Shu-Fen Liou

Inhibition of Proinflammatory Tumor Necrosis Factor-α-Induced Inducible Nitric-Oxide Synthase by Xanthine-Based 7-[2-[4-(2-Chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-1) and 7-[2-[4-(4-Nitrobenzene)piperazinyl]ethyl]-1, 3-dimethylxanthine (KMUP-3) in Rat Trachea: The Involvement of Soluble Guanylate Cyclase and Protein Kinase G

In the study of anti-proinflammation by 7-[2-[4-(2-chlorobenzene)piperazinyl] ethyl]-1,3-dimethylxanthine (KMUP-1) and 7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-3), exposure of rat tracheal smooth muscle cells (TSMCs) to tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, increased the expression of inducible nitric-oxide synthase (iNOS) and NO production and decreased the expression of soluble guanylate cyclase α1 (sGCα1), soluble guanylate cyclase β1 (sGCβ1), protein kinase G (PKG), and the release of cGMP in TSMCs. The cell-permeable cGMP analog 8-Br-cGMP, xanthine-based KMUP-1 and KMUP-3, and the phosphodiesterase 5 inhibitor zaprinast all inhibited TNF-α-induced increases of iNOS expression and NO levels and reversed TNF-α-induced decreases of sGCα1, sGCβ1, and PKG expression. These results imply that cGMP enhancers could have anti-proinflammatory potential in TSMCs. TNF-α also increased protein kinase A (PKA) expression and cAMP levels, cyclooxygenase-2 (COX-2) expression, and activated productions of prostaglandin (PG) E2 and 6-keto-PGF1α (stable PGI2 metabolite). Dexamethasone and N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398; a selective COX-2 inhibitor) attenuated TNF-α-induced expression of COX-2 and activated productions PGE2 and PGI2. However, KMUP-1 and KMUP-3 did not affect COX-2 activities and did not further enhance cAMP levels in the presence of TNF-α. It is suggested that TNF-α-induced increases of PKA expression and cAMP levels are mediated by releasing PGE2 and PGI2, the activation products of COX-2. In conclusion, xanthine-based KMUP-1 and KMUP-3 inhibit TNF-α-induced expression of iNOS in TSMCs, involving the sGC/cGMP/PKG expression pathway but without the involvement of COX-2.

KMUP‐1 attenuates isoprenaline‐induced cardiac hypertrophy in rats through NO/cGMP/PKG and ERK1/2/calcineurin A pathways

Background and purpose:  To determine whether KMUP‐1, a novel xanthine‐based derivative, attenuates isoprenaline (ISO)‐induced cardiac hypertrophy in rats, and if so, whether the anti‐hypertrophic effect is mediated by the nitric oxide (NO) pathway.

Inhibition of human prostate cancer cells proliferation by a selective alpha1-adrenoceptor antagonist labedipinedilol-A involves cell cycle arrest and apoptosis

In this research, we conducted an in vitro analysis to evaluate the prostate cancer cells response to labedipinedilol-A in order to determine the effect of this selective alpha(1)-adrenoceptor antagonist to suppress prostate cancer cell growth by affecting cell proliferation and apoptosis. Here, we report that treatment of androgen-sensitive (LNCaP) and androgen-insensitive (PC-3) prostate cancer cells with labedipinedilol-A inhibited cell proliferation in concentration-dependent and time-dependent manners. Moreover, norepinephrine-stimulated proliferation of both cell lines are markedly inhibited by labedipinedilol-A. The probable involvement of alpha(1)-adrenoceptors in this cellular response is suggested. Labedipinedilol-A-induced growth inhibition was associated with G(0)/G(1) arrest, and G(2)/M arrest depending upon concentrations. Cell cycle blockade was associated with reduced amounts of cyclin D1/2, cyclin E, Cdk2, Cdk4, and Cdk6 and increased levels of the Cdk inhibitory proteins (Cip1/p21 and Kip1/p27). In addition, labedipinedilol-A also induced apoptosis in PC-3 cells, as determined by using Hoechst 33342 staining, DNA fragmentation, and Annexin V staining assay. Furthermore, labedipinedilol-A triggered the mitochondrial apoptotic pathway, as indicated by increasing the expression of Bax, but decreasing the level of Bcl-2, resulting in mitochondrial membrane potential loss, cytochrome c release, and activation of caspase-9 and -3. We further investigated the role of MAPK cascades in the anti-proliferative and apoptosis effects of labedipinedilol-A, and confirmed that labedipinedilol-A could activate JNK1/2 but not p38 in both cell lines. Unlike JNK1/2, however, labedipinedilol-A treatment resulted in down-regulation of phospho-ERK1/2 expression. We concluded that labedipinedilol-A possessed the growth-suppressive and apoptotic effects on LNCaP and PC-3 cells by its alpha(1)-adrenoceptor blockade, and the apoptotic effects of labedipinedilol-A primarily through caspases and MAPKs mediated pathways.

Lercanidipine inhibits vascular smooth muscle cell proliferation and neointimal formation via reducing intracellular reactive oxygen species and inactivating Ras-ERK1/2 signaling.

Lercanidipine, a calcium channel antagonist, is currently employed in the treatment of essential hypertension and angina pectoris. The purpose of this study was to elucidate the anti-proliferative effect of lercanidipine and to investigate the molecular role of this agent. Both in vitro studies and in a balloon injury rat carotid artery model were employed to study the effect of lercanidipine on smooth muscle cell proliferation. Lercanidipine-inhibited rat vascular smooth muscle cell (VSMC) proliferation and migration in a dose-dependent manner following stimulation of VSMC cultures with 10% fetal bovine serum (FBS) and 20 ng/ml platelet-derived growth factor (PDGF)-BB. FBS- and PDGF-BB-stimulated intracellular Ras, MEK1/2, ERK1/2, proliferative cell nuclear antigen (PCNA), and Akt activations were significantly inhibited by lercanidipine; however, lercanidipine did not affect FBS- and PDGF-BB-induced STAT3 phosphorylation. Lercanidipine also inhibited PDGF-receptor beta chain phosphorylation and reactive oxygen species (ROS) production induced by PDGF-BB. Lercanidipine blocked the FBS-inducible progression through the G(0)/G(1) to the S-phase of the cell cycle in synchronized cells. In vivo, 14 days after balloon injury, treatment with 3 and 10 mg/kg lercanidipine resulted in significant inhibition of the neointima/media ratio. Suppression of neointima formation by lercanidipine was dependent on its influence on ERK1/2 phosphorylation. These results demonstrate that lercanidipine can suppress the proliferation of VSMCs via inhibiting cellular ROS, Ras-MEK1/2-ERK1/2, and PI3K-Akt pathways, and suggesting that it may have therapeutic relevance in the prevention of human restenosis.

Baicalein, an active component of Scutellaria baicalensis Georgi, prevents lysophosphatidylcholine-induced cardiac injury by reducing reactive oxygen species production, calcium overload and apoptosis via MAPK pathways.

BackgroundLysophosphatidylcholine (lysoPC), a metabolite from membrane phospholipids, accumulates in the ischemic myocardium and plays an important role in the development of myocardial dysfunction ventricular arrhythmia. In this study, we investigated if baicalein, a major component of Huang Qui, can protect against lysoPC-induced cytotoxicity in rat H9c2 embryonic cardiomyocytes.MethodsCell viability was detected by the MTT assay; ROS levels were assessed using DCFH-DA; and intracellular free calcium concentrations were assayed by spectrofluorophotometer. Cell apoptosis and necrosis were evaluated by the flow cytometry assay and Hoechst staining. Mitogen-Activated Protein Kinases (MAPKs), which included the ERK, JNK, and p38, and the apoptotic mechanisms including Bcl-2/Bax, caspase-3, caspase-9 and cytochrome c pathways were examined by Western blot analysis. The activation of MAPKs was examined by enzyme-linked immunosorbent assay.ResultsWe found that lysoPC induced death and apoptosis of H9c2 cells in a dose-dependent manner. Baicalein could prevent lysoPC-induced cell death, production of reactive oxygen species (ROS), and increase of intracellular calcium concentration in H9c2 cardiomyoctes. In addition, baicalein also inhibited lysoPC-induced apoptosis, with associated decreased pro-apoptotic Bax protein, increased anti-apoptotic Bcl-2 protein, resulting in an increase in the Bcl-2/Bax ratio. Finally, baicalein attenuated lysoPC-induced the expression of cytochrome c, casapase-3, casapase-9, and the phosphorylations of ERK1/2, JNK, and p38. LysoPC-induced ERK1/2, JNK, and p38 activations were inhibited by baicalein.ConclusionsBaicalein protects cardiomyocytes from lysoPC-induced apoptosis by reducing ROS production, inhibition of calcium overload, and deactivations of MAPK signaling pathways.

Inhibition of mitogen-mediated proliferation of rat vascular smooth muscle cells by labedipinedilol-A through PKC and ERK 1/2 pathway.

Labedipinedilol-A is a novel 1, 4-dihydropyridine type calcium antagonist with alpha-receptor blocking activity. This study investigates the effects of labedipinedilol-A on mitogen-induced proliferation of rat vascular smooth muscle cells (VSMCs). Labedipinedilol-As inhibition on cell proliferation was measured by the tetrazolium salt (XTT) test. Labedipinedilol-A dose-dependently inhibited mitogen-induced DNA synthesis, determined by the incorporation of 5-bromo-2′-deoxyuridine (BrdU). Labedipinedilol-A was also found capable of inhibiting the migration of VSMCs induced by PDGF-BB with an IC50 value of 5.6 μM. In accordance with these findings, labedipinedilol-A revealed blocking of the FBS-inducible progression through G0/G1 to S phase of the cell cycle in synchronized cells. Labedipinedilol-A appeared to cause inhibition of mitogens-induced PKC translocation, suggesting the probable involvement of protein kinase C (PKC) in this cellular response. Labedipinedilol-A reduced both intracellular Ca2+ and the phosphorylation of extracellular signal-regulated protein kinase 1/2 in PDGF-BB-stimulated VSMCs. It also suppressed the levels of proliferative cell nuclear antigen (PCNA) in VSMCs both time- and dose-dependently. These results indicate that labedipinedilol-A may inhibit cell proliferation by attenuating activation of the ERK 1/2 pathway, which is regulated by PKC and Ca2+, suggesting that it may have great potential in the prevention of progressive atherosclerosis.

San-Huang-Xie-Xin-Tang Prevents Rat Hearts from Ischemia/Reperfusion-Induced Apoptosis through eNOS and MAPK Pathways

San-Huang-Xie-Xin-Tang (SHXT) is a traditional Chinese medication consisting of three herbs, namely Coptidis rhizome, Scutellariae radix and Rhei rhizome. This study aimed to examine the cardioprotective effects of SHXT in a rat model of acute myocardial apoptosis induced by ischemia/reperfusion (I/R). Vehicle (intravenous saline) or SHXT (intravenous or oral) was administered prior to I/R (occlusion of left coronary artery for 45 min followed by reperfusion for 2 h). In the vehicle group, myocardial I/R caused myocardial infarction with increased plasma cardiac enzymes, severe arrhythmia and mortality. Myocardial apoptosis was induced by I/R as evidenced by DNA ladder and Bcl-2/Bax ratio. In the SHXT group, we found that SHXT significantly reduced plasma levels of cardiac enzymes, arrhythmia scores (from 5 ± 1 to 2 ± 1, P < .01) and mortality rate (from 53 to 0%, P < .01). In addition, pretreatment with intravenous SHXT reduced the infarct size dose-dependently when compared with the vehicle group (10 mg kg−1: 14.0 ± 0.2 versus 44.5 ± 5.0%, and 30 mg kg−1: 6.2 ± 1.2% versus 44.5 ± 5.0%, both P < .01). Similarly, oral administration of SHXT reduced the infarct size dose-dependently. Furthermore, SHXT markedly decreased the apoptosis induced by I/R with increased Bcl-2/Bax ratio. Finally, we found that SHXT counteracted the I/R-induced downstream signaling, resulting in increased myocardial eNOS expression and plasma nitrite, and decreased activation of ERK1/2, p38 and JNK. These data suggest that SHXT has cardioprotective effects against I/R-induced apoptosis, and that these effects are mediated, at least in part, by eNOS and MAPK pathways.

Labedipinedilol-A prevents lysophosphatidylcholine-induced vascular smooth muscle cell death through reducing reactive oxygen species production and anti-apoptosis

OBJECTIVE Labedipinedilol-A, a novel calcium antagonist, has been previously demonstrated to have pleiotropic protective effects in the cardiovascular system. This study aimed to investigate its cytoprotective effects in rat vascular smooth muscle cells (VSMCs) treated with lysophosphatidylcholine (lysoPC), a key lipid component mediating atherogenesis. METHODS AND RESULTS VSMCs were incubated with lysoPC with or without labedipinedilol-A pretreatment to determine its effects on lysoPC-induced cell death, Ca(2+) influx, oxidative stress, MAPK signaling and apoptosis. Labedipinedilol-A attenuated lysoPC-induced cell death and Ca(2+) influx. It also reduced reactive oxygen species (ROS) production evoked by lysoPC and down-regulated expressions of NAD(P)H oxidase subunits, Nox1 and Rac1. Moreover, it inhibited lysoPC-induced phosphorylation of MAPK including ERK1/2, JNK, and p38. It mitigated the dissipation of mitochondrial transmembrane potential induced by lysoPC. Lastly, labedipinedilol-A inhibited lysoPC-induced apoptosis with attenuation of caspase-3/-9 activations and modulation of Bax/Bcl-2 protein expressions. CONCLUSION Labedipinedilol-A can suppress lysoPC-induced VSMCs death via reducing ROS production and anti-apoptosis. These protective effects are potentially mediated through the inhibition of Ca(2+) influx, down-regulation of the NAD(P)H oxidase subunits (Nox1/Rac1) and MAPK signaling, and attenuation of mitochondrial depolarization. Thus, labedipinedilol-A may have a valuable role in the preventing atherosclerosis associated with hyperlipidemia.

KMUP-1 Suppresses RANKL-Induced Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss: Roles of MAPKs, Akt, NF-κB and Calcium/Calcineurin/NFATc1 Pathways

Background KMUP-1 is a xanthine derivative with inhibitory activities on the phosphodiesterase (PDE) 3,4 and 5 isoenzymes to suppress the degradation of cyclic AMP and cyclic GMP. However, the effects of KMUP-1 on osteoclast differentiation are still unclear. In this study, we investigated whether KMUP-1 inhibits osteoclastogenesis induced by RANKL in RAW 264.7 cells and bone loss induced by ovariectomy in mice, and the underlying mechanisms. Principal Findings In vitro, KMUP-1 inhibited RANKL-induced TRAP activity, the formation of multinucleated osteoclasts and resorption-pit formation. It also inhibited key mediators of osteoclastogenesis including IL-1β, IL-6, TNF-α and HMGB1. In addition, KMUP-1 inhibited RANKL-induced activation of signaling molecules (Akt, MAPKs, calcium and NF-κB), mRNA expression of osteoclastogensis-associated genes (TRAP, MMP-9, Fra-1, and cathepsin K) and transcription factors (c-Fos and NFATc1). Furthermore, most inhibitory effects of KMUP-1 on RANKL-mediated signal activations were reversed by a protein kinase A inhibitor (H89) and a protein kinase G inhibitor (KT5823). In vivo, KMUP-1 prevented loss of bone mineral content, preserved serum alkaline phosphate and reduced serum osteocalcin in ovariectomized mice. Conclusions KMUP-1 inhibits RANKL-induced osteoclastogenesis in vitro and protects against ovariectomy-induced bone loss in vivo. These effects are mediated, at least in part, by cAMP and cGMP pathways. Therefore, KMUP-1 may have a role in pharmacologic therapy of osteoporosis.

Isoeugenodilol inhibits smooth muscle cell proliferation and neointimal thickening after balloon injury via inactivation of ERK1/2 pathway

The purpose of this study was to determine the efficacy and the possible mechanism of action of the synthesized drug isoeugenodilol (a new third-generation beta-adrenoceptor blocker) on the growth factor-induced proliferation of cultured rat vascular smooth muscle cells (VSMCs) and neointimal formation in a rat carotid arterial balloon injury model. Isoeugenodilol significantly inhibited 10% FBS, 20 ng/ml PDGF-BB, and 20 ng/ml vascular endothelial growth factor (VEGF)-induced proliferation. In accordance with these findings, isoeugenodilol revealed blocking of the FBS-inducible progression through the G(0)/G(1) to the S phase of the cell cycle in synchronized cells. Neointimal formation, measured 14 days after injury, was reduced by the oral administration of isoeugenodilol (10 mg/kg/day). In an in vitro assay, isoeugenodilol inhibited the migration of VSMCs stimulated by PDGF-BB. These findings indicate that isoeugenodilol shows an inhibitory potency on neointimal formation due to inhibition of both migration and proliferation of VSMCs. In addition, isoeugenodilol in concentration-dependent manner decreased the levels of phosphorylated ERK1/2 in both VSMCs and balloon-injured carotid arteries. The levels of phosphorylated MEK1/2 and Pyk2 as well as intracellular Ca(2+) and reactive oxygen species (ROS) were in concentration-dependent manner reduced by isoeugenodilol. Taken together, these results indicate that isoeugenodilol may suppress mitogen-stimulated proliferation and migration partially through inhibiting cellular ROS and calcium, and hence, through activation of the Pyk2-ERK1/2 signal pathway. This suggests that isoeugenodilol has potential for the prevention of atherosclerosis and restenosis.