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Biochemical Pharmacology (v.85, #2)

Editorial Board (pp. iii).

Rhythm and blues: Animal models of epilepsy and depression comorbidity by S. Alisha Epps; David Weinshenker (pp. 135-146).
Clinical evidence shows a strong, bidirectional comorbidity between depression and epilepsy that is associated with decreased quality of life and responsivity to pharmacotherapies. At present, the neurobiological underpinnings of this comorbidity remain hazy. To complicate matters, anticonvulsant drugs can cause mood disturbances, while antidepressant drugs can lower seizure threshold, making it difficult to treat patients suffering from both depression and epilepsy. Animal models have been created to untangle the mechanisms behind the relationship between these disorders and to serve as screening tools for new therapies targeted to treat both simultaneously. These animal models are based on chemical interventions (e.g. pentylenetetrazol, kainic acid, pilocarpine), electrical stimulations (e.g. kindling, electroshock), and genetic/selective breeding paradigms (e.g. genetically epilepsy-prone rats (GEPRs), genetic absence epilepsy rat from Strasbourg (GAERS), WAG/Rij rats, swim lo-active rats (SwLo)). Studies on these animal models point to some potential mechanisms that could explain epilepsy and depression comorbidity, such as various components of the dopaminergic, noradrenergic, serotonergic, and GABAergic systems, as well as key brain regions, like the amygdala and hippocampus. These models have also been used to screen possible therapies. The purpose of the present review is to highlight the importance of animal models in research on comorbid epilepsy and depression and to explore the contributions of these models to our understanding of the mechanisms and potential treatments for these disorders.

Keywords: Epilepsy; Depression; Animal model; Comorbidity


Opportunities for functional selectivity in GPCR antibodies by David R. Webb; Tracy M. Handel; Anke Kretz-Rommel; Raymond C. Stevens (pp. 147-152).
Monoclonal antibodies (mAbs) have been used for decades as tools to probe the biology and pharmacology of receptors in cells and tissues. They are also increasingly being developed for clinical purposes against a broad range of targets, albeit to a lesser extent for G-protein-coupled receptors (GPCRs) relative to other therapeutic targets. Recent pharmacological, structural and biophysical data have provided a great deal of new insight into the molecular details, complexity and regulation of GPCR function. Whereas GPCRs used to be viewed as having either “on” or “off” conformational states, it is now recognized that their structures may be finely tuned by ligands and other interacting proteins, leading to the selective activation of specific signaling pathways. This information coupled with new technologies for the selection of mAbs targeting GPCRs will be increasingly deployed for the development of highly selective mAbs that recognize conformational determinants leading to novel therapeutics.

Keywords: GPCR; Ligand induced conformation; Biased signaling; Monoclonal antibody; Therapeutic antibody


Emerging opportunities for allosteric modulation of G-protein coupled receptors by Ching-I. Anderson Wang; Richard J. Lewis (pp. 153-162).
Their ubiquitous nature, wide cellular distribution and versatile molecular recognition and signalling help make G-protein binding receptors (GPCRs) the most important class of membrane proteins in clinical medicine, accounting for ∼40% of all current therapeutics. A large percentage of current drugs target the endogenous ligand binding (orthosteric) site, which are structurally and evolutionarily conserved, particularly among members of the same GPCR subfamily. With the recent advances in GPCR X-ray crystallography, new opportunities for developing novel subtype selective drugs have emerged. Given the increasing recognition that the extracellular surface conformation changes in response to ligand binding, it is likely that all GPCRs possess an allosteric site(s) capable of regulating GPCR signalling. Allosteric sites are less structurally conserved than their corresponding orthosteric site and thus provide new opportunities for the development of more selective drugs. Constitutive oligomerisation (dimerisation) identified in many of the GPCRs investigated, adds another dimension to the structural and functional complexity of GPCRs. In this review, we compare 60 crystal structures of nine GPCR subtypes (rhodopsin, ß2-AR, ß1-AR, A2a-AR, CXCR4, D3R, H1R, M2R, M3R) across four subfamilies of Class A GPCRs, and discuss mechanisms involved in receptor activation and potential allosteric binding sites across the highly variable extracellular surface of these GPCRs. This analysis has identified a new extracellular salt bridge (ESB-2) that might be exploited in the design of allosteric modulators.

Keywords: Abbreviations; GPCR; G-protein coupled receptor; ß; 2; -AR; ß; 2; -adrenoceptor; ß; 1; -AR; ß; 1; -adrenoceptor; A; 2a; -AR; A; 2a; - adenosine receptor; CXCR4; CXCR4 chemokine receptor; D; 3; R; dopamine 3 receptor; H; 1; R; histamine 1 receptor; M; 2; R; M; 2; muscarinic receptor; M; 3; R; M; 3; muscarinic receptor; TMH; transmembrane helices; ECL; extracellular loop; ESB; extracellular salt bridgeG-protein coupled receptor; X-ray crystallography; Allosteric modulation; Oligomerisation; Orthosteric binding site


Nitrogen-containing bisphosphonates induce apoptosis of hematopoietic tumor cells via inhibition of Ras signaling pathways and Bim-mediated activation of the intrinsic apoptotic pathway by Masanobu Tsubaki; Tatsuki Itoh; Takao Satou; Motohiro Imano; Makiko Komai; Naoki Ogawa; Junji Mukai; Shozo Nishida (pp. 163-172).
Nitrogen-containing bisphosphonates (N-BPs) induce apoptosis in tumor cells by inhibiting the prenylation of small G-proteins. However, the details of the apoptosis-inducing mechanism remain obscure. The present study showed that the induction of apoptosis by N-BPs in hematopoietic tumor cells is mediated by mitochondrial apoptotic signaling pathways, which are activated by the suppression of geranylgeranyl pyrophosphate (GGPP) biosynthesis. Furthermore, N-BPs decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK) and mTOR via suppression of Ras prenylation and enhanced Bim expression. The present results indicated that N-BPs induce apoptosis by decreasing the mitochondrial transmembrane potential, increasing the activation of caspase-9 and caspase-3, and enhancing Bim expression through inhibition of the Ras/MEK/ERK and Ras/mTOR pathways. The accumulation of N-BPs in bones suggests that they may act more effectively on tumors that have spread to bones or on Ras-variable tumors. This is the first study to show that the specific molecular pathways of N-BP-induced apoptosis.

Keywords: Nitrogen-containing bisphosphonates; Bim; Ras; ERK1/2; mTOR


Ibandronate increases the expression of the pro-apoptotic gene FAS by epigenetic mechanisms in tumor cells by R. Thaler; S. Spitzer; H. Karlic; C. Berger; K. Klaushofer; F. Varga (pp. 173-185).
There is growing evidence that aminobisphosphonates like ibandronate show anticancer activity by an unknown mechanism. Biochemically, they prevent posttranslational isoprenylation of small GTPases, thus inhibiting their activity. In tumor cells, activated RAS-GTPase, the founding member of the gene family, down-regulates the expression of the pro-apoptotic gene FAS via epigenetic DNA-methylation by DNMT1. We compared ibandronate treatment in neoplastic human U-2 osteosarcoma and in mouse CCL-51 breast cancer cells as well as in the immortalized non-neoplastic MC3T3-E1 osteoblastic cells. Ibandronate attenuated cell proliferation in all cell lines tested. In the neoplastic cells we found up-regulation of caspases suggesting apoptosis. Further we found stimulation of FAS-expression as a result of epigenetic DNA demethylation that was due to down-regulation of DNMT1, which was rescued by re-isoprenylation by both geranylgeranyl-pyrophosphate and farnesylpyrophosphate. In contrast, ibandronate did not affect FAS and DNMT1 expression in MC3T3-E1 non-neoplastic cells. Data suggest that bisphosphonates via modulation of the activity of small-GTPases induce apoptosis in neoplastic cells by DNA-CpG-demethylation and stimulation of FAS-expression. In conclusion the shown epigenetic mechanism underlying the anti-neoplastic activity of farnesyl-transferase-inhibition, also explains the clinical success of other drugs, which target this pathway.

Keywords: Bisphosphonates; Ibandronate; Epigenetic DNA-methylation; Small GTPases; FAS; apoptosis


Loss of O6-methylguanine-DNA methyltransferase confers collateral sensitivity to carmustine in topoisomerase II-mediated doxorubicin resistant triple negative breast cancer cells by Selina Raguz; Caroline Adams; Nahal Masrour; Sabeena Rasul; Panagiotis Papoutsoglou; Yunhui Hu; Giulia Cazzanelli; Yuan Zhou; Naina Patel; Charles Coombes; Ernesto Yagüe (pp. 186-196).
Triple-negative breast cancer is characterized by aggressive tumours whose cells lack oestrogen and progesterone receptors and do not over-express HER2. It accounts for approximately 10–15% of breast cancer cases. We sought to generate a cellular model of chemotherapy drug resistance for this type of disease to provide the tools for the development of new therapies. Doxorubicin is a component of some chemotherapy regimes used to treat this form of cancer but resistance preventing disease eradication frequently occurs, mainly due to over-expression of drug transporters such as P-glycoprotein. CALDOX cells were generated by exposure of CAL51 to doxorubicin. Resistance to doxorubicin did not involve drug transporters, as the both parental and resistant cells accumulated doxorubicin to comparable levels. CALDOX cells had slower proliferation rate and an extended G1 cell cycle stage than the parental line, mainly due to an intrinsic activation of CDNK1 (p21), but this cell cycle block was not involved in the mechanism of resistance. CALDOX cells had reduced levels of TOP2A (topoisomerase IIα) and were cross resistant to the topoisomerase II inhibitors etoposide and mitoxantrone. CALDOX cells showed collateral sensitivity to carmustine due to the lack of O6-methylguanine-DNA-methyltransferase (MGMT) expression, related to the hypermethylation of its promoter. The collateral sensitivity of CALDOX cells to carmustine provides the rationale to evaluate MGMT promoter methylation status to design better therapeutic strategies for triple negative breast cancer.

Keywords: Triple negative breast cancer; Collateral sensitivity; TOP2A; MGMT


Elevation of cysteine consumption in tamoxifen-resistant MCF-7 cells by Chang Seon Ryu; Hui Chan Kwak; Ji-Yoon Lee; Soo Jin Oh; Nguyen Thi Thuy Phuong; Keon Wook Kang; Sang Kyum Kim (pp. 197-206).
Tamoxifen (TAM) resistance is a main cause of therapeutic failure in breast cancers. Although methionine dependency is a phenotypic characteristic of tumor cells, the role of sulfur amino acid metabolism in chemotherapy resistance remains to be elucidated. This study compared metabolite profiles of sulfur amino acid metabolism from methionine to taurine or glutathione (GSH) between normal MCF-7 and TAM-resistant MCF-7 (TAMR-MCF-7) cells. TAMR-MCF-7 cells showed elevated levels and activities of enzymes involved in both transsulfuration from methionine to cysteine and metabolism of cysteine to GSH and taurine. Cysteine concentrations in TAMR-MCF-7 cells and medium conditioned by cell culture for 42h were markedly decreased, while GSH, hypotaurine, and taurine concentrations in the medium were increased. These results show that TAMR-MCF-7 cells display enhanced cysteine utilization. The addition of propargylglycine, a specific cystathionine γ-lyase inhibitor, and buthionine sulfoximine, a specific γ-glutamylcysteine ligase inhibitor, to TAMR-MCF-7 cells, but not to MCF-7 cells, resulted in cytotoxicity after sulfur amino acid deprivation. These results suggest that cell viability of TAMR-MCF-7 cells is affected by inhibition of sulfur amino acid metabolism, particularly cysteine synthesis from homocysteine and GSH synthesis from cysteine. Additionally, the S-adenosylmethionine/ S-adenosylhomocysteine ratio, an index of transmethylation potential, in TAMR-MCF-7 cells increased to ∼3.6-fold relative to that in MCF-7 cells, a finding that may result from upregulation of methionine adenosyltransferase IIa and S-adenosylhomocysteine hydrolase. In conclusion, this study suggests that TAMR-MCF-7 cells display enhanced cysteine utilization for synthesis of GSH and taurine, and are sensitive to inhibition of cysteine metabolism.

Keywords: Abbreviations; BHMT; betaine-homocysteine methyltransferase; CβS; cystathionine beta-synthase; CγL; cystathionine γ-lyase; CDO; cysteine dioxygenase; MS; methionine synthase; MTHFR; methylene tetrahydrofolate reductase; GCL; γ-glutamylcysteine ligase; SAH; S; -adenosylhomocysteine; TAM; Tamoxifen; SAAD; sulfur amino acid-deprivedBreast cancer; Cysteine; Glutathione; Tamoxifen resistance; Sulfur amino acid metabolism


Contributions of rat Ctr1 to the uptake and toxicity of copper and platinum anticancer drugs in dorsal root ganglion neurons by Johnson J. Liu; Yaeseul Kim; Fang Yan; Qi Ding; Virginia Ip; Nancy N. Jong; Julian F.B. Mercer; Mark J. McKeage (pp. 207-215).
Dorsal root ganglion (DRG) neurons are affected by platinum-induced neurotoxicity and neurodegenerative processes associated with disturbed copper homeostasis and transport. This study aimed to understand the role of copper transporter 1 (Ctr1) in the uptake and toxicity of copper and platinum drugs in cultured rat DRG neurons, and the functional activities of rat Ctr1 (rCtr1) as a membrane transporter of copper and platinum drugs. Heterologous expression of rCtr1 in HEK293 cells (HEK/rCtr1 cells) increased the uptake and cytotoxicity of copper, oxaliplatin, cisplatin and carboplatin, in comparison to isogenic vector-transfected control cells. Cultured rat DRG neurons endogenously expressed rCtr1 protein on their neuronal cell body plasma membranes and cytoplasm, and displayed substantial capacity for taking up copper, but were resistant to copper toxicity. The uptake of copper by both cultured rat DRG neurons and HEK/rCtr1 cells was saturable and inhibited by cold temperature, silver and zinc, consistent with it being mediated by rCtr1. Cultured rat DRG neurons accumulated platinum during their exposure to oxaliplatin and were sensitive to oxaliplatin cytotoxicity. The accumulation of platinum by both cultured rat DRG neurons and HEK/rCtr1 cells, during oxaliplatin exposure, was saturable and temperature dependent, but was inhibited by copper only in HEK/rCtr1 cells. In conclusion, rCtr1 can transport copper and platinum drugs, and sensitizes cells to their cytotoxicities. DRG neurons display substantial capacity for accumulating copper via a transport process mediated by rCtr1, but appear able to resist copper toxicity and use alternative mechanisms to take up oxaliplatin.

Keywords: Copper transporter 1; Copper uptake; Dorsal root ganglion neurons; Platinum anticancer drug; Peripheral neurotoxicity


Clot penetration and retention by plasminogen activators promote fibrinolysis by O.A. Marcos-Contreras; K. Ganguly; A. Yamamoto; R. Shlansky-Goldberg; D.B. Cines; V.R. Muzykantov; J.-C. Murciano (pp. 216-222).
Administered Plasminogen Activators, e.g. Activase® or tPA, Reteplase® or Ret and Tenectase® or TNK relate with the clot structure depending on their properties. tPA and TNK react with fibrin with high affinity and get retained in the clot surface. However Ret has a low fibrin affinity and it diffuses well within the clot, producing faster clot dissolution and somehow getting protected from plasma inhibitors, such as TAFIa and PAI-1.Tissue-type plasminogen activator (tPA) remains the sole thrombolytic approved by the FDA for the treatment of pulmonary embolism (PE). tPA has not been replaced by third generation plasminogen activators, e.g. Reteplase (Ret) and Tenecteplase (TNK) that circulate with longer life-spans and in theory should have more extended potency in vivo. One reason for this paradox is the inability to assign units of activity to plasminogen activators based on specific biologically relevant standards, which impairs objective comparison. Here, we compare clot permeation, retention and fibrinolytic activities of tPA, TNK and Ret in vitro and clot composition over time with outcome in a mouse model of disseminated pulmonary microembolism (ME). When clots were incubated in the continuous presence of drug, tPA, TNK and Ret lysed fibrin clots identically in the absence of PA inhibitor-1 ( e.g. PAI-1). Ret, which has lower fibrin affinity and greater susceptibility to inhibition by PAI-1 than tPA, was less effective in lysing plasma clots, while TNK was less effective when the fibrin content of the clots was enhanced. However, when clots were afforded only brief exposure to drug, as occurs in vivo, Ret showed more extensive clot permeation, greater retention and lysis than tPA or TNK. These results were reproduced in vivo in a mouse model of ME. These studies indicate the need for more relevant tests of plasminogen activator activity in vitro and in vivo and they show that clot permeation and retention are important potential predictors of clinical utility.

Keywords: Clot retention; Fibrinolysis; In vivo; Plasminogen activator; Plasminogen activator inhibitors; Pulmonary embolism


Proteasome inhibition attenuates heart failure during the late stages of pressure overload through alterations in collagen expression by Yuedong Ma; Yili Chen; Yang Yang; Baolin Chen; Dan Liu; Zhaojun Xiong; Chengxi Zhang; Yugang Dong (pp. 223-233).
Although the role of the ubiquitin–proteasome system (UPS) in cardiac hypertrophy induced by pressure overload has been consistently studied, the fundamental importance of the UPS in cardiac fibrosis has received much less attention. Our previous study found that proteasome inhibitor (MG132) treatment attenuated cardiac fibrosis and heart failure during the early and middle stages of pressure overload. However, the effects of this inhibitor on late-stage pressure overload hearts remain unclear and controversial. The present study was designed to investigate the effects and possible mechanisms of MG132 on cardiac fibrosis and dysfunction during the late stages of pressure overload. Male Sprague Dawley rats with abdominal aortic constriction (AAC) or a sham operation received an intraperitoneal injection of MG132 (0.1mgkg−1day−1) or vehicle for 16 weeks. Left ventricular (LV) function, collagen deposition and Ang II levels were evaluated at study termination. Ang II-stimulated adult rat cardiac fibroblasts were utilized to examine the effects of MG132 on collagen synthesis and the relationship between the renin–angiotensin–aldosterone system (RAAS) and the UPS. MG132 treatment attenuated ventricular dysfunction by suppressing cardiac fibrosis rather than inhibiting cardiac hypertrophy during the late-stages of pressure overload. We also found that Ang II activates UPS in the heart and MG132 attenuates Ang II-induced collagen synthesis via suppression of the NF-κB/TGF-β/Smad2 signaling pathways. Proteasome inhibition therefore could provide a new promising therapeutic strategy to prevent cardiac fibrosis and progression of heart failure even during the late-stages of pressure overload.

Keywords: Late stage of pressure overload; Heart failure; Collagen expression; Proteasome inhibitor; NF-κB


Mesalamine modulates intercellular adhesion through inhibition of p-21 activated kinase-1 by Vineeta Khare; Alex Lyakhovich; Kyle Dammann; Michaela Lang; Melanie Borgmann; Boris Tichy; Sarka Pospisilova; Gloria Luciani; Christoph Campregher; Rayko Evstatiev; Maren Pflueger; Harald Hundsberger; Christoph Gasche (pp. 234-244).
(a) PAK1 orchestrates mesalamine activity, (b) mesalamine inhibits PAK1; increases membranous E-cadherin and β-catenin; modulates cell adhesionMesalamine (5-ASA) is widely used for the treatment of ulcerative colitis, a remitting condition characterized by chronic inflammation of the colon. Knowledge about the molecular and cellular targets of 5-ASA is limited and a clear understanding of its activity in intestinal homeostasis and interference with neoplastic progression is lacking. We sought to identify molecular pathways interfered by 5-ASA, using CRC cell lines with different genetic background. Microarray was performed for gene expression profile of 5-ASA-treated and untreated cells (HCT116 and HT29). Filtering and analysis of data identified three oncogenic pathways interfered by 5-ASA: MAPK/ERK pathway, cell adhesion and β-catenin/Wnt signaling. PAK1 emerged as a consensus target of 5-ASA, orchestrating these pathways. We further investigated the effect of 5-ASA on cell adhesion. 5-ASA increased cell adhesion which was measured by cell adhesion assay and transcellular-resistance measurement. Moreover, 5-ASA treatment restored membranous expression of adhesion molecules E-cadherin and β-catenin. Role of PAK1 as a mediator of mesalamine activity was validated in vitro and in vivo. Inhibition of PAK1 by RNA interference also increased cell adhesion. PAK1 expression was elevated in APCmin polyps and 5-ASA treatment reduced its expression. Our data demonstrates novel pharmacological mechanism of mesalamine in modulation of cell adhesion and role of PAK1 in APCmin polyposis. We propose that inhibition of PAK1 expression by 5-ASA can impede with neoplastic progression in colorectal carcinogenesis. The mechanism of PAK1 inhibition and induction of membranous translocation of adhesion proteins by 5-ASA might be independent of its known anti-inflammatory action.

Keywords: Abbreviations; CRC; colorectal cancer; IBD; inflammatory bowel diseases; UC; ulcerative colitis; AJ; adherens junction; PAK1; p21 activated kinase 1PAK1; Mesalamine; Beta-catenin; E-cadherin; Cell adhesion


Superoxide anion mediates the L-selectin down-regulation induced by non-steroidal anti-inflammatory drugs in human neutrophils by Maria Domínguez-Luis; Ada Herrera-García; Maria Arce-Franco; Estefania Armas-González; Marta Rodríguez-Pardo; Fabian Lorenzo-Díaz; Manuel Feria; Susana Cadenas; Francisco Sánchez-Madrid; Federico Díaz-González (pp. 245-256).
Non-steroidal anti-inflammatory drugs (NSAIDs) induce the shedding of L-selectin in human neutrophils through a mechanism still not well understood. In this work we studied both the functional effect of NSAIDs on the neutrophils/endothelial cells dynamic interaction, and the potential involvement of reactive oxygen species (ROS) in the NSAIDs-mediated down-regulation of L-selectin. When human neutrophils were incubated with diclofenac, a significant reduction in the number of cells that rolled on activated endothelial cells was observed. Different NSAIDs (flufenamic acid, meclofenamic acid, diclofenac, indomethacin, nimesulide, flurbiprofen, meloxicam, phenylbutazone, piroxicam, ketoprofen and aspirin) caused variable increase in neutrophil intracellular ROS concentration, which was inversely proportional to the change produced in L-selectin surface expression. Pre-incubation of neutrophils with superoxide dismutase, but not with catalase, showed both a significant protective effect on the L-selectin down-regulation induced by several NSAIDs and a diminished effect of diclofenac on neutrophil rolling. Interestingly, diclofenac and flufenamic acid but not piroxicam significantly increased the extracellular superoxide anion production by neutrophils, and inhibition of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity with diphenyleneiodonium prevented the down-regulation of L-selectin by diclofenac. In accordance with these results, neutrophils from patients with chronic granulomatous disease, a hereditary disease in which neutrophils show a reduced capacity to form superoxide radicals, exhibited a lower down-regulation of L-selectin (IC50: 15.3μg/ml) compared to normal controls (IC50: 5.6μg/ml) in response to diclofenac.A group of NSAIDs is capable of interfering with the ability of neutrophils to interact with endothelial cells by triggering L-selectin-shedding through the NADPH-oxidase-dependent generation of superoxide anion at the plasma membrane.

Keywords: Non-steroidal anti-inflammatory drugs; Neutrophils; L-selectin; Superoxide anion


Reversal of the deleterious effects of chronic dietary HFCS-55 intake by PPAR-δ agonism correlates with impaired NLRP3 inflammasome activation by Massimo Collino; Elisa Benetti; Mara Rogazzo; Raffaella Mastrocola; Muhammed M. Yaqoob; Manuela Aragno; Christoph Thiemermann; Roberto Fantozzi (pp. 257-264).
Although high-fructose corn syrup (HFCS-55) is the major sweetener in foods and soft-drinks, its potential role in the pathophysiology of diabetes and obesity (“diabesity”) remains unclear. Peroxisome-proliferator activated receptor (PPAR)-δ agonists have never been tested in models of sugar-induced metabolic abnormalities. This study was designed to evaluate (i) the metabolic and renal consequences of HFCS-55 administration (15%wt/vol in drinking water) for 30 weeks on male C57Bl6/J mice and (ii) the effects of the selective PPAR-δ agonist GW0742 (1mg/kg/day for 16 weeks) in this condition. HFCS-55 caused (i) hyperlipidemia, (ii) insulin resistance, and (iii) renal injury/inflammation. In the liver, HFCS-55 enhanced the expression of fructokinase resulting in hyperuricemia and caused abnormalities in known insulin-driven signaling events. In the kidney, HFCS-55 enhanced the expression of the NLRP3 (nucleotide-binding domain and leucine-rich-repeat-protein 3) inflammasome complex, resulting in caspase-1 activation and interleukin-1β production. All of the above effects of HFCS-55 were attenuated by the specific PPAR-δ agonist GW0742. Thus, we demonstrate for the first time that the specific PPAR-δ agonist GW0742 attenuates the metabolic abnormalities and the renal dysfunction/inflammation caused by chronic HFCS-55 exposure by preventing upregulation of fructokinase (liver) and activation of the NLRP3 inflammasome (kidney).

Keywords: High-fructose corn syrup; PPAR-δ; GW0742; Insulin resistance; Inflammation


Influence of sildenafil and tadalafil on the enzyme- and transporter-inducing effects of bosentan and ambrisentan in LS180 cells by Johanna Weiss; Dirk Theile; Adriana Spalwisz; Jürgen Burhenne; Klaus-Dieter Riedel; Walter Emil Haefeli (pp. 265-273).
The combinations of the endothelin-1 receptor antagonists bosentan or ambrisentan with the phosphodiesterase 5 inhibitors sildenafil or tadalafil are current standard therapies of advanced pulmonary arterial hypertension. However, these drugs have a number of drug interactions. Changes of bosentan pharmacokinetics by sildenafil are attributed to reduced hepatic uptake as a consequence of inhibition of organic anion transporting polypeptides. We therefore tested in vitro the hypothesis that sildenafil and tadalafil reduce the enzyme- and transporter-inducing effects of bosentan or ambrisentan by preventing cellular access. Although intracellular concentrations of bosentan and ambrisentan (measured by high pressure liquid chromatography coupled with tandem mass-spectrometry) after four days of incubation of LS180 cells were lower when sildenafil or tadalafil were present, quantification of mRNA expression in these cells by real-time reverse transcription polymerase chain reaction revealed that bosentan and ambrisentan-mediated induction was stable or even increased in combination with sildenafil or tadalafil. For the drug transporter P-glycoprotein this was confirmed at the protein and functional level with highly significant correlations between P-gp mRNA, protein, and function. Moreover, using a reporter gene assay in LS180 cells, our study demonstrates for the first time that tadalafil is a potent, ambrisentan a weak, and sildenafil no activator of pregnane X receptor. In conclusion, our study demonstrates that although sildenafil and tadalafil indeed reduce intracellular concentrations of bosentan and ambrisentan in LS180 cells, they do not mitigate the inducing effects of these endothelin-1 receptor antagonists.

Keywords: Bosentan; Ambrisentan; Sildenafil; Tadalafil; Induction


TNF-α-mediated NF-κB survival signaling impairment by cisplatin enhances JNK activation allowing synergistic apoptosis of renal proximal tubular cells by Giulia Benedetti; Lisa Fredriksson; Bram Herpers; John Meerman; Bob van de Water; Marjo de Graauw (pp. 274-286).
Cisplatin-induced nephrotoxicity is an important limiting factor for cisplatin use. Tumor necrosis factor-α (TNF-α) is known to contribute to cisplatin-induced nephrotoxicity by inducing an inflammatory process aggravating the primary injury, thereby resulting in acute kidney injury (AKI). The present study investigates the pathways synergistically activated by cisplatin and TNF-α responsible for TNF-α-enhanced cisplatin-induced renal cell injury. To do so, immortalized renal proximal tubular epithelial cells (IM-PTECs) were co-treated with TNF-α and cisplatin. Under these conditions, cisplatin induced dose-dependent apoptosis in IM-PTECs, which was significantly enhanced by TNF-α. Transcriptomic analysis revealed that cisplatin inhibited the typical TNF-α response and cisplatin/TNF-α treatment up-regulated cell death pathways while it down-regulated survival pathways compared to cisplatin alone. In concordance, the gene expression levels of kidney injury markers combined with activation of specific inflammatory mediators were enhanced by cisplatin/TNF-α treatment, resembling the in vivo cisplatin-induced nephrotoxicity response. Furthermore, combined cisplatin/TNF-α treatment inhibited NF-κB nuclear translocation and NF-κB-mediated gene transcription leading to enhanced and prolonged JNK and c-Jun phosphorylation. JNK sustained activation further inhibited NF-κB signaling via a feedback loop mechanism. This led to an alteration in the transcription of the NF-κB-induced anti-apoptotic genes c-IAP2, Bcl-XL, Bruce and Bcl2 and pro-apoptotic genes Bfk and Xaf1 and consequently to sensitization of the IM-PTECs toward cisplatin/TNF-α-induced toxicity. In conclusion, our findings support a model whereby renal cells exposed to both cisplatin and TNF-α switch into a more pro-apoptotic and inflammatory program by altering their NF-κB/JNK/c-Jun balance.

Keywords: Nephrotoxicity; Cisplatin; TNF-α; NF-κB; JNK
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