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Biochemical Pharmacology (v.81, #4)

Editorial Advisory Board (pp. iii).

Implications of genome wide association studies for the understanding of type 2 diabetes pathophysiology by John R. Petrie; Ewan R. Pearson; Calum Sutherland (pp. 471-477).

This review summarises recent discoveries in the genetics of type 2 diabetes and discusses whether the preponderance of “β-cell” genes has implications for the importance of insulin resistance in the pathophysiology of the condition.The rapid rise in prevalence of type 2 diabetes mellitus (T2DM) has been driven by changes in environmental factors – primarily increased caloric intake and reduced energy expenditure – resulting in reduced whole body insulin sensitivity (often termed insulin resistance). Insulin resistance has been proposed to be a major driver of progression to T2DM. However, of 38 individual susceptibility loci for T2DM recently identified by genome wide association studies, by far the majority code for proteins involved in β-cell function. In this review, we discuss the possible reasons for the paucity of insulin resistance genes and ask whether the new genetic susceptibility data should focus attention on β-cell targets in the development of therapies for T2DM.

Keywords: Abbreviations; GWAS; genome wide association study; HOMA; homeostasis modelling assessment; IGI; insulinogenic index; OGTT; oral glucose tolerance test; T2DM; type 2 diabetesDiabetes; Gene; Insulin; Beta cell; Signalling

Synergistic antimicrotubule therapy for prostate cancer by Vaishali Pannu; Prasanthi Karna; Hari Krishna Sajja; Deep Shukla; Ritu Aneja (pp. 478-487).

Prostate cancer has been widely viewed as a chemoresistant neoplasm. Perhaps, the most prevalent antimicrotubule strategy involves docetaxel administration at its maximum-tolerated dose (MTD). Although the goal is to obtain total eradication of cancer cells, debilitating toxicities are presented by docetaxel therapy, including myelosuppression, immunosuppression, gastrointestinal toxicity and peripheral neuropathy. In addition, solubility limitations necessitate infusion of high-doses intravenously once or twice a week followed by a rest period, which allows recovery of normal proliferating cells to counter-balance efficacy. An emerging notion is that more of a toxic drug at its MTD is not necessarily better. It is likely that combinatorial antimicrotubule therapy with drugs occupying different sites on tubulin may enhance efficacy while reducing toxicity. Here we show that bromonoscapine (EM011), a microtubule-modulating noscapine analog, displays synergism with docetaxel as seen by cell viability and proliferation assays. Cell-cycle data demonstrated that lower dose-levels of docetaxel (25nM) in combination with EM011 caused an additive increase in proapoptotic activity. Since docetaxel alone caused severe mitotic arrest followed by mitotic slippage and endoreduplication, we strategized a sequential treatment regime that involved initial pretreatment with docetaxel followed by addition of EM011 to maximize mitotic arrest and subsequent apoptosis. In vivo studies with docetaxel and EM011 in combination showed a marked inhibition of tumor growth compared to docetaxel or EM011 as single-agents. Our studies suggest the potential usefulness of EM011 in the clinic to enhance docetaxel activity. This would reduce toxicity, thus improving the quality of life of docetaxel-treated patients.

Keywords: Key words; Tubulin-binding; Synergy; Polyploidy; Apoptosis; Docetaxel

ABC50 modulates sensitivity of HL60 leukemic cells to endoplasmic reticulum (ER) stress-induced cell death by Yongmao Yu; Yicheng Zhang; Ziping Zhu; Stuart A. Berger (pp. 488-497).

ABC50 promotes the association of Met-tRNA with the eIF2 complex. eIF2α represents a key initiation control point. In response to cellular stress of various kinds, eiF2α is phosphorylated by key kinases which suppress translation initiation. ABC50 promotes the association of Met-tRNA with eIF2, enhancing translation.ABC50 (aka ABCF1) is a member of the ATP Binding Cassette protein family. ABC50 stimulates complex formation between eIF2, GTP and Met-tRNA implicating it in translation initiation. Econazole (Ec) is an imidazole anti-fungal that induces endoplasmic reticulum (ER) stress in mammalian cells by promoting ER Ca²⁺ depletion and sustained protein synthesis inhibition. HL60 cells selected for Ec resistance were found to exhibit a multi-drug resistance phenotype associated specifically with ER stress. Differential Display was used to identify ABC50 as an overexpressed gene in resistant cells. ABC50 knockdown (KD) in Ec-resistant HL60 cells partially restored Ec sensitivity. In parental HL60 cells, ABC50 KD increased sensitivity to Ec, thapsigargin and tunicamycin but not to serum withdrawal or etoposide. ABC50 overexpression (OE) partially and specifically decreased sensitivity to ER stress agents. ABC50 KD or OE had no effect on ROS generation by Ec, ER Ca²⁺ stores or thapsigargin-stimulated influx. Increased eIF2α phosphorylation in response to ER stress was observed in the KD cells while decreased phosphorylation was observed in the OE cells. Ribosomal content was reduced in ABC50 KD cells and increased in OE cells. Knockdown suppressed protein synthesis while OE increased it. Protein synthesis was sustained in ABC50 OE cells exposed to Ec. ABC50 OE promoted ER stress resistance and increased antibody production in the hybridoma GK1.5 suggesting it may be useful for the overproduction of specific proteins. Taken together, these results indicate that ABC50 modulates sensitivity to Ec and other ER stress agents primarily through its effects on protein synthesis.

Keywords: Abbreviations; ABC50; ATP Binding Cassette family protein 50; Ec; econazole; eIF; Eukaryotic Initiation Factor; ER; endoplasmic reticulum; Eto; etoposide; ROS; reactive oxygen species; shRNA; short hairpin RNA; Tg; thapsigargin; Tu; tunicamycinABC50; Econazole; ER stress; Protein synthesis

Valproic acid perturbs hematopoietic homeostasis by inhibition of erythroid differentiation and activation of the myelo-monocytic pathway by Sébastien Chateauvieux; Serge Eifes; Franck Morceau; Christina Grigorakaki; Michael Schnekenburger; Estelle Henry; Mario Dicato; Marc Diederich (pp. 498-509).

As a histone deacetylase inhibitor, valproic acid (VPA) is a candidate for anticancer therapy. Besides, VPA exhibits various mechanisms of action and its effects on the molecular basis of hematopoiesis remain unclear. To study the effects of VPA on the hematopoietic system, we performed microarray analysis using K562 cells treated with 1mM VPA over a 72h time course. The association between gene ontology (GO) terms and the lists of differentially expressed genes was tested using the Bioconductor package GOstats. Enrichment analysis for cellular differentiation pathways was performed based on manually curated gene lists. Results from microarray analysis were confirmed by studying cell differentiation features at the molecular and cellular levels using other hematopoietic cell lines as well as hematopoietic stem/progenitor CD34⁺ cells. Microarray analysis revealed 3440 modulated genes in the presence of VPA. Genes involved in the granulo-monocytic differentiation pathway were up-regulated while genes of the erythroid pathway were down-regulated. This was confirmed by analyzing erythrocytic and myeloid membrane markers and lineage-related gene expression in HEL, MEG01, HL60 as well as CD34⁺ cells. Moreover, GATA-1 and its co-factors (FOG1, SP1) were down-regulated, while myelopoiesis activator PU.1 was up-regulated, in agreement with an inhibition of erythropoiesis. Our functional profiling and cell phenotyping approach demonstrates that VPA is able to alter hematopoietic homeostasis by modifying the cell population balance in the myeloid compartment. This may lead to a potential failure of erythropoiesis in patients with cancer or chronic inflammatory diseases having a well-described propensity to anemia.

Keywords: Differentiation; HDAC inhibitor; Hematopoiesis; Inflammation; Stem cell

Methotrexate induces apoptosis through p53/p21-dependent pathway and increases E-cadherin expression through downregulation of HDAC/EZH2 by Wen-Yu Huang; Pei-Ming Yang; Yu-Fan Chang; Victor E. Marquez; Ching-Chow Chen (pp. 510-517).

Methotrexate (MTX) is a dihydrofolate reductase (DHFR) inhibitor widely used as an anticancer drug in different kinds of human cancers. Here we investigated the anti-tumor mechanism of MTX against non-small cell lung cancer (NSCLC) A549 cells. MTX not only inhibited in vitro cell growth via induction of apoptosis, but also inhibited tumor formation in animal xenograft model. RNase protection assay (RPA) and RT-PCR demonstrated its induction of p53 target genes including DR5, p21, Puma and Noxa. Moreover, MTX promoted p53 phosphorylation at Ser15 and acetylaion at Lys373/382, which increase its stability and expression. The apoptosis and inhibition of cell viability induced by MTX were dependent on p53 and, partially, on p21. In addition, MTX also increased E-cadherin expression through inhibition of histone deacetylase (HDAC) activity and downregulation of polycomb group protein enhancer of zeste homologue 2 (EZH2). Therefore, the anticancer mechanism of MTX acts through initiation of p53-dependent apoptosis and restoration of E-cadherin expression by downregulation of HDAC/EZH2.

Keywords: Methotrexate; p53; p21; HDAC; EZH2

Mitogen-activated protein kinases ERK 1/2- and p38-GATA4 pathways mediate the Ang II-induced activation of FGF2 gene in neonatal rat cardiomyocytes by Wenjie Tang; Youzhen Wei; Kang Le; Zhi Li; Yingxia Bao; Jie Gao; Fangyan Zhang; Shaorui Cheng; Peiqing Liu (pp. 518-525).

Several genes, including fibroblast growth factor 2 (FGF2), are up-regulated in the hypertrophic heart. However, the molecular mechanisms responsible for the angiotensin II (Ang II)-induced activation of FGF2 in cardiomyocyte hypertrophy are largely unknown. The purpose of this study was to determine the signaling cascades underlying the Ang II-induced transcriptional activation of FGF2 in neonatal rat cardiomyocytes. Real-time quantitative RT-PCR and Western blot showed that Ang II upregulates FGF2 expression and that these effects were attenuated by U0126 or SB203580, but not by SP600125. Deletion analyses revealed that the region between −845 and −666 is essential for Ang II-induced FGF2 promoter activity. The existence of an atypical GATA4-binding motif, located at position −752, was identified using electrophoretic mobility shift assay (EMSA). Using both EMSA and chromatin immunoprecipitation (ChIP) analyses, we also showed that Ang II increases binding of GATA4 to DNA, and that this effect is attenuated in the presence of U0126 or SB203580, but not in the presence of SP600125. GATA4 siRNA significantly reduced Ang II-induced FGF2 mRNA levels. Together, these results indicate that binding of GATA4 to DNA is increased by Ang II via extracellular signal-regulated protein kinase 1/2 (ERK 1/2) and p38 kinase, which increases FGF2 gene expression in neonatal rat cardiomyocytes.

Keywords: FGF2; Promoter; Transcription factor; GATA4; Cardiac hypertrophy

Pioglitazone abrogates cyclosporine-evoked hypertension via rectifying abnormalities in vascular endothelial function by Mahmoud M. El-Mas; Hanan M. El-Gowelli; Khaled S. Abd-Elrahman; Evan I. Saad; Abdel-Galil A. Abdel-Galil; Abdel A. Abdel-Rahman (pp. 526-533).

Biochemical and oxidative vascular changes that contribute to the hypertensive action of cyclosporine. These effects are abrogated by concurrent administration of pioglitazone.In addition to insulin sensitization, the thiazolidenedione drug pioglitazone exhibits favorable circulatory effects. Here, we hypothesized that pioglitazone protects against the hypertension and related vascular derangements caused by the immunosuppressant drug cyclosporine (CSA). Compared with vehicle (olive oil)-treated rats, chronic treatment with CSA (20mg/kg/day s.c., for 14 days) increased blood pressure (BP), reduced the aortic protein expression of phosphorylated eNOS (p-eNOS), and impaired responsiveness of isolated aortas to endothelium-dependent vasorelaxations induced by carbachol. The effects of CSA on BP, aortic p-eNOS, and carbachol relaxations were abolished upon concurrent administration of pioglitazone (2.5mg/kg/day). Serum levels of adiponectin, an adipose tissue-derived adipokine, were not altered by CSA but showed significant elevations in rats treated with pioglitazone or pioglitazone plus CSA. The possibility that alterations in the antioxidant and/or lipid profile contributed to the CSA-pioglitazone BP interaction was investigated. Pioglitazone abrogated the oxidative (aortic superoxide dismutase), lipid peroxidation (aortic malondialdyde), and dyslipidemic (serum LDL levels and LDL/HDL ratio) effects of CSA. Histologically, CSA caused focal disruption in the endothelial lining of the aorta and this effect disappeared in rats co-treated with pioglitazone. Collectively, pioglitazone abrogates the hypertensive effect of CSA via ameliorating detrimental changes in vascular endothelial NOS/NO pathway and oxidative and lipid profiles caused by CSA.

Keywords: Cyclosporine; Pioglitazone; Blood pressure; Endothelium-dependent relaxations; Oxidative stress

The peroxisome proliferator-activated receptor β/δ (PPARβ/δ) agonist GW501516 prevents TNF-α-induced NF-κB activation in human HaCaT cells by reducing p65 acetylation through AMPK and SIRT1 by Emma Barroso; Elena Eyre; Xavier Palomer; Manuel Vázquez-Carrera (pp. 534-543).

Nuclear factor (NF)-κB is a ubiquitously expressed transcription factor controlling the expression of numerous genes involved in inflammation. The aim of this study was to evaluate whether activation of the peroxisome proliferator-activated receptor (PPAR) β/δ prevented TNF-α-induced NF-κB activation in human HaCaT keratinocytes and, if so, to determine the mechanism involved. The PPARβ/δ agonist GW501516 inhibited the increase caused by TNF-α in the mRNA levels of the NF-κB target genes interleukin 8 (IL-8), TNF-α and thymic stromal lymphopoietin (TSLP). Likewise, GW501516 prevented the increase in NF-κB DNA-binding activity observed in cells exposed to TNF-α. The reduction in NF-κB activity following GW501516 treatment in cells stimulated with TNF-α did not involve either increased IκBα protein levels or a reduction in the translocation of the p65 subunit of NF-κB. In contrast, GW501516 treatment decreased TNF-α-induced p65 acetylation. Acetylation of p65 is mainly regulated by p300, a transcriptional co-activator that binds to and acetylates p65. Of note, AMP kinase (AMPK) activation phosphorylates p300 and reduces its binding to p65. GW501516 increased AMPK phosphorylation and the subsequent p300 phosphorylation, leading to a marked reduction in the association between p65 and this transcriptional co-activator. In addition, treatment with the PPARβ/δ agonist increased SIRT1 protein levels. Finally, the reduction in IL-8 mRNA levels following GW501516 treatment in TNF-α-stimulated cells was abolished in the presence of the PPARβ/δ antagonist GSK0660, the AMPK inhibitor compound C and the SIRT1 inhibitor sirtinol, indicating that the effects of GW501516 on NF-κB activity were dependent on PPARβ/δ, AMPK and SIRT1, respectively.

Keywords: Abbreviations; AMPK; AMP-activated protein kinase; NF-κB; nuclear factor κB; PPAR; peroxisome proliferator-activated receptors; SIRT1; silent information regulator T1; TNFα; tumor necrosis factor α

Eriodictyol: A flavonoid antagonist of the TRPV1 receptor with antioxidant activity by Mateus Fortes Rossato; Gabriela Trevisan; Cristiani Isabel Banderó Walker; Jonatas Zeni Klafke; Ana Paula de Oliveira; Jardel Gomes Villarinho; Ricardo Basso Zanon; Luiz Fernando Freire Royes; Margareth Linde Athayde; Marcus Vinicius Gomez; Juliano Ferreira (pp. 544-551).

Eriodictyol acts as an antagonist of the TRPV1 receptor and as an antioxidant; it induces antinociception without some of the side effects and limitations such as hyperthermia that are expected for TRPV1 antagonists.The transient potential vanilloid 1 receptor (TRPV1) is a calcium-permeable channel responsible for the transduction and modulation of acute and chronic pain signaling. As such, this receptor is a potential target for the treatment of a number of pain disorders. However, AMG517, a TRPV1 antagonist, presents several clinical limitations that include the induction of severe hyperthermia. The aim of this study was to investigate the possible interaction of the flavonoid eriodictyol with the TRPV1 receptor and to determine its putative antinociceptive and hyperthermic effects. Eriodictyol was able to displace [³H]-resiniferatoxin binding (IC₅₀=47; 21–119nM) and to inhibit calcium influx mediated by capsaicin (IC₅₀=44; 16–125nM), suggesting that eriodictyol acts as a TRPV1 antagonist. Moreover, eriodictyol induced antinociception in the intraplantar capsaicin test, with maximal inhibition of 49±10 and 64±4% for oral (ID₅₀=2.3; 1.1–5.7mg/kg) and intrathecal (ID₅₀=2.2; 1.7–2.9nmol/site) administration, respectively. Eriodictyol did not induce any change in body temperature or locomotor activity. Orally administered eriodictyol (4.5mg/kg) prevented the nociception induced by intrathecal injections of capsaicin, as well as the non-protein thiol loss and 3-nitrotyrosine (3-NT) formation induced by capsaicin in spinal cord. Eriodictyol also reduced the thermal hyperalgesia and mechanical allodynia elicited by complete Freund's adjuvant (CFA) paw injection. In conclusion, eriodictyol acts as an antagonist of the TRPV1 receptor and as an antioxidant; it induces antinociception without some of the side effects and limitations such as hyperthermia that are expected for TRPV1 antagonists.

Keywords: 3-NT; Binding; Calcium influx; Eriodictyol; Pain; TRPV1; Thiol

Design and in vitro characterization of PAC1/VPAC1-selective agonists with potent neuroprotective effects by Ngoc-Duc Doan; Steve Bourgault; Agnieszka Dejda; Myriam Létourneau; Michel Detheux; David Vaudry; Hubert Vaudry; David Chatenet; Alain Fournier (pp. 552-561).

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide that exerts a large array of actions in the central nervous system and periphery. Through the activation of PAC1 and VPAC1, PACAP is able to exert neuroprotective, as well as anti-inflammatory effects, two phenomena involved in the pathogenesis and the progression of neurodegenerative diseases. The aim of the current study was to provide insights into the molecular arrangement of the amino terminus of PACAP and to develop new potent and selective PAC1/VPAC1 agonists promoting neuronal survival. We have synthesized a series of PACAP derivatives and measured their binding affinity and their ability to induce intracellular calcium mobilization for each receptor, i.e. PAC1, VPAC1, and VPAC2. Ultimately, analogs with an improved pharmacological profile were evaluated in an in vitro model of neuronal loss. Results showed that introduction of a hydroxyproline or an alanine moiety, respectively, at position 2 or 7 generated derivatives without significant VPAC2 agonistic activity. Moreover, the structure–activity relationship study suggests the presence of common (Asx-turn like) and distinct (different N-capping type) secondary structures that might be responsible for receptor recognition, selectivity and activation. Finally, evaluation of the neuroprotective activity of [Ala⁷]PACAP27 and [Hyp²]PACAP27 demonstrated their ability to protect potently human dopaminergic SH-SY5Y neuroblasts against the toxicity of MPP⁺, in pre- and co-treatment experiments. These new pharmacological and structural data should prove useful for the rational design of PACAP-derived compounds that could be putative therapeutic agents for the treatment of neurodegenerative diseases.

Keywords: Abbreviations; CNS; central nervous system; MPP; ⁺; 1-methyl-4-phenylpyridinium; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidePACAP; Structure–activity; PAC1/VPAC1 selectivity; Neuroprotection; Parkinson's disease

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Biochemical Pharmacology (v.81, #4)

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Biochemical Pharmacology (v.81, #4)