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Biochemical Pharmacology (v.81, #7)
Evaluating the role of Toll-like receptors in diseases of the central nervous system by Michael Carty; Andrew G. Bowie (pp. 825-837).
A key part of the innate immune system is a network of pattern recognition receptors (PRRs) and their associated intracellular signalling pathways. Toll-like receptors (TLRs) are one such group of PRRs that detect pathogen associated molecular patterns (PAMPs). Activation of the TLRs with their respective agonists results in the activation of intracellular signalling pathways leading to the expression of proinflammatory mediators and anti-microbial effector molecules. Activation of the innate immune system through TLRs also triggers the adaptive immune response, resulting in a comprehensive immune program to eradicate invading pathogens. It is now known that immune surveillance and inflammatory responses occur in the central nervous system (CNS). Furthermore it is becoming increasingly clear that TLRs have a role in such CNS responses and are also implicated in the pathogenesis of a number of conditions in the CNS, such as Alzheimer's, stroke and multiple sclerosis. This is likely due to the generation of endogenous TLR agonists in these conditions which amplifies a detrimental neurotoxic inflammatory response. However TLRs in some situations can be neuroprotective, if triggered in a favourable context. This review aims to examine the recent literature on TLRs in the CNS thus demonstrating their importance in a range of infectious and non-infectious diseases of the brain.
Keywords: Abbreviations; AD; Alzheimer's disease; ALRs; Aim2 like receptors; Aβ; Amyloidβ; BBB; blood brain barrier; CARD; caspase activating and recruitment domain; CB; Cerebral malaria; CFA; complete freunds adjuvant; CNS; central nervous system; CpG; cytosine phosphate guanosine; DAMPs; danger associates molecular patterns; DCs; dendritic cells; dsRNA; double-stranded RNA; EAE; experimental autoimmune encephalitis; EGF; epidermal growth factor; ERK; extracellular signal-regulated kinase; FADD; Fas-associated death domain; FAβ; fibrillar Aβ; FPRL1; formyl peptide receptor-like 1; GBS; group B streptococcus; GPI; Glycosylphosphatidylinositol; HMGB1; high mobility group box 1 protein; HSE; Herpes simplex virus induced encephalitis; HSP; heat shock proteins; HSV; Herpes simplex virus; IL-1β; Interleukin-1β; IL-6; Interleukin-6; iNOS; inducible nitric oxide synthase; IRAK; IL-1 receptor–associated kinases; IRF; Interferon Regulatory Factor; JNK; c-Jun N-terminal kinase; KO; knockout; LPS; lipopolysaccharide; Mal; MyD88 adaptor-like protein; MAPK; Mitogen-activated protein kinase; MMPs; matrix metalloproteinases; MS; multiple sclerosis; MyD88; Myeloid differentiation factor 88; NACHT; domain present in NAIP, CIITA, HET-E, TP-1; NAP1; NAK-associated protein 1; NFκB; Nuclear factor κB; NLRs; Nod-like Receptors; NO; nitric oxide; NPCs; neural progenitor cells; PAMP; pathogen-associated molecular pattern; polyI:C; polyinosine-deoxycytidylic acid; PRRs; pattern recognition receptors; RIP1; receptor interacting protein 1; RLRs; Rig like receptors RLRs; ROS; reactive oxygen species; SARM; sterile α-and armadillo-motif containing protein; SINTBAD; similar to NAP1 TBK1 adaptor; TAK1; transforming growth factor-b-activated protein kinase 1; TANK; TRAF family member-associated NFκB activator; TBK1; TANK-binding kinase; TIR; Toll/IL-1 receptor; TLR; Toll-like receptor; TNFα; tumour necrosis factor α; TRADD; TNF-R-associated death domain; TRAF6; tumor necrosis factor receptor associated factor 6; TRAM; TRIF-related adaptor protein; TRIF; TIR domain-containing adaptor; VEGF; vascular endothelial growth factor; WT; wild typeInnate immunity; Toll-like receptor; Infectious; Non-infectious; Diseases; Central nervous system
Evaluating the role of Toll-like receptors in diseases of the central nervous system by Michael Carty; Andrew G. Bowie (pp. 825-837).
A key part of the innate immune system is a network of pattern recognition receptors (PRRs) and their associated intracellular signalling pathways. Toll-like receptors (TLRs) are one such group of PRRs that detect pathogen associated molecular patterns (PAMPs). Activation of the TLRs with their respective agonists results in the activation of intracellular signalling pathways leading to the expression of proinflammatory mediators and anti-microbial effector molecules. Activation of the innate immune system through TLRs also triggers the adaptive immune response, resulting in a comprehensive immune program to eradicate invading pathogens. It is now known that immune surveillance and inflammatory responses occur in the central nervous system (CNS). Furthermore it is becoming increasingly clear that TLRs have a role in such CNS responses and are also implicated in the pathogenesis of a number of conditions in the CNS, such as Alzheimer's, stroke and multiple sclerosis. This is likely due to the generation of endogenous TLR agonists in these conditions which amplifies a detrimental neurotoxic inflammatory response. However TLRs in some situations can be neuroprotective, if triggered in a favourable context. This review aims to examine the recent literature on TLRs in the CNS thus demonstrating their importance in a range of infectious and non-infectious diseases of the brain.
Keywords: Abbreviations; AD; Alzheimer's disease; ALRs; Aim2 like receptors; Aβ; Amyloidβ; BBB; blood brain barrier; CARD; caspase activating and recruitment domain; CB; Cerebral malaria; CFA; complete freunds adjuvant; CNS; central nervous system; CpG; cytosine phosphate guanosine; DAMPs; danger associates molecular patterns; DCs; dendritic cells; dsRNA; double-stranded RNA; EAE; experimental autoimmune encephalitis; EGF; epidermal growth factor; ERK; extracellular signal-regulated kinase; FADD; Fas-associated death domain; FAβ; fibrillar Aβ; FPRL1; formyl peptide receptor-like 1; GBS; group B streptococcus; GPI; Glycosylphosphatidylinositol; HMGB1; high mobility group box 1 protein; HSE; Herpes simplex virus induced encephalitis; HSP; heat shock proteins; HSV; Herpes simplex virus; IL-1β; Interleukin-1β; IL-6; Interleukin-6; iNOS; inducible nitric oxide synthase; IRAK; IL-1 receptor–associated kinases; IRF; Interferon Regulatory Factor; JNK; c-Jun N-terminal kinase; KO; knockout; LPS; lipopolysaccharide; Mal; MyD88 adaptor-like protein; MAPK; Mitogen-activated protein kinase; MMPs; matrix metalloproteinases; MS; multiple sclerosis; MyD88; Myeloid differentiation factor 88; NACHT; domain present in NAIP, CIITA, HET-E, TP-1; NAP1; NAK-associated protein 1; NFκB; Nuclear factor κB; NLRs; Nod-like Receptors; NO; nitric oxide; NPCs; neural progenitor cells; PAMP; pathogen-associated molecular pattern; polyI:C; polyinosine-deoxycytidylic acid; PRRs; pattern recognition receptors; RIP1; receptor interacting protein 1; RLRs; Rig like receptors RLRs; ROS; reactive oxygen species; SARM; sterile α-and armadillo-motif containing protein; SINTBAD; similar to NAP1 TBK1 adaptor; TAK1; transforming growth factor-b-activated protein kinase 1; TANK; TRAF family member-associated NFκB activator; TBK1; TANK-binding kinase; TIR; Toll/IL-1 receptor; TLR; Toll-like receptor; TNFα; tumour necrosis factor α; TRADD; TNF-R-associated death domain; TRAF6; tumor necrosis factor receptor associated factor 6; TRAM; TRIF-related adaptor protein; TRIF; TIR domain-containing adaptor; VEGF; vascular endothelial growth factor; WT; wild typeInnate immunity; Toll-like receptor; Infectious; Non-infectious; Diseases; Central nervous system
Decoy receptor 3: A pleiotropic immunomodulator and biomarker for inflammatory diseases, autoimmune diseases and cancer by Wan-Wan Lin; Shie-Liang Hsieh (pp. 838-847).
Recently, several decoy molecules belonging to tumor necrosis factor receptor superfamily (TNFRSF) have been identified, including decoy receptor 1 (DcR1), decoy receptor 2 (DcR2), and decoy receptor 3 (DcR3). One of the tumor necrosis factor superfamily (TNFSF) members, TNF-related apoptosis-inducing ligand (TRAIL), binds to DcR1 and DcR2, which are membranous receptors with a truncated cytoplasmic domain, thus unable to transduce TRAIL-mediated signaling. In contrast to DcR1 and DcR2, DcR3 is a soluble receptor capable of neutralizing the biological effects of three other TNFSF members: Fas ligand (FasL/TNFSF6/CD95L), LIGHT (TNFSF14) and TNF-like molecule 1A (TL1A/TNFSF15). Since FasL is a potent apoptosis- and inflammation-inducing factor, LIGHT is involved in apoptosis and inflammation, and TL1A is a T cell costimulator and is involved in gut inflammation, DcR3 can be defined as an immunomodulator on the basis of its neutralizing effects on FasL, LIGHT, and TL1A. Initial studies demonstrated that DcR3 expression is elevated in tumors cells; however, later work showed that DcR3 expression is also upregulated in inflammatory diseases, where serum DcR3 levels correlate with disease progression. In addition to its neutralizing effect, DcR3 also acts as an effector molecule to modulate cell function via ‘non-decoy’ activities. This review focuses on the immunomodulatory effects of DcR3 via ‘decoy’ and ‘non-decoy’ functions, and discusses the potential of DcR3 as a biomarker to predict cancer invasion and inflammation progression. We also discuss the possible utility of recombinant DcR3 as a therapeutic agent to control autoimmune diseases, as well as the potential to attenuate tumor progression by inhibiting DcR3 expression.
Keywords: Inflammation; Autoimmune; Cancer; TNF
Decoy receptor 3: A pleiotropic immunomodulator and biomarker for inflammatory diseases, autoimmune diseases and cancer by Wan-Wan Lin; Shie-Liang Hsieh (pp. 838-847).
Recently, several decoy molecules belonging to tumor necrosis factor receptor superfamily (TNFRSF) have been identified, including decoy receptor 1 (DcR1), decoy receptor 2 (DcR2), and decoy receptor 3 (DcR3). One of the tumor necrosis factor superfamily (TNFSF) members, TNF-related apoptosis-inducing ligand (TRAIL), binds to DcR1 and DcR2, which are membranous receptors with a truncated cytoplasmic domain, thus unable to transduce TRAIL-mediated signaling. In contrast to DcR1 and DcR2, DcR3 is a soluble receptor capable of neutralizing the biological effects of three other TNFSF members: Fas ligand (FasL/TNFSF6/CD95L), LIGHT (TNFSF14) and TNF-like molecule 1A (TL1A/TNFSF15). Since FasL is a potent apoptosis- and inflammation-inducing factor, LIGHT is involved in apoptosis and inflammation, and TL1A is a T cell costimulator and is involved in gut inflammation, DcR3 can be defined as an immunomodulator on the basis of its neutralizing effects on FasL, LIGHT, and TL1A. Initial studies demonstrated that DcR3 expression is elevated in tumors cells; however, later work showed that DcR3 expression is also upregulated in inflammatory diseases, where serum DcR3 levels correlate with disease progression. In addition to its neutralizing effect, DcR3 also acts as an effector molecule to modulate cell function via ‘non-decoy’ activities. This review focuses on the immunomodulatory effects of DcR3 via ‘decoy’ and ‘non-decoy’ functions, and discusses the potential of DcR3 as a biomarker to predict cancer invasion and inflammation progression. We also discuss the possible utility of recombinant DcR3 as a therapeutic agent to control autoimmune diseases, as well as the potential to attenuate tumor progression by inhibiting DcR3 expression.
Keywords: Inflammation; Autoimmune; Cancer; TNF
FNC, a novel nucleoside analogue inhibits cell proliferation and tumor growth in a variety of human cancer cells by Qiang Wang; Xiaobing Liu; Qingduan Wang; Yan Zhang; Jinhua Jiang; Xiaohe Guo; Qingxia Fan; Liyun Zheng; Xuejun Yu; Ning Wang; Zhenliang Pan; Chuanjun Song; Wenqing Qi; Junbiao Chang (pp. 848-855).
Inhibition of cellular DNA synthesis is a strategy to block cancer cell division. Nucleoside analogues can incorporate into DNA and terminate DNA strand elongation. So far, several nucleoside analogues have been successfully used as anticancer drugs. FNC, 2′-deoxy-2′-β-fluoro-4′-azidocytidine is a novel cytidine analogue which demonstrated potent activity against hepatitis C virus (HCV). To investigate the therapeutic potential of FNC in human cancers we studied its activity in a number of cancer cells in vitro and in vivo. FNC potently inhibited cell proliferation with an IC50 of 0.95–4.55μM in a variety of aggressive human cancer cell lines including B-cell non-Hodgkin's lymphomas, lung adenocarcinoma and acute myeloid leukemia. Cells treated with FNC exhibited G1 and S cell cycle arrest at high and low dose, respectively, which confirms the mechanism of action of nucleoside analogues. Treatment of B-NHL cell lines with FNC induced apoptosis in a dose and time dependent manner. Finally, mouse xenograft models of hepatocarcinoma (H22), sarcoma (S180) and gastric carcinoma (SGC7901) demonstrated that FNC had significant tumor growth inhibition activity in a dose-dependent manner with low toxicity. Together, our results suggest that FNC may be a valuable therapy in cancer patients and warrant early phase clinical trial evaluation.
Keywords: Nucleoside analogues; Apoptosis; DNA synthesis; FNC; Tumor growth inhibition
FNC, a novel nucleoside analogue inhibits cell proliferation and tumor growth in a variety of human cancer cells by Qiang Wang; Xiaobing Liu; Qingduan Wang; Yan Zhang; Jinhua Jiang; Xiaohe Guo; Qingxia Fan; Liyun Zheng; Xuejun Yu; Ning Wang; Zhenliang Pan; Chuanjun Song; Wenqing Qi; Junbiao Chang (pp. 848-855).
Inhibition of cellular DNA synthesis is a strategy to block cancer cell division. Nucleoside analogues can incorporate into DNA and terminate DNA strand elongation. So far, several nucleoside analogues have been successfully used as anticancer drugs. FNC, 2′-deoxy-2′-β-fluoro-4′-azidocytidine is a novel cytidine analogue which demonstrated potent activity against hepatitis C virus (HCV). To investigate the therapeutic potential of FNC in human cancers we studied its activity in a number of cancer cells in vitro and in vivo. FNC potently inhibited cell proliferation with an IC50 of 0.95–4.55μM in a variety of aggressive human cancer cell lines including B-cell non-Hodgkin's lymphomas, lung adenocarcinoma and acute myeloid leukemia. Cells treated with FNC exhibited G1 and S cell cycle arrest at high and low dose, respectively, which confirms the mechanism of action of nucleoside analogues. Treatment of B-NHL cell lines with FNC induced apoptosis in a dose and time dependent manner. Finally, mouse xenograft models of hepatocarcinoma (H22), sarcoma (S180) and gastric carcinoma (SGC7901) demonstrated that FNC had significant tumor growth inhibition activity in a dose-dependent manner with low toxicity. Together, our results suggest that FNC may be a valuable therapy in cancer patients and warrant early phase clinical trial evaluation.
Keywords: Nucleoside analogues; Apoptosis; DNA synthesis; FNC; Tumor growth inhibition
7-Chloro-6-piperidin-1-yl-quinoline-5,8-dione (PT-262), a novel ROCK inhibitor blocks cytoskeleton function and cell migration by Chih-Chien Tsai; Huei-Fang Liu; Kai-Cheng Hsu; Jinn-Moon Yang; Chinpiao Chen; Kuang-Kai Liu; Tzu-Sheng Hsu; Jui-I. Chao (pp. 856-865).
The 5,8-quinolinediones are precursors for producing multiple types of bioactive products. In this study, we investigated a new compound derived from 5,8-quinolinediones, 7-chloro-6-piperidin-1-yl-quinoline-5,8-dione (designated as PT-262), which markedly induced cytoskeleton remodeling and migration inhibition in lung carcinoma cells. Comparison with various cytoskeleton inhibitors, including paclitaxel, colchicine and phallacidin, the cell morphology following treatment with PT-262 was similar to phallacidin on the cell elongation and abnormal actin polymerization. However, PT-262 did not directly bind to actin filaments. ROCK (Rho-associated coiled-coil forming protein kinase) is a downstream effector of RhoA to mediate the phosphorylation of myosin light chain (MLC) and cytoskeleton reorganization. The RhoA–ROCK–MLC pathway has been shown to promote cancer cell migration and metastasis. Interestingly, PT-262 was more effective on inhibiting ROCK kinase activities than specific ROCK inhibitors Y-27632 and H-1152. PT-262 induced cytoskeleton remodeling and migration inhibition in A549 lung carcinoma cells. The total MLC and phosphorylated MLC proteins and stress fibers were blocked after treatment with PT-262. Nonetheless, the RhoA protein and GTPase activity were not altered by PT-262. A computational model suggests that PT-262 interacts with the ATP-binding site of ROCK protein. Together, these findings demonstrate that PT-262 is a new ROCK inhibitor.
Keywords: Abbreviations; ROCK; Rho-associated coiled-coil forming protein kinase; MLC; myosin light chain; MBS; myosin binding subunit; DMSO; dimethyl sulfoxide; PI; propidium iodide; MTT; 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide; FBS; fetal bovine serum; ERK; extracellular signal-regulated kinase; PBS; phosphate-buffered saline; F-actin; actin filamentPT-262; ROCK; Cell migration; Cytoskeleton; Lung cancer
7-Chloro-6-piperidin-1-yl-quinoline-5,8-dione (PT-262), a novel ROCK inhibitor blocks cytoskeleton function and cell migration by Chih-Chien Tsai; Huei-Fang Liu; Kai-Cheng Hsu; Jinn-Moon Yang; Chinpiao Chen; Kuang-Kai Liu; Tzu-Sheng Hsu; Jui-I. Chao (pp. 856-865).
The 5,8-quinolinediones are precursors for producing multiple types of bioactive products. In this study, we investigated a new compound derived from 5,8-quinolinediones, 7-chloro-6-piperidin-1-yl-quinoline-5,8-dione (designated as PT-262), which markedly induced cytoskeleton remodeling and migration inhibition in lung carcinoma cells. Comparison with various cytoskeleton inhibitors, including paclitaxel, colchicine and phallacidin, the cell morphology following treatment with PT-262 was similar to phallacidin on the cell elongation and abnormal actin polymerization. However, PT-262 did not directly bind to actin filaments. ROCK (Rho-associated coiled-coil forming protein kinase) is a downstream effector of RhoA to mediate the phosphorylation of myosin light chain (MLC) and cytoskeleton reorganization. The RhoA–ROCK–MLC pathway has been shown to promote cancer cell migration and metastasis. Interestingly, PT-262 was more effective on inhibiting ROCK kinase activities than specific ROCK inhibitors Y-27632 and H-1152. PT-262 induced cytoskeleton remodeling and migration inhibition in A549 lung carcinoma cells. The total MLC and phosphorylated MLC proteins and stress fibers were blocked after treatment with PT-262. Nonetheless, the RhoA protein and GTPase activity were not altered by PT-262. A computational model suggests that PT-262 interacts with the ATP-binding site of ROCK protein. Together, these findings demonstrate that PT-262 is a new ROCK inhibitor.
Keywords: Abbreviations; ROCK; Rho-associated coiled-coil forming protein kinase; MLC; myosin light chain; MBS; myosin binding subunit; DMSO; dimethyl sulfoxide; PI; propidium iodide; MTT; 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide; FBS; fetal bovine serum; ERK; extracellular signal-regulated kinase; PBS; phosphate-buffered saline; F-actin; actin filamentPT-262; ROCK; Cell migration; Cytoskeleton; Lung cancer
Induction of integrin β3 in PGE2-stimulated adhesion of mastocytoma P-815 cells to the Arg-Gly-Asp-enriched fragment of fibronectin by Yasuyo Okada; Jun-ichi Nishikawa; Masanori Semma; Atsushi Ichikawa (pp. 866-872).
We previously demonstrated that prostaglandin (PG) E2 stimulates adhesion of mastocytoma P-815 cells (P-815 cells) to the Arg-Gly-Asp (RGD)-enriched matrix via the PGE2 receptor subtype EP4 [Hatae N, Kita A, Tanaka S, Sugimoto Y, Ichikawa A. Induction of adherent activity in mastocytoma P-815 cells by the cooperation of two prostaglandin E2 receptor subtypes, EP3 and EP4. J Biol Chem 2003;278:17977–81]. Here we investigated the role of various integrin subtypes in the induction of adherent activity in PGE2-stimulated P-815 cells. FACS analysis showed that P-815 cells express high levels of integrin α4, α5, β1 and β2 subunits and moderate levels of integrin αIIb, αv, β3 and β7 subunits. When treated with PGE2, the EP4 agonist ONO-AE1-329 or the cell permeable cAMP analogue, 8-Br-cAMP, P-815 cells showed markedly increased cell surface expression of integrin αIIb, αv and β3 subunits, and these expressions were significantly reduced by addition of the protein synthesis inhibitor cycloheximide. Along with increased cell surface expression, mRNA and protein levels of the integrin β3 subunit, but not of integrin αIIb and αv subunits, were simultaneously elevated. On the other hand, adhesion of P-815 cells in response to PGE2 or 8-Br-cAMP was abolished by antibodies specific for integrin αv and β3 subunits, but not by antibodies for integrin α4, α5, β1, β2 and β7 subunits. Moreover, treatment with tirofiban, an integrin αIIbβ3 antagonist, or eptifibatide, an integrin αvβ3/αIIbβ3 antagonist resulted in a decrease in adhesion of P-815 cells in response to PGE2 or 8-Br-cAMP. These results suggest that de novo synthesis of the integrin β3 subunit plays a pivotal role in PGE2-induced adhesion of P-815 cells to the RGD-enriched matrix through EP4-mediated cAMP signaling.
Keywords: Mastocytoma P-815 cells; Prostaglandin E; 2; Adhesion; Integrin; Fibronectin
Induction of integrin β3 in PGE2-stimulated adhesion of mastocytoma P-815 cells to the Arg-Gly-Asp-enriched fragment of fibronectin by Yasuyo Okada; Jun-ichi Nishikawa; Masanori Semma; Atsushi Ichikawa (pp. 866-872).
We previously demonstrated that prostaglandin (PG) E2 stimulates adhesion of mastocytoma P-815 cells (P-815 cells) to the Arg-Gly-Asp (RGD)-enriched matrix via the PGE2 receptor subtype EP4 [Hatae N, Kita A, Tanaka S, Sugimoto Y, Ichikawa A. Induction of adherent activity in mastocytoma P-815 cells by the cooperation of two prostaglandin E2 receptor subtypes, EP3 and EP4. J Biol Chem 2003;278:17977–81]. Here we investigated the role of various integrin subtypes in the induction of adherent activity in PGE2-stimulated P-815 cells. FACS analysis showed that P-815 cells express high levels of integrin α4, α5, β1 and β2 subunits and moderate levels of integrin αIIb, αv, β3 and β7 subunits. When treated with PGE2, the EP4 agonist ONO-AE1-329 or the cell permeable cAMP analogue, 8-Br-cAMP, P-815 cells showed markedly increased cell surface expression of integrin αIIb, αv and β3 subunits, and these expressions were significantly reduced by addition of the protein synthesis inhibitor cycloheximide. Along with increased cell surface expression, mRNA and protein levels of the integrin β3 subunit, but not of integrin αIIb and αv subunits, were simultaneously elevated. On the other hand, adhesion of P-815 cells in response to PGE2 or 8-Br-cAMP was abolished by antibodies specific for integrin αv and β3 subunits, but not by antibodies for integrin α4, α5, β1, β2 and β7 subunits. Moreover, treatment with tirofiban, an integrin αIIbβ3 antagonist, or eptifibatide, an integrin αvβ3/αIIbβ3 antagonist resulted in a decrease in adhesion of P-815 cells in response to PGE2 or 8-Br-cAMP. These results suggest that de novo synthesis of the integrin β3 subunit plays a pivotal role in PGE2-induced adhesion of P-815 cells to the RGD-enriched matrix through EP4-mediated cAMP signaling.
Keywords: Mastocytoma P-815 cells; Prostaglandin E; 2; Adhesion; Integrin; Fibronectin
UVB light regulates expression of antioxidants and inflammatory mediators in human corneal epithelial cells by Adrienne T. Black; Marion. K. Gordon; Diane E. Heck; Michael A. Gallo; Debra L. Laskin; Jeffrey D. Laskin (pp. 873-880).
The cornea is highly sensitive to ultraviolet B (UVB) light-induced oxidative stress, a process that results in the production of inflammatory mediators which have been implicated in tissue injury. In the present studies, we characterized the inflammatory response of human corneal epithelial cells to UVB (2.5–25mJ/cm2). UVB caused a dose-dependent increase in the generation of reactive oxygen species in the cells. This was associated with increases in mRNA expression of the antioxidants Cu,Zn superoxide dismutase (SOD), Mn-SOD, catalase and heme oxygenase-1 (HO-1), as well as the glutathione S-transferases (GST), GSTA1-2, GSTA3, GSTA4, GSTM1, and mGST2. UVB also upregulated expression of the proinflammatory cytokines, IFNγ, IL-1β, TGFβ and TNFα, and enzymes important in prostaglandin (PG) biosynthesis including cyclooxygenase-2 (COX-2) and the PG synthases mPGES-2, PGDS, PGFS and thromboxane synthase, and in leukotriene biosynthesis including 5-lipoxygenase (5-LOX), 15-LOX-2, and the epidermal and platelet forms of 12-LOX. UVB was found to activate JNK and p38 MAP kinases in corneal epithelial cells; ERK1/2 MAP kinase was found to be constitutively active, and its activity increased following UVB treatment. Inhibition of p38 blocked UVB-induced expression of TNFα, COX-2, PGDS and 15-LOX-2, while JNK inhibition suppressed TNFα and HO-1. These data indicate that UVB modulates corneal epithelial cell expression of antioxidants and proinflammatory mediators by distinct mechanisms. Alterations in expression of these mediators are likely to be important in regulating inflammation and protecting the cornea from UVB-induced oxidative stress.
Keywords: Abbreviations; COX-1; cyclooxygenase-1; COX-2; cyclooxygenase-2; FLAP; 5-lipoxygenase activating protein; GST; glutathione; S; -transferase; mGST; microsomal GST; HO-1; heme oxygenase-1; LTA; 4; hydrolase; leukotriene A; 4; hydrolase; LTC; 4; synthase; leukotriene C; 4; synthase; LOX; lipoxygenase; 5-LOX; 5-lipoxygenase; 12-LOX-epi; 12-lipoxygenase-epidermal type; 12-LOX-plt; 12-lipoxygenase platelet-type; IFN; interferon; IL; interleukin; PGE; 2; prostaglandin E; 2; mPGES-1; microsomal PGE; 2; synthase-1; mPGES-2; microsomal PGE; 2; synthase-2; PGD; 2; prostaglandin D; 2; PGDS; PGD; 2; synthase; PGF; 2; prostaglandin F; 2; PGFS; PGF; 2; synthase; PGI; 2; prostaglandin I; 2; PGIS; PGI; 2; synthase; PGH; 2; prostaglandin H; 2; SOD; superoxide dismutase; TXA; 2; thromboxane A; 2; TXAS; TXA; 2; synthase; TGFβ; transforming growth factor beta; TNFα; tumor necrosis factor alphaUltraviolet light; Ocular; Oxidative stress; Prostaglandins; Leukotrienes
UVB light regulates expression of antioxidants and inflammatory mediators in human corneal epithelial cells by Adrienne T. Black; Marion. K. Gordon; Diane E. Heck; Michael A. Gallo; Debra L. Laskin; Jeffrey D. Laskin (pp. 873-880).
The cornea is highly sensitive to ultraviolet B (UVB) light-induced oxidative stress, a process that results in the production of inflammatory mediators which have been implicated in tissue injury. In the present studies, we characterized the inflammatory response of human corneal epithelial cells to UVB (2.5–25mJ/cm2). UVB caused a dose-dependent increase in the generation of reactive oxygen species in the cells. This was associated with increases in mRNA expression of the antioxidants Cu,Zn superoxide dismutase (SOD), Mn-SOD, catalase and heme oxygenase-1 (HO-1), as well as the glutathione S-transferases (GST), GSTA1-2, GSTA3, GSTA4, GSTM1, and mGST2. UVB also upregulated expression of the proinflammatory cytokines, IFNγ, IL-1β, TGFβ and TNFα, and enzymes important in prostaglandin (PG) biosynthesis including cyclooxygenase-2 (COX-2) and the PG synthases mPGES-2, PGDS, PGFS and thromboxane synthase, and in leukotriene biosynthesis including 5-lipoxygenase (5-LOX), 15-LOX-2, and the epidermal and platelet forms of 12-LOX. UVB was found to activate JNK and p38 MAP kinases in corneal epithelial cells; ERK1/2 MAP kinase was found to be constitutively active, and its activity increased following UVB treatment. Inhibition of p38 blocked UVB-induced expression of TNFα, COX-2, PGDS and 15-LOX-2, while JNK inhibition suppressed TNFα and HO-1. These data indicate that UVB modulates corneal epithelial cell expression of antioxidants and proinflammatory mediators by distinct mechanisms. Alterations in expression of these mediators are likely to be important in regulating inflammation and protecting the cornea from UVB-induced oxidative stress.
Keywords: Abbreviations; COX-1; cyclooxygenase-1; COX-2; cyclooxygenase-2; FLAP; 5-lipoxygenase activating protein; GST; glutathione; S; -transferase; mGST; microsomal GST; HO-1; heme oxygenase-1; LTA; 4; hydrolase; leukotriene A; 4; hydrolase; LTC; 4; synthase; leukotriene C; 4; synthase; LOX; lipoxygenase; 5-LOX; 5-lipoxygenase; 12-LOX-epi; 12-lipoxygenase-epidermal type; 12-LOX-plt; 12-lipoxygenase platelet-type; IFN; interferon; IL; interleukin; PGE; 2; prostaglandin E; 2; mPGES-1; microsomal PGE; 2; synthase-1; mPGES-2; microsomal PGE; 2; synthase-2; PGD; 2; prostaglandin D; 2; PGDS; PGD; 2; synthase; PGF; 2; prostaglandin F; 2; PGFS; PGF; 2; synthase; PGI; 2; prostaglandin I; 2; PGIS; PGI; 2; synthase; PGH; 2; prostaglandin H; 2; SOD; superoxide dismutase; TXA; 2; thromboxane A; 2; TXAS; TXA; 2; synthase; TGFβ; transforming growth factor beta; TNFα; tumor necrosis factor alphaUltraviolet light; Ocular; Oxidative stress; Prostaglandins; Leukotrienes
Aurora inhibitor MLN8237 in combination with docetaxel enhances apoptosis and anti-tumor activity in mantle cell lymphoma by Wenqing Qi; Laurence S. Cooke; Xiaobing Liu; Lisa Rimsza; Denise J. Roe; Ann Manziolli. Daniel O. Persky; Thomas P. Miller; Daruka Mahadevan (pp. 881-890).
Auroras (A and B) are oncogenic serine/threonine kinases that play key roles in the mitotic phase of the eukaryotic cell cycle. Analysis of the leukemia lymphoma molecular profiling project (LLMPP) database indicates Aurora over-expression correlates with poor prognosis. A tissue microarray (TMA) composed of 20 paired mantle cell lymphoma (MCL) patients demonstrated >75% of patients had high levels Aurora expression. Aurora A and B were also found elevated in 13 aggressive B-NHL cell lines. MLN8237, an Aurora inhibitor induced G2/M arrest with polyploidy and abrogated Aurora A and histone-H3 phosphorylation. MLN8237 inhibited aggressive B-NHL cell proliferation at an IC50 of 10–50nM and induced apoptosis in a dose- and time-dependent manner. Low dose combinations of MLN8237+docetaxel enhanced apoptosis by ∼3–4-fold in cell culture compared to single agents respectively. A mouse xenograft model of MCL demonstrated that MLN8237 (10 or 30mg/kg) or docetaxel (10mg/kg) alone had modest anti-tumor activity. However, MLN8237 plus docetaxel demonstrated a statistically significant tumor growth inhibition and enhanced survival compared to single agent therapy. Together, our results suggest that MLN8237 plus docetaxel may represent a novel therapeutic strategy that could be evaluated in early phase trials in relapsed/refractory aggressive B-cell NHL.
Keywords: Non-Hodgkin lymphoma (NHL); Mantle cell lymphoma (MCL); Aurora A and B; Aurora Inhibitor; MLN8237; Docetaxel
Aurora inhibitor MLN8237 in combination with docetaxel enhances apoptosis and anti-tumor activity in mantle cell lymphoma by Wenqing Qi; Laurence S. Cooke; Xiaobing Liu; Lisa Rimsza; Denise J. Roe; Ann Manziolli. Daniel O. Persky; Thomas P. Miller; Daruka Mahadevan (pp. 881-890).
Auroras (A and B) are oncogenic serine/threonine kinases that play key roles in the mitotic phase of the eukaryotic cell cycle. Analysis of the leukemia lymphoma molecular profiling project (LLMPP) database indicates Aurora over-expression correlates with poor prognosis. A tissue microarray (TMA) composed of 20 paired mantle cell lymphoma (MCL) patients demonstrated >75% of patients had high levels Aurora expression. Aurora A and B were also found elevated in 13 aggressive B-NHL cell lines. MLN8237, an Aurora inhibitor induced G2/M arrest with polyploidy and abrogated Aurora A and histone-H3 phosphorylation. MLN8237 inhibited aggressive B-NHL cell proliferation at an IC50 of 10–50nM and induced apoptosis in a dose- and time-dependent manner. Low dose combinations of MLN8237+docetaxel enhanced apoptosis by ∼3–4-fold in cell culture compared to single agents respectively. A mouse xenograft model of MCL demonstrated that MLN8237 (10 or 30mg/kg) or docetaxel (10mg/kg) alone had modest anti-tumor activity. However, MLN8237 plus docetaxel demonstrated a statistically significant tumor growth inhibition and enhanced survival compared to single agent therapy. Together, our results suggest that MLN8237 plus docetaxel may represent a novel therapeutic strategy that could be evaluated in early phase trials in relapsed/refractory aggressive B-cell NHL.
Keywords: Non-Hodgkin lymphoma (NHL); Mantle cell lymphoma (MCL); Aurora A and B; Aurora Inhibitor; MLN8237; Docetaxel
Taurine suppresses doxorubicin-triggered oxidative stress and cardiac apoptosis in rat via up-regulation of PI3-K/Akt and inhibition of p53, p38-JNK by Joydeep Das; Jyotirmoy Ghosh; Prasenjit Manna; Parames C. Sil (pp. 891-909).
The objective of the present study was to investigate the signaling mechanisms involved in the beneficial role of taurine against doxorubicin-induced cardiac oxidative stress. Male rats were administered doxorubicin. Hearts were collected 3 weeks after the last dose of doxorubicin and were analyzed. Doxorubicin administration retarded the growth of the body and the heart and caused injury in the cardiac tissue because of increased oxidative stress. Similar experiments with doxorubicin showed reduced cell viability, increased ROS generation, intracellular Ca2+ and DNA fragmentation, disrupted mitochondrial membrane potential and apoptotic cell death in primary cultured neonatal rat cardiomyocytes. Signal transduction studies showed that doxorubicin increased p53, JNK, p38 and NFκB phosphorylation; decreased the levels of phospho ERK and Akt; disturbed the Bcl-2 family protein balance; activated caspase 12, caspase 9 and caspase 3; and induced cleavage of the PARP protein. However, taurine treatment or cardiomyocyte incubation with taurine suppressed all of the adverse effects of doxorubicin. Studies with several inhibitors, including PS-1145 (an IKK inhibitor), SP600125 (a JNK inhibitor), SB203580 (a p38 inhibitor) and LY294002 (a PI3-K/Akt inhibitor), demonstrated that the mechanism of taurine-induced cardio protection involves activation of specific survival signals and PI3-K/Akt as well as the inhibition of p53, JNK, p38 and NFκB. These novel findings suggest that taurine might have clinical implications for the prevention of doxorubicin-induced cardiac oxidative stress.
Keywords: Abbreviations; CAT; catalase; DOX; doxorubicin; GSH; glutathione; GSSG; glutathione disulfide; GPx; glutathione peroxidase; HDL; high-density lipoprotein; LDH; lactate dehydrogenase; MDA; malondialdehyde; MAPKs; mitogen-activated protein kinases; NF-κB; nuclear factor kappa B; PI3-K; phosphatidylinositol 3-kinase; ROS; reactive oxygen species; SOD; superoxide dismutase; TAU; taurineDoxorubicin; Cardiac oxidative stress; Reactive oxygen species; p53; NF-κB and MAPK; PI3-K/Akt; Apoptosis; Taurine; Antioxidant; Cell survival
Taurine suppresses doxorubicin-triggered oxidative stress and cardiac apoptosis in rat via up-regulation of PI3-K/Akt and inhibition of p53, p38-JNK by Joydeep Das; Jyotirmoy Ghosh; Prasenjit Manna; Parames C. Sil (pp. 891-909).
The objective of the present study was to investigate the signaling mechanisms involved in the beneficial role of taurine against doxorubicin-induced cardiac oxidative stress. Male rats were administered doxorubicin. Hearts were collected 3 weeks after the last dose of doxorubicin and were analyzed. Doxorubicin administration retarded the growth of the body and the heart and caused injury in the cardiac tissue because of increased oxidative stress. Similar experiments with doxorubicin showed reduced cell viability, increased ROS generation, intracellular Ca2+ and DNA fragmentation, disrupted mitochondrial membrane potential and apoptotic cell death in primary cultured neonatal rat cardiomyocytes. Signal transduction studies showed that doxorubicin increased p53, JNK, p38 and NFκB phosphorylation; decreased the levels of phospho ERK and Akt; disturbed the Bcl-2 family protein balance; activated caspase 12, caspase 9 and caspase 3; and induced cleavage of the PARP protein. However, taurine treatment or cardiomyocyte incubation with taurine suppressed all of the adverse effects of doxorubicin. Studies with several inhibitors, including PS-1145 (an IKK inhibitor), SP600125 (a JNK inhibitor), SB203580 (a p38 inhibitor) and LY294002 (a PI3-K/Akt inhibitor), demonstrated that the mechanism of taurine-induced cardio protection involves activation of specific survival signals and PI3-K/Akt as well as the inhibition of p53, JNK, p38 and NFκB. These novel findings suggest that taurine might have clinical implications for the prevention of doxorubicin-induced cardiac oxidative stress.
Keywords: Abbreviations; CAT; catalase; DOX; doxorubicin; GSH; glutathione; GSSG; glutathione disulfide; GPx; glutathione peroxidase; HDL; high-density lipoprotein; LDH; lactate dehydrogenase; MDA; malondialdehyde; MAPKs; mitogen-activated protein kinases; NF-κB; nuclear factor kappa B; PI3-K; phosphatidylinositol 3-kinase; ROS; reactive oxygen species; SOD; superoxide dismutase; TAU; taurineDoxorubicin; Cardiac oxidative stress; Reactive oxygen species; p53; NF-κB and MAPK; PI3-K/Akt; Apoptosis; Taurine; Antioxidant; Cell survival
ERK1/2 deactivation enhances cytoplasmic Nur77 expression level and improves the apoptotic effect of fenretinide in human liver cancer cells by Hui Yang; Yuqiang Nie; Yuyuan Li; Yu-Jui Yvonne Wan (pp. 910-916).
Fenretinide, a synthetic retinoid, is a promising anticancer agent based on many in vitro, animal, and chemoprevention clinical trial studies. However, cells such as HepG2 human liver cancer cells are resistant to the apoptotic effect of fenretinide. Previously, we have shown that fenretinide-induced apoptosis is Nur77 dependent, and the sensitivity of the cancer cells to fenretinide-induced apoptosis is positively associated with cytoplasmic enrichment of Nur77. The goal of current study was to identify means to modulate nuclear export of Nur77 in order to improve the efficacy of fenretinide. Fenretinide treatment deactivated ERK1/2 in Huh7 cells, but activated ERK1/2 in HepG2 cells, which was positively associated with the sensitivity of cells to the apoptotic effect of fenretinide. Neither fenretinide nor ERK1/2 inhibitor PD98059 alone could affect the survival of HepG2 cells, but the combination of both induced cell death and increased caspase 3/7 activity. In fenretinide sensitive Huh7 cells, activation of ERK1/2 by epidermal growth factor (EGF) prevented fenretinide-induced cell death and caspase 3/7 induction. In addition, modulation of ERK1/2 changed the intracellular localization of Nur77. Fenretinide/PD98059-induced cell death of HepG2 cell was positively associated with induction and cytoplasmic location as well as mitochondria enrichment of Nur77. The effect was specific for ERK1/2 because other mitogen activated protein kinases such as P38, Akt, and JNK did not have correlated changes in their phosphorylation levels. Taken together, the current study demonstrates that ERK1/2-modulated Nur77 intracellular location dictates the efficacy of fenretinide-induced apoptosis.
Keywords: Fenretinide; Nur77; Apoptosis; ERK1/2; ROS; HCC
ERK1/2 deactivation enhances cytoplasmic Nur77 expression level and improves the apoptotic effect of fenretinide in human liver cancer cells by Hui Yang; Yuqiang Nie; Yuyuan Li; Yu-Jui Yvonne Wan (pp. 910-916).
Fenretinide, a synthetic retinoid, is a promising anticancer agent based on many in vitro, animal, and chemoprevention clinical trial studies. However, cells such as HepG2 human liver cancer cells are resistant to the apoptotic effect of fenretinide. Previously, we have shown that fenretinide-induced apoptosis is Nur77 dependent, and the sensitivity of the cancer cells to fenretinide-induced apoptosis is positively associated with cytoplasmic enrichment of Nur77. The goal of current study was to identify means to modulate nuclear export of Nur77 in order to improve the efficacy of fenretinide. Fenretinide treatment deactivated ERK1/2 in Huh7 cells, but activated ERK1/2 in HepG2 cells, which was positively associated with the sensitivity of cells to the apoptotic effect of fenretinide. Neither fenretinide nor ERK1/2 inhibitor PD98059 alone could affect the survival of HepG2 cells, but the combination of both induced cell death and increased caspase 3/7 activity. In fenretinide sensitive Huh7 cells, activation of ERK1/2 by epidermal growth factor (EGF) prevented fenretinide-induced cell death and caspase 3/7 induction. In addition, modulation of ERK1/2 changed the intracellular localization of Nur77. Fenretinide/PD98059-induced cell death of HepG2 cell was positively associated with induction and cytoplasmic location as well as mitochondria enrichment of Nur77. The effect was specific for ERK1/2 because other mitogen activated protein kinases such as P38, Akt, and JNK did not have correlated changes in their phosphorylation levels. Taken together, the current study demonstrates that ERK1/2-modulated Nur77 intracellular location dictates the efficacy of fenretinide-induced apoptosis.
Keywords: Fenretinide; Nur77; Apoptosis; ERK1/2; ROS; HCC
The NADPH oxidase inhibitor VAS2870 impairs cell growth and enhances TGF-β-induced apoptosis of liver tumor cells by Patricia Sancho; Isabel Fabregat (pp. 917-924).
Liver tumor cells show several molecular alterations which favor pro-survival signaling. Among those, we have proposed the NADPH oxidase NOX1 as a prosurvival signal for liver tumor cells. On the one side, we have described that FaO rat hepatoma cells show NOX1-dependent partial resistance to apoptosis induced by Transforming Growth Factor beta (TGF-β). On the other side, we have shown that FaO cells, as well as different human hepatocellular carcinoma (HCC) cell lines, are able to proliferate in the absence of serum through the activation of a NOX1-dependent signaling pathway. The aim of this work was to analyze the effects of NADPH oxidase pharmacological inhibition in liver tumor cells using the inhibitor VAS2870. This compound inhibits dose-dependently autocrine increase of cell number in FaO rat hepatoma cells, and almost completely blocked ROS production and thymidine incorporation when used at 25μM. Such inhibitory effect on autocrine growth is coincident with lower mRNA levels of EGFR (Epidermal Growth Factor Receptor) and its ligand TGF-α (Transforming Growth Factor-alpha), and decreased phosphorylation of the EGFR itself and other downstream targets, such as SRC or AKT. Moreover, NADPH oxidase pharmacological inhibition also effectively attenuates serum-dependent growth and phosphorylation of AKT and ERK. Importantly, these inhibitory effects on either autocrine or serum-dependent cell growth are observed in several human HCC cell lines. Finally, we have observed that VAS2870 is also effective in enhancing apoptosis induced by a physiological stimulus, such as TGF-β. In summary, NADPH oxidase pharmacological inhibition could be considered a promising tool in the treatment of liver cancer.
Keywords: NADPH oxidases; NOX; Reactive Oxygen Species (ROS); Proliferation; Hepatocellular carcinoma (HCC)
The NADPH oxidase inhibitor VAS2870 impairs cell growth and enhances TGF-β-induced apoptosis of liver tumor cells by Patricia Sancho; Isabel Fabregat (pp. 917-924).
Liver tumor cells show several molecular alterations which favor pro-survival signaling. Among those, we have proposed the NADPH oxidase NOX1 as a prosurvival signal for liver tumor cells. On the one side, we have described that FaO rat hepatoma cells show NOX1-dependent partial resistance to apoptosis induced by Transforming Growth Factor beta (TGF-β). On the other side, we have shown that FaO cells, as well as different human hepatocellular carcinoma (HCC) cell lines, are able to proliferate in the absence of serum through the activation of a NOX1-dependent signaling pathway. The aim of this work was to analyze the effects of NADPH oxidase pharmacological inhibition in liver tumor cells using the inhibitor VAS2870. This compound inhibits dose-dependently autocrine increase of cell number in FaO rat hepatoma cells, and almost completely blocked ROS production and thymidine incorporation when used at 25μM. Such inhibitory effect on autocrine growth is coincident with lower mRNA levels of EGFR (Epidermal Growth Factor Receptor) and its ligand TGF-α (Transforming Growth Factor-alpha), and decreased phosphorylation of the EGFR itself and other downstream targets, such as SRC or AKT. Moreover, NADPH oxidase pharmacological inhibition also effectively attenuates serum-dependent growth and phosphorylation of AKT and ERK. Importantly, these inhibitory effects on either autocrine or serum-dependent cell growth are observed in several human HCC cell lines. Finally, we have observed that VAS2870 is also effective in enhancing apoptosis induced by a physiological stimulus, such as TGF-β. In summary, NADPH oxidase pharmacological inhibition could be considered a promising tool in the treatment of liver cancer.
Keywords: NADPH oxidases; NOX; Reactive Oxygen Species (ROS); Proliferation; Hepatocellular carcinoma (HCC)
Artepillin C, as a PPARγ ligand, enhances adipocyte differentiation and glucose uptake in 3T3-L1 cells by Sun-Sil Choi; Byung-Yoon Cha; Kagami Iida; Young-Sil Lee; Takayuki Yonezawa; Toshiaki Teruya; Kazuo Nagai; Je-Tae Woo (pp. 925-933).
Artepillin C promotes adipocyte differentiation and glucose uptake in part by direct binding to PPARγ.The nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ plays an important role in adipocyte differentiation. Its ligands, including thiazolidinediones, improve insulin sensitivity in type 2 diabetes. We investigated the effects of artepillin C, an ingredient of Baccharis dracunculifolia, on adipogenesis and glucose uptake using 3T3-L1 cells. In PPARγ ligand-binding assays, artepillin C exhibited binding affinity toward PPARγ. Artepillin C dose-dependently enhanced adipocyte differentiation of 3T3-L1 cells. As a result of the artepillin C-induced adipocyte differentiation, the gene expression of PPARγ and its target genes, such as aP2, adiponectin and glucose transporter (GLUT) 4, was increased. These increases were abolished by cotreatment with GW9662, a PPARγ antagonist. In mature 3T3-L1 adipocytes, artepillin C significantly enhanced the basal and insulin-stimulated glucose uptake. These effects were decreased by cotreatment with a PI3K inhibitor. Although artepillin C had no effects on the insulin signaling cascade, artepillin C enhanced the expression and plasma membrane translocation of GLUT1 and GLUT4 in mature adipocytes. In conclusion, these findings suggest that artepillin C promotes adipocyte differentiation and glucose uptake in part by direct binding to PPARγ, which could be the basis of the pharmacological benefits of green propolis intake in reducing the risk of type 2 diabetes.
Keywords: Artepillin C; 3T3-L1 cells; Adipocyte differentiation; PPARγ ligand; Glucose uptake
Artepillin C, as a PPARγ ligand, enhances adipocyte differentiation and glucose uptake in 3T3-L1 cells by Sun-Sil Choi; Byung-Yoon Cha; Kagami Iida; Young-Sil Lee; Takayuki Yonezawa; Toshiaki Teruya; Kazuo Nagai; Je-Tae Woo (pp. 925-933).
Artepillin C promotes adipocyte differentiation and glucose uptake in part by direct binding to PPARγ.The nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ plays an important role in adipocyte differentiation. Its ligands, including thiazolidinediones, improve insulin sensitivity in type 2 diabetes. We investigated the effects of artepillin C, an ingredient of Baccharis dracunculifolia, on adipogenesis and glucose uptake using 3T3-L1 cells. In PPARγ ligand-binding assays, artepillin C exhibited binding affinity toward PPARγ. Artepillin C dose-dependently enhanced adipocyte differentiation of 3T3-L1 cells. As a result of the artepillin C-induced adipocyte differentiation, the gene expression of PPARγ and its target genes, such as aP2, adiponectin and glucose transporter (GLUT) 4, was increased. These increases were abolished by cotreatment with GW9662, a PPARγ antagonist. In mature 3T3-L1 adipocytes, artepillin C significantly enhanced the basal and insulin-stimulated glucose uptake. These effects were decreased by cotreatment with a PI3K inhibitor. Although artepillin C had no effects on the insulin signaling cascade, artepillin C enhanced the expression and plasma membrane translocation of GLUT1 and GLUT4 in mature adipocytes. In conclusion, these findings suggest that artepillin C promotes adipocyte differentiation and glucose uptake in part by direct binding to PPARγ, which could be the basis of the pharmacological benefits of green propolis intake in reducing the risk of type 2 diabetes.
Keywords: Artepillin C; 3T3-L1 cells; Adipocyte differentiation; PPARγ ligand; Glucose uptake
PPARγ activation redirects macrophage cholesterol from fecal excretion to adipose tissue uptake in mice via SR-BI by Sue-Anne Toh; John S. Millar; Jeffrey Billheimer; Ilia Fuki; Snehal U. Naik; Colin Macphee; Max Walker; Daniel J. Rader (pp. 934-941).
PPARγ agonists, used in the treatment of Type 2 diabetes, can raise HDL-cholesterol, therefore could potentially stimulate macrophage-to-feces reverse cholesterol transport (RCT). We aimed to test whether PPARγ activation promotes macrophage RCT in vivo. Macrophage RCT was assessed in mice using cholesterol loaded/3H-cholesterol labeled macrophages. PPARγ agonist GW7845 (20mg/kg/day) did not change3H-tracer plasma appearance, but surprisingly decreased fecal3H-free sterol excretion by 43% ( P<0.01) over 48h. Total free cholesterol efflux from macrophages to serum (collected from control and GW7845 groups) was not different, although ABCA1-mediated efflux was significantly higher with GW7845. To determine the effect of PPARγ activation on HDL cholesterol uptake by different tissues, the metabolic fate of HDL labeled with3H-cholesteryl ether (CE) was also measured. We observed two-fold increase in HDL derived3H-CE uptake by adipose tissue ( P<0.005) with concomitant 22% decrease in HDL derived3H-CE uptake by the liver ( P<0.05) in GW7845 treated wild type mice. This was associated with a significant increase in SR-BI protein expression in adipose tissue, but not liver. The same experiment in SR-BI knockout mice, showed no difference in HDL derived3H-CE uptake by adipose tissue or liver. In conclusion, PPARγ activation decreases the fecal excretion of macrophage derived cholesterol in mice. This is not due to inhibition of cholesterol efflux from macrophages, but rather involves redirection of effluxed cholesterol from liver towards adipose tissue uptake via SR-BI. This represents a novel mechanism for regulation of RCT and may extend the therapeutic implications of these ligands.
Keywords: Abbreviations; HDL-C; high density lipoprotein cholesterol; CVD; cardiovascular disease; RCT; reverse cholesterol transport; ABCA1; ATP binding cassette transporter A1; ABCG1; ATP binding cassette transporter G1; SR-BI; scavenger receptor type-BIPPARγ; HDL; Reverse cholesterol transport; Adipose tissue; SR-BI
PPARγ activation redirects macrophage cholesterol from fecal excretion to adipose tissue uptake in mice via SR-BI by Sue-Anne Toh; John S. Millar; Jeffrey Billheimer; Ilia Fuki; Snehal U. Naik; Colin Macphee; Max Walker; Daniel J. Rader (pp. 934-941).
PPARγ agonists, used in the treatment of Type 2 diabetes, can raise HDL-cholesterol, therefore could potentially stimulate macrophage-to-feces reverse cholesterol transport (RCT). We aimed to test whether PPARγ activation promotes macrophage RCT in vivo. Macrophage RCT was assessed in mice using cholesterol loaded/3H-cholesterol labeled macrophages. PPARγ agonist GW7845 (20mg/kg/day) did not change3H-tracer plasma appearance, but surprisingly decreased fecal3H-free sterol excretion by 43% ( P<0.01) over 48h. Total free cholesterol efflux from macrophages to serum (collected from control and GW7845 groups) was not different, although ABCA1-mediated efflux was significantly higher with GW7845. To determine the effect of PPARγ activation on HDL cholesterol uptake by different tissues, the metabolic fate of HDL labeled with3H-cholesteryl ether (CE) was also measured. We observed two-fold increase in HDL derived3H-CE uptake by adipose tissue ( P<0.005) with concomitant 22% decrease in HDL derived3H-CE uptake by the liver ( P<0.05) in GW7845 treated wild type mice. This was associated with a significant increase in SR-BI protein expression in adipose tissue, but not liver. The same experiment in SR-BI knockout mice, showed no difference in HDL derived3H-CE uptake by adipose tissue or liver. In conclusion, PPARγ activation decreases the fecal excretion of macrophage derived cholesterol in mice. This is not due to inhibition of cholesterol efflux from macrophages, but rather involves redirection of effluxed cholesterol from liver towards adipose tissue uptake via SR-BI. This represents a novel mechanism for regulation of RCT and may extend the therapeutic implications of these ligands.
Keywords: Abbreviations; HDL-C; high density lipoprotein cholesterol; CVD; cardiovascular disease; RCT; reverse cholesterol transport; ABCA1; ATP binding cassette transporter A1; ABCG1; ATP binding cassette transporter G1; SR-BI; scavenger receptor type-BIPPARγ; HDL; Reverse cholesterol transport; Adipose tissue; SR-BI
Flavonoid conjugates interact with organic anion transporters (OATs) and attenuate cytotoxicity of adefovir mediated by organic anion transporter 1 (OAT1/SLC22A6) by Chi Chun Wong; Nigel P. Botting; Caroline Orfila; Nawaf Al-Maharik; Gary Williamson (pp. 942-949).
Flavonoids are conjugated by phase II enzymes in humans to form glucuronidated and sulfated metabolites that are excreted in urine via the kidney. In this study, we examined the interaction between metabolites of quercetin and isoflavonoids found in vivo with human organic anion transporters 1 (OAT1) and 3 (OAT3) and their potential in attenuating OAT-induced cytotoxicity of adefovir. Accumulation of flavonoid conjugates was studied in human embryonic kidney 293H cells overexpressing OAT1 or OAT3. OAT1-overexpressing cells exhibited an increased uptake of the sulfated conjugates, genistein-4′- O-sulfate and quercetin-3′- O-sulfate. OAT3-overexpressing cells demonstrated enhanced uptake of glucuronide conjugates, such as daidzein-7- O-glucuronide, genistein-7- O-glucuronide, glycitein-7- O-glucuronide and quercetin-3′- O-glucuronide. Position of conjugation was important since quercetin-3- O-glucuronide and quercetin-7- O-glucuronide were poorly transported. Kinetic analysis revealed high affinity uptake of quercetin-3′- O-sulfate by OAT1 ( Km=1.73μM; Vmax=105pmol/min/mg). OAT3 transported isoflavone glucuronides with lower affinity ( Km=7.9–19.1μM) but with higher Vmax (171–420pmol/min/mg). Quercetin-3′- O-sulfate strongly inhibited OAT1-mediated p-aminohippuric acid uptake with an IC50 of 1.22μM. Transport of 5-carboxyfluorescein by OAT3 was potently inhibited by quercetin-3- O-glucuronide, quercetin-3′- O-glucuronide and quercetin-3′- O-sulfate (IC50=0.43–1.31μM). In addition, quercetin-3′- O-sulfate was shown to effectively reduce OAT1-mediated cytotoxicity of adefovir, an antiviral drug, in a dose-dependent manner. These data suggest that OAT1 and OAT3 are responsible for basolateral uptake of flavonoid conjugates in kidney, and flavonoid conjugates inhibit OAT1 and OAT3 activity at physiologically relevant concentrations. Interaction with OATs limits systemic availability of flavonoids and may be a mechanism of food–drug interaction via inhibition of renal uptake.
Keywords: Abbreviations; BCRP; breast cancer resistance protein; MRP; multidrug resistance protein; OAT; organic anion transporterBioavailability; Food–drug interaction; Isoflavones; Organic anion transporters; Quercetin
Flavonoid conjugates interact with organic anion transporters (OATs) and attenuate cytotoxicity of adefovir mediated by organic anion transporter 1 (OAT1/SLC22A6) by Chi Chun Wong; Nigel P. Botting; Caroline Orfila; Nawaf Al-Maharik; Gary Williamson (pp. 942-949).
Flavonoids are conjugated by phase II enzymes in humans to form glucuronidated and sulfated metabolites that are excreted in urine via the kidney. In this study, we examined the interaction between metabolites of quercetin and isoflavonoids found in vivo with human organic anion transporters 1 (OAT1) and 3 (OAT3) and their potential in attenuating OAT-induced cytotoxicity of adefovir. Accumulation of flavonoid conjugates was studied in human embryonic kidney 293H cells overexpressing OAT1 or OAT3. OAT1-overexpressing cells exhibited an increased uptake of the sulfated conjugates, genistein-4′- O-sulfate and quercetin-3′- O-sulfate. OAT3-overexpressing cells demonstrated enhanced uptake of glucuronide conjugates, such as daidzein-7- O-glucuronide, genistein-7- O-glucuronide, glycitein-7- O-glucuronide and quercetin-3′- O-glucuronide. Position of conjugation was important since quercetin-3- O-glucuronide and quercetin-7- O-glucuronide were poorly transported. Kinetic analysis revealed high affinity uptake of quercetin-3′- O-sulfate by OAT1 ( Km=1.73μM; Vmax=105pmol/min/mg). OAT3 transported isoflavone glucuronides with lower affinity ( Km=7.9–19.1μM) but with higher Vmax (171–420pmol/min/mg). Quercetin-3′- O-sulfate strongly inhibited OAT1-mediated p-aminohippuric acid uptake with an IC50 of 1.22μM. Transport of 5-carboxyfluorescein by OAT3 was potently inhibited by quercetin-3- O-glucuronide, quercetin-3′- O-glucuronide and quercetin-3′- O-sulfate (IC50=0.43–1.31μM). In addition, quercetin-3′- O-sulfate was shown to effectively reduce OAT1-mediated cytotoxicity of adefovir, an antiviral drug, in a dose-dependent manner. These data suggest that OAT1 and OAT3 are responsible for basolateral uptake of flavonoid conjugates in kidney, and flavonoid conjugates inhibit OAT1 and OAT3 activity at physiologically relevant concentrations. Interaction with OATs limits systemic availability of flavonoids and may be a mechanism of food–drug interaction via inhibition of renal uptake.
Keywords: Abbreviations; BCRP; breast cancer resistance protein; MRP; multidrug resistance protein; OAT; organic anion transporterBioavailability; Food–drug interaction; Isoflavones; Organic anion transporters; Quercetin
Effect of its deaminated metabolite, 2′,2′-difluorodeoxyuridine, on the transport and toxicity of gemcitabine in HeLa cells by L.S. Hodge; M.E. Taub; T.S. Tracy (pp. 950-956).
Rate of gemcitabine (dFdC) uptake via equilibrative nucleoside transporters (ENT) is decreased in the presence of its metabolite (dFdU), but intracellular retention is ultimately increased.Gemcitabine is a pyrimidine analog effective against many solid tumors. Following intravenous administration, deaminases in the plasma rapidly convert the parent compound, gemcitabine, to its deaminated metabolite, 2′,2′-difluorodeoxyuridine (dFdU), resulting in an elimination half-life for gemcitabine of 8min. The half-life of dFdU, however, is upwards of 14h, yielding plasma concentrations that are frequently 10–20-fold higher than that of gemcitabine. The uptake of gemcitabine into tumor cells is facilitated by both concentrative (hCNT) and equilibrative (hENT) nucleoside transporters. Recently, it was observed that dFdU is a substrate for hCNT as well. The purpose of this study was to investigate the effects of dFdU on gemcitabine uptake and efflux via hENT1 and hENT2 in HeLa cells. Our results suggest that dFdU is a substrate for both hENT1 and hENT2 as well as a competitive inhibitor of gemcitabine transport at concentrations >100-fold lower than those typically achieved in plasma (IC50=0.45 and 1.2μM for hENT1/2 and hENT2, respectively). However, inhibition of gemcitabine uptake is time-dependent, as dFdU limits gemcitabine uptake into HeLa cells by more than 80% during short (<20s) incubation periods but increases net gemcitabine retention as incubation length increases. While dFdU enhances the accumulation of gemcitabine by up to 1.5-fold following a 60min incubation, dFdU did not enhance gemcitabine cytotoxicity. In conclusion, this is the first report of an interaction between dFdU and gemcitabine suggesting that the deaminated metabolite may play an important role in the disposition of gemcitabine in tumor cells.
Keywords: Gemcitabine; Equilibrative nucleoside transporter; Difluorodeoxyuridine; HeLa cells
Effect of its deaminated metabolite, 2′,2′-difluorodeoxyuridine, on the transport and toxicity of gemcitabine in HeLa cells by L.S. Hodge; M.E. Taub; T.S. Tracy (pp. 950-956).
Rate of gemcitabine (dFdC) uptake via equilibrative nucleoside transporters (ENT) is decreased in the presence of its metabolite (dFdU), but intracellular retention is ultimately increased.Gemcitabine is a pyrimidine analog effective against many solid tumors. Following intravenous administration, deaminases in the plasma rapidly convert the parent compound, gemcitabine, to its deaminated metabolite, 2′,2′-difluorodeoxyuridine (dFdU), resulting in an elimination half-life for gemcitabine of 8min. The half-life of dFdU, however, is upwards of 14h, yielding plasma concentrations that are frequently 10–20-fold higher than that of gemcitabine. The uptake of gemcitabine into tumor cells is facilitated by both concentrative (hCNT) and equilibrative (hENT) nucleoside transporters. Recently, it was observed that dFdU is a substrate for hCNT as well. The purpose of this study was to investigate the effects of dFdU on gemcitabine uptake and efflux via hENT1 and hENT2 in HeLa cells. Our results suggest that dFdU is a substrate for both hENT1 and hENT2 as well as a competitive inhibitor of gemcitabine transport at concentrations >100-fold lower than those typically achieved in plasma (IC50=0.45 and 1.2μM for hENT1/2 and hENT2, respectively). However, inhibition of gemcitabine uptake is time-dependent, as dFdU limits gemcitabine uptake into HeLa cells by more than 80% during short (<20s) incubation periods but increases net gemcitabine retention as incubation length increases. While dFdU enhances the accumulation of gemcitabine by up to 1.5-fold following a 60min incubation, dFdU did not enhance gemcitabine cytotoxicity. In conclusion, this is the first report of an interaction between dFdU and gemcitabine suggesting that the deaminated metabolite may play an important role in the disposition of gemcitabine in tumor cells.
Keywords: Gemcitabine; Equilibrative nucleoside transporter; Difluorodeoxyuridine; HeLa cells