Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
Biochemical Pharmacology (v.77, #5)
RLIP76: A novel glutathione-conjugate and multi-drug transporter by Sharad S. Singhal; Sushma Yadav; Cherice Roth; Jyotsana Singhal (pp. 761-769).
RLIP76, a stress-responsive, multi-functional protein with multi-specific transport activity towards glutathione-conjugates (GS-E) and chemotherapeutic agents, is frequently over-expressed in malignant cells. Our recent studies suggest that it plays a prominent anti-apoptotic role selectively in cancer cells. We have previously shown that RLIP76 accounts for up to 80% of the transport of GS-E and blocking the RLIP76-mediated transport of GS-E in cells results in the accumulation of pro-apoptotic endogenous electrophiles and on-set of apoptosis. Here we demonstrate that when RLIP76 mediate transport of GS-E is abrogated either by anti-RLIP76 IgG or accumulation of 4-hydroxynonenal (4-HNE) and its GSH-conjugate (GS-HNE) occurs and a massive apoptosis is observed in cells, indicate that the inhibition of RLIP76 transport activity at the cell surface is sufficient for observed anti-tumor activity. RLIP76 is linked with certain cellular functions including membrane plasticity and movement (as a primary ‘effector’ in the Ral pathway, perhaps functioning as a GTPase activating protein, or GAP), and as a component of clathrin-coated pit-mediated receptor-ligand endocytosis—a process that mediates movement of membrane vesicles.
Keywords: Abbreviations; RLIP76 (RalBP1); Ral-interacting protein; GSH; glutathione; GS-E; glutathione-electrophile-conjugates; DNP-SG; dinitrophenyl S-glutathione; DOX; doxorubicin; 4HNE; 4-hydroxy-t-nonenal; IOVs; in-side-out vesicles; MDR; multi-drug-resistance; Pgp; P-glycoprotein; MRP1; multi-drug-resistance associated protein; SCLC; small cell lung cancer; NSCLC; non-small cell lung cancer; POB1; partner of RalBP1; AP-2; adaptor proteinRLIP76; Cancer; Drug-resistance; Glutathione-conjugate transport; Xenografts
Inhibition of CHK1 kinase by Gö6976 converts 8-chloro-adenosine-induced G2/M arrest into S arrest in human myelocytic leukemia K562 cells by Xiu-Zhen Jia; Sheng-Yong Yang; Jing Zhou; Shu-Yan Li; Ju-Hua Ni; Guo-Shun An; Hong-Ti Jia (pp. 770-780).
8-Chloro-cAMP (8-Cl-cAMP) and its metabolite 8-chloro-adenosine (8-Cl-Ado) inhibit cell growth by 8-Cl-Ado-converted 8-Cl-ATP that targets cell-cycle control and RNA metabolism. However, the cell-cycle checkpoint pathways remain to be identified. Recent studies have shown that 8-Cl-cAMP administration and 8-Cl-Ado exposure may damage chromosomal DNA in vivo and in vitro. In this study, we demonstrate that 8-Cl-Ado-induced DNA damage activates G2/M phase checkpoint, which is associated with ATM-activated CHK1-CDC25C-CDC2 pathway joined by BRCA1-CHK1 branch in apoptosis-resistant human myelocytic leukemia K562 (p53-null) cells. Inhibition of CHK1 kinase by Gö6976, an inhibitor of CHK1 activity, can promote DNA damage and lead to the activation of CHK2, converting G2/M checkpoint into intra-S-phase checkpoint in which two parallel branches, the ATM-CHK2-CDC25A-CDK2 and the ATM-NBS1/SMC1 cascades, are involved. These observations may provide aid in better understanding of the mechanisms of 8-Cl-cAMP and 8-Cl-Ado actions and in potential design of the combined therapy.
Keywords: 8-Chloro-adenosine; DNA damage; G2/M checkpoint; Intra-S-phase checkpoint; CHK1 inhibitor/Gö6976; Human myelocytic leukemia K562 cell
Inhibiting the function of ABCB1 and ABCG2 by the EGFR tyrosine kinase inhibitor AG1478 by Zhi Shi; Amit K. Tiwari; Suneet Shukla; Robert W. Robey; In-Wha Kim; Smitaben Parmar; Susan E. Bates; Qiu-Sheng Si; Curtis S. Goldblatt; Ioana Abraham; Li-Wu Fu; Suresh V. Ambudkar; Zhe-Sheng Chen (pp. 781-793).
The tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) is a potent and specific EGFR tyrosine kinase inhibitor (TKI); its promising pre-clinical results have led to clinical trials. Overexpression of ATP-binding cassette (ABC) transporters such as ABCB1, ABCC1 and ABCG2 is one of the main causes of multidrug resistance (MDR) and usually results in the failure of cancer chemotherapy. However, the interaction of AG1478 with these ABC transporters is still unclear. In the present study, we have investigated this interaction and found that AG1478 has differential effects on these transporters. In ABCB1-overexpressing cells, non-toxic doses of AG1478 were found to partially inhibit resistance to ABCB1 substrate anticancer drugs as well as increase intracellular accumulation of [3H]-paclitaxel. Similarly, in ABCG2-overexpressing cells, AG1478 significantly reversed resistance to ABCG2 substrate anticancer drugs and increased intracellular accumulation of [3H]-mitoxantrone as well as fluorescent compound BODIPY-prazosin. AG1478 also profoundly inhibited the transport of [3H]-E217βG and [3H]-methotrexate by ABCG2. We also found that AG1478 slightly stimulated ABCB1 ATPase activity and significantly stimulated ABCG2 ATPase activity. Interestingly, AG1478 did not inhibit the photolabeling of ABCB1 or ABCG2 with [125I]-iodoarylazidoprazosin. Additionally, AG1478 did not alter the sensitivity of parental, ABCB1- or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drug and had no effect on the function of ABCC1. Overall, we conclude that AG1478 is able to inhibit the function of ABCB1 and ABCG2, with a more pronounced effect on ABCG2. Our findings provide valuable contributions to the development of safer and more effective EGFR TKIs for use as anticancer agents in the clinic.
Keywords: EGFR tyrosine kinase inhibitor; Multidrug resistance; ABCB1; ABCG2
CCL5 increases lung cancer migration via PI3K, Akt and NF-κB pathways by Chun-Yin Huang; Yi-Chin Fong; Chun-Yi Lee; Meng-Yi Chen; Hsiao-Chi Tsai; Horng-Chaung Hsu; Chih-Hsin Tang (pp. 794-803).
CCL5 (previously called RANTES) is in the CC-chemokine family and plays a crucial role in the migration and metastasis of human cancer cells. Besides, integrins are the major adhesive molecules in mammalian cells. Here we found CCL5 increased the migration and cell surface expression of αvβ3 integrin in human lung cancer cells (A549 cells). CCL5 stimulation increased phosphorylation of the p85α subunit of phosphatidylinositol 3-kinase (PI3K) and serine 473 of Akt. Also, we found that PI3K inhibitor (Ly294002) or Akt inhibitor suppressed CCL5-induced migration activities and integrin expression of A549 cells. Transfection of cells with p85 or Akt mutant also reduced CCL5-mediated cancer migration. In addition, treatment of A549 cells with CCL5 induced IκB kinase α/β (IKK α/β) phosphorylation, IκB phosphorylation, p65 Ser536 phosphorylation, and κB-luciferase activity. Furthermore, the CCL5-mediated increases in p65 Ser536 phosphorylation were inhibited by Ly294002 and Akt inhibitor. Taken together, our results suggest that CCL5 acts through PI3K/Akt, which in turn activates IKKα/β and NF-κB, resulting in the activation of αvβ3 integrin and contributing to the migration of human lung cancer cells.
Keywords: Abbreviations; ECM; extracellular matrix; PI3K; phosphatidylinositol 3-kinase; IKK α/β; IκB kinase α/β; NF-κB; nuclear factor-κBCCL5; Lung cancer; Migration; Akt; PI3K
Cooperation between Apo2L/TRAIL and bortezomib in multiple myeloma apoptosis by Patricia Balsas; Nuria López-Royuela; Patricia Galán-Malo; Alberto Anel; Isabel Marzo; Javier Naval (pp. 804-812).
The proteasome inhibitor bortezomib is currently an important drug for treatment of relapsed and refractory multiple myeloma (MM) and for elderly patients. However, cells from some patients show resistance to bortezomib. We have evaluated the possibility of improving bortezomib therapy with Apo2L/TRAIL, a death ligand that induces apoptosis in MM but not in normal cells. Results indicate that cotreatment with low doses of bortezomib significantly increased apoptosis of MM cells showing partial sensitivity to Apo2L/TRAIL. Bortezomib treatment did not significantly alter plasma membrane amount of DR4 and DR5 but increased Apo2L/TRAIL-induced caspase-8 and caspase-3 activation. Apo2L/TRAIL reverted bortezomib-induced up-regulation of β-catenin, Mcl-1 and FLIP, associated with the enhanced cytotoxicity of combined treatment. More important, some cell lines displaying resistance to bortezomib were sensitive to Apo2L/TRAIL-induced apoptosis. A cell line made resistant by continuous culture of RPMI 8226 cells in the presence of bortezomib (8226/7B) was highly sensitive to Apo2L/TRAIL-induced apoptosis. Moreover, RPMI 8226 cells overexpressing Mcl-1 (8226/Mcl-1) or Bcl-xL (8226/Bcl-xL) also showed enhanced resistance to bortezomib, but co-treatment with Apo2L/TRAIL reverted this resistance. These results indicate that Apo2L/TRAIL can cooperate with bortezomib to induce apoptosis in myeloma cells and can be an useful adjunct for MM therapy.
Keywords: Abbreviations; MM; multiple myeloma; MTT; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; PS; phosphatidylserine; Δ; Ψ; m; mitochondrial transmembrane potential; DiOC; 6; (3); 3,3′-dihexyloxacarbocyanine iodide; TMRE; tetramethylrhodamine ethyl esterBortezomib; TRAIL; Mcl-1; Bcl-x; L; Apoptosis; Myeloma
TGFβ1 antagonistic peptides inhibit TGFβ1-dependent angiogenesis by Simona Serratì; Francesca Margheri; Marco Pucci; Anna Rita Cantelmo; Rosaria Cammarota; Javier Dotor; Francisco Borràs-Cuesta; Gabriella Fibbi; Adriana Albini; Mario Del Rosso (pp. 813-825).
The role of transforming growth factor beta (TGFβ) in tumor promotion and in angiogenesis is context-dependent. While TGFβ prevents tumor growth and angiogenesis in early phases of tumor development, evidence is accumulating about its pro-angiogenic and tumor promotion activities in late-stages of tumor progression. Here we have studied, in an experimental context previously reported to disclose the pro-angiogenic effects of TGFβ, the blocking activity of TGFβ antagonist peptides. In agreement with previous results, we have observed that TGFβ exerts a powerful pro-angiogenic activity on human normal dermal microvascular endothelial cells (MVEC), by promoting invasion and capillary morphogenesis in Matrigel. No apoptotic activity of TGFβ was observed. By RT-PCR we have shown that TGFβ up-regulates expression not only of plasminogen activator inhibitor type-1 (PAI-1), but also of the urokinase-type plasminogen activator receptor (uPAR), whose inhibition by specific antibodies blunted the TGFβ angiogenic response in vitro. The SMAD2/3 and FAK signaling pathways were activated by TGFβ in MVEC, as an early and late response, respectively. The use of two different TGFβ1 antagonist peptides, derived from TGFβ type III receptor sequence and 15-mer phage display technology, inhibited the signaling and pro-angiogenic response in vitro, as well as uPAR and PAI-1 up-regulation of MVEC following TGFβ challenge. The anti-angiogenic properties of both inhibitors were evident also in the in vivo TGFβ Matrigel Sponge Assay. These results may be relevant to develop a potentially fruitful strategy for the therapy of late-stage-associated tumor angiogenesis.
Keywords: TGFβ; Angiogenesis; uPAR; PAI-1; SMAD
Activation and modulation of 72kDa matrix metalloproteinase-2 by peroxynitrite and glutathione by Serena Viappiani; Adrian C. Nicolescu; Andrew Holt; Grzegorz Sawicki; Bryan D. Crawford; Hernando León; Tyler van Mulligen; Richard Schulz (pp. 826-834).
Matrix metalloproteinase-2 (MMP-2) has emerged as a key protease in various pathologies associated with oxidative stress, including myocardial ischemia-reperfusion, heart failure or inflammation. Peroxynitrite (ONOO−), an important effector of oxidative stress, was reported to activate some full length MMP zymogens, particularly in the presence of glutathione (GSH), but whether this occurs for MMP-2 is unknown. Treating MMP-2 zymogen with ONOO− resulted in a concentration-dependent regulation of MMP-2, with 0.3–1μM ONOO− increasing and 30–100μM ONOO− attenuating enzyme activity. The enzyme's Vmax was also significantly increased by 1μM ONOO−. Comparable responses to ONOO− treatment were observed using the intracellular target of MMP-2, troponin I (TnI). GSH at 100μM attenuated the effects of ONOO− on MMP-2. Mass spectrometry revealed that ONOO− can oxidize and, in the presence of GSH, S-glutathiolate the MMP-2 zymogen or a synthetic peptide containing the cysteine-switch motif in the enzyme's autoinhibitory domain. These results suggest that ONOO− and GSH can modulate the activity of 72kDa MMP-2 by modifying the cysteine residue in the autoinhibitory domain of the zymogen, a process that may be relevant to pathophysiological conditions associated with increased oxidative stress.
Keywords: Abbreviations; APMA; 4-aminophenylmercuric acetate; DPN; decomposed peroxynitrite; DTT; dithiothreitol; ESI-QTOF; electrospray ionization-quadrupole-time of flight tandem mass spectrometry; GSH; glutathione; IAM; iodoacetamide; MALDI-TOF; matrix-assisted laser desorption ionization-time of flight mass spectrometry; MMP; matrix metalloproteinase; ONOO; −; peroxynitrite; TnI; troponin IMatrix metalloproteinase-2; Oxidative stress; Peroxynitrite; S; -glutathiolation; Troponin I
RhoA downstream of Gq and G12/13 pathways regulates protease-activated receptor-mediated dense granule release in platelets by Jianguo Jin; Yingying Mao; Dafydd Thomas; Soochong Kim; James L. Daniel; Satya P. Kunapuli (pp. 835-844).
Platelet secretion is an important physiological event in hemostasis. The protease-activated receptors, PAR 1 and PAR 4, and the thromboxane receptor activate the G12/13 pathways, in addition to the Gq pathways. Here, we investigated the contribution of G12/13 pathways to platelet dense granule release. 2MeSADP, which does not activate G12/13 pathways, does not cause dense granule release in aspirin-treated platelets. However, supplementing 2MeSADP with YFLLRNP (60μM), as selective activator of G12/13 pathways, resulted in dense granule release. Similarly, supplementing PLC activation with G12/13 stimulation also leads to dense granule release. These results demonstrate that supplemental signaling from G12/13 is required for Gq-mediated dense granule release and that ADP fails to cause dense granule release because the platelet P2Y receptors, although activate PLC, do not activate G12/13 pathways. When RhoA, downstream signaling molecule in G12/13 pathways, is blocked, PAR-mediated dense granule release is inhibited. Furthermore, ADP activated RhoA downstream of Gq and upstream of PLC. Finally, RhoA regulated PKCδ T505 phosphorylation, suggesting that RhoA pathways contribute to platelet secretion through PKCδ activation. We conclude that G12/13 pathways, through RhoA, regulate dense granule release and fibrinogen receptor activation in platelets.
Keywords: Platelet secretion; Dense granule; G; 12/13; ADP; RhoA
Teupolioside, a phenylpropanoid glycosides of Ajuga reptans, biotechnologically produced by IRBN22 plant cell line, exerts beneficial effects on a rodent model of colitis by Rosanna Di Paola; Emanuela Esposito; Emanuela Mazzon; Luisa Riccardi; Rocco Caminiti; Roberto Dal Toso; Giovanna Pressi; Salvatore Cuzzocrea (pp. 845-857).
The aim of the present study was to examine the effects of phenylpropanoid glycoside, teupolioside, biotechnologically produced by IRBN22 Ajuga reptans cell line, in rats subjected to experimental colitis.Colitis was induced in rats by intracolonic instillation of dinitrobenzene sulfonic acid (DNBS). Teupolioside was administered daily orally (0.2 or 2mgkg−1). On Day 4, animals were sacrificed and tissues were taken for histological and biochemical analysis.Four days after DNBS administration, colon TNF-α and IL-1β productions were increased, associated with colon damage. Neutrophil infiltration, by myeloperoxidase activity, in the mucosa was associated with up-regulation of ICAM-1 and P-selectin and high levels of malondialdehyde. Immunohistochemistry for nitrotyrosine and poly (ADP-ribose) polymerase (PARP) showed an intense staining in the inflamed colon. Biochemical methods and zymography were used to analyze MMP-9 and -2 activities in colon tissues from DNBS-injured rats. Treatment with teupolioside significantly reduced the appearance of diarrhoea and the loss of body weight. This was associated with a remarkable amelioration in the disruption of the colonic architecture and a significant reduction in colonic myeloperoxidase activity and malondialdehyde levels. Teupolioside also reduced the pro-inflammatory cytokines release, the appearance of nitrotyrosine and PARP immunoreactivity in the colon and reduced the up-regulation of ICAM-1 and the expression of P-selectin. Therefore, teupolioside also reduced proMMP-9 and -2 activity induced in the colon by DNBS administration.The results of this study suggested that administration of teupolioside may be beneficial for treatment of inflammatory bowel disease.
Keywords: Teupolioside; Colitis; Adhesion molecules; MMP9 (matrix metallopeptidase 9)
Ursodeoxycholic acid induces glutathione synthesis through activation of PI3K/Akt pathway in HepG2 cells by Sakiko Arisawa; Kumiki Ishida; Natsumi Kameyama; Jun Ueyama; Ai Hattori; Yasuaki Tatsumi; Hisao Hayashi; Motoyoshi Yano; Kazuhiko Hayashi; Yoshiaki Katano; Hidemi Goto; Kenji Takagi; Shinya Wakusawa (pp. 858-866).
Ursodeoxycholic acid (UDCA) is widely recognized as an effective compound in the treatment of chronic hepatitis and is known to modulate the redox state of the liver accompanied by an increase of GSH. In the present study, to access the antioxidative effect of UDCA and to clarify the molecular basis of the action on GSH level, we evaluated its effects in HepG2 cells exposed to excessive iron. UDCA inhibited both a decrease in the GSH level and an increase in the reactive oxygen species caused by excessive iron in the cells. UDCA increased the gene expression of the catalytic- and modifier-units of glutamine-cysteine ligase (GCL), which is a key enzyme in GSH synthesis. We further investigated the effect of UDCA on the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and obtained results showing that UDCA-induced increase in the GSH level was prevented by LY294002, a PI3K inhibitor. In addition, Western blot analysis of Akt showed that, while the total Akt level remained unchanged, the phosphorylated Akt level was increased by UDCA, and this increase was also prevented by LY294002. Moreover, UDCA promoted the translocation of a transcription factor, nuclear factor-E2-related factor-2 (Nrf2), into the nucleus, and this action was inhibited by LY294002. From these results, it was indicated that UDCA increased the GSH synthesis through an activation of the PI3K/Akt/Nrf2 pathway. This may be a primary mechanism of antioxidative action of UDCA concerned with its therapeutic effectiveness in chronic hepatitis.
Keywords: Abbreviations; ROS; reactive oxygen species; UDCA; ursodeoxycholic acid; CHC; chronic hepatitis C; ALT; alanine aminotransferase; PI3K; phosphatidylinositol 3-kinase; Nrf2; nuclear factor-E2-related factor-2; GCL; glutamine-cysteine ligase; GSHS; GSH synthetase; CDCFH; dichlorodihydrofluorescein; mBCl; monochlorobimane; PCR; polymerase chain reaction; ERK; extracellular signal-regulated protein kinase; MAPK; mitogene-activated protein kinase; GAPDH; glycerine aldehyde dehydrogenaseUrsodeoxycholic acid; Reactive oxygen species; Glutathione; PI3K/Akt pathway; Nrf2
The peripheral pro-nociceptive state induced by repetitive inflammatory stimuli involves continuous activation of protein kinase A and protein kinase C epsilon and its NaV1.8 sodium channel functional regulation in the primary sensory neuron by Cristiane Flora Villarreal; Daniela Sachs; Mani Indiana Funez; Carlos Amílcar Parada; Fernando de Queiroz Cunha; Sérgio Henrique Ferreira (pp. 867-877).
In the present study, the participation of the NaV1.8 sodium channel was investigated in the development of the peripheral pro-nociceptive state induced by daily intraplantar injections of PGE2 in rats and its regulation in vivo by protein kinase A (PKA) and protein kinase C epsilon (PKCɛ) as well. In the prostaglandin E2 (PGE2)-induced persistent hypernociception, the NaV1.8 mRNA in the dorsal root ganglia (DRG) was up-regulated. The local treatment with dipyrone abolished this persistent hypernociception but did not alter the NaV1.8 mRNA level in the DRG. Daily intrathecal administrations of antisense NaV1.8 decreased the NaV1.8 mRNA in the DRG and reduced ongoing persistent hypernociception. Once the persistent hypernociception had been abolished by dipyrone, but not by NaV1.8 antisense treatment, a small dose of PGE2 restored the hypernociceptive plateau. These data show that, after a period of recurring inflammatory stimuli, an intense and prolonged nociceptive response is elicited by a minimum inflammatory stimulus and that this pro-nociceptive state depends on NaV1.8 mRNA up-regulation in the DRG. In addition, during the persistent hypernociceptive state, the PKA and PKCɛ expression and activity in the DRG are up-regulated and the administration of the PKA and PKCɛ inhibitors reduce the hypernociception as well as the NaV1.8 mRNA level. In the present study, we demonstrated that the functional regulation of the NaV1.8 mRNA by PKA and PKCɛ in the primary sensory neuron is important for the development of the peripheral pro-nociceptive state induced by repetitive inflammatory stimuli and for the maintenance of the behavioral persistent hypernociception.
Keywords: Inflammatory pain; Chronic pain; Na; V; 1.8 sodium channel; PKA; PKCɛ; Primary sensory neuron
Interleukin 17 sustains rather than induces inflammation by Francesco Maione; Nikolaos Paschalidis; Nicola Mascolo; Neil Dufton; Mauro Perretti; Fulvio D’Acquisto (pp. 878-887).
Interleukin (IL)17 is a novel cytokine that has been suggested to play a key role in sustaining chronic inflammation in autoimmune diseases. Since its discovery, much attention has been given to mediators and factors responsible for the development of IL-17-producing cells while very few studies have investigated the inflammatory properties of this cytokine. Here we aimed to characterize the inflammatory properties of IL-17 and to establish if this cytokine per se can initiate an inflammatory reaction or if its main role is to contribute to the exacerbation of ongoing inflammation. To this aim we used two different mouse models of inflammation: the paw oedema and the airpouch. Interestingly, injection of IL-17 in the hind paw did not cause oedema while administration in the pre-inflamed tissues of 6-day-old air pouches induced a long lasting inflammatory reaction that was sustained between 4 and 24h post-injection. Phenotypic analysis of cellular infiltrates demonstrated selective PMN recruitment in exudates and pouch lining tissues. This event was accompanied by induction of a distinct set of pro-inflammatory cytokines including IL-1β, IL-6, TNF-α and chemokines KC and MCP-1. Co-administration of a neutralizing anti-KC antibody with IL-17 significantly reduced its inflammatory response suggesting a key role for this chemokine in mediating the inflammatory effects of IL-17. In conclusion these results demonstrate that IL-17 does not initiate an inflammatory reaction while, if injected in pre-inflamed tissues, is able to further amplify biochemical and cellular events characteristic of the early stages of the inflammatory reaction.
Keywords: Interleukin-17; Inflammation; Airpouch; Paw oedema; Th17
Inhibitory modulation of the mitochondrial permeability transition by minocycline by Anne Gieseler; Adrian Tilman Schultze; Kathleen Kupsch; Mohammad Fahad Haroon; Gerald Wolf; Detlef Siemen; Peter Kreutzmann (pp. 888-896).
The semi-synthetic tetracycline derivative minocycline exerts neuroprotective properties in various animal models of neurodegenerative disorders. Although anti-inflammatory and anti-apoptotic effects are reported to contribute to the neuroprotective action, the exact molecular mechanisms underlying the beneficial properties of minocycline remain to be clarified. We analyzed the effects of minocycline in a cell culture model of neuronal damage and in single-channel measurements on isolated mitoplasts. Treatment of neuron-enriched cortical cultures with rotenone, a high affinity inhibitor of the mitochondrial complex I, resulted in a deregulation of the intracellular Ca2+-dynamics, as recorded by live cell imaging. Minocycline (100μM) and cyclosporin A (2μM), a known inhibitor of the mitochondrial permeability transition pore, decreased the rotenone-induced Ca2+-deregulation by 60.9% and 37.6%, respectively. Investigations of the mitochondrial permeability transition pore by patch-clamp techniques revealed for the first time a dose-dependent reduction of the open probability by minocycline (IC50=190nM). Additionally, we provide evidence for the high antioxidant potential of MC in our model. In conclusion, the present data substantiate the beneficial properties of minocycline as promising neuroprotectant by its inhibitory activity on the mitochondrial permeability transition pore.
Keywords: Abbreviations; CsA; cyclosporin A; DFF; 2′,7′-difluorofluorescein; DIV; days; in vitro; DMEM; Dulbecco’s Modified Eagle Medium; DMSO; dimethylsulfoxide; DPPH; 2,2-diphenyl-1-picrylhydrazyl; Fluo-4 AM; Fluo-4 pentaacetoxy-methylester; GFAP; glial fibrillary acid protein; HEPES; N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; MC; minocycline; mPTP; mitochondrial permeability transition pore; MTT; 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide; NMDA; N-methyl-; d; -aspartate; P; o; probability of being in the open state; PBS; phosphate-buffered saline; PD; Parkinson’s disease; RLM; rat liver mitochondria; ROI; region of interestPrimary cortical neurons; Rotenone; Mitochondrial permeability transition; Minocycline; Patch-clamp
Expression and transcriptional regulation of ABC transporters and cytochromes P450 in hCMEC/D3 human cerebral microvascular endothelial cells by Sandrine Dauchy; Florence Miller; Pierre-Olivier Couraud; Richard J. Weaver; Babette Weksler; Ignacio-Andres Romero; Jean-Michel Scherrmann; Isabelle De Waziers; Xavier Declèves (pp. 897-909).
We investigated the expression of genes encoding ABC transporters, cytochromes P450 (CYPs) and some transcription factors in the hCMEC/D3 immortalized human cerebral microvascular endothelial cell line, a promising in vitro model of the human BBB, and we compared these expressions to a non-brain endothelial cell line (HUVEC) and freshly human brain microvessels. qRT-PCR showed that the MDR1, BCRP, MRP1, MRP3, MRP4 and MRP5 genes were expressed and that the main CYP gene was CYP2U1 in hCMEC/D3. The pattern of ABC and CYPs gene expression in hCMEC/D3 differed from HUVEC which did not express MDR1. Moreover, expression of P-gp and BCRP was lower in hCMEC/D3 than in human brain microvessels but remain functional as shown by rhodamine 123 efflux assay. The gene encoding the aryl hydrocarbon receptor (AhR), a transcription factor that regulates the expression of some ABC and CYPs was highly expressed in hCMEC/D3 and HUVEC, while the pregnane-X-receptor (PXR) and the constitutive androstane receptor (CAR) were barely detected. We investigated the function of the AhR-mediated regulatory pathway in hCMEC/D3 by treating them with the AhR agonist TCDD. The expressions of two AhR-target genes, CYP1A1 and CYP1B1, were increased 26-fold and 28-fold. But the expressions of ABC transporter genes were not significantly altered. We have thus determined the pattern of expression of the genes encoding ABC transporters, CYPs and three transcription factors in hCMEC/D3 and shown that the AhR pathway might afford an original functional transport and metabolic pattern in cerebral endothelial cells that is different from other peripheral endothelial cells.
Keywords: Abbreviations; ABC; ATP-binding cassette; AhR; aryl hydrocarbon receptor; BBB; blood–brain barrier; BCRP; breast cancer resistance protein; CAR; constitutive androstane receptor; CNS; central nervous system; CYP; cytochromes P450; MDR; multidrug resistance; MRP; multidrug resistance-associated protein; P-gp; P-glycoprotein; PXR; pregnane xenobiotic receptorhCMEC/D3 cell line; ABC transporters; CYP; AhR; Blood–brain barrier
Cross-species comparison of in vivo PK/PD relationships for second-generation antisense oligonucleotides targeting apolipoprotein B-100 by Rosie Z. Yu; Kristina M. Lemonidis; Mark J. Graham; John E. Matson; Rosanne M. Crooke; Diane L. Tribble; Mark K. Wedel; Arthur A. Levin; Richard S. Geary (pp. 910-919).
Similar exposure-response relationships of ISIS 301012 in human ApoB transgenic mice and in humans.▪The in vivo pharmacokinetics/pharmacodynamics of 2′- O-(2-methoxyethyl) (2′-MOE) modified antisense oligonucleotides (ASOs), targeting apolipoprotein B-100 (apoB-100), were characterized in multiple species. The species-specific apoB antisense inhibitors demonstrated target apoB mRNA reduction in a drug concentration and time-dependent fashion in mice, monkeys, and humans. Consistent with the concentration-dependent decreases in liver apoB mRNA, reductions in serum apoB, and LDL-C, and total cholesterol were concurrently observed in animal models and humans. Additionally, the long duration of effect after cessation of dosing correlated well with the elimination half-life of 2′-MOE modified apoB ASOs studied in mice ( t1/2≅20 days) and humans ( t1/2≅30 days) following parental administrations. The plasma concentrations of ISIS 301012, observed in the terminal elimination phase of both mice and monkeys were in equilibrium with liver. The partition ratios between liver and plasma were similar, approximately 6000:1, across species, and thus provide a surrogate for tissue exposure in humans. Using an inhibitory Emax model, the ASO liver EC50s were 101±32, 119±15, and 300±191μg/g of ASO in high-fat-fed (HF) mice, transgenic mice containing the human apoB transgene, and monkeys, respectively. The estimated liver EC50 in man, extrapolated from trough plasma exposure, was 81±122μg/g. Therefore, extraordinary consistency of the exposure–response relationship for the apoB antisense inhibitor was observed across species, including human. The cross-species PK/PD relationships provide confidence in the use of pharmacology animal models to predict human dosing for second-generation ASOs targeting the liver.
Keywords: Abbreviations; ASO; antisense oligonucleotide; ApoB-100; apolipoprotein B-100; VLDL-C; very low density lipoprotein cholesterol; IDL-C; intermediate density lipoprotein cholesterol; LDL-C; low-density lipoprotein cholesterol; HF-fed; high-fat diet fed; IACUC; Institutional Animal Care and Use Committee; s.c.; subcutaneous; i.p.; intraperitoneal; ELISA; enzyme-linked immunosorbent assay; LLOQ; the lower limit of quantitation; CGE; capillary gel electrophoresis; SPE; solid phase extraction; MOE; 2′-; O; -(2-methoxy) ethylPharmacokinetics; Pharmacodynamics; ApoB; Lipid lowering; Cross-species; Antisense
The mechanism causing the difference in kinetic properties between rat CYP2D4 and human CYP2D6 in the oxidation of dextromethorphan and bufuralol by Shizuo Narimatsu; Daichi Kazamori; Kazufumi Masuda; Takashi Katsu; Yoshihiko Funae; Shinsaku Naito; Hironori Nakura; Shigeru Yamano; Nobumitsu Hanioka (pp. 920-931).
The capacity to oxidize bufuralol (BF) and dextromethorphan (DEX) was compared kinetically between human CYP2D6 and four rat CYP2D (CYP2D1, -2D2, -2D3 and -2D4) isoenzymes in a yeast cell expression system. In BF 1″-hydroxylation and DEX O-demethylation, only CYP2D4 showed hook-shaped Eadie–Hofstee plots, the other four CYP2D enzymes exhibiting linear plots. In DEX N-demethylation, rat CYP2D2 did not show any detectable activity under the conditions used, whereas the other four enzymes yielded linear Eadie–Hofstee plots. To elucidate the mechanisms causing the nonlinear kinetics, four CYP2D4 mutants, CYP2D4-F109I, -V123F, -L216F and -A486F, were prepared. CYP2D4-V123F, -L216F and -A486F yielded linear or linear-like Eadie–Hofstee plots for BF 1″-hydroxylation, whereas only CYP2D4-A486F exhibited linear plots for DEX O-demethylation. The substitution of Phe-109 by isoleucine did not have any effect on the oxidative capacity of CYP2D4 for either BF or DEX. These results suggest that the introduction of phenylalanine in the active-site cavity of CYP2D4 simplifies complicated interactions between the substrates and the amino acid residues, but the mechanisms causing the simplification differ between BF and DEX.
Keywords: CYP2D4; CYP2D6; Bufuralol; Dextromethorphan; Nonlinear kinetics; Site-directed mutagenesis