Tsu-Shing Wang

Induction of Heme Oxygenase 1 and Inhibition of Tumor Necrosis Factor α-Induced Intercellular Adhesion Molecule Expression by Andrographolide in EA.hy926 Cells

Andrographolide is the most abundant diterpene lactone in Andrographis paniculata, which is widely used as a traditional medicine in Southeast Asia. Heme oxygenase 1 (HO-1) is an antioxidant enzyme encoded by a stress-responsive gene. HO-1 has been reported to inhibit the expression of adhesion molecules in vascular endothelial cells (EC). Intercellular adhesion molecule (ICAM-1) is an inflammatory biomarker that is involved in the adhesion of monocytes to EC. In this study, we investigated the effect of andrographolide on the expression of ICAM-1 induced by tumor necrosis factor alpha (TNF-alpha) in EA.hy926 cells and the possible mechanisms involved. Andrographolide (2.5-7.5 microM) inhibited the TNF-alpha-induced expression of ICAM-1 in a dose-dependent manner and resulted in a decrease in HL-60 cell adhesion to EA.hy926 cells (p < 0.05). In parallel, andrographolide significantly induced the expression of HO-1 in a concentration-dependent fashion (p < 0.05). Andrographolide increased the rate of nuclear translocation of nuclear factor erythroid 2-related 2 (Nrf2) and induced antioxidant response element-luciferase reporter activity. Transfection with HO-1-specific small interfering RNA knocked down HO-1 expression, and the inhibition of expression of ICAM-1 by andrographolide was significantly reversed. These results suggest that stimulation of Nrf2-dependent HO-1 expression is involved in the suppression of TNF-alpha-induced ICAM-1 expression exerted by andrographolide.

Bioavailability of andrographolide and protection against carbon tetrachloride-induced oxidative damage in rats

Andrographolide, a bioactive diterpenoid, is identified in Andrographis paniculata. In this study, we investigated the pharmacokinetics and bioavailability of andrographolide in rats and studied whether andrographolide enhances antioxidant defense in a variety of tissues and protects against carbon tetrachloride-induced oxidative damage. After a single 50-mg/kg administration, the maximum plasma concentration of andrographolide was 1μM which peaked at 30min. The bioavailability of andrographolide was 1.19%. In a hepatoprotection study, rats were intragastrically dosed with 30 or 50mg/kg andrographolide for 5 consecutive days. The results showed that andrographolide up-regulated glutamate cysteine ligase (GCL) catalytic and modifier subunits, superoxide dismutase (SOD)-1, heme oxygenase (HO)-1, and glutathione (GSH) S-transferase (GST) Ya/Yb protein and mRNA expression in the liver, heart, and kidneys. The activity of SOD, GST, and GSH reductase was also increased in rats dosed with andrographolide (p<0.05). Immunoblot analysis and EMSA revealed that andrographolide increased nuclear Nrf2 contents and Nrf2 binding to DNA, respectively. After the 5-day andrographolide treatment, one group of animals was intraperitoneally injected with carbon tetrachloride (CCl4) at day 6. Andrographolide pretreatment suppressed CCl4-induced plasma aminotransferase activity and hepatic lipid peroxidation (p<0.05). These results suggest that andrographolide is quickly absorbed in the intestinal tract in rats with a bioavailability of 1.19%. Andrographolide protects against chemical-induced oxidative damage by up-regulating the gene transcription and activity of antioxidant enzymes in various tissues.

Oxidative modifications of proteins by sodium arsenite in human umbilical vein endothelial cells

Epidemiologic studies have demonstrated that chronic arsenic exposure is associated with the incidence of chronic diseases. This association is partly related to the increase in reactive oxygen species (ROS) overload and protein oxidation that result from arsenic exposure. In this study, we intended to identify proteins susceptible to oxidative carbonylation by sodium arsenite and the impact of carbonylation on the function of these proteins in human umbilical vein endothelial cells (HUVECs). The 2,4‐dinitrophenylhydrazine (DNPH) dot‐blot assay revealed that arsenite (0–50 μM) dose‐dependently increased protein carbonylation. Consistent with these findings, the cellular ROS level as measured by 2′,7′‐dichlorofluorescein diacetate (DCHF‐DA) assay was increased in cells exposed to arsenite. By two‐dimensional gel electrophoresis and matrix assist laser desorption ionization time of flight mass spectrometry (MALDI‐TOF/MS), one glycolytic enzyme, enolase‐α, two cytoskeleton proteins, fascin (F‐actin associated protein) and vimentin, and two protein quality control proteins, HSC70 (heat‐shock cognate protein 70), and PDIA3 (protein disulfide isomerase family A, member 3) were identified to be arsenic‐sensitive carbonlyated proteins. Accompanied by carbonylation, enolase‐α activity was dose‐dependently decreased and the F‐actin filament network was disturbed. Taken together, our results suggest that arsenite exposure results in the generation of carbonylated proteins, and the resultant changes in energy metabolism and in the cytoskeletal network may partly lead to cell damage.

Cigarette smoke extract induces expression of cell adhesion molecules in HUVEC via actin filament reorganization

Epidemiologic studies have shown a strong association between cigarette smoking and cardiovascular diseases. Various oxidative species and free radicals are produced during cigarette smoking and these lead to endothelial dysfunction and inflammation. Expression of adhesion molecules, such as intercellular adhesion molecule‐1 (ICAM‐1), E‐selectin, and vascular cell adhesion molecule‐1, and adhesion of leukocytes are present in atherosclerosis. We showed previously that a nonfractionated cigarette smoke extract (CSE) induces surface expression of ICAM‐1 and E‐selectin in human umbilical vein endothelial cells (HUVEC). We then investigated the role of the MAPKs (ERK1/2, JNK, and p38) and AP‐1 and the role of actin cytoskeleton reorganization in the CSE‐induced expression of ICAM‐1 and E‐selectin. Western blot analysis showed that CSE treatment rapidly and significantly caused phosphorylation of JNK and ERK1/2 but not of p38. Cytochalasin D (an actin filament disruptor) partially inhibited CSE‐induced ICAM‐1 and E‐selectin surface expression. However, inhibitors of ERK1/2 (PD98059) and JNK (SP600125) did not attenuate the CSE‐induced ICAM‐1 and E‐selectin surface expression. The results of electrophoretic mobility shift assay showed that CSE enhanced AP‐1 binding activity. Therefore, CSE activated AP‐1 and upregulated ICAM‐1 and E‐selectin surface expression in HUVEC seem to be via an MAPK‐independent pathway. Moreover, the dynamic reorganization of the actin cytoskeleton seems to be required for the CSE‐induced surface expression of ICAM‐1 and E‐selectin. Environ. Mol. Mutagen., 2009.

Galangin ameliorates cisplatin-induced nephrotoxicity by attenuating oxidative stress, inflammation and cell death in mice through inhibition of ERK and NF-kappaB signaling

&NA; Cisplatin is a chemotherapeutic agent widely used in the treatment of various cancers. However, cisplatin can induce nephrotoxicity and neurotoxicity, limiting its dosage and usage. Galangin, a natural flavonol, has been found to exhibit anti‐oxidant and anti‐inflammatory effects in vivo. Here, we investigated the effects of galangin on cisplatin‐induced acute kidney injury (AKI) and its molecular mechanisms in mice. Galangin administration reduced the cisplatin‐induced oxidative stress by decreasing renal MDA and 3‐NT formations. Galangin administration also increased renal anti‐oxidative enzyme activities (SOD, GPx, and CAT) and GSH levels depleted by cisplatin. Furthermore, galangin administration inactivated stress‐induced Nrf2 protein and its downstream products, HO‐1 and GCLC. In terms of the inflammatory response, galangin administration reduced I&kgr;B&agr; phosphorylation, NF‐&kgr;B phosphorylation and nuclear translocation, and then inhibited cisplatin‐induced secretions of pro‐inflammatory TNF‐&agr;, IL‐1&bgr; and IL‐6. In addition, cisplatin‐induced ERK and p38 phosphorylations were inhibited by galangin administration. In terms of cell death, galangin administration reduced levels of p53, pro‐apoptotic Bax and activated caspase‐3 to inhibit the cisplatin‐induced apoptosis. Galangin administration also reduced the expression levels of RIP1 and RIP3 to inhibit cisplatin‐induced RIP1/RIP3‐dependent necroptosis. Therefore, galangin administration significantly ameliorates cisplatin‐induced nephrotoxicity by attenuating oxidative stress, inflammation, and cell death through inhibitions of ERK and NF‐&kgr;B signaling pathways. Galangin might be a potential adjuvant for clinical cisplatin therapy. HighlightsGalangin alleviates cisplatin‐induced acute kidney injury through ERK and NF‐kappaB signaling pathways.Galangin reduces cisplatin‐induced oxidative stress and inflammatory response.Galangin suppresses cisplatin‐induced cell death, apoptosis and necroptosis.

Docosahexaenoic acid inhibits 12- O -tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

Fascin-1, an actin-bundling protein, plays an important role in cancer cell migration and invasion; however, the underlying mechanism remains unclear. On the basis of a 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell migration model, it was shown that TPA increased fascin-1 mRNA and protein expression and fascin-1-dependent cell migration. TPA dose- and time-dependently increased PKCδ and STAT3α activation and GSK3β phosphorylation; up-regulated Wnt-1, β-catenin, and STAT3α expression; and increased the nuclear translocation of β-catenin and STAT3α. Rottlerin, a PKCδ inhibitor, abrogated the increases in STAT3α activation and β-catenin and fascin-1 expression. WP1066, a STAT3 inhibitor, suppressed TPA-induced STAT3α DNA binding activity and β-catenin expression. Knockdown of β-catenin attenuated TPA-induced fascin-1 and STAT3α expression as well as cell migration. In addition to MCF-7, migration of Hs578T breast cancer cells was inhibited by silencing fascin-1, β-catenin, and STAT3α expression as well. TPA also induced Wnt-1 expression and secretion, and blocking Wnt-1 signaling abrogated β-catenin induction. DHA pretreatment attenuated TPA-induced cell migration, PKCδ and STAT3α activation, GSK3β phosphorylation, and Wnt-1, β-catenin, STAT3α, and fascin-1 expression. Our results demonstrated that TPA-induced migration is likely associated with the PKCδ and Wnt-1 pathways, which lead to STAT3α activation, GSK3β inactivation, and β-catenin increase and up-regulation of fascin-1 expression. Moreover, the anti-metastatic potential of DHA is partly attributed to its suppression of TPA-activated PKCδ and Wnt-1 signaling.

IP3 and calcium signaling involved in the reorganization of the actin cytoskeleton and cell rounding induced by cigarette smoke extract in human endothelial cells.

Smoking increases the risk of cardiovascular disorders and leads to damage caused by inflammation and oxidative stress. The actin cytoskeleton is a key player in the response to inflammatory stimuli and is an early target of cellular oxidative stress. The purpose of this study was to investigate the changes in actin cytoskeleton dynamics in human endothelial EA.hy926 cells exposed to cigarette smoke extract (CSE). Immunostaining revealed that CSE exposure resulted in modification of the actin cytoskeleton and led to cell rounding in a dose‐ and time‐dependent manner. In addition, the intracellular calcium concentration was increased by treatment with CSE. Pretreatment with antioxidants (lipoic acid, glutathione, N‐acetyl cysteine, aminoguanidine, α‐tocopherol, and vitamin C) significantly attenuated the CSE‐induced actin cytoskeleton reorganization and cell rounding. Calcium ion chelators (EGTA, BAPTA‐AM AM) and a potent store‐operated calcium channel inhibitor (MRS 1845) also reduced CSE‐induced intracellular calcium changes and attenuated actin cytoskeleton reorganization and cell morphology change. Moreover, the CSE‐induced intracellular calcium increase was suppressed by pretreatment with the inositol trisphosphate receptor (IP3R) inhibitor xestospongin C, the phospholipase C (PLC) inhibitor U‐73122, and the protein kinase C (PKC) inhibitor GF109203X. These results suggest that reactive oxygen species production and intracellular calcium increase play an essential role in CSE‐induced actin disorganization and cell rounding through a PLC–IP3–PKC signaling pathway.

Treatment with Caffeic Acid and Resveratrol Alleviates Oxidative Stress Induced Neurotoxicity in Cell and Drosophila Models of Spinocerebellar Ataxia Type3

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a polyglutamine (polyQ) repeat in the protein ataxin-3 which is involved in susceptibility to mild oxidative stress induced neuronal death. Here we show that caffeic acid (CA) and resveratrol (Res) decreased reactive oxygen species (ROS), mutant ataxin-3 and apoptosis and increased autophagy in the pro-oxidant tert-butyl hydroperoxide (tBH)-treated SK-N-SH-MJD78 cells containing mutant ataxin-3. Furthermore, CA and Res improved survival and locomotor activity and decreased mutant ataxin-3 and ROS levels in tBH-treated SCA3 Drosophila. CA and Res also altered p53 and nuclear factor-κB (NF-κB) activation and expression in tBH-treated cell and fly models of SCA3, respectively. Blockade of NF-κB activation annulled the protective effects of CA and Res on apoptosis, ROS, and p53 activation in tBH-treated SK-N-SH-MJD78 cells, which suggests the importance of restoring NF-κB activity by CA and Res. Our findings suggest that CA and Res may be useful in the management of oxidative stress induced neuronal apoptosis in SCA3.

Borage oil supplementation decreases lipopolysaccharide-induced inflammation and skeletal muscle wasting in mice

Because in vitro data have shown gamma-linolenic acid (GLA) to be protective in LPS-induced macrophage inflammation and myotube atrophy, we explored the therapeutic value of borage oil (BO), a GLA rich oil, in LPS-induced inflammation and muscle wasting in C57BL/6JNarl mice. Supplementation with BO was more potent than supplementation with soybean oil (SO) in decreasing LPS-induced expression of pro-inflammatory cytokines and glutathione in serum and tissues. Notably, GLA did not reverse LPS-induced inflammatory cytokine expression in C2C12 myotubes transfected with a constitutively active mutant IκB kinase-β plasmid, which suggested the importance of the inhibition of nuclear factor-κB (NF-κB) activation by GLA. Moreover, BO prevented LPS-induced skeletal muscle weight loss as well as molecule expression of ubiquitin-proteasome pathway and the autophagy-lysosomal pathway which played a key role in skeletal muscle protein degradation. BO but not SO reduced the LPS-induced increase in toll-like receptor 4 (TLR4) expression and activation of mitogen-activated protein kinases (MAPKs) and NF-κB in gastrocnemius muscle. In summary, supplementation with BO is more effective than supplementation with SO in preventing LPS-induced inflammation and muscle wasting. Blockade of the TLR4/MAPKs/NF-κB pathway is crucial in the action of BO on LPS-induced inflammation and wasting in skeletal muscle.

18-Carbon polyunsaturated fatty acids via down-regulation NF-κB activation reduce lipopolysaccharide-induced myotube atrophy

Muscle atrophy often occurs in cachexia of various inflammation-related diseases. Systemic inflammation and the inflammatory pathway contribute to cachexia-induced muscle atrophy, which is associated with activation of two major protein degradation systems, the ubiquitin–proteasome pathway (UPP) and the autophagy–lysosomal pathway (ALP). The aim of this study was to explore the effects of linoleic acid, alpha-linolenic acid, and gamma-linolenic acid, 18-carbon polyunsaturated fatty acids (PUFAs), on lipopolysaccharide (LPS)-induced myotube atrophy and possible mechanisms of these actions. Our data demonstrated that these three test 18-carbon PUFAs significantly inhibited LPS-induced C2C12 myotube atrophy as well as activation of UPP and ALP, as evidenced by decreases in LPS-induced expression of muscle-specific ring finger protein 1, protein ubiquitination, and microtubule-associated protein 1 light chain 3B. Moreover, the 18-carbon PUFAs diminished LPS-induced expression of pro-inflammatory cytokines and phosphorylated mitogen-activated protein kinases as well as the transcriptional activity of nuclear factor-κB (NF-κB). Notably, the 18-carbon PUFAs did not reverse LPS-induced atrophy in C2C12 myotubes transfected with a constitutively active mutant IκB kinase-β plasmid, which suggests the importance of the inhibition of NF-κB activation by the 18-carbon PUFAs. Our findings suggest that the 18-carbon PUFAs are beneficial for improving cachexia-induced myotube atrophy in inflammation-related diseases.