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Biochemical Pharmacology (v.84, #6)
Insulin resistance in the brain: An old-age or new-age problem? by Ritchie Williamson; Alison McNeilly; Calum Sutherland (pp. 737-745).
Life expectancy is rising however with more people living longer there is a concomitant rise in the incidence of dementia. In addition to age-related cognitive decline there is a higher risk of going on to develop vascular dementia and Alzheimer's disease associated with aspects of modern lifestyle. Most worryingly, recent data reports accelerated cognitive decline in adolescents associated with poor diet (high fat and calorie intake). Thus the increase in dementia in ‘old-age’ may have as much to do with ‘new-age’ lifestyle as it does with normal ageing. It would seem wise therefore to investigate the molecular connections between lifestyle and cognitive decline in more detail. Epidemiological evidence suggests an increased risk of developing dementia (including Alzheimer's disease) in individuals with obesity and type 2 diabetes but also in those with poor insulin sensitivity without diabetes, implicating a mechanistic link between adiposity, insulin sensitivity and dementia. Insulin receptors are expressed in the brain and physiological roles for insulin in the CNS are starting to be delineated. Indeed disrupted neuronal insulin action may underlie the link between diabetes and neurodegenerative disorders. This review discusses the difficulties in quantifying insulin sensitivity of the brain and why it is vital that we develop technology for this purpose so that we can establish its role in this ‘new-age’ dementia. This has particular relevance to the design and interpretation of clinical trials in progress to assess potential benefits of insulin and insulin sensitisers on prevention of cognitive decline.
Keywords: Diabetes; Dementia; Insulin resistance
Insulin resistance in the brain: An old-age or new-age problem? by Ritchie Williamson; Alison McNeilly; Calum Sutherland (pp. 737-745).
Life expectancy is rising however with more people living longer there is a concomitant rise in the incidence of dementia. In addition to age-related cognitive decline there is a higher risk of going on to develop vascular dementia and Alzheimer's disease associated with aspects of modern lifestyle. Most worryingly, recent data reports accelerated cognitive decline in adolescents associated with poor diet (high fat and calorie intake). Thus the increase in dementia in ‘old-age’ may have as much to do with ‘new-age’ lifestyle as it does with normal ageing. It would seem wise therefore to investigate the molecular connections between lifestyle and cognitive decline in more detail. Epidemiological evidence suggests an increased risk of developing dementia (including Alzheimer's disease) in individuals with obesity and type 2 diabetes but also in those with poor insulin sensitivity without diabetes, implicating a mechanistic link between adiposity, insulin sensitivity and dementia. Insulin receptors are expressed in the brain and physiological roles for insulin in the CNS are starting to be delineated. Indeed disrupted neuronal insulin action may underlie the link between diabetes and neurodegenerative disorders. This review discusses the difficulties in quantifying insulin sensitivity of the brain and why it is vital that we develop technology for this purpose so that we can establish its role in this ‘new-age’ dementia. This has particular relevance to the design and interpretation of clinical trials in progress to assess potential benefits of insulin and insulin sensitisers on prevention of cognitive decline.
Keywords: Diabetes; Dementia; Insulin resistance
The opioid growth factor–opioid growth factor receptor axis: Homeostatic regulator of cell proliferation and its implications for health and disease by Patricia J. McLaughlin; Ian S. Zagon (pp. 746-755).
The opioid growth factor (OGF), chemically termed [Met5]-enkephalin, is an endogenous opioid peptide that interacts with the OGF receptor (OGFr) to delay the G1/S interface of the cell cycle by modulating cyclin-dependent inhibitory kinase (CKI) pathways. The OGF–OGFr axis is a tonically active, inhibitory pathway that is an important regulator during homeostasis and re-epithelialization, and plays a role in the onset and progression of autoimmune diseases and cancer. Modulation of the OGF–OGFr axis can be accomplished by a variety of pharmacological and molecular approaches including use of intermittent or continuous exposure to the opioid antagonist naltrexone, genetic manipulation of OGFr expression, and antibody neutralization of OGF. Clinically, OGF is a biological therapy that has potential application for treatment of cancer. Currently, naltrexone at low dosages is being evaluated for treatment of autoimmune diseases such as Crohn's and multiple sclerosis. High dosages of naltrexone are effective in reversing dry eye and accelerating the repair of corneal abrasions in normal and diabetic rats; these studies are under investigation in the clinical setting. Naltrexone also enhances full-thickness wound closure in animal models of Type 1 or Type 2 diabetes, and translation of this knowledge to the clinic is planned. In summary, understanding the OGF–OGFr axis as a homeostatic regulator of proliferation has substantial implications for maintaining human health and treatment of disease.
Keywords: Enkephalins; Cyclin-dependent inhibitory kinases; DNA synthesis; Naltrexone; Dry eye; Wound repair; Cancer
The opioid growth factor–opioid growth factor receptor axis: Homeostatic regulator of cell proliferation and its implications for health and disease by Patricia J. McLaughlin; Ian S. Zagon (pp. 746-755).
The opioid growth factor (OGF), chemically termed [Met5]-enkephalin, is an endogenous opioid peptide that interacts with the OGF receptor (OGFr) to delay the G1/S interface of the cell cycle by modulating cyclin-dependent inhibitory kinase (CKI) pathways. The OGF–OGFr axis is a tonically active, inhibitory pathway that is an important regulator during homeostasis and re-epithelialization, and plays a role in the onset and progression of autoimmune diseases and cancer. Modulation of the OGF–OGFr axis can be accomplished by a variety of pharmacological and molecular approaches including use of intermittent or continuous exposure to the opioid antagonist naltrexone, genetic manipulation of OGFr expression, and antibody neutralization of OGF. Clinically, OGF is a biological therapy that has potential application for treatment of cancer. Currently, naltrexone at low dosages is being evaluated for treatment of autoimmune diseases such as Crohn's and multiple sclerosis. High dosages of naltrexone are effective in reversing dry eye and accelerating the repair of corneal abrasions in normal and diabetic rats; these studies are under investigation in the clinical setting. Naltrexone also enhances full-thickness wound closure in animal models of Type 1 or Type 2 diabetes, and translation of this knowledge to the clinic is planned. In summary, understanding the OGF–OGFr axis as a homeostatic regulator of proliferation has substantial implications for maintaining human health and treatment of disease.
Keywords: Enkephalins; Cyclin-dependent inhibitory kinases; DNA synthesis; Naltrexone; Dry eye; Wound repair; Cancer
Development and therapeutic impact of HDAC6-selective inhibitors by Sabrina Dallavalle; Claudio Pisano; Franco Zunino (pp. 756-765).
Histone deacetylases (HDAC) play a key role in regulating gene expression by deacetylating histones. Some HDAC isoforms can also modulate the function of nonhistone proteins implicated in regulatory processes, and therefore HDACs are recognized as useful targets for therapeutic purposes. HDAC inhibitors have generated substantial interest as antitumor agents, because they induce various cellular effects, including apoptosis, cell cycle arrest and inhibition of angiogenesis. The nature of cellular response likely depends on the biological context and on the pattern of HDAC isoform inhibition. Various HDAC inhibitors belonging to different structural classes have been developed. Many inhibitors are characterized by a pan-HDAC inhibitory profile. The potential advantages of isoform-selective inhibitors over pan-HDAC inhibitors in terms of efficacy or toxicity remain to be defined. The emerging interest for HDAC6-selective inhibitors is related to the modulation of acetylation of nonhistone regulatory proteins implicated in cancer-relevant processes, including cell migration, metastasis, angiogenesis and stress-response pathways. This review is focused on the recent development of HDAC inhibitors, with particular reference to HDAC6-selective inhibitors, and the efforts and perspectives in optimization of their therapeutic applications.
Keywords: HDAC inhibitors; Histone deacetilase 6; Antitumor therapy; Drug combinations; Drug design
Development and therapeutic impact of HDAC6-selective inhibitors by Sabrina Dallavalle; Claudio Pisano; Franco Zunino (pp. 756-765).
Histone deacetylases (HDAC) play a key role in regulating gene expression by deacetylating histones. Some HDAC isoforms can also modulate the function of nonhistone proteins implicated in regulatory processes, and therefore HDACs are recognized as useful targets for therapeutic purposes. HDAC inhibitors have generated substantial interest as antitumor agents, because they induce various cellular effects, including apoptosis, cell cycle arrest and inhibition of angiogenesis. The nature of cellular response likely depends on the biological context and on the pattern of HDAC isoform inhibition. Various HDAC inhibitors belonging to different structural classes have been developed. Many inhibitors are characterized by a pan-HDAC inhibitory profile. The potential advantages of isoform-selective inhibitors over pan-HDAC inhibitors in terms of efficacy or toxicity remain to be defined. The emerging interest for HDAC6-selective inhibitors is related to the modulation of acetylation of nonhistone regulatory proteins implicated in cancer-relevant processes, including cell migration, metastasis, angiogenesis and stress-response pathways. This review is focused on the recent development of HDAC inhibitors, with particular reference to HDAC6-selective inhibitors, and the efforts and perspectives in optimization of their therapeutic applications.
Keywords: HDAC inhibitors; Histone deacetilase 6; Antitumor therapy; Drug combinations; Drug design
OSI-930 analogues as novel reversal agents for ABCG2-mediated multidrug resistance by Ye-Hong Kuang; Jay P. Patel; Kamlesh Sodani; Chung-Pu Wu; Li-Qiu Liao; Atish Patel; Amit K. Tiwari; Chun-Ling Dai; Xiang Chen; Li-Wu Fu; Suresh V. Ambudkar; Vijaya L. Korlipara; Zhe-Sheng Chen (pp. 766-774).
OSI-930, a dual c-Kit and KDR tyrosine kinase inhibitor, is reported to have undergone a Phase I dose escalation study in patients with advanced solid tumors. A series of fifteen pyridyl and phenyl analogues of OSI-930 were designed and synthesized. Extensive screening of these compounds led to the discovery that nitropyridyl and ortho-nitrophenyl analogues, VKJP1 and VKJP3, were effective in reversing ABC subfamily G member 2 (ABCG2) transporter-mediated multidrug resistance (MDR). VKJP1 and VKJP3 significantly sensitized ABCG2-expressing cells to established substrates of ABCG2 including mitoxantrone, SN-38, and doxorubicin in a concentration-dependent manner, but not to the non-ABCG2 substrate cisplatin. However, they were unable to reverse ABCB1- or ABCC1-mediated MDR indicating their selectivity for ABCG2. Western blotting analysis was performed to evaluate ABCG2 expression and it was found that neither VKJP1 nor VKJP3 significantly altered ABCG2 protein expression for up to 72h. [3H]-mitoxantrone accumulation study demonstrated that VKJP1 and VKJP3 increased the intracellular accumulation of [3H]-mitoxantrone, a substrate of ABCG2. VKJP1 and VKJP3 also remarkably inhibited the transport of [3H]-methotrexate by ABCG2 membrane vesicles. Importantly, both VKJP1 and VKJP3 were efficacious in stimulating the activity of ATPase of ABCG2 and inhibited the photoaffinity labeling of this transporter by its substrate [125I]-iodoarylazidoprazosin. The results suggested that VKJP1 and VKJP3, specifically inhibit the function of ABCG2 through direct interaction with its substrate binding site(s). Thus VKJP1 and VKJP3 represent a new class of drugs for reducing MDR in ABCG2 over-expressing tumors.
Keywords: Abbreviations; OSI-930; {N-(4-trifluoromethoxyphenyl)3-((quinolin-4-ylmethyl) amino) thiophene-2-carboxamide}; ABC; ATP binding cassette; ABCG2 (BCRP/MXR/ABCP); ABC subfamily G member 2; MDR; multidrug resistance; P-gp; P-glycoprotein; ABCCs (MRPs); ABC subfamily C member; RTK; receptor tyrosine kinases; KDR; kinase insert domain receptor; TKIs; tyrosine kinase inhibitors; IAAP; [; 125; I]-iodoarylazidoprazosin; FTC; fumitremorgin C; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMSO; dimethylsulfoxideOSI-930; ABC transporters; ABCG2; Multidrug resistance
OSI-930 analogues as novel reversal agents for ABCG2-mediated multidrug resistance by Ye-Hong Kuang; Jay P. Patel; Kamlesh Sodani; Chung-Pu Wu; Li-Qiu Liao; Atish Patel; Amit K. Tiwari; Chun-Ling Dai; Xiang Chen; Li-Wu Fu; Suresh V. Ambudkar; Vijaya L. Korlipara; Zhe-Sheng Chen (pp. 766-774).
OSI-930, a dual c-Kit and KDR tyrosine kinase inhibitor, is reported to have undergone a Phase I dose escalation study in patients with advanced solid tumors. A series of fifteen pyridyl and phenyl analogues of OSI-930 were designed and synthesized. Extensive screening of these compounds led to the discovery that nitropyridyl and ortho-nitrophenyl analogues, VKJP1 and VKJP3, were effective in reversing ABC subfamily G member 2 (ABCG2) transporter-mediated multidrug resistance (MDR). VKJP1 and VKJP3 significantly sensitized ABCG2-expressing cells to established substrates of ABCG2 including mitoxantrone, SN-38, and doxorubicin in a concentration-dependent manner, but not to the non-ABCG2 substrate cisplatin. However, they were unable to reverse ABCB1- or ABCC1-mediated MDR indicating their selectivity for ABCG2. Western blotting analysis was performed to evaluate ABCG2 expression and it was found that neither VKJP1 nor VKJP3 significantly altered ABCG2 protein expression for up to 72h. [3H]-mitoxantrone accumulation study demonstrated that VKJP1 and VKJP3 increased the intracellular accumulation of [3H]-mitoxantrone, a substrate of ABCG2. VKJP1 and VKJP3 also remarkably inhibited the transport of [3H]-methotrexate by ABCG2 membrane vesicles. Importantly, both VKJP1 and VKJP3 were efficacious in stimulating the activity of ATPase of ABCG2 and inhibited the photoaffinity labeling of this transporter by its substrate [125I]-iodoarylazidoprazosin. The results suggested that VKJP1 and VKJP3, specifically inhibit the function of ABCG2 through direct interaction with its substrate binding site(s). Thus VKJP1 and VKJP3 represent a new class of drugs for reducing MDR in ABCG2 over-expressing tumors.
Keywords: Abbreviations; OSI-930; {N-(4-trifluoromethoxyphenyl)3-((quinolin-4-ylmethyl) amino) thiophene-2-carboxamide}; ABC; ATP binding cassette; ABCG2 (BCRP/MXR/ABCP); ABC subfamily G member 2; MDR; multidrug resistance; P-gp; P-glycoprotein; ABCCs (MRPs); ABC subfamily C member; RTK; receptor tyrosine kinases; KDR; kinase insert domain receptor; TKIs; tyrosine kinase inhibitors; IAAP; [; 125; I]-iodoarylazidoprazosin; FTC; fumitremorgin C; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMSO; dimethylsulfoxideOSI-930; ABC transporters; ABCG2; Multidrug resistance
Targeted mutagenesis of the Vibrio fischeri flavin reductase FRase I to improve activation of the anticancer prodrug CB1954 by P.M. Swe; J.N. Copp; L.K. Green; C.P. Guise; A.M. Mowday; J.B. Smaill; A.V. Patterson; D.F. Ackerley (pp. 775-783).
Phase I/II cancer gene therapy trials of the Escherichia coli nitroreductase NfsB in partnership with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] have indicated that CB1954 toxicity is dose-limiting at concentrations far below the enzyme KM. Here we report that the flavin reductase FRase I from Vibrio fischeri is also a CB1954 nitroreductase, which has a substantially lower apparent KM than E. coli NfsB. To enhance the activity of FRase I with CB1954 we used targeted mutagenesis and an E. coli SOS reporter strain to engineer single- and multi-residue variants that possess a substantially reduced apparent KM and an increased kcat/ KM relative to the wild type enzyme. In a bacteria-delivered model for enzyme prodrug therapy, the engineered FRase I variants were able to kill human colon carcinoma (HCT-116) cells at significantly lower CB1954 concentrations than wild type FRase I or E. coli NfsB.
Keywords: CB1954; Nitroreductase; SOS response; Enzyme prodrug therapy; Directed evolution
Targeted mutagenesis of the Vibrio fischeri flavin reductase FRase I to improve activation of the anticancer prodrug CB1954 by P.M. Swe; J.N. Copp; L.K. Green; C.P. Guise; A.M. Mowday; J.B. Smaill; A.V. Patterson; D.F. Ackerley (pp. 775-783).
Phase I/II cancer gene therapy trials of the Escherichia coli nitroreductase NfsB in partnership with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] have indicated that CB1954 toxicity is dose-limiting at concentrations far below the enzyme KM. Here we report that the flavin reductase FRase I from Vibrio fischeri is also a CB1954 nitroreductase, which has a substantially lower apparent KM than E. coli NfsB. To enhance the activity of FRase I with CB1954 we used targeted mutagenesis and an E. coli SOS reporter strain to engineer single- and multi-residue variants that possess a substantially reduced apparent KM and an increased kcat/ KM relative to the wild type enzyme. In a bacteria-delivered model for enzyme prodrug therapy, the engineered FRase I variants were able to kill human colon carcinoma (HCT-116) cells at significantly lower CB1954 concentrations than wild type FRase I or E. coli NfsB.
Keywords: CB1954; Nitroreductase; SOS response; Enzyme prodrug therapy; Directed evolution
Notoginsenoside Ft1 promotes angiogenesis via HIF-1α mediated VEGF secretion and the regulation of PI3K/AKT and Raf/MEK/ERK signaling pathways by Kaikai Shen; Lili Ji; Chenyuan Gong; Yibo Ma; Li Yang; Yi Fan; Maoqi Hou; Zhengtao Wang (pp. 784-792).
Notoginsenoside Ft1 (Ft1) is a saponin isolated from Panax notoginseng, which has been used traditionally for the treatment of trauma injuries in East Asia. Here we show that Ft1 is a novel stimulator of angiogenesis. The results show that Ft1 induces proliferation, migration, and tube formation in cultured human umbilical vein endothelial cells (HUVECs). Ft1 increases translocalization of hypoxia-inducible factor-1α (HIF-1α) from cytoplasm to nuclei, where it binds to the vascular endothelial growth factor (VEGF) promoter, increasing the expression of VEGF mRNA and the subsequent secretion of the growth factor. Ft1 induces the activation of PI3K/AKT and Raf/MEK/ERK signaling pathways. Pharmacological inhibition with LY294002, wortmanin or PD98059 reduces Ft1-induced angiogenesis, indicating the important role played by these pathways. In addition, Ft1 induces phosphorylation of the mammalian target of rapamycin (mTOR), and siRNA-mediated mTOR knockdown decreases tube formation, proliferation, transport of HIF-1α into nuclei and VEGF mRNA expression in response to Ft1. Finally, in vivo, Ft1 promotes the formation of blood vessels in Matrigel plug and wound healing in mice. Taken together, the present results reveal that Ft1 stimulates angiogenesis via HIF-1α-mediated VEGF expression, with PI3K/AKT and Raf/MEK/ERK signaling cascades concurrently participating in the process.
Keywords: Notoginsenoside Ft1 (Ft1); Angiogenesis; Wound healing; Vascular endothelial growth factor (VEGF); Hypoxia-inducible factor-1α (HIF-1α)
Notoginsenoside Ft1 promotes angiogenesis via HIF-1α mediated VEGF secretion and the regulation of PI3K/AKT and Raf/MEK/ERK signaling pathways by Kaikai Shen; Lili Ji; Chenyuan Gong; Yibo Ma; Li Yang; Yi Fan; Maoqi Hou; Zhengtao Wang (pp. 784-792).
Notoginsenoside Ft1 (Ft1) is a saponin isolated from Panax notoginseng, which has been used traditionally for the treatment of trauma injuries in East Asia. Here we show that Ft1 is a novel stimulator of angiogenesis. The results show that Ft1 induces proliferation, migration, and tube formation in cultured human umbilical vein endothelial cells (HUVECs). Ft1 increases translocalization of hypoxia-inducible factor-1α (HIF-1α) from cytoplasm to nuclei, where it binds to the vascular endothelial growth factor (VEGF) promoter, increasing the expression of VEGF mRNA and the subsequent secretion of the growth factor. Ft1 induces the activation of PI3K/AKT and Raf/MEK/ERK signaling pathways. Pharmacological inhibition with LY294002, wortmanin or PD98059 reduces Ft1-induced angiogenesis, indicating the important role played by these pathways. In addition, Ft1 induces phosphorylation of the mammalian target of rapamycin (mTOR), and siRNA-mediated mTOR knockdown decreases tube formation, proliferation, transport of HIF-1α into nuclei and VEGF mRNA expression in response to Ft1. Finally, in vivo, Ft1 promotes the formation of blood vessels in Matrigel plug and wound healing in mice. Taken together, the present results reveal that Ft1 stimulates angiogenesis via HIF-1α-mediated VEGF expression, with PI3K/AKT and Raf/MEK/ERK signaling cascades concurrently participating in the process.
Keywords: Notoginsenoside Ft1 (Ft1); Angiogenesis; Wound healing; Vascular endothelial growth factor (VEGF); Hypoxia-inducible factor-1α (HIF-1α)
The antiplatelet activity of magnolol is mediated by PPAR-β/γ by Ching-Yu Shih; Tz-Chong Chou (pp. 793-803).
Activation of peroxisome proliferator-activated receptor (PPAR) isoforms (α, β/δ, and γ) is known to inhibit platelet aggregation. In the present study, we examined whether PPARs-mediated pathways contribute to the antiplatelet activity of magnolol, a compound purified from Magnolia officinalis. Magnolol (20–60μM) dose-dependently enhanced the activity and intracellular level of PPAR-β/γ in platelets. In the presence of selective PPAR-β antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibition of magnolol on collagen-induced platelet aggregation and intracellular Ca2+ mobilization was significantly reversed. Moreover, magnolol-mediated up-regulation of NO/cyclic GMP/PKG pathway and Akt phosphorylation leading to increase of eNOS activity were markedly abolished by blocking PPAR-β/γ activity. Additionally, magnolol significantly inhibited collagen-induced PKCα activation through a PPAR-β/γ and PKCα interaction manner. The arachidonic acid (AA) or collagen-induced thromboxane B2 formation and elevation of COX-1 activity caused by AA were also markedly attenuated by magnolol. However, these above effects of magnolol on platelet responses were strongly reduced by simultaneous addition of GSK0660 or GW9662, suggesting that PPAR-β/γ-mediated processes may account for magnolol-regulated antiplatelet mechanisms. Similarly, administration of PPAR-β/γ antagonists remarkably abolished the actions of magnolol in preventing platelet plug formation and prolonging bleeding time in mice. Taken together, we demonstrate for the first time that the antiplatelet and anti-thrombotic activities of magnolol are modulated by up-regulation of PPAR-β/γ-dependent pathways.
Keywords: Abbreviations; PPAR; peroxisome proliferator-activated receptor; AA; arachidonic acid; cyclic GMP; 3′,5′-cyclic monophosphate; NOS; nitric oxide synthase; NO; nitric oxide; PRP; platelet rich plasma; LDH; lactate dehydrogenase; TXA; 2; thromboxane A; 2; TXB; 2; thromboxane B; 2; PKCα; protein kinase Cα; PI3K; phosphatidylinositol-3 kinaseMagnolol; Peroxisome proliferator-activated receptors (PPARs); Platelet aggregation; Cyclic GMP; Protein kinase Cα; Nitric oxide
The antiplatelet activity of magnolol is mediated by PPAR-β/γ by Ching-Yu Shih; Tz-Chong Chou (pp. 793-803).
Activation of peroxisome proliferator-activated receptor (PPAR) isoforms (α, β/δ, and γ) is known to inhibit platelet aggregation. In the present study, we examined whether PPARs-mediated pathways contribute to the antiplatelet activity of magnolol, a compound purified from Magnolia officinalis. Magnolol (20–60μM) dose-dependently enhanced the activity and intracellular level of PPAR-β/γ in platelets. In the presence of selective PPAR-β antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibition of magnolol on collagen-induced platelet aggregation and intracellular Ca2+ mobilization was significantly reversed. Moreover, magnolol-mediated up-regulation of NO/cyclic GMP/PKG pathway and Akt phosphorylation leading to increase of eNOS activity were markedly abolished by blocking PPAR-β/γ activity. Additionally, magnolol significantly inhibited collagen-induced PKCα activation through a PPAR-β/γ and PKCα interaction manner. The arachidonic acid (AA) or collagen-induced thromboxane B2 formation and elevation of COX-1 activity caused by AA were also markedly attenuated by magnolol. However, these above effects of magnolol on platelet responses were strongly reduced by simultaneous addition of GSK0660 or GW9662, suggesting that PPAR-β/γ-mediated processes may account for magnolol-regulated antiplatelet mechanisms. Similarly, administration of PPAR-β/γ antagonists remarkably abolished the actions of magnolol in preventing platelet plug formation and prolonging bleeding time in mice. Taken together, we demonstrate for the first time that the antiplatelet and anti-thrombotic activities of magnolol are modulated by up-regulation of PPAR-β/γ-dependent pathways.
Keywords: Abbreviations; PPAR; peroxisome proliferator-activated receptor; AA; arachidonic acid; cyclic GMP; 3′,5′-cyclic monophosphate; NOS; nitric oxide synthase; NO; nitric oxide; PRP; platelet rich plasma; LDH; lactate dehydrogenase; TXA; 2; thromboxane A; 2; TXB; 2; thromboxane B; 2; PKCα; protein kinase Cα; PI3K; phosphatidylinositol-3 kinaseMagnolol; Peroxisome proliferator-activated receptors (PPARs); Platelet aggregation; Cyclic GMP; Protein kinase Cα; Nitric oxide
2-(2,4-dihydroxyphenyl)-5-( E)-propenylbenzofuran promotes endothelial nitric oxide synthase activity in human endothelial cells by Angela Ladurner; Atanas G. Atanasov; Elke H. Heiss; Lisa Baumgartner; Stefan Schwaiger; Judith M. Rollinger; Hermann Stuppner; Verena M. Dirsch (pp. 804-812).
Endothelial nitric oxide synthase (eNOS) mediates important vaso-protective and immunomodulatory effects. Aim of this study was to examine whether lignan derivatives isolated from the roots of the anti-inflammatory medicinal plant Krameria lappacea influence eNOS activity and endothelial nitric oxide (NO) release. The study was performed using cultured human umbilical vein endothelial cells (HUVECs) and HUVEC-derived EA.hy926 cells. Among the eleven isolated compounds only 2-(2,4-dihydroxyphenyl)-5-( E)-propenylbenzofuran (DPPB) was able to increase eNOS enzyme activity.DPPB (1–10μM) treatment for 24h induced a significant and dose-dependent increase in eNOS activity as determined by the [14C]l-arginine/[14C]l-citrulline conversion assay. Immunoblotting studies further revealed a time-dependent DPPB-induced increase in eNOS-Ser1177 and decrease in eNOS-Thr495 phosphorylation, as well as increased AMPK phosphorylation at Thr172, whereas Akt phosphorylation at Ser473 was not affected. Si-RNA-mediated knockdown of AMPK and inhibition of CaMKKβ by STO 609, as well as intracellular Ca2+ chelation by Bapta AM abolished the stimulating effect of DPPB on eNOS-Ser1177 and AMPK-Thr172 phosphorylation. Furthermore, we could show that DPPB increases intracellular Ca2+ concentrations assessed with the fluorescent dye Fluo-3-AM. DPPB enhances eNOS activity and endothelial NO release by raising intracellular Ca2+ levels and increases signaling through a CaMKKβ–AMPK dependent pathway.
Keywords: Endothelial nitric oxide synthase (eNOS); Ca; 2+; Benzofuran derivative; AMPK; CaMKKβ
2-(2,4-dihydroxyphenyl)-5-( E)-propenylbenzofuran promotes endothelial nitric oxide synthase activity in human endothelial cells by Angela Ladurner; Atanas G. Atanasov; Elke H. Heiss; Lisa Baumgartner; Stefan Schwaiger; Judith M. Rollinger; Hermann Stuppner; Verena M. Dirsch (pp. 804-812).
Endothelial nitric oxide synthase (eNOS) mediates important vaso-protective and immunomodulatory effects. Aim of this study was to examine whether lignan derivatives isolated from the roots of the anti-inflammatory medicinal plant Krameria lappacea influence eNOS activity and endothelial nitric oxide (NO) release. The study was performed using cultured human umbilical vein endothelial cells (HUVECs) and HUVEC-derived EA.hy926 cells. Among the eleven isolated compounds only 2-(2,4-dihydroxyphenyl)-5-( E)-propenylbenzofuran (DPPB) was able to increase eNOS enzyme activity.DPPB (1–10μM) treatment for 24h induced a significant and dose-dependent increase in eNOS activity as determined by the [14C]l-arginine/[14C]l-citrulline conversion assay. Immunoblotting studies further revealed a time-dependent DPPB-induced increase in eNOS-Ser1177 and decrease in eNOS-Thr495 phosphorylation, as well as increased AMPK phosphorylation at Thr172, whereas Akt phosphorylation at Ser473 was not affected. Si-RNA-mediated knockdown of AMPK and inhibition of CaMKKβ by STO 609, as well as intracellular Ca2+ chelation by Bapta AM abolished the stimulating effect of DPPB on eNOS-Ser1177 and AMPK-Thr172 phosphorylation. Furthermore, we could show that DPPB increases intracellular Ca2+ concentrations assessed with the fluorescent dye Fluo-3-AM. DPPB enhances eNOS activity and endothelial NO release by raising intracellular Ca2+ levels and increases signaling through a CaMKKβ–AMPK dependent pathway.
Keywords: Endothelial nitric oxide synthase (eNOS); Ca; 2+; Benzofuran derivative; AMPK; CaMKKβ
N- O-Isopropyl sulfonamido-based hydroxamates: Kinetic characterisation of a series of MMP-12/MMP-13 dual target inhibitors by Salvatore Santamaria; Elisa Nuti; Giovanni Cercignani; Luciana Marinelli; Valeria La Pietra; Ettore Novellino; Armando Rossello (pp. 813-820).
Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases known to play key roles in extracellular matrix (ECM) breakdown disorders, such as the two main forms of arthritis, rheumatoid arthritis (RA) and osteoarthritis (OA). MMP-13 (collagenase 3) is the leading MMP involved in cartilage degradation through its particular ability to cleave type-II collagen and as such plays a pivotal role in the pathogenesis of these diseases. Here we report the kinetic characterisation of N- O-isopropyl sulfonamido-based hydroxamates, potent inhibitors of MMP-13 and MMP-12, bearing different P1 and P1′ substituents. One of these compounds proved to be a potent (4≤ Ki≤5nM) slow-binding inhibitor towards MMP-13 and MMP-12, with very favourable low association (104M−1s−1) and dissociation constants (10−4s−1). Moreover, this compound exhibited a good selectivity for MMP-13 and MMP-12 over MMP-1, MMP-3, MMP-7, MMP-8 and, even to a minor extent, MMP-2. A molecular-docking study carried out using the experimentally-derived X-ray crystal structure of MMP-12 (PDB ID:3F17) revealed critical hydrogen bonding of the hydroxamate and the sulfonamide moieties with key active site residues. Since also MMP-12 is involved in RA, this MMP-13/MMP-12 dual target inhibitor could be a valid candidate for the treatment of this pathology.
Keywords: Enzyme kinetics; Matrix metalloproteinase; Slow-binding inhibition; Rheumatoid arthritis; MMP-13; MMP-12
N- O-Isopropyl sulfonamido-based hydroxamates: Kinetic characterisation of a series of MMP-12/MMP-13 dual target inhibitors by Salvatore Santamaria; Elisa Nuti; Giovanni Cercignani; Luciana Marinelli; Valeria La Pietra; Ettore Novellino; Armando Rossello (pp. 813-820).
Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases known to play key roles in extracellular matrix (ECM) breakdown disorders, such as the two main forms of arthritis, rheumatoid arthritis (RA) and osteoarthritis (OA). MMP-13 (collagenase 3) is the leading MMP involved in cartilage degradation through its particular ability to cleave type-II collagen and as such plays a pivotal role in the pathogenesis of these diseases. Here we report the kinetic characterisation of N- O-isopropyl sulfonamido-based hydroxamates, potent inhibitors of MMP-13 and MMP-12, bearing different P1 and P1′ substituents. One of these compounds proved to be a potent (4≤ Ki≤5nM) slow-binding inhibitor towards MMP-13 and MMP-12, with very favourable low association (104M−1s−1) and dissociation constants (10−4s−1). Moreover, this compound exhibited a good selectivity for MMP-13 and MMP-12 over MMP-1, MMP-3, MMP-7, MMP-8 and, even to a minor extent, MMP-2. A molecular-docking study carried out using the experimentally-derived X-ray crystal structure of MMP-12 (PDB ID:3F17) revealed critical hydrogen bonding of the hydroxamate and the sulfonamide moieties with key active site residues. Since also MMP-12 is involved in RA, this MMP-13/MMP-12 dual target inhibitor could be a valid candidate for the treatment of this pathology.
Keywords: Enzyme kinetics; Matrix metalloproteinase; Slow-binding inhibition; Rheumatoid arthritis; MMP-13; MMP-12
Niacin reduces plasma CETP levels by diminishing liver macrophage content in CETP transgenic mice by Zhaosha Li; Yanan Wang; Ronald J. van der Sluis; José W.A. van der Hoorn; Hans M.G. Princen; Miranda Van Eck; Theo J.C. Van Berkel; Patrick C.N. Rensen; Menno Hoekstra (pp. 821-829).
The anti-dyslipidemic drug niacin has recently been shown to reduce the hepatic expression and plasma levels of CETP. Since liver macrophages contribute to hepatic CETP expression, we investigated the role of macrophages in the CETP-lowering effect of niacin in mice. In vitro studies showed that niacin does not directly attenuate CETP expression in macrophages. Treatment of normolipidemic human CETP transgenic mice, fed a Western-type diet with niacin for 4 weeks, significantly reduced the hepatic cholesterol concentration (−20%), hepatic CETP gene expression (−20%), and plasma CETP mass (−30%). Concomitantly, niacin decreased the hepatic expression of CD68 (−44%) and ABCG1 (−32%), both of which are specific markers for the hepatic macrophage content. The decrease in hepatic CETP expression was significantly correlated with the reduction of hepatic macrophage markers. Furthermore, niacin attenuated atherogenic diet-induced inflammation in liver, as evident from decreased expression of TNF-alpha (−43%). Niacin similarly decreased the macrophage markers and absolute macrophage content in hyperlipidemic APOE*3-Leiden.CETP transgenic mice on a Western-type diet. In conclusion, niacin decreases hepatic CETP expression and plasma CETP mass by attenuating liver inflammation and macrophage content in response to its primary lipid-lowering effect, rather than by attenuating the macrophage CETP expression level.
Keywords: CETP; Niacin; Macrophages; Lipoprotein; Liver
Niacin reduces plasma CETP levels by diminishing liver macrophage content in CETP transgenic mice by Zhaosha Li; Yanan Wang; Ronald J. van der Sluis; José W.A. van der Hoorn; Hans M.G. Princen; Miranda Van Eck; Theo J.C. Van Berkel; Patrick C.N. Rensen; Menno Hoekstra (pp. 821-829).
The anti-dyslipidemic drug niacin has recently been shown to reduce the hepatic expression and plasma levels of CETP. Since liver macrophages contribute to hepatic CETP expression, we investigated the role of macrophages in the CETP-lowering effect of niacin in mice. In vitro studies showed that niacin does not directly attenuate CETP expression in macrophages. Treatment of normolipidemic human CETP transgenic mice, fed a Western-type diet with niacin for 4 weeks, significantly reduced the hepatic cholesterol concentration (−20%), hepatic CETP gene expression (−20%), and plasma CETP mass (−30%). Concomitantly, niacin decreased the hepatic expression of CD68 (−44%) and ABCG1 (−32%), both of which are specific markers for the hepatic macrophage content. The decrease in hepatic CETP expression was significantly correlated with the reduction of hepatic macrophage markers. Furthermore, niacin attenuated atherogenic diet-induced inflammation in liver, as evident from decreased expression of TNF-alpha (−43%). Niacin similarly decreased the macrophage markers and absolute macrophage content in hyperlipidemic APOE*3-Leiden.CETP transgenic mice on a Western-type diet. In conclusion, niacin decreases hepatic CETP expression and plasma CETP mass by attenuating liver inflammation and macrophage content in response to its primary lipid-lowering effect, rather than by attenuating the macrophage CETP expression level.
Keywords: CETP; Niacin; Macrophages; Lipoprotein; Liver
Screening for the efficacy on lipid accumulation in 3T3-L1 cells is an effective tool for the identification of new anti-diabetic compounds by Xiao-Yi Zeng; Xiu Zhou; Jun Xu; Stanley M.H. Chan; Charlie L. Xue; Juan C. Molero; Ji-Ming Ye (pp. 830-837).
Reducing lipid accumulation in insulin target tissues is critical for the treatment of type 2 diabetes. This study aimed to develop a biochemical assay in cells for high throughput (HTP) screening of anti-diabetic drugs by reducing lipid accumulation via different mechanisms. We designed a new method to extract triglyceride (TG) with KOH to allow biochemical quantification of TGs for HTP screening in 3T3-L1 cells. This new method was validated for its biochemical properties with identical results of TG obtained with or without KOH ( r2=0.9978, p<0.001) and a fourfold improvement in TG extraction recovery rate (88–95%, p<0.001) as compared to the conventional chloroform/methanol extraction (12–18%). The ability of this phenotype screening to capture potential anti-diabetic drugs was verified by pharmacological agents well known to alter lipid accumulation by different mechanisms including AMPK activators, fatty acid synthesis inhibitors, PPARγ activator and several lipogenic substrates. To further demonstrate the application of this screening tool for discovery of new anti-diabetic drugs, we screened >200 new candidates selected from Chinese medicine and identified 49 compounds from different classes which reduced TG content by >50% at 1μM or >75% at 10μM. Finally, we tested two selected leads (albiflorin and oxymatrine) in vivo and confirmed their efficacy in reducing visceral adiposity, glucose intolerance and hepatic steatosis in high fat-fed or high fructose-fed mice. Our results indicate that screening for the efficacy on lipid accumulation in cells by biochemical quantification of TGs with KOH extraction is an effective tool for the identification of new anti-diabetic compounds.
Keywords: Abbreviations; Abs; absorbance; ACC; acetyl-CoA carboxylase; AF; albiflorin; AMPK; AMP-activated protein kinase; ATG; arctigenin; BBR; berberine; BW; body weight; CH-Con; standard chow; C/M; chloroform/methanol; DMEM; Dulbecco's modified Eagles's medium; FA; fatty acid; FAS; fatty acid synthase; FBS; fetal bovine serum; FXR; farnesoid; ×; receptor; GTT; glucose tolerance test; HFD; high fat diet; HTP; high throughput; IBMX; 3-isobutyl-1-methylxanthine; ip; intraperitoneal; KOH; potassium hydroxide; LDH; lactate dehydrogenase; OA; oleanolic acid; OM; oxymatrine; ORO; Oil Red O; PPAR; peroxisome proliferator-activating receptor; PSG; penicillin-streptomycin-glutamine; RSG; rosiglitazone; SREBP-1c; sterol regulatory element-binding protein-1c; STD; standard; T2D; Type 2 diabetes; TCM; traditional Chinese medicine; TG; triglyceride; TOFA; 5-(tetradecyloxy)-2-furancarboxylic acid; TP; triterpenoid; Veh; vehicleDrug discovery; Phenotypic screening; Lipid accumulation; Insulin resistance; Obesity; Fatty liver
Screening for the efficacy on lipid accumulation in 3T3-L1 cells is an effective tool for the identification of new anti-diabetic compounds by Xiao-Yi Zeng; Xiu Zhou; Jun Xu; Stanley M.H. Chan; Charlie L. Xue; Juan C. Molero; Ji-Ming Ye (pp. 830-837).
Reducing lipid accumulation in insulin target tissues is critical for the treatment of type 2 diabetes. This study aimed to develop a biochemical assay in cells for high throughput (HTP) screening of anti-diabetic drugs by reducing lipid accumulation via different mechanisms. We designed a new method to extract triglyceride (TG) with KOH to allow biochemical quantification of TGs for HTP screening in 3T3-L1 cells. This new method was validated for its biochemical properties with identical results of TG obtained with or without KOH ( r2=0.9978, p<0.001) and a fourfold improvement in TG extraction recovery rate (88–95%, p<0.001) as compared to the conventional chloroform/methanol extraction (12–18%). The ability of this phenotype screening to capture potential anti-diabetic drugs was verified by pharmacological agents well known to alter lipid accumulation by different mechanisms including AMPK activators, fatty acid synthesis inhibitors, PPARγ activator and several lipogenic substrates. To further demonstrate the application of this screening tool for discovery of new anti-diabetic drugs, we screened >200 new candidates selected from Chinese medicine and identified 49 compounds from different classes which reduced TG content by >50% at 1μM or >75% at 10μM. Finally, we tested two selected leads (albiflorin and oxymatrine) in vivo and confirmed their efficacy in reducing visceral adiposity, glucose intolerance and hepatic steatosis in high fat-fed or high fructose-fed mice. Our results indicate that screening for the efficacy on lipid accumulation in cells by biochemical quantification of TGs with KOH extraction is an effective tool for the identification of new anti-diabetic compounds.
Keywords: Abbreviations; Abs; absorbance; ACC; acetyl-CoA carboxylase; AF; albiflorin; AMPK; AMP-activated protein kinase; ATG; arctigenin; BBR; berberine; BW; body weight; CH-Con; standard chow; C/M; chloroform/methanol; DMEM; Dulbecco's modified Eagles's medium; FA; fatty acid; FAS; fatty acid synthase; FBS; fetal bovine serum; FXR; farnesoid; ×; receptor; GTT; glucose tolerance test; HFD; high fat diet; HTP; high throughput; IBMX; 3-isobutyl-1-methylxanthine; ip; intraperitoneal; KOH; potassium hydroxide; LDH; lactate dehydrogenase; OA; oleanolic acid; OM; oxymatrine; ORO; Oil Red O; PPAR; peroxisome proliferator-activating receptor; PSG; penicillin-streptomycin-glutamine; RSG; rosiglitazone; SREBP-1c; sterol regulatory element-binding protein-1c; STD; standard; T2D; Type 2 diabetes; TCM; traditional Chinese medicine; TG; triglyceride; TOFA; 5-(tetradecyloxy)-2-furancarboxylic acid; TP; triterpenoid; Veh; vehicleDrug discovery; Phenotypic screening; Lipid accumulation; Insulin resistance; Obesity; Fatty liver
p23 co-chaperone protects the aryl hydrocarbon receptor from degradation in mouse and human cell lines by Phuong Minh Nguyen; Depeng Wang; Yu Wang; Yanjie Li; James A. Uchizono; William K. Chan (pp. 838-850).
The aryl hydrocarbon receptor (AhR) is a ligand-sensitive transcription factor which is responsible for most 2,3,7,8-tetrachlorodibenzo- p-dioxin toxicities. Without ligand, the AhR complex is cytoplasmic and contains p23. Our objective was to investigate whether the wild type p23 levels are important for the AhR function. We generated eight p23-specific knockdown stable cell lines via either electroporation or lentiviral infection. Five of these stable cell lines were generated from a mouse hepatoma cell line (Hepa1c1c7) and three were from human hepatoma and cervical cell lines (Hep3B and HeLa). All of them expressed lower AhR protein levels, leading to reduced ligand-induced, DRE-driven downstream activity. The AhR protein levels in p23-specific knockdown stable cells were reversed back to wild type levels after exogenous p23 was introduced. Reduction of the AhR protein levels in these stable cells was caused by a decrease in the AhR message levels and an increase of the AhR protein degradation in the absence of ligand. This ligand-independent degradation of AhR was not reversed by MG132, suggesting that the 26S proteasome was not responsible for the degradation. In addition, MG132 could not protect AhR from the ligand-induced degradation in both mouse and human p23-knockdown stable cells.
Keywords: Abbreviations; WT; wild type; NC; negative control knockdown stable; p23kd1-8; p23 knockdown stable 1-8; AhR; aryl hydrocarbon receptor; Arnt; aryl hydrocarbon receptor nuclear translocator; DRE; dioxin response elementp23 co-chaperone; Aryl hydrocarbon receptor; AhR; Protein degradation; Dioxin
p23 co-chaperone protects the aryl hydrocarbon receptor from degradation in mouse and human cell lines by Phuong Minh Nguyen; Depeng Wang; Yu Wang; Yanjie Li; James A. Uchizono; William K. Chan (pp. 838-850).
The aryl hydrocarbon receptor (AhR) is a ligand-sensitive transcription factor which is responsible for most 2,3,7,8-tetrachlorodibenzo- p-dioxin toxicities. Without ligand, the AhR complex is cytoplasmic and contains p23. Our objective was to investigate whether the wild type p23 levels are important for the AhR function. We generated eight p23-specific knockdown stable cell lines via either electroporation or lentiviral infection. Five of these stable cell lines were generated from a mouse hepatoma cell line (Hepa1c1c7) and three were from human hepatoma and cervical cell lines (Hep3B and HeLa). All of them expressed lower AhR protein levels, leading to reduced ligand-induced, DRE-driven downstream activity. The AhR protein levels in p23-specific knockdown stable cells were reversed back to wild type levels after exogenous p23 was introduced. Reduction of the AhR protein levels in these stable cells was caused by a decrease in the AhR message levels and an increase of the AhR protein degradation in the absence of ligand. This ligand-independent degradation of AhR was not reversed by MG132, suggesting that the 26S proteasome was not responsible for the degradation. In addition, MG132 could not protect AhR from the ligand-induced degradation in both mouse and human p23-knockdown stable cells.
Keywords: Abbreviations; WT; wild type; NC; negative control knockdown stable; p23kd1-8; p23 knockdown stable 1-8; AhR; aryl hydrocarbon receptor; Arnt; aryl hydrocarbon receptor nuclear translocator; DRE; dioxin response elementp23 co-chaperone; Aryl hydrocarbon receptor; AhR; Protein degradation; Dioxin
Pharmacological characterization of α-elapitoxin-Al2a from the venom of the Australian pygmy copperhead ( Austrelaps labialis): An atypical long-chain α-neurotoxin with only weak affinity for α7 nicotinic receptors by Francesca Marcon; Mathieu Leblanc; Irina Vetter; Richard J. Lewis; Pierre Escoubas; Graham M. Nicholson (pp. 851-863).
Despite the in vivo lethality of venom, neurotoxicity has not previously been considered a significant complication of envenoming by the Australian pygmy copperhead ( Austrelaps labialis). However, recent evidence has emerged demonstrating that this venom contains potent presynaptic and postsynaptic neurotoxicity. The present study describes the isolation and pharmacological characterization of the first postsynaptic neurotoxin, α-EPTX-Al2a, from the venom of A. labialis. α-EPTX-Al2a (8072.77Da) caused a concentration-dependent block of twitch contractions and a complete block of responses to cholinergic agonists in the chick biventer cervicis nerve–muscle preparation. This action is consistent with postjunctional neurotoxicity. Monovalent tiger snake antivenom prevented the onset of neurotoxicity if applied prior to toxin administration, but was only able to partially reverse neurotoxicity once muscle paralysis had developed. α-EPTX-Al2a produced a potent pseudo-irreversible antagonism of chick muscle nicotinic acetylcholine receptors (nAChRs), with an estimated pA2 value of 7.902 ( KB=12.5nM). Interestingly, the toxin only produced a modest block of neuronal α7 nAChRs, with an IC50 of 1.2μM, and failed to inhibit ganglionic α3β2/α3β4 nAChRs in a fluorescence-based FLIPR assay using SH-SY5Y cells. α-EPTX-Al2a contained 75 amino acid residues with five disulfide bonds that had significant homology to classical long-chain α-neurotoxins. While α-EPTX-Al2a retains most pharmacophore residues critical for binding to muscle-type (α1)2βγδ nAChRs it lacks the key Ala28 and Arg36 residues important for α7 nAChR affinity. Given that A. labialis venom contains both irreversible presynaptic and postsynaptic neurotoxins, clinicians need to be aware of potential neurotoxic complications associated with pygmy copperhead envenomation.
Keywords: Abbreviations; ACh; acetylcholine; ANOVA; analysis of variance; CBCNM; chick biventer cervicis nerve–muscle; CCh; carbachol; MALDI-TOF; matrix-assisted laser desorption/ionization time-of-flight; nAChR; nicotinic acetylcholine receptor; EPTX; elapitoxin; FPLC; fast perfusion liquid chromatography; RP-HPLC; reversed-phase high-pressure liquid chromatography; SPAN; snake presynaptic sPLA; 2; neurotoxin; TFA; trifluoroacetic acid; TSAV; monovalent tiger snake antivenom; t; 90; time to 90% neuromuscular blockade; V; e; elution volume; V; o; void volumeSnake toxin; Long-chain α-neurotoxin; α-elapitoxin-Al2a; Austrelaps labialis; (α1); 2; βγδ nicotinic acetylcholine receptors; α7 nicotinic acetylcholine receptors; α3* nicotinic acetylcholine receptors
Pharmacological characterization of α-elapitoxin-Al2a from the venom of the Australian pygmy copperhead ( Austrelaps labialis): An atypical long-chain α-neurotoxin with only weak affinity for α7 nicotinic receptors by Francesca Marcon; Mathieu Leblanc; Irina Vetter; Richard J. Lewis; Pierre Escoubas; Graham M. Nicholson (pp. 851-863).
Despite the in vivo lethality of venom, neurotoxicity has not previously been considered a significant complication of envenoming by the Australian pygmy copperhead ( Austrelaps labialis). However, recent evidence has emerged demonstrating that this venom contains potent presynaptic and postsynaptic neurotoxicity. The present study describes the isolation and pharmacological characterization of the first postsynaptic neurotoxin, α-EPTX-Al2a, from the venom of A. labialis. α-EPTX-Al2a (8072.77Da) caused a concentration-dependent block of twitch contractions and a complete block of responses to cholinergic agonists in the chick biventer cervicis nerve–muscle preparation. This action is consistent with postjunctional neurotoxicity. Monovalent tiger snake antivenom prevented the onset of neurotoxicity if applied prior to toxin administration, but was only able to partially reverse neurotoxicity once muscle paralysis had developed. α-EPTX-Al2a produced a potent pseudo-irreversible antagonism of chick muscle nicotinic acetylcholine receptors (nAChRs), with an estimated pA2 value of 7.902 ( KB=12.5nM). Interestingly, the toxin only produced a modest block of neuronal α7 nAChRs, with an IC50 of 1.2μM, and failed to inhibit ganglionic α3β2/α3β4 nAChRs in a fluorescence-based FLIPR assay using SH-SY5Y cells. α-EPTX-Al2a contained 75 amino acid residues with five disulfide bonds that had significant homology to classical long-chain α-neurotoxins. While α-EPTX-Al2a retains most pharmacophore residues critical for binding to muscle-type (α1)2βγδ nAChRs it lacks the key Ala28 and Arg36 residues important for α7 nAChR affinity. Given that A. labialis venom contains both irreversible presynaptic and postsynaptic neurotoxins, clinicians need to be aware of potential neurotoxic complications associated with pygmy copperhead envenomation.
Keywords: Abbreviations; ACh; acetylcholine; ANOVA; analysis of variance; CBCNM; chick biventer cervicis nerve–muscle; CCh; carbachol; MALDI-TOF; matrix-assisted laser desorption/ionization time-of-flight; nAChR; nicotinic acetylcholine receptor; EPTX; elapitoxin; FPLC; fast perfusion liquid chromatography; RP-HPLC; reversed-phase high-pressure liquid chromatography; SPAN; snake presynaptic sPLA; 2; neurotoxin; TFA; trifluoroacetic acid; TSAV; monovalent tiger snake antivenom; t; 90; time to 90% neuromuscular blockade; V; e; elution volume; V; o; void volumeSnake toxin; Long-chain α-neurotoxin; α-elapitoxin-Al2a; Austrelaps labialis; (α1); 2; βγδ nicotinic acetylcholine receptors; α7 nicotinic acetylcholine receptors; α3* nicotinic acetylcholine receptors
Antioxidant sulforaphane and sensitizer trinitrobenzene sulfonate induce carboxylesterase-1 through a novel element transactivated by nuclear factor-E2 related factor-2 by Yi-Tzai Chen; Deshi Shi; Dongfang Yang; Bingfang Yan (pp. 864-871).
Carboxylesterase-1 (CES1), the most versatile human carboxylesterase, plays critical roles in drug metabolism and lipid mobilization. This enzyme is highly induced by antioxidants and sensitizers in various cell lines. These compounds are known to activate nuclear factor-E2 related factor-2 (Nrf2) by reacting to kelch-like ECH-associated protein-1 (Keap1). The aims of this study were to determine whether antioxidant sulforaphane (SFN) and sensitizer trinitrobenzene sulfonate (TNBS) target Keap1 similarly and whether they use the same element for CES1 induction. Cells over-expressing Keap1 were treated with TNBS or SFN and the formation of disulfide bonds among Keap1 molecules were determined. SFN promoted intramolecular disulfide formation whereas TNBS promoted intermolecular disulfide formation of Keap1. Two elements, sensitizing/antioxidant response element (S/ARE) and ARE4, were identified to support Nrf2 in the regulated expression of CES1A1. Both elements were bound by Nrf2, however, the S/ARE element supported, whereas the ARE4 element repressed Nrf2 transactivation. The repression required higher amounts of Nrf2, suggesting that the transactivation through the S/ARE element dominates the trans-repression through the ARE4 element under normal antioxidative condition. These findings conclude that compounds, although triggering the Keap1-Nrf2 pathway, may differ in the mode of reacting with Keap1. These findings also conclude that both positive and negative Nrf2 elements exist even within the same gene, and such opposing mechanisms provide fine-tuning in transcriptional regulation by the Keap1-Nrf2 pathway. High levels of CES1 are linked to lipid retention. Excessive induction of CES1 by antioxidants and sensitizers likely provides a mechanism for potential detrimental effect on human health.
Keywords: Abbreviations; AKR1C2; aldo-keto reductase-1C2; ARE; antioxidant response element; CES; carboxylesterase; ChIP; chromatin-immunoprecipitation; CYP; cytochrome P450; DMEM; Dulbecco's Modified Eagles Medium; EMSA; electrophoretic mobility shift assay; FBS; fetal bovine serum; GAPDH; glyceradehyde-3-phosphate dehydrogenase; GCLC; glutamate-cysteine ligase catalytic subunit; GCLM; glutamate-cysteine ligase regulatory subunit; NQO1; NAD(P)H:quinone oxidoreductase; Nrf2; nuclear factor-E2 related factor-2; PBS; phosphate buffered saline; RT-qPCR; reverse transcription-quantitative polymerase chain reaction; S/ARE; sensitizing/antioxidant response element; SFN; sulforaphane; TNBS; trinitrobenzene sulfonate; WME; William's medium ECarboxylesterase; Keap1-Nrf2 pathway; Negative and positive AREs; Keap1 reactivity; Antioxidants; Skin sensitizers
Antioxidant sulforaphane and sensitizer trinitrobenzene sulfonate induce carboxylesterase-1 through a novel element transactivated by nuclear factor-E2 related factor-2 by Yi-Tzai Chen; Deshi Shi; Dongfang Yang; Bingfang Yan (pp. 864-871).
Carboxylesterase-1 (CES1), the most versatile human carboxylesterase, plays critical roles in drug metabolism and lipid mobilization. This enzyme is highly induced by antioxidants and sensitizers in various cell lines. These compounds are known to activate nuclear factor-E2 related factor-2 (Nrf2) by reacting to kelch-like ECH-associated protein-1 (Keap1). The aims of this study were to determine whether antioxidant sulforaphane (SFN) and sensitizer trinitrobenzene sulfonate (TNBS) target Keap1 similarly and whether they use the same element for CES1 induction. Cells over-expressing Keap1 were treated with TNBS or SFN and the formation of disulfide bonds among Keap1 molecules were determined. SFN promoted intramolecular disulfide formation whereas TNBS promoted intermolecular disulfide formation of Keap1. Two elements, sensitizing/antioxidant response element (S/ARE) and ARE4, were identified to support Nrf2 in the regulated expression of CES1A1. Both elements were bound by Nrf2, however, the S/ARE element supported, whereas the ARE4 element repressed Nrf2 transactivation. The repression required higher amounts of Nrf2, suggesting that the transactivation through the S/ARE element dominates the trans-repression through the ARE4 element under normal antioxidative condition. These findings conclude that compounds, although triggering the Keap1-Nrf2 pathway, may differ in the mode of reacting with Keap1. These findings also conclude that both positive and negative Nrf2 elements exist even within the same gene, and such opposing mechanisms provide fine-tuning in transcriptional regulation by the Keap1-Nrf2 pathway. High levels of CES1 are linked to lipid retention. Excessive induction of CES1 by antioxidants and sensitizers likely provides a mechanism for potential detrimental effect on human health.
Keywords: Abbreviations; AKR1C2; aldo-keto reductase-1C2; ARE; antioxidant response element; CES; carboxylesterase; ChIP; chromatin-immunoprecipitation; CYP; cytochrome P450; DMEM; Dulbecco's Modified Eagles Medium; EMSA; electrophoretic mobility shift assay; FBS; fetal bovine serum; GAPDH; glyceradehyde-3-phosphate dehydrogenase; GCLC; glutamate-cysteine ligase catalytic subunit; GCLM; glutamate-cysteine ligase regulatory subunit; NQO1; NAD(P)H:quinone oxidoreductase; Nrf2; nuclear factor-E2 related factor-2; PBS; phosphate buffered saline; RT-qPCR; reverse transcription-quantitative polymerase chain reaction; S/ARE; sensitizing/antioxidant response element; SFN; sulforaphane; TNBS; trinitrobenzene sulfonate; WME; William's medium ECarboxylesterase; Keap1-Nrf2 pathway; Negative and positive AREs; Keap1 reactivity; Antioxidants; Skin sensitizers