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O33 Oxidative stress induced both apoptosis and necroptosis in endothelial cells
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Abstracts / Biochemical Pharmacology 139 (2017) 105–141
on aorta in comparison to 3-(3-hydroxyphenyl) propionic acid. Some colonic metabolites of quercetin are able to decrease the arterial blood pressure in rats. Future experiments are necessary to verify whether these effects can be long-lasting and clinically relevant. doi:10.1016/j.bcp.2017.06.096
O32 Curcumin induces expression of 15-hydroxyprostaglandin dehydrogenase in normal gastric mucosa RGM-1 cells and mouse stomach in vivo: A potential role of AP-1 Jeong-Hwa Woo a, Jong-Min Park b, Young-Joon Surh b, Hye-Kyung Na a a Sungshin Women’s University, Seoul, Republic of Korea, b Seoul National University, Seoul, Republic of Korea Overproduction of prostaglandin E2 (PGE2) has been reported to be implicated in carcinogenesis. The intracellular level of PGE2 is regulated not only by its biosynthesis, but also by the degradation process. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme that catalyzes the inactivation of PGE2. In the present study, we found that curcumin, a yellow coloring agent present in the rhizome of Curcuma longa Linn (Zingiberaceae), induced expression of 15-PGDH at both the protein and mRNA levels in normal rat gastric mucosa cells (RGM-1). By using deletion constructs of the 15-PGDH promoter, we have found that activator protein-1 (AP-1) is the most essential transcription factor responsible for curcumin-induced upregulation of 15-PGDH expression. In addition, oral administration of curcumin increased the expression of 15-PGDH in the mouse stomach. Curcumin enhanced the expression of c-Jun and c-Fos in the nuclear fraction of RGM-1 cells and the mouse stomach in vivo. Knockdown of c-jun, a gene encoding one of the major components of AP-1, suppressed curcumin-induced expression of 15-PGDH. In addition, curcumin increased phosphorylation of ERK1/2 and JNK. Curcumin-induced 15-PGDH expression was abrogated by the pharmacological inhibition of the aforementioned kinases. In contrast, tetrahydrocurcumin which lacks the a,b-unsaturated carbonyl group failed to induce expression of 15-PGDH, suggesting that the electrophilic a,b-unsaturated carbonyl group of curcumin is essential for its induction of 15-PGDH expression in RGM-1 cells. Taken together, our study suggests that curcumin-induced upregulation of 15-PGDH in RGM-1 cells and mouse stomach through activation of AP-1 may contribute to chemopreventive effects of this phytochemical on inflammation-associated gastric carcinogenesis. doi:10.1016/j.bcp.2017.06.097
O33 Oxidative stress induced both apoptosis and necroptosis in endothelial cells Xiuping Chen, Wenwen Zhao University of Macau, Macau, China Oxidative stress plays an essential role in mediating endothelial cell death while the underlying mechanisms remain elusive. Previous reports suggested that ROS overproduction induced both apoptosis and necrosis. Herein, the pro-death effect of tert-butyl hydroperoxide (t-BHP), an alternative for H2O2, on human umbilical vein endothelial cells was investigated with a low concentration (50 lM) of t-BHP (t-BHPL) and a high concentration (500 lM) of tBHP (t-BHPH). T-BHPL induced caspase-dependent apoptosis and ROS generation, which was inhibited by N-acetyl-L-cysteine (NAC). Furthermore, NADPH oxidase inhibitor diphenyleneiodonium (DPI),
NOX4 siRNA, and NOX4 inhibitor GKT137831 reduced t-BHPLinduced ROS generation while mitochondrial respiratory chain inhibitors rotenone (Rot), 2-thenoyltrifluoroacetone (TTFA), and antimycin A (AA) failed to do so. NOX4 overexpression resulted in increased ROS generation and Akt expression but decreased sensitivity to tBHPL. In contrast, RIP1 inhibitor necrostatin-1, MLKL inhibitor necrosulfonamide and silencing RIP1, RIP3, and MLKL inhibited t-BHPHinduced cell death while pan-caspase inhibitor Z-VAD-FMK showed no effect. t-BHPH-induced ROS production was inhibited by TTFA, AA and Rot while DPI showed no effect. T-BHPH induced RIP1/RIP3 interaction, which was decreased by Rot, TTFA, and AA. Silencing RIP1 and RIP3, but not MLKL, inhibited t-BHPH-induced mitochondrial membrane potential (MMP) decrease and ROS production. In summary, t-BHP induced both apoptosis and necroptosis in endothelial cells which was mediated by ROS. ROS derived from NADPH oxidase and mitochondria contributed to t-BHPL and t-BHPH-induced apoptosis and necroptosis, respectively. Acknowledgments: This study was supported by the Science and Technology Development Fund of Macau (FDCT) (078/2016/A2) and the Research Fund of University of Macau (MYRG2016-00043ICMS-QRCM). doi:10.1016/j.bcp.2017.06.098
Session 12: Translating novel pathways – Cardiovascular disease I O34 Endothelium-independent vasorelaxant effect of Thymus linearis via inhibition of phosphodiesterases in porcine coronary artery Alamgeer Alamgeer a, Cyril Auger b, P. Chabert b, C. Lugnier b, V.B. Schini-Kerth b a University of Sargodha, Sargodha, Pakistan, b University of Strasbourg, Strasbourg, France Thymus linearis Benth. indigenous to Pakistan has been traditionally used for the treatment of various diseases including hypertension. The aim of the study was to investigate the vasorelaxant effect of Thymus linearis in porcine coronary artery rings. Aqueousmethanolic extract and various fractions (butanol, ethyacetate and aqueous) of Thymus linearis were prepared. The effects of extract and fractions were assessed using coronary artery rings and various pharmacological tools were applied to characterize the underlying mechanism of active fraction (butanol). The inhibitory effect of butanolic fraction was also studied on phosphodiesterases using radio enzymatic assay. Phytochemical studies were also carried out using GCMS. The aqueous-methanolic extract of Thymus linearis elicited similar relaxations in coronary artery rings with and without endothelium in a dose-dependent manner. Among the fractions butanolic fraction of Thymus linearis (TLB) was found to be the most potent. TLB significantly potentiated the relaxation induced by forskolin, isoproterenol, sodium nitroprusside and atrial natriuretic peptide whereas the relaxation induced by YC-1, BAY 41-2272, levcromakalim and 1-EBIO was unaffected by TLB. Pretreatment with TLB inhibited the contraction induced by KCl, CaCl2 and U46619 in endothelium denuded coronary artery rings. Furthermore, TLB significantly inhibited the PDE activity (PDE1CaM activated PDE2, PDE3, PDE4 AND PDE5) in a concentration-dependent manner. GCMS analysis revealed the presence of thymol and Carvacrolas as major constituents. In conclusion, TLB induced endothelium-independent relaxations in porcine coronary artery rings by acting directly at the vascular smooth muscle to increase concentrations of both cyclic AMP and cyclic GMP via inhibition of various PDEs. doi:10.1016/j.bcp.2017.06.099
Abstracts / Biochemical Pharmacology 139 (2017) 105–141
on aorta in comparison to 3-(3-hydroxyphenyl) propionic acid. Some colonic metabolites of quercetin are able to decrease the arterial blood pressure in rats. Future experiments are necessary to verify whether these effects can be long-lasting and clinically relevant. doi:10.1016/j.bcp.2017.06.096
O32 Curcumin induces expression of 15-hydroxyprostaglandin dehydrogenase in normal gastric mucosa RGM-1 cells and mouse stomach in vivo: A potential role of AP-1 Jeong-Hwa Woo a, Jong-Min Park b, Young-Joon Surh b, Hye-Kyung Na a a Sungshin Women’s University, Seoul, Republic of Korea, b Seoul National University, Seoul, Republic of Korea Overproduction of prostaglandin E2 (PGE2) has been reported to be implicated in carcinogenesis. The intracellular level of PGE2 is regulated not only by its biosynthesis, but also by the degradation process. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme that catalyzes the inactivation of PGE2. In the present study, we found that curcumin, a yellow coloring agent present in the rhizome of Curcuma longa Linn (Zingiberaceae), induced expression of 15-PGDH at both the protein and mRNA levels in normal rat gastric mucosa cells (RGM-1). By using deletion constructs of the 15-PGDH promoter, we have found that activator protein-1 (AP-1) is the most essential transcription factor responsible for curcumin-induced upregulation of 15-PGDH expression. In addition, oral administration of curcumin increased the expression of 15-PGDH in the mouse stomach. Curcumin enhanced the expression of c-Jun and c-Fos in the nuclear fraction of RGM-1 cells and the mouse stomach in vivo. Knockdown of c-jun, a gene encoding one of the major components of AP-1, suppressed curcumin-induced expression of 15-PGDH. In addition, curcumin increased phosphorylation of ERK1/2 and JNK. Curcumin-induced 15-PGDH expression was abrogated by the pharmacological inhibition of the aforementioned kinases. In contrast, tetrahydrocurcumin which lacks the a,b-unsaturated carbonyl group failed to induce expression of 15-PGDH, suggesting that the electrophilic a,b-unsaturated carbonyl group of curcumin is essential for its induction of 15-PGDH expression in RGM-1 cells. Taken together, our study suggests that curcumin-induced upregulation of 15-PGDH in RGM-1 cells and mouse stomach through activation of AP-1 may contribute to chemopreventive effects of this phytochemical on inflammation-associated gastric carcinogenesis. doi:10.1016/j.bcp.2017.06.097
O33 Oxidative stress induced both apoptosis and necroptosis in endothelial cells Xiuping Chen, Wenwen Zhao University of Macau, Macau, China Oxidative stress plays an essential role in mediating endothelial cell death while the underlying mechanisms remain elusive. Previous reports suggested that ROS overproduction induced both apoptosis and necrosis. Herein, the pro-death effect of tert-butyl hydroperoxide (t-BHP), an alternative for H2O2, on human umbilical vein endothelial cells was investigated with a low concentration (50 lM) of t-BHP (t-BHPL) and a high concentration (500 lM) of tBHP (t-BHPH). T-BHPL induced caspase-dependent apoptosis and ROS generation, which was inhibited by N-acetyl-L-cysteine (NAC). Furthermore, NADPH oxidase inhibitor diphenyleneiodonium (DPI),
NOX4 siRNA, and NOX4 inhibitor GKT137831 reduced t-BHPLinduced ROS generation while mitochondrial respiratory chain inhibitors rotenone (Rot), 2-thenoyltrifluoroacetone (TTFA), and antimycin A (AA) failed to do so. NOX4 overexpression resulted in increased ROS generation and Akt expression but decreased sensitivity to tBHPL. In contrast, RIP1 inhibitor necrostatin-1, MLKL inhibitor necrosulfonamide and silencing RIP1, RIP3, and MLKL inhibited t-BHPHinduced cell death while pan-caspase inhibitor Z-VAD-FMK showed no effect. t-BHPH-induced ROS production was inhibited by TTFA, AA and Rot while DPI showed no effect. T-BHPH induced RIP1/RIP3 interaction, which was decreased by Rot, TTFA, and AA. Silencing RIP1 and RIP3, but not MLKL, inhibited t-BHPH-induced mitochondrial membrane potential (MMP) decrease and ROS production. In summary, t-BHP induced both apoptosis and necroptosis in endothelial cells which was mediated by ROS. ROS derived from NADPH oxidase and mitochondria contributed to t-BHPL and t-BHPH-induced apoptosis and necroptosis, respectively. Acknowledgments: This study was supported by the Science and Technology Development Fund of Macau (FDCT) (078/2016/A2) and the Research Fund of University of Macau (MYRG2016-00043ICMS-QRCM). doi:10.1016/j.bcp.2017.06.098
Session 12: Translating novel pathways – Cardiovascular disease I O34 Endothelium-independent vasorelaxant effect of Thymus linearis via inhibition of phosphodiesterases in porcine coronary artery Alamgeer Alamgeer a, Cyril Auger b, P. Chabert b, C. Lugnier b, V.B. Schini-Kerth b a University of Sargodha, Sargodha, Pakistan, b University of Strasbourg, Strasbourg, France Thymus linearis Benth. indigenous to Pakistan has been traditionally used for the treatment of various diseases including hypertension. The aim of the study was to investigate the vasorelaxant effect of Thymus linearis in porcine coronary artery rings. Aqueousmethanolic extract and various fractions (butanol, ethyacetate and aqueous) of Thymus linearis were prepared. The effects of extract and fractions were assessed using coronary artery rings and various pharmacological tools were applied to characterize the underlying mechanism of active fraction (butanol). The inhibitory effect of butanolic fraction was also studied on phosphodiesterases using radio enzymatic assay. Phytochemical studies were also carried out using GCMS. The aqueous-methanolic extract of Thymus linearis elicited similar relaxations in coronary artery rings with and without endothelium in a dose-dependent manner. Among the fractions butanolic fraction of Thymus linearis (TLB) was found to be the most potent. TLB significantly potentiated the relaxation induced by forskolin, isoproterenol, sodium nitroprusside and atrial natriuretic peptide whereas the relaxation induced by YC-1, BAY 41-2272, levcromakalim and 1-EBIO was unaffected by TLB. Pretreatment with TLB inhibited the contraction induced by KCl, CaCl2 and U46619 in endothelium denuded coronary artery rings. Furthermore, TLB significantly inhibited the PDE activity (PDE1CaM activated PDE2, PDE3, PDE4 AND PDE5) in a concentration-dependent manner. GCMS analysis revealed the presence of thymol and Carvacrolas as major constituents. In conclusion, TLB induced endothelium-independent relaxations in porcine coronary artery rings by acting directly at the vascular smooth muscle to increase concentrations of both cyclic AMP and cyclic GMP via inhibition of various PDEs. doi:10.1016/j.bcp.2017.06.099