Publications by authors named "Tong-Shin Chang"

42 Publications

Diclofenac impairs autophagic flux via oxidative stress and lysosomal dysfunction: Implications for hepatotoxicity.

Redox Biol 2020 10 12;37:101751. Epub 2020 Oct 12.

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, Republic of Korea. Electronic address:

Treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with various side effects, including cardiovascular and hepatic disorders. Studies suggest that mitochondrial damage and oxidative stress are important mediators of toxicity, yet the underlying mechanisms are poorly understood. In this study, we identified that some NSAIDs, including diclofenac, inhibit autophagic flux in hepatocytes. Further detailed studies demonstrated that diclofenac induced a reactive oxygen species (ROS)-dependent increase in lysosomal pH, attenuated cathepsin activity and blocked autophagosome-lysosome fusion. The reactivation of lysosomal function by treatment with clioquinol or transfection with the transcription factor EB restored lysosomal pH and thus autophagic flux. The production of mitochondrial ROS is critical for this process since scavenging ROS reversed lysosomal dysfunction and activated autophagic flux. The compromised lysosomal activity induced by diclofenac also inhibited the fusion with and degradation of mitochondria by mitophagy. Diclofenac-induced cell death and hepatotoxicity were effectively protected by rapamycin. Thus, we demonstrated that diclofenac induces the intracellular ROS production and lysosomal dysfunction that lead to the suppression of autophagy. Impaired autophagy fails to maintain mitochondrial integrity and aggravates the cellular ROS burden, which leads to diclofenac-induced hepatotoxicity.
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http://dx.doi.org/10.1016/j.redox.2020.101751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7575798PMC
October 2020

Identification of 6'-β-fluoro-homoaristeromycin as a potent inhibitor of chikungunya virus replication.

Eur J Med Chem 2020 Feb 9;187:111956. Epub 2019 Dec 9.

Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, 08826, South Korea. Electronic address:

We have reported on aristeromycin (1) and 6'-fluorinated-aristeromycin analogues (2), which are active against RNA viruses such as Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), Zika virus (ZIKV), and Chikungunya virus (CHIKV). However, these exhibit substantial cytotoxicity. As this cytotoxicity may be attributed to 5'-phosphorylation, we designed and synthesized one-carbon homologated 6'-fluorinated-aristeromycin analogues. This modification prevents 5'-phosphorlyation by cellular kinases, whereas the inhibitory activity towards S-adenosyl-l-homocysteine (SAH) hydrolase will be retained. The enantiomerically pure 6'-fluorinated-5'-homoaristeromycin analogues 3a-e were synthesized via the electrophilic fluorination of the silyl enol ether with Selectfluor, using a base-build up approach as the key steps. All synthesized compounds exhibited potent inhibitory activity towards SAH hydrolase, among which 6'-β-fluoroadenosine analogue 3a was the most potent (IC = 0.36 μM). Among the compounds tested, 6'-β-fluoro-homoaristeromycin 3a showed potent antiviral activity (EC = 0.12 μM) against the CHIKV, without noticeable cytotoxicity up to 250 μM. Only 3a displayed anti-CHIKV activity, whereas both3a and 3b inhibited SAH hydrolase with similar IC values (0.36 and 0.37 μM, respectively), which suggested that 3a's antiviral activity did not merely depend on the inhibition of SAH hydrolase. This is further supported by the fact that the antiviral effect was specific for CHIKV and some other alphaviruses and none of the homologated analogues inhibited other RNA viruses, such as SARS-CoV, MERS-CoV, and ZIKV. The potent inhibition and high selectivity index make 6'-β-fluoro-homoaristeromycin (3a) a promising new template for the development of antivirals against CHIKV, a serious re-emerging pathogen that has infected millions of people over the past 15 years.
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http://dx.doi.org/10.1016/j.ejmech.2019.111956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115507PMC
February 2020

Poncirin Inhibits Osteoclast Differentiation and Bone Loss through Down-Regulation of NFATc1 and .

Biomol Ther (Seoul) 2020 Jul;28(4):337-343

Lab of Cell Differentiation Research, College of Oriental Medicine, Gachon University, Seongnam 13120, Republic of Korea.

Activation of osteoclast and inactivation of osteoblast result in loss of bone mass with bone resorption, leading to the pathological progression of osteoporosis. The receptor activator of NF-κB ligand (RANKL) is a member of the TNF superfamily, and is a key mediator of osteoclast differentiation. A flavanone glycoside isolated from the fruit of , poncirin has anti-allergic, hypocholesterolemic, anti-inflammatory and anti-platelet activities. The present study investigates the effect of poncirin on osteoclast differentiation of RANKL-stimulated RAW264.7 cells. We observed reduced formation of RANKL-stimulated TRAP-positive multinucleated cells (a morphological feature of osteoclasts) after poncirin exposure. Real-time qPCR analysis showed suppression of the RANKL-mediated induction of key osteoclastogenic molecules such as NFATc1, TRAP, c-Fos, MMP9 and cathepsin K after poncirin treatment. Poncirin also inhibited the RANKL-mediated activation of NF-κB and, notably, JNK, without changes in ERK and p38 expression in RAW264.7 cells. Furthermore, we assessed the efficacy of poncirin in the lipopolysaccharide (LPS)-induced bone erosion model. Evaluating the micro-CT of femurs revealed that bone erosion in poncirin treated mice was markedly attenuated. Our results indicate that poncirin exerts anti-osteoclastic effects and by suppressing osteoclast differentiation. We believe that poncirin is a promising candidate for inflammatory bone loss therapeutics.
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http://dx.doi.org/10.4062/biomolther.2018.216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327144PMC
July 2020

Design, Synthesis, and Anti-RNA Virus Activity of 6'-Fluorinated-Aristeromycin Analogues.

J Med Chem 2019 07 20;62(13):6346-6362. Epub 2019 Jun 20.

Research Institute of Pharmaceutical Sciences, College of Pharmacy , Seoul National University , Seoul 151-742 , Korea.

The 6'-fluorinated aristeromycins were designed as dual-target antiviral compounds aimed at inhibiting both the viral RNA-dependent RNA polymerase (RdRp) and the host cell -adenosyl-l-homocysteine (SAH) hydrolase, which would indirectly target capping of viral RNA. The introduction of a fluorine at the 6'-position enhanced the inhibition of SAH hydrolase and the activity against RNA viruses. The adenosine and -methyladenosine analogues showed potent inhibition against SAH hydrolase, while only the adenosine derivatives exhibited potent antiviral activity against all tested RNA viruses such as Middle East respiratory syndrome-coronavirus (MERS-CoV), severe acute respiratory syndrome-coronavirus, chikungunya virus, and/or Zika virus. 6',6'-Difluoroaristeromycin () showed the strongest antiviral effect for MERS-CoV, with a ∼2.5 log reduction in infectious progeny titer in viral load reduction assay. The phosphoramidate prodrug also demonstrated potent broad-spectrum antiviral activity, possibly by inhibiting the viral RdRp. This study shows that 6'-fluorinated aristeromycins can serve as starting points for the development of broad-spectrum antiviral agents that target RNA viruses.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075649PMC
July 2019

AMRI-59 functions as a radiosensitizer via peroxiredoxin I-targeted ROS accumulation and apoptotic cell death induction.

Oncotarget 2017 Dec 9;8(69):114050-114064. Epub 2017 Dec 9.

Division of Applied Radiation Bioscience, Korea Institute of Radiological and Medical Sciences, Seoul, Korea.

Previously, we identified AMRI-59 as a specific pharmaceutical inhibitor of peroxiredoxin (PRX) I enzyme activity. In this study, we examined whether AMRI-59 acts as a radiosensitizer in non-small cell lung cancer cells using clonogenic assays. The intracellular mechanisms underlying the radiosensitization effect of AMRI-59 were determined via immunoblotting in addition to measurement of ROS generation, mitochondrial potential and cell death. AMRI-59 activity was examined by co-treating nude mice with the compound and γ-ionizing radiation (IR), followed by measurement of tumor volumes and apoptosis. The dose enhancement ratios of 30 μM AMRI-59 in NCI-H460 and NCI-H1299 were 1.51 and 2.12, respectively. Combination of AMRI-59 with IR augmented ROS production and mitochondrial potential disruption via enhancement of PRX I oxidation, leading to increased expression of γH2AX, a DNA damage marker, and suppression of ERK phosphorylation, and finally, activation of caspase-3. Notably, inhibition of ROS production prevented ERK suppression, and blockage of ERK in combination with AMRI-59 and IR led to enhanced caspase-3 activation and apoptosis. In a xenograft assay using NCI-H460 and NCI-H1299, combined treatment with AMRI-59 and IR delayed tumor growth by 26.98 and 14.88 days, compared with controls, yielding enhancement factors of 1.73 and 1.37, respectively. Taken together, the results indicate that AMRI-59 functions as a PRX I-targeted radiosensitizer by inducing apoptosis through activation of the ROS/γH2AX/caspase pathway and suppression of ERK.
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http://dx.doi.org/10.18632/oncotarget.23114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768385PMC
December 2017

Protective Role of Mitochondrial Peroxiredoxin III against UVB-Induced Apoptosis of Epidermal Keratinocytes.

J Invest Dermatol 2017 06 12;137(6):1333-1342. Epub 2017 Feb 12.

Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea; College of Pharmacy, Ewha Womans University, Seoul, Republic of Korea. Electronic address:

UVB light induces generation of reactive oxygen species, ultimately leading to skin cell damage. Mitochondria are a major source of reactive oxygen species in UVB-irradiated skin cells, with increased levels of mitochondrial reactive oxygen species having been implicated in keratinocyte apoptosis. Peroxiredoxin III (PrxIII) is the most abundant and potent HO-removing enzyme in the mitochondria of most cell types. Here, the protective role of PrxIII against UVB-induced apoptosis of epidermal keratinocytes was investigated. Mitochondrial HO levels were differentiated from other types of ROS using mitochondria-specific fluorescent HO indicators. Upon UVB irradiation, PrxIII-knockdown HaCaT human keratinocytes and PrxIII-deficient (PrxIII) mouse primary keratinocytes exhibited enhanced accumulation of mitochondrial HO compared with PrxIII-expressing controls. Keratinocytes lacking PrxIII were subsequently sensitized to apoptosis through mitochondrial membrane potential loss, cardiolipin oxidation, cytochrome c release, and caspase activation. Increased UVB-induced epidermal tissue damage in PrxIII mice was attributable to increased caspase-dependent keratinocyte apoptosis. Our findings show that mitochondrial HO is a key mediator in UVB-induced apoptosis of keratinocytes and that PrxIII plays a critical role in protecting epidermal keratinocytes against UVB-induced apoptosis through eliminating mitochondrial HO. These findings support the concept that reinforcing mitochondrial PrxIII defenses may help prevent UVB-induced skin damage such as inflammation, sunburn, and photoaging.
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http://dx.doi.org/10.1016/j.jid.2017.01.027DOI Listing
June 2017

Identification of cytoprotective constituents of the flower buds of Tussilago farfara against glucose oxidase-induced oxidative stress in mouse fibroblast NIH3T3 cells and human keratinocyte HaCaT cells.

Arch Pharm Res 2016 Apr 16;39(4):474-480. Epub 2016 Mar 16.

College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, 03760, Korea.

A new cytoprotective compound, 1-[(4S)-3,4-dihydro-4-hydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl]-ethanone (1) was isolated from the flower buds of Tussilago farfara L. (Compositae), together with eight known compounds, 3,4-dicaffeoyl isoquinic acid (2), trans-cinnamic acid (3), 4-hydroxyacetophenone (4), 4,5-dicaffeoylquinic acid methyl ester (5), 3,5-dicaffeoylquinic acid methyl ester (6), 4-hydroxybenzoic acid (7), isoquercetrin (8), and ligucyperonol (9). Compounds 2-4 were found in this plant for the first time. The isolates 1-9, were tested for their cytoprotective activities against glucose oxidase-induced oxidative stress in mouse fibroblast NIH3T3 cells and human keratinocyte HaCaT cells. Among them, 1 and 3 showed significant cytoprotective activities as determined by MTT assay and lactate dehydrogenase leakage, indicating their possibility as the potent cytoprotective agents. The structure of 1 was determined by spectroscopic data analysis including 1D- and 2D-NMR experiments, and its absolute configuration was elucidated by a circular dichroism.
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http://dx.doi.org/10.1007/s12272-016-0730-zDOI Listing
April 2016

Sulfiredoxin inhibitor induces preferential death of cancer cells through reactive oxygen species-mediated mitochondrial damage.

Free Radic Biol Med 2016 Feb 23;91:264-74. Epub 2015 Dec 23.

Department of Life Science and the Research Center for Cellular Homeostasis, Ewha Womans University, Seoul 120-750, South Korea. Electronic address:

Recent studies have shown that many types of cancer cells have increased levels of reactive oxygen species (ROS) and enhance antioxidant capacity as an adaptation to intrinsic oxidative stress, suggesting that cancer cells are more vulnerable to oxidative insults and are more dependent on antioxidant systems compared with normal cells. Thus, disruption of redox homeostasis caused by a decline in antioxidant capacity may provide a method for the selective death of cancer cells. Here we show that ROS-mediated selective death of tumor cells can be caused by inhibiting sulfiredoxin (Srx), which reduces hyperoxidized peroxiredoxins, leading to their reactivation. Srx inhibitor increased the accumulation of sulfinic peroxiredoxins and ROS, which led to oxidative mitochondrial damage and caspase activation, resulting in the death of A549 human lung adenocarcinoma cells. Srx depletion also inhibited the growth of A549 cells like Srx inhibition, and the cytotoxic effects of Srx inhibitor were considerably reversed by Srx overexpression or antioxidants such as N-acetyl cysteine and butylated hydroxyanisol. Moreover, Srx inhibitor rendered tumorigenic ovarian cells more susceptible to ROS-mediated death compared with nontumorigenic cells and significantly suppressed the growth of A549 xenografts without acute toxicity. Our results suggest that Srx might serve as a novel therapeutic target for cancer treatment based on ROS-mediated cell death.
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http://dx.doi.org/10.1016/j.freeradbiomed.2015.12.023DOI Listing
February 2016

A concise synthesis of tubuphenylalanine and epi-tubuphenylalanine via a diastereoselective Mukaiyama aldol reaction of silyl ketene acetal.

Org Biomol Chem 2016 Jan 26;14(3):913-9. Epub 2015 Nov 26.

College of Pharmacy & Graduate School of Pharmaceutical Sciences, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul, 03760, Republic of Korea.

We have developed a straightforward and auxiliary-free synthetic route towards tBu-tubuphenylalanine (tBu-Tup) and tBu-epi-tubuphenylalanine (tBu-epi-Tup), which are the key components of tubulysins and their analogs. A Lewis acid-mediated diastereoselective Mukaiyama aldol reaction using silyl ketene acetal and N-Boc-L-phenylalaninal provided γ-amino-β-hydroxyl-α-methyl esters, which were deoxygenated to γ-amino-α-methyl esters under Barton-McCombie deoxygenation conditions. Notably, the desired tBu-Tup and tBu-epi-Tup were obtained in good overall yields in four steps.
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http://dx.doi.org/10.1039/c5ob02239hDOI Listing
January 2016

Effective Killing of Cancer Cells Through ROS-Mediated Mechanisms by AMRI-59 Targeting Peroxiredoxin I.

Antioxid Redox Signal 2016 Mar 18;24(8):453-69. Epub 2015 Dec 18.

1 Graduate School of Pharmaceutical Sciences, Ewha Womans University , Seoul, Republic of Korea.

Aims: The intrinsic increase of reactive oxygen species (ROS) production in cancer cells after malignant transformation frequently induces redox adaptation, leading to enhanced antioxidant capacity. Peroxiredoxin I (PrxI), an enzyme responsible for eliminating hydrogen peroxide, has been found to be elevated in many types of cancer cells. Since overexpression of PrxI promoted cancer cells' survival and resistance to chemotherapy and radiotherapy, PrxI has been proposed as a therapeutic target for anticancer drugs. In this study, we aimed to investigate the anticancer efficacy of a small molecule inhibitor of PrxI.

Results: By a high-throughput screening approach, we identified AMRI-59 as a potent inhibitor of PrxI. AMRI-59 increased cellular ROS, leading to the activation of both mitochondria- and apoptosis signal-regulated kinase-1-mediated signaling pathways, resulting in apoptosis of A549 human lung adenocarcinoma. AMRI-59 caused no significant changes in ROS level, proliferation, and apoptosis of PrxI-knockdown A549 cells by RNA interference. PrxI overexpression or N-acetylcysteine pretreatment abrogated AMRI-59-induced cytotoxicity in A549 cells. AMRI-59 rendered tumorigenic ovarian cells more susceptible to ROS-mediated death compared with nontumorigenic cells. Moreover, significant antitumor activity of AMRI-59 was observed in mouse tumor xenograft model implanted with A549 cells with no apparent acute toxicity.

Innovation: This study offers preclinical proof-of-concept for AMRI-59, a lead small molecule inhibitor of PrxI, as an anticancer agent.

Conclusions: Our results highlight a promising strategy for cancer therapy that preferentially eradicates cancer cells by targeting the PrxI-mediated redox-dependent survival pathways.
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http://dx.doi.org/10.1089/ars.2014.6187DOI Listing
March 2016

Resveratrol inhibits collagen-induced platelet stimulation through suppressing NADPH oxidase and oxidative inactivation of SH2 domain-containing protein tyrosine phosphatase-2.

Free Radic Biol Med 2015 Dec 22;89:842-51. Epub 2015 Oct 22.

Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120-750, Republic of Korea. Electronic address:

Reactive oxygen species (ROS) produced upon collagen stimulation are implicated in propagating various platelet-activating pathways. Among ROS-producing enzymes, NADPH oxidase (NOX) is largely responsible for collagen receptor-dependent ROS production. Therefore, NOX has been proposed as a novel target for the development of antiplatelet agent. We here investigate whether resveratrol inhibits collagen-induced NOX activation and further examine the effects of resveratrol on ROS-dependent signaling pathways in collagen-stimulated platelets. Collagen-induced superoxide anion production in platelets was inhibited by resveratrol. Resveratrol suppressed collagen-induced phosphorylation of p47(phox), a major regulatory subunit of NOX. Correlated with the inhibitory effects on NOX, resveratrol protected SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) from ROS-mediated inactivation and subsequently attenuated the specific tyrosine phosphorylation of key components (spleen tyrosine kinase, Vav1, Bruton's tyrosine kinase, and phospholipase Cγ2) for collagen receptor signaling cascades. Resveratrol also inhibited downstream responses such as cytosolic calcium elevation, P-selectin surface exposure, and integrin-αIIbβ3 activation. Furthermore, resveratrol inhibited platelet aggregation and adhesion in response to collagen. The antiplatelet effects of resveratrol through the inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2 suggest that resveratrol is a potential compound for prevention and treatment of thrombovascular diseases.
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http://dx.doi.org/10.1016/j.freeradbiomed.2015.10.413DOI Listing
December 2015

Inhibitory effect of brazilin on osteoclast differentiation and its mechanism of action.

Int Immunopharmacol 2015 Dec 1;29(2):628-634. Epub 2015 Oct 1.

Lab of Cell Differentiation Research, College of Korean Medicine, Gachon University, Seongnam 13120, Republic of Korea. Electronic address:

Brazilin isolated from Caesalpinia sappan has long been known as a natural red pigment. Our study evaluated the inhibitory effect of brazilin on osteoclast differentiation and investigated its mechanism of action. Our results demonstrated that brazilin inhibited receptor activator of nuclear factor kappa-B ligand (RANKL)-mediated osteoclast differentiation in RAW264.7 cells in a dose-dependent manner, without any evidence of cytotoxicity. The mRNA expression of tartrate-resistant acid phosphatase (TRAP), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), matrix metalloproteinase 9 (MMP-9), and cathepsin K in RANKL-treated RAW264.7 cells was inhibited by brazilin treatment. Brazilin also decreased RANKL-induced expression of inflammatory mediator genes such as inducible nitric oxide synthase, iNOS; cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 and inhibited extracellular signal-regulated kinases (ERK) and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) p65 phosphorylation in RANKL-stimulated RAW264.7 cells. A lipopolysaccharide (LPS)-induced osteoporosis study was also performed to assess the effects of brazilin in vivo. Micro-computed tomography (CT) analysis of the femurs showed that LPS treatment causes bone loss in mice, but it was significantly attenuated after co-treatment with brazilin (100mg/kg). Therefore, brazilin may have therapeutic potential in preventing bone loss.
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http://dx.doi.org/10.1016/j.intimp.2015.09.018DOI Listing
December 2015

Recent Advances in Anticancer Chemotherapeutics based upon Azepine Scaffold.

Anticancer Agents Med Chem 2016 ;16(5):539-57

College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea.

In the recent few years, the emergence of heterocyclic ring-containing anti-cancer agents has gained a great deal of attention among medicinal chemists. Among these, azepine-based compounds are particularly becoming attractive recently. In this Focus Review, we highlight the recent advancements in the development of azepine-based anti-cancer compounds since the year 2000.
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http://dx.doi.org/10.2174/1871520615666150916091750DOI Listing
December 2016

Structure-Activity Relationships of Neplanocin A Analogues as S-Adenosylhomocysteine Hydrolase Inhibitors and Their Antiviral and Antitumor Activities.

J Med Chem 2015 Jun 5;58(12):5108-20. Epub 2015 Jun 5.

†Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea.

On the basis of the potent inhibitory activity of neplanocin A (1) against S-adenosylhomocysteine (AdoHcy) hydrolase, we analyzed the comprehensive structure-activity relationships by modifying the adenine and carbasugar moiety of 1 to find the pharmacophore in the active site of the enzyme. The introduction of 7-deazaadenine instead of adenine eliminated the inhibitory activity against the AdoHcy hydrolase, while 3-deazaadenine maintained the inhibitory activity of the enzyme, indicating that N-7 is essential for its role as a hydrogen bonding acceptor. The substitution of hydrogen at the 6'-position with fluorine increased the inhibitory activity of the enzyme. The one-carbon homologation at the 5'-position generally decreased the inhibitory activity of the enzyme, indicating that steric repulsion exists. A molecular docking study also supported these experimental data. In this study, 6'-fluoroneplanocin A (2) was the most potent inhibitor of AdoHcy hydrolase (IC50 = 0.24 μM). It showed a potent anti-VSV activity (EC50 = 0.43 μM) and potent anticancer activity in all the human tumor cell lines tested.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00553DOI Listing
June 2015

Peroxiredoxin II is an antioxidant enzyme that negatively regulates collagen-stimulated platelet function.

J Biol Chem 2015 May 23;290(18):11432-42. Epub 2015 Mar 23.

From the Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120-750 and

Collagen-induced platelet signaling is mediated by binding to the primary receptor glycoprotein VI (GPVI). Reactive oxygen species produced in response to collagen have been found to be responsible for the propagation of GPVI signaling pathways in platelets. Therefore, it has been suggested that antioxidant enzymes could down-regulate GPVI-stimulated platelet activation. Although the antioxidant enzyme peroxiredoxin II (PrxII) has emerged as having a role in negatively regulating signaling through various receptors by eliminating H2O2 generated upon receptor stimulation, the function of PrxII in collagen-stimulated platelets is not known. We tested the hypothesis that PrxII negatively regulates collagen-stimulated platelet activation. We analyzed PrxII-deficient murine platelets. PrxII deficiency enhanced GPVI-mediated platelet activation through the defective elimination of H2O2 and the impaired protection of SH2 domain-containing tyrosine phosphatase 2 (SHP-2) against oxidative inactivation, which resulted in increased tyrosine phosphorylation of key components for the GPVI signaling cascade, including Syk, Btk, and phospholipase Cγ2. Interestingly, PrxII-mediated antioxidative protection of SHP-2 appeared to occur in the lipid rafts. PrxII-deficient platelets exhibited increased adhesion and aggregation upon collagen stimulation. Furthermore, in vivo experiments demonstrated that PrxII deficiency facilitated platelet-dependent thrombus formation in injured carotid arteries. This study reveals that PrxII functions as a protective antioxidant enzyme against collagen-stimulated platelet activation and platelet-dependent thrombosis.
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http://dx.doi.org/10.1074/jbc.M115.644260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4416848PMC
May 2015

Kaempferol suppresses collagen-induced platelet activation by inhibiting NADPH oxidase and protecting SHP-2 from oxidative inactivation.

Free Radic Biol Med 2015 Jun 31;83:41-53. Epub 2015 Jan 31.

Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Women's University, Seoul 120-750, Republic of Korea. Electronic address:

Reactive oxygen species (ROS) generated upon collagen stimulation act as second messengers to propagate various platelet-activating events. Among the ROS-generating enzymes, NADPH oxidase (NOX) plays a prominent role in platelet activation. Thus, NOX has been suggested as a novel target for anti-platelet drug development. Although kaempferol has been identified as a NOX inhibitor, the influence of kaempferol on the activation of platelets and the underlying mechanism have never been investigated. Here, we studied the effects of kaempferol on NOX activation, ROS-dependent signaling pathways, and functional responses in collagen-stimulated platelets. Superoxide anion generation stimulated by collagen was significantly inhibited by kaempferol in a concentration-dependent manner. More importantly, kaempferol directly bound p47(phox), a major regulatory subunit of NOX, and significantly inhibited collagen-induced phosphorylation of p47(phox) and NOX activation. In accordance with the inhibition of NOX, ROS-dependent inactivation of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) was potently protected by kaempferol. Subsequently, the specific tyrosine phosphorylation of key components (Syk, Vav1, Btk, and PLCγ2) of collagen receptor signaling pathways was suppressed by kaempferol. Kaempferol also attenuated downstream responses, including cytosolic calcium elevation, P-selectin surface exposure, and integrin-αIIbβ3 activation. Ultimately, kaempferol inhibited platelet aggregation and adhesion in response to collagen in vitro and prolonged in vivo thrombotic response in carotid arteries of mice. This study shows that kaempferol impairs collagen-induced platelet activation through inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2. This effect suggests that kaempferol has therapeutic potential for the prevention and treatment of thrombovascular diseases.
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http://dx.doi.org/10.1016/j.freeradbiomed.2015.01.018DOI Listing
June 2015

Reactive oxygen species play a critical role in collagen-induced platelet activation via SHP-2 oxidation.

Antioxid Redox Signal 2014 Jun 30;20(16):2528-40. Epub 2014 Jan 30.

1 Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Womans University , Seoul, South Korea .

Aims: The collagen-stimulated generation of reactive oxygen species (ROS) regulates signal transduction in platelets, although the mechanism is unclear. The major targets of ROS include protein tyrosine phosphatases (PTPs). ROS-mediated oxidation of the active cysteine site in PTPs abrogates the PTP catalytic activity. The aim of this study was to elucidate whether collagen-induced ROS generation leads to PTP oxidation, which promotes platelet stimulation.

Results: SH2 domain-containing PTP-2 (SHP-2) is oxidized in platelets by ROS produced upon collagen stimulation. The oxidative inactivation of SHP-2 leads to the enhanced tyrosine phosphorylation of spleen tyrosine kinase (Syk), Vav1, and Bruton's tyrosine kinase (Btk) in the linker for the activation of T cells signaling complex, which promotes the tyrosine phosphorylation-mediated activation of phospholipase Cγ2 (PLCγ2). Moreover, we found that, relative to wild-type platelets, platelets derived from glutathione peroxidase 1 (GPx1)/catalase double-deficient mice showed enhanced cellular ROS levels, oxidative inactivation of SHP-2, and tyrosine phosphorylation of Syk, Vav1, Btk, and PLCγ2 in response to collagen, which subsequently led to increased intracellular calcium levels, degranulation, and integrin αIIbβ3 activation. Consistent with these findings, GPx1/catalase double-deficiency accelerated the thrombotic response in FeCl3-injured carotid arteries.

Innovation: The present study is the first to demonstrate that SHP-2 is targeted by ROS produced in collagen-stimulated platelets and suggests that a novel mechanism for the regulation of platelet activation by ROS is due to oxidative inactivation of SHP-2.

Conclusion: We conclude that collagen-induced ROS production leads to SHP-2 oxidation, which promotes platelet activation by upregulating tyrosine phosphorylation-based signal transduction.
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http://dx.doi.org/10.1089/ars.2013.5337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4025609PMC
June 2014

Processed Panax ginseng, Sun Ginseng Increases Type I Collagen by Regulating MMP-1 and TIMP-1 Expression in Human Dermal Fibroblasts.

J Ginseng Res 2012 Jan;36(1):61-7

Lab of Cell Differentiation Research, College of Oriental Medicine, Gachon University, Seongnam 461-701, Korea.

In the present study, effects of sun ginseng (SG) on the collagen synthesis and the proliferation of dermal fibroblast were investigated. Collagen synthesis was measured by assaying procollagen type I C-peptide production. In addition, the level of matrix metalloproteinase (MMP)-1 was assessed by western blot analysis. SG suppressed the MMP-1 protein level in a dose-dependent manner. In contrast, SG dose-dependently increased tissue inhibitors of MMP (TIMP)-1 production in fibroblasts. SG increased type I collagen production directly and/or indirectly by reducing MMP-1 and stimulating TIMP-1 production in human dermal fibroblasts. SG dose-dependently induced fibroblast proliferation and this, in turn, can trigger more collagen production. These results suggest that SG may be a potential pharmacological agent with anti-aging properties in cultured human skin fibroblast.
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http://dx.doi.org/10.5142/jgr.2012.36.1.61DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3659568PMC
January 2012

Sulfiredoxin protein is critical for redox balance and survival of cells exposed to low steady-state levels of H2O2.

J Biol Chem 2012 Jan 15;287(1):81-89. Epub 2011 Nov 15.

Division of Life and Pharmaceutical Sciences and Center for Cell Signaling and Drug Discovery Research, Ewha Womans University, Seoul 120-750, Korea; College of Pharmacy, Ewha Womans University, Seoul, 120-750, Korea. Electronic address:

Sulfiredoxin (Srx) is an enzyme that catalyzes the reduction of cysteine sulfinic acid of hyperoxidized peroxiredoxins (Prxs). Having high affinity toward H2O2, 2-Cys Prxs can efficiently reduce H2O2 at low concentration. We previously showed that Prx I is hyperoxidized at a rate of 0.072% per turnover even in the presence of low steady-state levels of H2O2. Here we examine the novel role of Srx in cells exposed to low steady-state levels of H2O2, which can be achieved by using glucose oxidase. Exposure of low steady-state levels of H2O2 (10-20 μm) to A549 or wild-type mouse embryonic fibroblast (MEF) cells does not lead to any significant change in oxidative injury because of the maintenance of balance between H2O2 production and elimination. In contrast, loss-of-function studies using Srx-depleted A549 and Srx-/- MEF cells demonstrate a dramatic increase in extra- and intracellular H2O2, sulfinic 2-Cys Prxs, and apoptosis. Concomitant with hyperoxidation of mitochondrial Prx III, Srx-depleted cells show an activation of mitochondria-mediated apoptotic pathways including mitochondria membrane potential collapse, cytochrome c release, and caspase activation. Furthermore, adenoviral re-expression of Srx in Srx-depleted A549 or Srx-/- MEF cells promotes the reactivation of sulfinic 2-Cys Prxs and results in cellular resistance to apoptosis, with enhanced removal of H2O2. These results indicate that Srx functions as a novel component to maintain the balance between H2O2 production and elimination and then protects cells from apoptosis even in the presence of low steady-state levels of H2O2.
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http://dx.doi.org/10.1074/jbc.M111.316711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249139PMC
January 2012

Methods for detection and measurement of hydrogen peroxide inside and outside of cells.

Mol Cells 2010 Jun 4;29(6):539-49. Epub 2010 Jun 4.

Department of Life Science, Division of Life and Pharmaceutical Sciences, and Center for Cell Signaling and Drug Discovery Research, Ewha Womans University, Seoul, 120-750, Korea.

Hydrogen peroxide (H(2)O(2)) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of its production. Characterization of the cellular functions of H(2)O(2) requires measurement of its concentration selectively in the presence of other oxygen metabolites and with spatial and temporal fidelity in live cells. For the measurement of H(2)O(2) in biological fluids, several sensitive methods based on horseradish peroxidase and artificial substrates (such as Amplex Red and 3,5,3'5'-tetramethylbenzidine) or on ferrous oxidation in the presence of xylenol orange (FOX) have been developed. For measurement of intracellular H(2)O(2), methods based on dihydro compounds such as 2',7'-dichlorodihydrofluorescein that fluoresce on oxidation are used widely because of their sensitivity and simplicity. However, such probes react with a variety of cellular oxidants including nitric oxide, peroxynitrite, and hypochloride in addition to H(2)O(2). Deprotection reaction-based probes (PG1 and PC1) that fluoresce on H(2)O(2)-specific removal of a boronate group rather than on nonspecific oxidation have recently been developed for selective measurement of H(2)O(2) in cells. Furthermore, a new class of organelle-targetable fluorescent probes has been devised by joining PG1 to a substrate of SNAP-tag. Given that SNAP-tag can be genetically targeted to various subcellular organelles, localized accumulation of H(2)O(2) can be monitored with the use of SNAP-tag bioconjugation chemistry. However, given that both dihydro- and deprotection-based probes react irreversibly with H(2)O(2), they cannot be used to monitor transient changes in H(2)O(2) concentration. This drawback has been overcome with the development of redox-sensitive green fluorescent protein (roGFP) probes, which are prepared by the introduction of two redox-sensitive cysteine residues into green fluorescent protein; the oxidation of these residues to form a disulfide results in a conformational change of the protein and altered fluorogenic properties. Such genetically encoded probes react reversibly with H(2)O(2) and can be targeted to various compartments of the cell, but they are not selective for H(2)O(2) because disulfide formation in roGFP is promoted by various cellular oxidants. A new type of H(2)O(2)-selective, genetically encoded, and reversible fluorescent probe, named HyPer, was recently prepared by insertion of a circularly permuted yellow fluorescent protein (cpYFP) into the bacterial peroxide sensor protein OxyR.
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http://dx.doi.org/10.1007/s10059-010-0082-3DOI Listing
June 2010

Mitochondrial reactive oxygen species originating from Romo1 exert an important role in normal cell cycle progression by regulating p27(Kip1) expression.

Free Radic Res 2009 Aug;43(8):729-37

Laboratory of Molecular Cell Biology, Graduate School of Medicine, Korea University College of Medicine, Korea University, Seoul, 136-705, Republic of Korea.

Reactive oxygen species (ROS) steady-state levels are required for entry into the S phase of the cell cycle in normal cells, as well as in tumour cells. However, the contribution of mitochondrial ROS to normal cell proliferation has not been well investigated thus far. A previous report showed that Romo1 was responsible for the high ROS levels in tumour cells. Here, we show that endogenous ROS generated by Romo1 are indispensable for cell cycle transition from G1 to S phase in normal WI-38 human lung fibroblasts. The ROS level in these cells was down-regulated by Romo1 knockdown, resulting in cell cycle arrest in the G1 phase. This arrest was associated with an increase in the level of p27(Kip1). These results demonstrate that mitochondrial ROS generated by Romo1 expression is required for normal cell proliferation and it is suggested that Romo1 plays an important role in redox signalling during normal cell proliferation.
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http://dx.doi.org/10.1080/10715760903038432DOI Listing
August 2009

Pretreatment with interferon-gamma protects microglia from oxidative stress via up-regulation of Mn-SOD.

Free Radic Biol Med 2009 Apr 9;46(8):1204-10. Epub 2009 Feb 9.

Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China.

Microglial cells, resident macrophage-like immune cells in the brain, are exposed to intense oxidative stress under various pathophysiological conditions. For self-defense against oxidative injuries, microglial cells must be equipped with antioxidative mechanisms. In this study, we investigated the regulation of antioxidant enzyme systems in microglial cells by interferon-gamma (IFN-gamma) and found that pretreatment with IFN-gamma for 20 h protected microglial cells from the toxicity of various reactive species such as hydrogen peroxide (H(2)O(2)), superoxide anion, 4-hydroxy-2(E)-nonenal, and peroxynitrite. The cytoprotective effect of IFN-gamma pretreatment was abolished by the protein synthesis inhibitor cycloheximide. In addition, treatment of microglial cells with both IFN-gamma and H(2)O(2) together did not protect them from the H(2)O(2)-evoked toxicity. These results imply that protein synthesis is required for the protection by IFN-gamma. Among various antioxidant enzymes such as manganese or copper/zinc superoxide dismutase (Mn-SOD or Cu/Zn-SOD), catalase, and glutathione peroxidase (GPx), only Mn-SOD was up-regulated in IFN-gamma-pretreated microglial cells. Transfection with siRNA of Mn-SOD abolished both up-regulation of Mn-SOD expression and protection from H(2)O(2) toxicity by IFN-gamma pretreatment. Furthermore, whereas the activities of Mn-SOD and catalase were up-regulated by IFN-gamma pretreatment, those of Cu/Zn-SOD and GPx were not. These results indicate that IFN-gamma pretreatment protects microglial cells from oxidative stress via selective up-regulation of the level of Mn-SOD and activity of Mn-SOD and catalase.
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http://dx.doi.org/10.1016/j.freeradbiomed.2009.01.027DOI Listing
April 2009

Sulfiredoxin Translocation into Mitochondria Plays a Crucial Role in Reducing Hyperoxidized Peroxiredoxin III.

J Biol Chem 2009 Mar 28;284(13):8470-7. Epub 2009 Jan 28.

College of Pharmacy, Division of Life & Pharmaceutical Sciences, and Center for Cell Signaling & Drug Discovery Research, Ewha Womans University, Science Building C, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, Korea.

The mitochondria are the major intracellular source of reactive oxygen species (ROS), which are generated during cellular respiration. The role of peroxiredoxin (Prx) III, a 2-Cys Prx family member, in the scavenging of mitochondrial H(2)O(2) has recently been emphasized. While eliminating H(2)O(2), Prx can become overoxidized and inactivated by modifying the active cysteine into cysteine sulfinic acid (Cys-SO(2)H). When 2-Cys Prxs are inactivated in vitro, sulfiredoxin (Srx) reduces the cysteine sulfinic acid to cysteines. However, whereas Srx is localized in the cytoplasm, Prx III is present exclusively in the mitochondria. Although Srx reduces sulfinic Prx III in vitro, it remains unclear whether the reduction of Prx III in cells is actually mediated by Srx. Our gain- and loss-of-function experiments show that Srx is responsible for reducing not only sulfinic cytosolic Prxs (I and II) but also sulfinic mitochondrial Prx III. We further demonstrate that Srx translocates from the cytosol to mitochondria in response to oxidative stress. Overexpression of mitochondrion-targeted Srx promotes the regeneration of sulfinic Prx III and results in cellular resistance to apoptosis, with enhanced elimination of mitochondrial H(2)O(2) and decreased rates of mitochondrial membrane potential collapse. These results indicate that Srx plays a crucial role in the reactivation of sulfinic mitochondrial Prx III and that its mitochondrial translocation is critical in maintaining the balance between mitochondrial H(2)O(2) production and elimination.
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http://dx.doi.org/10.1074/jbc.M808981200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2659205PMC
March 2009

Molecular mechanism of the reduction of cysteine sulfinic acid of peroxiredoxin to cysteine by mammalian sulfiredoxin.

J Biol Chem 2006 May 24;281(20):14400-7. Epub 2006 Mar 24.

Laboratory of Cell Signaling, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

Among many proteins with cysteine sulfinic acid (Cys-SO2H) residues, the sulfinic forms of certain peroxiredoxins (Prxs) are selectively reduced by sulfiredoxin (Srx) in the presence of ATP. All Srx enzymes contain a conserved cysteine residue. To elucidate the mechanism of the Srx-catalyzed reaction, we generated various mutants of Srx and examined their interaction with PrxI, their ATPase activity, and their ability to reduce sulfinic PrxI. Our results suggest that three surface-exposed amino acid residues, corresponding to Arg50, Asp57, and Asp79 of rat Srx, are critical for substrate recognition. The presence of the sulfinic form (but not the reduced form) of PrxI induces the conserved cysteine of Srx to take the gamma-phosphate of ATP and then immediately transfers the phosphate to the sulfinic moiety of PrxI to generate a sulfinic acid phosphoryl ester (Prx-Cys-S(=O)OPO3(2-)). This ester is reductively cleaved by a thiol molecule (RSH) such as GSH, thioredoxin, and dithiothreitol to produce a disulfide-S-monoxide (Prx-Cys-S(=O)-S-R). The disulfide-S-monoxide is further reduced through the oxidation of three thiol equivalents to complete the catalytic cycle and regenerate Prx-Cys-SH.
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http://dx.doi.org/10.1074/jbc.M511082200DOI Listing
May 2006

2-Cys peroxiredoxin function in intracellular signal transduction: therapeutic implications.

Trends Mol Med 2005 Dec 9;11(12):571-8. Epub 2005 Nov 9.

Center for Cell Signaling Research and Division of Molecular Life Science, Ewha Womans University, Seoul 120-750, Korea.

H(2)O(2) is a reactive oxygen species that has drawn much interest because of its role as a second messenger in receptor-mediated signaling. Mammalian 2-Cys peroxiredoxins have been shown to eliminate efficiently the H(2)O(2) generated in response to receptor stimulation. 2-Cys peroxiredoxins are members of a novel peroxidase family that catalyze the H(2)O(2) reduction reaction in the presence of thioredoxin, thioredoxin reductase and NADPH. Several lines of evidence suggest that 2-Cys peroxiredoxins have dual roles as regulators of the H(2)O(2) signal and as defenders of oxidative stress. In particular, 2-Cys peroxiredoxin appears to provide selective, specific and localized control of receptor-mediated signal transduction. Thus, the therapeutic potential of 2-Cys peroxiredoxins is clear for diseases, such as cancer and cardiovascular diseases, that involve reactive oxygen species.
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http://dx.doi.org/10.1016/j.molmed.2005.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185838PMC
December 2005

Notch interferes with the scaffold function of JNK-interacting protein 1 to inhibit the JNK signaling pathway.

Proc Natl Acad Sci U S A 2005 Oct 22;102(40):14308-13. Epub 2005 Sep 22.

National Creative Research Initiative Center for Cell Death, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea.

The transmembrane protein Notch is cleaved by gamma-secretase to yield an active form, Notch intracellular domain (Notch-IC), in response to the binding of ligands, such as Jagged. Notch-IC contributes to the regulation of a variety of cellular events, including cell fate determination during embryonic development as well as cell growth, differentiation, and survival. We now show that Notch1-IC suppresses the scaffold activity of c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP1) in the JNK signaling pathway. Notch1-IC physically associated with the JNK binding domain of JIP1 and thereby interfered with the interaction between JIP1 and JNK. JIP1 mediated the activation of JNK1 induced by glucose deprivation in mouse embryonic fibroblasts, and ectopic expression of Notch1-IC inhibited JNK activation and apoptosis triggered by glucose deprivation. Taken together, these findings suggest that Notch1-IC negatively regulates the JNK pathway by disrupting the scaffold function of JIP1.
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http://dx.doi.org/10.1073/pnas.0501600102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1242280PMC
October 2005

Effects of DK-002, a synthesized (6aS,cis)-9,10-Dimethoxy-7,11b-dihydro-indeno[2,1-c]chromene-3,6a-diol, on platelet activity.

Life Sci 2006 Feb 8;78(10):1091-7. Epub 2005 Sep 8.

Department of Preventive Pharmacy, College of Pharmacy, Seoul National University, Seoul 151-742, Korea.

In the present study, the mechanism of antiplatelet activity of DK-002, a synthesized (6aS,cis)-9,10-Dimethoxy-7,11b-dihydro-indeno[2,1-c]chromene-3,6a-diol, was investigated. DK-002 inhibited the thrombin, collagen, and ADP-induced rat platelet aggregation in a concentration-dependent manner, with IC50 values of 120, 27, and 47 microM, respectively. DK-002 also inhibited thrombin-induced dense granule secretion, thromboxane A2 synthesis, and [Ca2+]i elevation in platelets. DK-002 did not show any significant effect on ADP-induced inhibition of cyclic AMP elevation by prostaglandin E1, but DK-002 was confirmed to inhibit ADP-induced [Ca2+]i elevation and shape change. DK-002 inhibited 4-bromo-A23187-induced [Ca2+]i elevation in the presence of creatine phosphate/creatine phosphokinase (CP/CPK, a ADP scavenging system) and indomethacin (a specific inhibitor of cyclooxygenase). DK-002 also inhibited Ca2+ mobilization in thrombin- or 4-bromo-A23187-stimulated platelets through its inhibitory effects on both Ca2+ release from intracellular stores and Ca2+ influx, in the presence of CP/CPK and indomethacin. Taken together, the present study shows that DK-002 has inhibitory effects on stimulation of platelets, and suggests that its antiplatelet activity might be related to the inhibitory mechanism on Ca2+ mobilization in stimulated platelets.
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http://dx.doi.org/10.1016/j.lfs.2005.06.017DOI Listing
February 2006

Controlled elimination of intracellular H(2)O(2): regulation of peroxiredoxin, catalase, and glutathione peroxidase via post-translational modification.

Antioxid Redox Signal 2005 May-Jun;7(5-6):619-26

Laboratory of Cell Signaling, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.

The predominant enzymes responsible for elimination of hydrogen peroxide (H(2)O(2)) in cells are peroxiredoxins (Prxs), catalase, and glutathione peroxidases (GPxs). Evidence suggests that catalytic activities of certain isoforms of these H(2)O(2)-eliminating enzymes are extensively regulated via posttranslational modification. Prx I and Prx II become inactivated when phosphorylated on Thr(90) by cyclin B-dependent kinase Cdc2. In addition, the active-site cysteine of Prx I-IV undergoes a reversible sulfinylation (oxidation to cysteine sulfinic acid) in cells. Desulfinylation (reduction to cysteine) is achieved by a novel enzyme named sulfiredoxin. c-Abl and Arg nonreceptor protein tyrosine kinases associate with catalase in cells treated with H(2)O(2) by mechanisms involving the SH3 domains of the kinases and the Pro(293)PheAsnPro motif of catalase and activate catalase by phosphorylating it on Tyr(231) and Tyr(386). Similarily, GPx1 is activated by c-Abl- and Arg-mediated phosphorylation. The tyrosine phosphorylation is critical for ubiquitination-dependent degradation of catalase.
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http://dx.doi.org/10.1089/ars.2005.7.619DOI Listing
September 2005

Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins.

Curr Opin Cell Biol 2005 Apr;17(2):183-9

Laboratory of Cell Signaling, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

Hydrogen peroxide (H2O2) accumulates transiently in various cell types stimulated with peptide growth factors and participates in receptor signaling by oxidizing the essential cysteine residues of protein tyrosine phosphatases and the lipid phosphatase PTEN. The reversible inactivation of these phosphatases by H2O2 is likely required to prevent futile cycles of phosphorylation-dephosphorylation of proteins and phosphoinositides. The accumulation of H2O2 is possible even in the presence of large amounts of the antioxidant enzymes peroxiredoxin I and II in the cytosol, probably because of a built-in mechanism of peroxiredoxin inactivation that is mediated by H2O2 and reversed by an ATP-dependent reduction reaction catalyzed by sulfiredoxin.
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http://dx.doi.org/10.1016/j.ceb.2005.02.004DOI Listing
April 2005

Antiplatelet activity of BRX-018, (6aS,cis)-malonic acid 3-acetoxy-6a9-bis-(2-methoxycarbonyl-acetoxy)-6,6a,7,11b-tetrahydro-indeno[2,1-c]chromen-10-yl ester methylester.

Thromb Res 2005 27;115(4):309-18. Epub 2004 Oct 27.

College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea.

Brazilin (7,11b-dihydrobenz[b]indeno[1,2-d]pyran-3,6a,9,10 (6H)-tetrol), the major component of Caesalpinia sappan L., was reported to show antiplatelet activity through the inhibition of phospholipase A2 (PLA2) activity and the increase in intracellular free Ca2+ concentration ([Ca2+]i). To search more potential antiplatelet agent, brazilin derivatives were synthesized and examined for their effects on the platelet aggregation. Among those compounds, BRX-018, (6aS,cis)-Malonic acid 3-acetoxy-6a9-bis-(2-methoxycarbonyl-acetoxy)-6,6a,7,11b-tetrahydro-indeno[2,1-c]chromen-10-yl ester methylester, was confirmed as one of the potential antiplatelet agents. In the present study, we investigated the antiplatelet mechanism of BRX-018. BRX-018 inhibited the thrombin-, collagen-, and ADP-induced rat platelet aggregation in a concentration-dependent manner, with IC50 values of 35, 15, and 25 microM, respectively. BRX-018 also inhibited thrombin-induced dense granule secretion, thromboxane A2 (TXA2) synthesis, and [Ca2+]i elevation in platelets. BRX-018 was also found to inhibit A23187-induced [Ca2+]i and aggregation in the presence of apyrase (ADP scavenger) but not in the presence of both apyrase and indomethacin (a specific inhibitor of cyclooxygenase, COX). Although BRX-018 significantly inhibited arachidonic acid (AA)-induced aggregation and TXA2 synthesis, it had no significant inhibitory effect on cyclooxygenase activity in vitro. In contrast, BRX-018 inhibited the activity of purified PLA2. Dixon plot showed that this inhibition was mixed type with an inhibition constant of Ki=23 microM. Taken together, the present study suggests that BRX-018 may be a promising antiplatelet agent and that its antiplatelet activity may be based on the inhibitory mechanisms on TXA2 synthesis in stimulated platelets.
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http://dx.doi.org/10.1016/j.thromres.2004.09.018DOI Listing
August 2005