cells. In addition, EXP3179 inhibited (P<0.05) both phorbol myristate acetate-stimulated p47phox translocation from cytosol to membranes and protein kinase C activity. Affinity experiments and enzymatic assays confirmed that EXP3179 inhibited several protein kinase C isoforms. EXP3179 also inhibited (P<0.05) phorbol myristate acetate-stimulated MMP-9 secretion. In a study performed in 153 hypertensive patients, phagocytic NADPH oxidase activity was lower (P<0.05) in losartan-treated compared with untreated patients and in patients treated with other angiotensin II type 1 receptor antagonists or with angiotensin-converting enzyme inhibitors. Plasma levels of MMP-9 were lower (P<0.05) in losartan-treated hypertensives compared with the other group of patients. Thus, EXP3179 acts as a blocker of the NADPH oxidase in phagocytic cells by a potential mechanism that targets the protein kinase C signaling pathway. This effect can be involved in reduced MMP-9 secretion by these cells. It is proposed that the EXP3179 metabolite may confer to losartan the specific capacity to reduce oxidative stress mediated by phagocytic cells in hypertensive patients.