Biomedical subjects

Sheng-Chu Kuo

Publications and source records attributed to Sheng-Chu Kuo.

At least 37 records · Page 2Linked to original sources

PubMed2005

YC-1 suppresses constitutive nuclear factor-kappaB activation and induces apoptosis in human prostate cancer cells.

Although the indazole compound, YC-1, is reported to exert anticancer activities in several cancer cell types, its target and mechanism of action have not been well explored. The objectives of this study were to ascertain whether YC-1 directly induces apoptosis in prostate cancer cells and to explore the mechanism(s) whereby YC-1 causes cell death. Hormone-refractory metastatic human prostate cancer PC-3 cells were selected for this study. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay indicated that YC-1 suppresses growth of PC-3 cells in a concentration-dependent and time-dependent manner. Apoptosis was determined using 4',6-diamidino-2-phenylindole staining, and cell cycle progression was examined by FACScan flow cytometry. YC-1 treatment showed chromatin condensation and increased the percentage of PC-3 cells in the hypodiploid sub-G0-G1 phase, indicative of apoptosis. Additionally, exposure to YC-1 was found to induce activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase. Translocation and activation of nuclear factor-kappaB (NF-kappaB) were determined by immunofluorescent staining and ELISA, respectively. The results showed that YC-1 abolished constitutive nuclear translocation and activation of NF-kappaB/p65. Furthermore, inhibition of inhibitor of kappaBalpha (IkappaBalpha) phosphorylation and accumulation of IkappaBalpha were observed. The antitumor effects of YC-1 were evaluated by measuring the growth of tumor xenografts in YC-1-treated severe combined immunodeficient mice. The volumes of PC-3 tumors produced in severe combined immunodeficient mice were observed to decline significantly after treatment with YC-1 compared with vehicle controls. We concluded that the antitumor effects of YC-1 in PC-3 cells include the induction of apoptosis and the suppression of NF-kappaB activation. Given these unique actions, further investigations of the effects of YC-1 against hormone-refractory prostate cancer are warranted.

Adenocarcinoma↗

PubMed2005

A potential role of YC-1 on the inhibition of cytokine release in peripheral blood mononuclear leukocytes and endotoxemic mouse models.

To evaluate the anti-sepsis potential of YC-1, we have examined the effect of YC-1 on the regulation of cytokine production in human leukocytes and endotoxemic mice. The data demonstrated that YC-1 showed a preferential inhibition on proinflammatory cytokine production without inhibition of cell growth or induction of cytotoxicity in human leukocytes. On the other hand, in the septic mouse model, treatment with an intraperitoneal application of LPS caused a cumulative death within 27 hours. The post-treatment administration of YC-1 significantly increased the survival rate in endotoxemic mice. Furthermore, several mediators were detected and the data showed that YC-1 profoundly blocked LPS-induced NO as well as TNF-alpha production, and prevented lung damage by histological examination. Samples from the animal model showed that LPS-induced NF-kappaB/DNA binding activity and consequent up-regulation of iNOS expression in tissues were abolished by post-administration of YC-1. Furthermore, YC-1, by itself, did not modify cGMP content while significantly inhibit LPS-induced cGMP formation, suggesting that YC-1-mediated effect was not through a cGMP-elevating pathway. Taken together, it is evident that the post-treatment administration of YC-1 after LPS application significantly inhibits NF-kappaB activation, iNOS expression, NO over-production, and cytokine release reaction resulting in an improved survival rate in endotoxemic mice. It is suggested that YC-1 may be a potential agent for the therapeutic treatment of sepsis.

Animals↗

PubMed2004

The blockade of cyclopiazonic acid-induced store-operated Ca2+ entry pathway by YC-1 in neutrophils.

In the presence of external Ca2+, pretreatment of neutrophils with 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) inhibited the cyclopiazonic acid (CPA)-induced [Ca2+](i) elevation in a concentration- but not a time-dependent manner, while YC-1 had no effect on the Ca2+ signals in a Ca2+-free medium. YC-1 failed to inhibit ATP- and interleukin-8 (IL-8)-induced [Ca2+](i) changes. Addition of YC-1 after cell activation strongly inhibited the CPA-induced [Ca2+](i) changes. In a classical Ca2+ readdition protocol, a similar extent inhibition of Ca2+ spike by YC-1 introduced either prior to or after CPA stimulation was obtained. In rat neutrophils, mRNA for endothelial differentiation gene (edg)1, edg5, edg6 and edg8, the putative targets for sphingosine 1-phosphate (S1P), could be detected. However, S1P was found to have little effect on Ca(2+) signals. YC-1 did not inhibit but enhanced the sphingosine-induced [Ca2+](i) changes. Inhibition by YC-1 of CPA-induced [Ca2+](i) changes was not prevented by 7-nitroindazole and N-(3-aminomethyl)benzylacetamidine (1400W), two nitric oxide synthase (NOS) inhibitors, by aristolochic acid, a phospholipase A(2) inhibitor, or by suspension in a Na(+)-deprived medium. YC-1 did not affect the mitochondrial membrane potential. Moreover, YC-1 did not alter [Ca2+](i) changes in response to ionomycin after CPA and formyl-Met-Leu-Phe (fMLP) stimulation in a Ca2+-free medium. YC-1 had no effect on the basal [Ca2+](i) level, the pharmacologically isolated plasma membrane Ca2+-ATPase activity, and Ba2+ entry into CPA-activated cells. YC-1 alone resulted in the accumulation of actin filaments in neutrophils, while significantly reduced the intensity of actin filament staining in the subsequent activation with CPA. These results indicate that YC-1 inhibited CPA-activated store-operated Ca2+ entry (SOCE) probably through the direct blockade of channel activation and/or the disruption of the integrity of the actin cytoskeleton necessary for supporting Ca2+ entry pathway in neutrophils.

Actins↗

PubMed2004

YC-1 [3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl Indazole] exhibits a novel antiproliferative effect and arrests the cell cycle in G0-G1 in human hepatocellular carcinoma cells.

This study delineates the antiproliferative activities and in vivo efficacy of YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole] in human hepatocellular carcinoma cells. YC-1 inhibited the growth of HA22T and Hep3B cells in a concentration-dependent manner without significant cytotoxicity. YC-1 induced G(1) phase arrest in the cell cycle, as detected by an increase in the proportion of cells in the G(1) phase using FAC-Scan flow cytometric analysis. It was further shown that cGMP, p42/p44 mitogen-activated protein kinase, or AKT kinase-mediated signaling pathways did not contribute to the YC-1-induced effect. Of note, YC-1 induced a dramatic increase in the expression of cyclin-dependent kinase (CDK)-inhibitory protein, p21(CIP1/WAP1), and a modest increase in p27(KIP1). The association of p21(CIP1/WAP1) with CDK2 was markedly increased in cells responsive to YC-1. YC-1 did not modify the expression of cyclin D1, cyclin E, CDK2, or CDK4. In a corollary in vivo study, YC-1 induced dose-dependent inhibition of tumor growth in mice inoculated with HA22T cells. Immunohistochemical analysis revealed an inverse relationship between the staining of p21(CIP1/WAF) and the staining of Ki-67, a cell proliferation marker. Based on the results reported herein, we suggest that YC-1 induces cell cycle arrest and inhibits tumor growth both in vitro and in vivo via the up-regulation of p21(CIP1/WAP1) expression in HA22T cells. Because of this, YC-1 is a potential antitumor agent worthy of further investigation.

Antineoplastic Agents↗

PubMed2004

2-Benzyloxybenzaldehyde inhibits formyl peptide-stimulated increase in intracellular Ca2+ in neutrophils mainly by blocking Ca2+ entry.

2-Benzyloxybenzaldehyde (CCY1a) inhibited the formyl-Met-Leu-Phe (fMLP)-induced elevation of cytosolic [Ca(2+)]() ([Ca(2+)](i)) in rat neutrophils. The late plateau phase, but not the initial Ca(2+) spike, of the fMLP-induced [Ca(2+)](i) change was inhibited by CCY1a. In the absence of external Ca(2+), CCY1a had no appreciable effect on either the fMLP- or cyclopiazonic acid (CPA)-induced [Ca(2+)](i) elevation. CCY1a failed to inhibit [Ca(2+)](i) changes induced by N-ethylmaleimide, GEA3162, ionomycin or sphingosine, but slightly inhibited the Ca(2+) signals elicited by ATP or interleukin-8 (IL-8). In a classical Ca(2+) readdition protocol, addition of CCY1a after cell activation strongly inhibited the [Ca(2+)](i) response to fMLP, whilst that to CPA was only slightly reduced. CCY1a nearly abrogated the fMLP-stimulated Mn(2+) influx but was less effective on the CPA-induced response. CCY1a attenuated the levels of tyrosine-phosphorylated bands in the 70-85 kDa molecular mass range. CCY1a had no effect on the basal [Ca(2+)](i) level, the pharmacologically isolated plasma membrane Ca(2+)-ATPase activity or on the mitochondrial membrane potential. Thus, CCY1a blocks fMLP-induced Ca(2+) entry into neutrophils probably by blocking the relevant Ca(2+) channel directly or, alternatively, indirectly through the attenuation of tyrosine phosphorylation of some cellular proteins.

Animals↗

PubMed2004

The indazole derivative YD-3 inhibits thrombin-induced vascular smooth muscle cell proliferation and attenuates intimal thickening after balloon injury.

Proliferation of vascular smooth muscle cells (VSMCs) is postulated to be one of the key events in the pathogenesis of atherosclerosis and restenosis. We investigated whether YD-3, a lowmolecular weight, non-peptide compound, could modulate proliferation of VSMCs in vitro and restenosis after balloon angioplasty in vivo. We examined the effect of YD-3 on thrombininduced VSMC proliferation by [(3)H]thymidine incorporation assay. The data demonstrated that YD-3 inhibited VSMC proliferation in a concentration-dependent manner. To define the mechanisms of YD-3 action, we found that YD-3 showed a profound inhibition on thrombin-induced Ras and ERK1/2 activities by using Western blotting analysis. Furthermore, oral administration of YD-3 exhibited a marked reduction in neointimal thickness using the carotid injury model in rats. Using immunochemical detection, our experiments also revealed that YD-3 significantly suppressed expression of the PAR-1 receptor, and markedly inhibited PAR-1-activating peptide (SFLLRN)-induced VSMC proliferation in a concentration-dependent manner. These results suggest that YD-3 inhibits thrombin-induced VSMC growth via the Ras- and ERK1/2-mediated signaling pathway. Moreover, YD-3 also shows a developmental potential in the treatment of atherosclerosis and restenosis after vascular injury.

Administration, Oral↗

PubMed2004

The inhibitory mechanism of YC-1, a benzyl indazole, on smooth muscle cell proliferation: an in vitro and in vivo study.

The pharmacological mechanisms of a synthetic compound 1-benzyl-3-(5'-hydroxymethyl-2'-furyl) indazole (YC-1) in preventing smooth muscle cell proliferation remains to be elucidated. The present study was aimed to explore the effects of YC-1 on certain molecules responsible for cell proliferation, including transforming growth factor (TGF)-beta1, soluble guanylyl cyclase (sGC) and focal adhesion kinase (FAK). The in vivo assay was correlated to the in vitro results of YC-1 on vascular stenosis. YC-1 was applied topically via a pluronic gel onto the balloon-injured rat carotid arteries, which were then harvested two weeks later for histological analysis. Our in vitro results showed that TGF-beta1 was suppressed by YC-1 by 50%. The translational level of sGC was threefold activated by YC-1 while the transcription level of sGC was increased up to 24-fold. FAK, the molecule responsible for cell proliferation and migration, was suppressed by YC-1 on the translational levels for 72%. These in vitro results were in consistent with the in vivo observation that the area ratio of neointima to media was reduced by YC-1. This study provides insights into the pharmacological mechanisms of YC-1 in preventing abnormal smooth muscle cell proliferation and thus supports the use of YC-1 as an adjuvant therapy for balloon injury-induced restenosis.

Administration, Topical↗

PubMed2003

High-performance liquid chromatography determination of methionine adenosyltransferase activity using catechol-O-methyltransferase-coupled fluorometric detection.

A nonradioactive, sensitive, rapid, and specific method for the determination of methionine adenosyltransferase activity has been established. In this method, the methyl group of S-adenosyl-L-methionine was enzymatically transferred to esculetin with the aid of catechol-O-methyltransferase and then the resulting scopoletin was extracted with n-hexane:ethyl acetate (7:3, v/v) and measured by high-performance liquid chromatography with Si 60 column and fluorometric detection with excitation and emission wavelengths at 347 and 415 nm, respectively. The detection limit for scopoletin was about 100 fmol. Using this method to determine MAT activity in HL-60 cells required only about 2.5 microg of protein and the incubation time needed for enzymatic reaction is less than 30 min. The HPLC analysis procedure took only 5 min per sample. The kinetic study showed that MAT in HL-60 cells exhibited negative cooperativity with a Hill coefficient of 0.5. The values of K(m) and V(max) were 6.1+/-0.3 microM and 135.4+/-1.5 nmol AdoMet formed/mg protein/h, respectively.

Calibration↗

PubMed2003

A phenylacetate derivative, SCK6, inhibits cell proliferation via G1 cell cycle arrest and apoptosis.

Phenylacetate is a differentiation agent and has anticancer activity with relatively low toxicity. In the present study, we examined the anticancer effect of six synthetic phenylacetate derivatives in human lung cancer cells in our search for more effective phenylacetate analogous. Results showed that the antiproliferative effects of these synthetic compounds were stronger than those of phenylacetate, and that N-butyl-2-(2-fluorolphenyl)acetamide (SCK6) is the most potent compound. To address the mechanism of the antiproliferative effect of SCK6, cell cycle analysis was performed. Result showed that SCK6 (1 mM) induced G(1) arrest in CH27 cells. Western blot analysis of G(1) phase regulatory proteins demonstrated that the protein levels of cyclin-dependent kinase 2 (Cdk2), Cdk4, Cyclin E and Cyclin D3 were decreased after treatment with SCK6 but not those of Cdk6, Cyclin D1 and D2. In contrast, SCK6 increased the protein levels of p53 and p21(CIP1/WAF1). Data from in situ terminal transferase-mediated dUTP-fluorescensin nick end-labeling (TUNEL) assay and DNA fragmentation analysis demonstrated that SCK6 induced apoptotic cell death in CH27 cells. This SCK6-induced apoptosis was accompanied by a downregulation of Bcl-2 protein and activation of the caspase-9 cascade. Overexpression of Bcl-2 by adeno-Bcl-2 vector infection significantly inhibited SCK6-induced apoptosis. Moreover, treatment with caspase inhibitors also markedly reduced cell death induced by SCK6. Taken together, these results suggest that downregulation of G(1)-associated Cdks and cyclins and upregulation of p53 and p21(CIP1/WAF1) may contribute to SCK6-mediated G(1)-phase arrest. Furthermore, the decrease in Bcl-2 and the activation of caspase-9/caspase-3 may be the effector mechanism through which SCK6 induces apoptosis.

Acetamides↗

PubMed2003

Investigation of the cellular mechanism of inhibition of formyl-methionyl-leucyl-phenylalanine-induced superoxide anion generation in rat neutrophils by 2-benzyloxybenzaldehyde.

The inhibition of formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O2(.-)) generation by 2-benzyloxybenzaldehyde (CCY1a) was investigated in rat neutrophils, and the underlying mechanism of this inhibition was assessed. CCY1a concentration-dependently inhibited O2(.-) generation (IC(50)=18.5+/-4.3 microM). In cell-free systems, CCY1a failed to alter O2(.-) generation during dihydroxyfumaric acid autoxidation, in phorbol 12-myristate 13-acetate (PMA)-activated neutrophil particulate NADPH oxidase preparations, or during arachidonic acid-induced NADPH oxidase activation. CCY1a increased cellular cyclic AMP (cAMP) levels in a time- and concentration-dependent manner, and this cAMP-elevating effect was inhibited by the adenylyl cyclase inhibitor 9-(tetrahydro-2'-furyl)adenine (SQ22536), adenosine deaminase (ADA), and the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline. In neutrophils, inhibition of O2(.-) generation by CCY1a was partially reversed by the protein kinase A inhibitor (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-l][1,6]benzodiazocine-10-carboxylic acid, hexyl ester (KT5720). CCY1a did not affect fMLP-induced p38 mitogen-activated protein kinase phosphorylation, but concentration-dependently attenuated the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt (IC(50) about 31.3 and 19.4 microM, respectively). The plateau phase, but not the initial spike, of fMLP-induced [Ca2+](i) changes was inhibited by CCY1a in a concentration-dependent manner. CCY1a inhibition of Ca2+ entry, ERK, and Akt phosphorylation was not prevented by SQ22536 or ADA. fMLP-induced phospholipase D (PLD) activation was inhibited by CCY1a (IC(50)=13.9+/-2.0 microM). ADA and KT5720 did not prevent the inhibition of PLD activation by CCY1a. Collectively, these results indicate that the inhibition by CCY1a of fMLP-induced O2(.-) generation in rat neutrophils can probably be attributed to the increase in cAMP levels, and to the blockade of Ca2+ entry, suppression of Akt, and PLD activation via cAMP-independent mechanisms.

Animals↗

PubMed2003

Enhancement of long-term potentiation by a potent nitric oxide-guanylyl cyclase activator, 3-(5-hydroxymethyl-2-furyl)-1-benzyl-indazole.

Nitric oxide (NO) is known to affect synaptic plasticity in various regions of the brain via the cGMP-cGMP-dependent protein kinase (PKG) pathway. We found that a novel compound 3-(5-hydroxymethyl-2-furyl)-1-benzyl-indazole (YC-1), a drug known to modulate the response of soluble guanylyl cyclase to NO, greatly potentiates long-term potentiation (LTP). This compound markedly enhanced the induction of LTP in rat hippocampal and amygdala slices by weak tetanic stimulation. The potentiation of LTP by YC-1 was greatly reduced by NO synthase inhibitor Ng-nitro-l-arginine-methylester, guanylyl cyclase inhibitor 1 H-[1,2,4]-oxadiazolo(4,3-a)-quinoxalin-1-one, and PKG inhibitor (9S,10R,12R)-2,3,9,10,11,12, hexahydro-10-methoxy-2,9-dimethyl-1-ox0-9.12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-I][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT5823). In addition, mitogen-activated protein kinase kinase inhibitor 2'-amino-3'-methoxyflavone (PD98059) also markedly inhibited LTP potentiating action of YC-1. Intracellular increase of Ca2+ concentration derived from N-methyl-d-aspartate and glutamate metabotropic receptors contributes to the potentiating action of YC-1. Concurrent perfusion of YC-1 and NO donor sodium nitroprusside for a short time period resulted in the induction of LTP by stimuli at a frequency as low as 0.02 Hz. Incubation of unstimulated hippocampal slices with YC-1 plus nitroprusside increased the immunofluorescence of phospho-extracellular signal-regulated kinase (ERK) and phospho-cAMP response element binding protein (CREB). Furthermore, the Western blot shows that the phosphorylation of ERKs 1 and 2 and CREB of unstimulated hippocampal slices was increased by YC-1 plus nitroprusside, which was inhibited by KT5823. The NO-cGMP-PKG-ERK signaling pathway thus plays important role in the potentiation of LTP by YC-1.

Animals↗

PubMed2003

The role of PAR4 in thrombin-induced thromboxane production in human platelets.

There are two protease-activated receptors (PARs), PAR1 and PAR4, in human platelets. It has been suggested that PAR1 mediates platelet responses to low concentrations of thrombin, whereas PAR4 mediates signaling only at high concentrations. In the present study, we used a selective PAR4 blocker, YD-3, to investigate the role of PAR4 in thrombin-induced thromboxane formation in human platelets. YD-3 completely prevented thromboxane production by either a low concentration of thrombin (0.1 U/ml) or the PAR4 agonist peptide GYPGKF. In contrast, YD-3 did not affect thromboxane production caused by the PAR1 agonist peptide SFLLRN, collagen or arachidonic acid. YD-3 also decreased [(3) H]arachidonic acid release from thrombin-stimulated platelets. Moreover, desensitization of platelets with GYPGKF prevented low thrombin-induced thromboxane formation. The decreased thromboxane production by YD-3 is linked to inhibition of calcium influx in thrombin-stimulated platelets. These results suggest that PAR4 plays an important role in the regulation of thromboxane formation in platelets responding to thrombin through prolonged elevation of [Ca(2+)](i) and activation of phospholipase A(2). These data also indicate that PAR4 can be activated by relatively low concentrations of thrombin in human platelets. The selective inhibition of thrombin-induced thromboxane production by YD-3 may be of therapeutic benefit for thrombotic diseases.

Arachidonic Acid↗

PubMed2002

4-Fluoro-N-butylphenylacetamide: a synthetic phenylacetate derivative that upregulates Bcl-X(S), activates caspase cascade and induces apoptosis in human squamous lung cancer CH27 cells.

Phenylacetate (PA) and related aromatic fatty acids induce antiproliferation and differentiation of cancer cell; they have potent anti-tumor properties with relatively low toxicity. To search for more potent analogues of PA, PA derivatives have been synthesized. In this study, we investigated the effects of six synthetic PA derivatives on the growth of human lung cancer cells. Results showed that the anti-proliferative effects of these synthetic compounds were strong than those of PA, 4-fluoro-N-butylphenylacetamide (H6) is the most potent compound. 4,6-Diamidino-2-phenylindole (DAPI) staining, in situ TUNEL assay and DNA gel electrophoresis analysis indicated that a marked reduction in the number of CH27 cells with H6 was related to the induction of apoptosis. The apoptosis triggered by H6 was accompanied by up-regulation of Bcl-X(S), accumulation of cytosolic cytochrome c and activation of caspase cascade (caspase-9 and -3). Furthermore, H6 induces proteolytic cleavage of poly (ADP-ribose) polymerase, which followed the appearance of caspase activity and preceded DNA fragmentation. Pretreatment with caspase inhibitors markedly inhibited H6-induced caspase activity and apoptosis. These results suggest that H6 may induce apoptosis through a Bcl-X(S) and caspase-dependent mechanism.

Acetamides↗

PubMed2002

1-Benzyl-3-(5'-hydroxymethyl-2'-furyl)indazole (YC-1) derivatives as novel inhibitors against sodium nitroprusside-induced apoptosis.

Antiapoptotic agents based on 1-benzyl-3-(5'-hydroxymethyl-2'-furyl)indazole (22, YC-1) derivatives were explored for effective treatment of sepsis and septic shock. We found that compound 22, 1-benzyl-3-(5'-methoxymethyl-2'-furyl)indazole (27), and 1-phenyl-3-(5'-hydroxymethyl-2'-furyl)indazole (23) were the most effective inhibitors of sodium nitroprusside-induced vascular smooth muscle cell apoptosis. These three compounds are proposed as potential therapeutic agents for the treatment of sepsis.

Administration, Oral↗

PubMed2002

2-Benzyloxybenzaldehyde inhibits formyl-methionyl-leucyl-phenylalanine stimulation of phospholipase D activation in rat neutrophils.

2-Benzyloxybenzaldehyde (CCY1a) inhibited the formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated phospholipase D (PLD)-mediated products, phosphatidic acid (PA) and phosphatidylethanol (PEt) formation in rat neutrophils in a concentration-dependent manner with IC(50) values of 15.8+/-2.5 and 13.9+/-2.0 microM, respectively. The underlying cellular signaling mechanism of CCY1a inhibition was investigated. CCY1a inhibited the plateau phase but not the initial Ca(2+) spike of fMLP-stimulated Ca(2+) signal. CCY1a did not inhibit the [Ca(2+)](i) change in Ca(2+)-free medium in response to fMLP, but inhibited the [Ca(2+)](i) change by the subsequent addition of Ca(2+). In addition, CCY1a treatment attenuated the fMLP-induced protein tyrosine phosphorylation. The membrane translocation of ADP-ribosylation factor (ARF) and Rho A proteins in neutrophils stimulated with fMLP was attenuated by CCY1a in a concentration-dependent manner. In a cell-free system, neither the membrane association of ARF and Rho A caused by GTPgammaS nor the phorbol myristate acetate-stimulated membrane translocation of Rho A was suppressed significantly by CCY1a. These results indicate that the attenuation of protein tyrosine phosphorylation, blockade of Ca(2+) entry, and the suppression of ARF and Rho A membrane translocation are probably obligatory for the CCY1a inhibition of PLD activity in rat neutrophils in response to fMLP.

ADP-Ribosylation Factors↗

PubMed2002

Inhibition of formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst by cirsimaritin involves inhibition of phospholipase D signaling in rat neutrophils.

In this study, the cellular localization of the inhibitory effect of a natural flavonoid cirsimaritin against formyl-methionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst in rat neutrophils was investigated. Cirsimaritin concentration-dependently inhibited the superoxide anion (O(*-)(2))generation and O(2) consumption (IC(50) 11.5+/-2.2 micro M and 17.0+/-3.9 micro M, respectively) of neutrophils. Cirsimaritin did not reduce, but slightly enhanced the O(*-)(2) generation in phorbol 12-myristate 13-acetate (PMA)-activated or arachidonic acid-stimulated NADPH oxidase preparation as well as during the autoxidation of dihydroxyfumaric acid. Cirsimaritin did not elevate cellular cAMP levels, and only partially inhibited the fMLP-induced [Ca(2+)](i) changes in the presence or absence of extracellular Ca(2+). The phosphorylation of protein tyrosine, extracellular signal-regulated protein kinase and Akt caused by fMLP were attenuated by cirsimaritin in a concentration-dependent manner. In contrast, cirsimaritin had no effect on the phosphorylation of p38 mitogen-activated protein kinase. Cirsimaritin produced a concentration-dependent reduction in the formation of phosphatidic acid and phosphatidylethanol, in the presence of ethanol, from fMLP-stimulated neutrophils (IC(50) 15.1+/-6.5 micro M and 15.6+/-3.4 micro M, respectively), but did not affect the phosphatidylethanol formation in response to PMA. Under the similar concentration range, cirsimaritin attenuated the membrane translocation of ARF and Rho A. However, the GTPgammaS-stimulated membrane-associated ARF and Rho in cell lysate were unaffected by cirsimaritin. Collectively, these results indicate that the inhibition of fMLP-induced respiratory burst by cirsimaritin in rat neutrophils is likely mainly through the blockade of phospholipase D signaling pathway.

Animals↗

PubMed2002

Inhibition of lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages by a synthetic carbazole, LCY-2-CHO.

In activated macrophages, large amounts of nitric oxide (NO) are generated by inducible nitric oxide synthase (iNOS). This is an important mechanism in macrophage-induced cytotoxicity and inflammation. In the present study, a synthetic carbazole compound, 9-(2-chlorobenzyl)-9H-carbazole-3-carbaldehyde (LCY-2-CHO), was found to have an inhibitory effect on lipopolysaccharide (LPS)-stimulated NO generation in RAW 264.7 macrophages (IC50 value of 1.3+/-0.4 microM). LCY-2-CHO did not induce cytotoxicity and had a negligible effect on iNOS activity. To explore the mechanism of inhibition of NO generation by LCY-2-CHO, the expression of the iNOS gene was examined. LCY-2-CHO abolished the LPS-induced expression of both iNOS protein and mRNA in a parallel concentration-dependent manner with IC50 values similar to those required for inhibition of NO generation. LCY-2-CHO did not enhance the degradation of iNOS mRNA. In cells transiently transfected with an iNOS promoter-chloramphenicol acetyltransferase (CAT) reporter construct, LCY-2-CHO attenuated the LPS-induced iNOS promoter activity. However, LCY-2-CHO had no effect on the degradation of IkappaB-alpha or IkappaB-beta, DNA binding activity, or transcriptional activity of nuclear factor-kappaB (NF-kappaB). These results indicate that LCY-2-CHO inhibits NO generation via a decrease in the transcription of iNOS mRNA through a signaling pathway that does not involve NF-kappaB activation.

Animals↗

PubMed2002

Marked decrease of cyclosporin absorption caused by phellamurin in rats.

Phellamurin is a flavonoid glycoside that is abundant in the leaves of Phellodendron wilsonii Hayata et Kanehira (Rutaceae). In vitro everted rat intestine study indicated that phellamurin inhibited intestinal P-glycoprotein in a dose-dependent manner. In order to investigate the effect of phellamurin on cyclosporin absorption and disposition, rats were given cyclosporin (5 mg/kg) with or without phellamurin in a parallel design. Fluorescence polarization immunoassay was used to determine the blood concentration of cyclosporin. Unanticipatedly, our results indicated that the coadministration of phellamurin significantly decreased the Cmax of cyclosporin by 77 % and reduced the AUC(0-infinity) of cyclosporin by 56 %. This indicated that a serious interaction occurred between phellamurin with cyclosporin. To ensure the efficacy of cyclosporin, we suggest that the coadministration of phellamurin or Phellodendron wilsonii with cyclosporin should be avoided.

Animals↗