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Biochemical Pharmacology (v.85, #4)

Editorial Board (pp. iii).

Estrogen receptor signaling as a target for novel breast cancer therapeutics by Jack-Michel Renoir; Véronique Marsaud; Gwendal Lazennec (pp. 449-465).

Schematic representation of some cytoplasmic, membrane and nuclear estrogen receptor (ERα) targets: estradiol (E2) activation of ERα signaling is indicated by blue arrows; activation/enhancement of targetable proteins is indicated by green arrows, and inhibition with red T symbol.In breast cancer (BC) epithelial cells, the mitogenic action of estradiol is transduced through binding to two receptors, ERα and ERβ, which act as transcription factors. Anti-estrogens (AEs) and aromatase inhibitors (AIs) are used clinically to arrest the estrogen-dependent growth of BC. In the case of AE or AI resistance, Herceptin or lapatinib may be used to inhibit growth factors. Estrogen effects are mediated not only through nuclear ERs but also through cytoplasmic/membrane ERs and G-protein-coupled ERs. These estrogen-binding systems associate with various proteins that direct cell cycle signaling, proliferation and survival. The partners of nuclear ER include SRC1-3, HDACs and ERβ itself as well as newly identified proteins, such as E6-AP, LKB1, PELP1, PAX-2 and FOXA1. The partners of extra-nuclear ERα include PI3K and the tyrosine kinase Src. These various factors are all potential targets for therapeutic intervention. In addition, BC proliferation is enhanced by insulin and EGF, which stimulate signaling through the MAPK and PI3K/AKT pathways by activation of the IGF-1R and EGFR axes, respectively. These pathways are tightly interconnected with ER-activated signaling, and membrane ERα forms complexes with Src and PI3K. Chemokine-mediated signaling also modulates the estrogen response. Inhibiting these pathways with specific inhibitors or activating some of the pathways by gene manipulation may be therapeutically valuable for arresting BC cell cycle progression and for inducing apoptosis to antagonize hormone-resistance. Here, we review some newly identified putatively targetable ER partners and highlight the need to develop tumor-targeting drug carrier systems affecting both the tumor cells and the tumor environment.

Keywords: Abbreviations; AE; anti-estrogen; AI; aromatase inhibitors; AIB1/ACTR/p/CIP/RAC3/TRAM-1/SRC-3; amplified in breast cancer 1/activator of thyroid hormone receptor/p300/CBP-interacting protein/receptor-associated coactivator 3/thyroid hormone receptor activator molecule; 1/SRC-3; steroid receptor coactivator-3; AKT/PKB; protein kinase B; AP1; activator protein-1; BC; breast cancer; Bcl2; B cell lymphoma 2; CARM1; coactivator-associated arginine methyl-transferase 1; DRIP/TRAP; vitamin D receptor interacting protein/thyroid hormone receptor-associated proteins; EGF; epidermal growth factor; Erk-1 and Erk-2; extracellular signal-regulated kinases 1 and 2; 17β-estradiol; (17β)-estra-1,3,5(10)-triene-3,17β-diol; ER; estradiol receptor; FAK; focal adhesion protein kinase; gossypol; 2,2′-bis-(formyl-1,6,7-trihydroxy-5-isopropyl)-3-methylnaphtalene; HER/Erb-B; human epidermal growth factor receptor/avian erythroblastosis oncogene B; IGF; insulin growth factor; IRS1; insulin receptor substrate 1; LSD1/KDM1A; lysine-specific demethylase 1; MAPK; mitogen-activated protein kinase; MIBE; ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]but-2-enoate; mTOR; Mammalian target of rapamycin; NR; nuclear receptor; PAX-2; Paired Box-2 gene product; PDK1; 3-phospho-inositide-dependent protein kinase; PELP1/MNAR; proline-,glutamic acid-, and leucine-rich protein1/modulator of the non-genomic action of nuclear receptor; PI3K; phosphoinositide-3 kinase; PR; progesterone receptor; PRMT1; protein arginine methyltransferase; PTEN; phosphatase and tensin homolog; PTM; post-translational modification; Src; sarcoma virus tyrosine kinase; SERM; selective estradiol receptor modulator; SERD; selective estradiol receptor downregulator; SDF-1; stromal cell-derived factor-1; SHARP; SMRT/HDAC1 associated repressor protein; SMRT; silencing mediator for retinoid and thyroid hormone receptor; SP1; specificity protein 1; SWI/SNF; switch/sucrose nonfermentable chromatin remodeler; TRAP220; thyroid receptor-associated protein complex 220; kDa component; uPA; urokinase-type plasminogen activatorBreast cancer; Estrogen receptors; Growth factors; Hormone resistance; Therapeutic targets

P2Y purinergic receptors: New targets for analgesic and antimigraine drugs by Giulia Magni; Stefania Ceruti (pp. 466-477).

Millions of individuals worldwide suffer from acute and, more severely, chronic pain conditions (e.g., neuropathic pain, and migraine). The latter bear tremendous personal, familial, and social costs, since sufferers and their relatives undergo a complete turnaround of their lives with the search of relief from pain becoming their pivotal thought. Sadly, to date no effective pharmacological approaches are available which can alleviate chronic pain significantly or in the long run in all patients. The current central strategy for the development of new and effective painkillers lies in the hypothesis that cellular and/or molecular players in nociception must exists that are not targeted by “classical” analgesics, and therefore researchers have put tremendous efforts into the in-depth analysis of the pathways leading to pain development and maintenance over time. In this complex scenario, two outsiders are now taking the center stage: glial cells in sensory ganglia and in the central nervous system, thanks to their ability to communicate with neurons and to modulate their firing, and the purinergic system. Extracellular purine and pyrimidine nucleotides are involved in the physiology of virtually every body district, and their extracellular concentrations massively increase under pathological situations, suggesting that they might represent potential targets for the modulation of disease-associated symptoms, like pain. Here, we provide an overview of the present knowledge of the role of nucleotides in nociception, with a particular emphasis on G protein-coupled P2Y receptors and their involvement in the communication between first- and second-order neurons in sensory nerve pathways and surrounding glial cells.

Keywords: Nucleotides; P2 receptors; Satellite glial cells; Microglia; Migraine; Algogens

TGF-β as a therapeutic target in high grade gliomas – Promises and challenges by Justin V. Joseph; Veerakumar Balasubramaniyan; Annemiek Walenkamp; Frank A.E. Kruyt (pp. 478-485).

Transforming growth factor-β (TGF-β) is a cytokine with a key role in tissue homeostasis and cancer. TGF-β elicits both tumor suppressive and tumor promoting functions during cancer progression, in a wide range of cancers. Here, we review the tumor promoting function of TGF-β and its possible promise as a therapeutic target in high grade gliomas, including glioblastoma multiforme (GBM), a disease with very poor prognosis. TGF-β signaling is highly active in high grade gliomas and elevated TGF-β activity has been associated with poor clinical outcome in this deadly disease. Common features of GBMs include fast cell proliferation, invasion into normal brain parenchyma, hypoxia, high angiogenic – and immunosuppressive activity, characteristics that all have been linked to activation of the TGF-β pathway. TGF-β signaling has also been connected with the cancer stem cell (CSC) phenotype in GBM. CSCs represent a subset of GBM cells thought to be responsible for tumor initiation, progression and relapse of disease. Following the description of these different properties of TGF-β signaling and the underlying mechanisms identified thus far, the promise of TGF-β targeted therapy in malignant gliomas is discussed. Several drugs targeting TGF-β signaling have been developed that showed potent antitumor activity in preclinical models. A number of agents are currently evaluated in early clinical studies in glioma patients. Available results of these studies are highlighted and a perspective on the promise of TGF-β-targeted therapy is given.

Keywords: TGF-β; Glioma; Cancer stem cells; Microenvironment; Therapy

β-Phenylethyl isothiocyanate reverses platinum resistance by a GSH-dependent mechanism in cancer cells with epithelial-mesenchymal transition phenotype by Wen-jing Wu; Yan Zhang; Zhao-lei Zeng; Xiao-bing Li; Kai-shun Hu; Hui-yan Luo; Jing Yang; Peng Huang; Rui-hua Xu (pp. 486-496).

Platinum (Pt)-based chemotherapy is an important regimen in the clinical treatment of cancer, but development of drug resistance presents a major challenge. One key mechanism involved in resistance to Pt drugs is the decrease of intracellular Pt due to the drug efflux through the glutathione (GSH)-mediated export, and this is particularly significant in cancer cells with stem-cell like properties. In the present study, we showed that two Pt-resistant human cancer cell lines exhibited stem-cell like EMT properties, had high cellular GSH and accumulated significantly less cellular Pt compared to their parental cells, and failed to undergo apoptosis when exposed to Pt at the drug concentrations toxic to the parental cells. Importantly, we found that the natural compound β-phenylethyl isothiocyanate (PEITC) was able to effectively abolish this drug resistant mechanism by effective depletion of cellular GSH, leading to a significant increase in cellular Pt as well as DNA-bound Pt. A combination of PEITC and Pt showed a striking synergistic anticancer activity both in vitro and in vivo, as evidenced by an increase in drug-induced apoptosis, a loss of colony formation capacity, and significant suppression of tumor growth in mice. Taken together, our study shows a promising therapeutic strategy to overcome drug resistance to platinum-based chemotherapy and may potentially have broad implications in clinical treatment of cancer.

Keywords: Chemo-resistance; PEITC; Glutathione; EMT; Chemotherapy

Identification and characterization of inhibitors of cytoplasmic 5′-nucleotidase cN-II issued from virtual screening by Lars Petter Jordheim; Zsuzsanna Marton; Moez Rhimi; Emeline Cros-Perrial; Corinne Lionne; Suzanne Peyrottes; Charles Dumontet; Nushin Aghajari; Laurent Chaloin (pp. 497-506).

Clinical and preclinical observations have lead to the hypothesis that 5′-nucleotidase cN-II could constitute a therapeutic target in oncology, either per se or to increase the activity of cytotoxic nucleoside analogs.To identify potential cN-II inhibitors, we performed in silico screening of freely available chemical databases, in vitro enzymatic assays with recombinant cN-II, soaking experiments with crystals of truncated cN-II as well as biological evaluation of selected compounds, alone or in combination with cytotoxic nucleoside analogs, on cancer cells.The top ranked compounds from virtual screening included an anthraquinone derivative (AdiS) that were shown to block the enzyme activity with a K_i of 2.0mM. Soaking experiments performed with crystals of truncated cN-II allowed to obtain crystallographic data at a resolution of 2.9Å and indicating interaction between AdiS and F354/I152 situated in the effector site 1 of cN-II. In addition, this compound exhibited different levels of cytotoxicity in vitro on several cancer cell lines and increased the induction of apoptosis in RL cells incubated with 0.5 or 1.5μM cladribine, 0.05μM clofarabine or 30μM fludarabine. Finally, AdiS showed synergy with cladribine and additivity with clofarabine.This study showed that virtual screening is a useful tool for the identification of potent cN-II inhibitors, and our biological results indicated interesting activity for one lead compound that can be further developed as therapeutics.

Keywords: cN-II; Virtual screening; Enzymatic assay; Inhibitor; Crystallography

Design, synthesis and characterization of novel small molecular inhibitors of ephrin-B2 binding to EphB4 by Srinivas Duggineni; Sayantan Mitra; Roberta Noberini; Xiaofeng Han; Nan Lin; Yan Xu; Wang Tian; Jing An; Elena B. Pasquale; Ziwei Huang (pp. 507-513).

EphB4 is a member of the large Eph receptor tyrosine kinase family. By interacting with its preferred ligand ephrin-B2, which is also a transmembrane protein, EphB4 plays a role in a variety of physiological and pathological processes ranging from bone remodeling to cancer malignancy. EphB4–ephrin-B2 binding occurs at sites of contact between cells. Ephrin-B2 causes EphB4 clustering and increased kinase activity to generate downstream signals that affect cell behavior. Previous work identified a high-affinity antagonistic peptide that targets EphB4, named TNYL-RAW. This peptide is 15 amino acid long, has a molecular weight of ∼1700Da and binds to the ephrin-binding pocket of EphB4. Here we report the structure-based design and chemical synthesis of two novel small molecules of ∼600–700Da, which were designed starting from the small and functionally critical C-terminal portion of the TNYL-RAW peptide. These compounds inhibit ephrin-B2 binding to EphB4 at low micromolar concentrations. Additionally, although the ephrin-B2 ligand can interacts with multiple other Eph receptors besides EphB4, the two compounds retain the high selectivity of the TNYL-RAW peptide in targeting EphB4. TNYL-RAW peptide displacement experiments using the more potent of the two compounds, compound5, suggest a competitive mode of inhibition. These EphB4 antagonistic compounds can serve as promising templates for the further development of small molecule drugs targeting EphB4.

Keywords: Small molecular inhibitors; Eph receptors; Structure-based drug design; Tumor angiogenesis; Protein–protein interactions

The Pim kinase inhibitor SGI-1776 decreases cell surface expression of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and drug transport by Pim-1-dependent and -independent mechanisms by Karthika Natarajan; Jasjeet Bhullar; Suneet Shukla; Mehmet Burcu; Zhe-Sheng Chen; Suresh V. Ambudkar; Maria R. Baer (pp. 514-524).

Overexpression of the ATP-binding cassette (ABC) drug efflux proteins P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) on malignant cells is associated with inferior chemotherapy outcomes. Both, ABCB1 and ABCG2, are substrates of the serine/threonine kinase Pim-1; Pim-1 knockdown decreases their cell surface expression, but SGI-1776, the first clinically tested Pim inhibitor, was shown to reverse drug resistance by directly inhibiting ABCB1-mediated transport. We sought to characterize Pim-1-dependent and -independent effects of SGI-1776 on drug resistance. SGI-1776 at the Pim-1-inhibitory and non-cytotoxic concentration of 1μM decreased the IC₅₀s of the ABCG2 and ABCB1 substrate drugs in cytotoxicity assays in resistant cells, with no effect on the IC₅₀ of non-substrate drug, nor in parental cells. SGI-1776 also increased apoptosis of cells overexpressing ABCG2 or ABCB1 exposed to substrate chemotherapy drugs and decreased their colony formation in the presence of substrate, but not non-substrate, drugs, with no effect on parental cells. SGI-1776 decreased ABCB1 and ABCG2 surface expression on K562/ABCB1 and K562/ABCG2 cells, respectively, with Pim-1 overexpression, but not HL60/VCR and 8226/MR20 cells, with lower-level Pim-1 expression. Finally, SGI-1776 inhibited uptake of ABCG2 and ABCB1 substrates in a concentration-dependent manner irrespective of Pim-1 expression, inhibited ABCB1 and ABCG2 photoaffinity labeling with the transport substrate [¹²⁵I]iodoarylazidoprazosin ([¹²⁵I]IAAP) and stimulated ABCB1 and ABCG2 ATPase activity. Thus SGI-1776 decreases cell surface expression of ABCB1 and ABCG2 and inhibits drug transport by Pim-1-dependent and -independent mechanisms, respectively. Decrease in ABCB1 and ABCG2 cell surface expression mediated by Pim-1 inhibition represents a novel mechanism of chemosensitization.

Keywords: SGI-1776; Pim-1 kinase; ABCG2; ABCB1; Multidrug resistance; Leukemia

Off-target effects of thrombolytic drugs: apolipoprotein A-I proteolysis by alteplase and tenecteplase by Monica Gomaraschi; Alice Ossoli; Cecilia Vitali; Silvia Pozzi; Laura Vitali Serdoz; Cristina Pitzorno; Gianfranco Sinagra; Guido Franceschini; Laura Calabresi (pp. 525-530).

The administration of thrombolytic drugs is of proven benefit in a variety of clinical conditions requiring acute revascularization, including acute myocardial infarction (AMI), ischemic stroke, pulmonary embolism, and venous thrombosis. Generated plasmin can degrade non-target proteins, including apolipoprotein A-I (apoA-I), the major protein constituent of high-density lipoproteins (HDL). Aim of the present study was to compare the extent of apoA-I proteolytic degradation in AMI patients treated with two thrombolytic drugs, alteplase and the genetically engineered t-PA variant tenecteplase. ApoA-I degradation was evaluated in sera from 38 AMI patients treated with alteplase or tenecteplase. In vitro, apoA-I degradation was tested by incubating control sera or purified HDL with alteplase or tenecteplase at different concentrations (5–100μg/ml). Treatment with alteplase and tenecteplase results in apoA-I proteolysis; the extent of apoA-I degradation was more pronounced in alteplase-treated patients than in tenecteplase-treated patients. In vitro, the extent of apoA-I proteolysis was higher in alteplase-treated sera than in tenecteplase-treated sera, in the whole drug concentration range. No direct effect of the two thrombolytic agents on apoA-I degradation was observed. In addition to apoA-I, apoA-IV was also degraded by the two thrombolytic agents and again proteolytic degradation was higher with alteplase than tenecteplase. In conclusion, this study indicates that both alteplase and tenecteplase cause plasmin-mediated proteolysis of apoA-I, with alteplase resulting in a greater apoA-I degradation than tenecteplase, potentially causing a transient impairment of HDL atheroprotective functions.

Keywords: Abbreviations; AMI; acute myocardial infarction; apoA-I; apolipoprotein A-I; BMI; body mass index; CK; creatine kinase; CK-MB; CK myocardial band; CRP; C-reactive protein; HDL; high density lipoproteins; LDL; low density lipoproteins; RCT; reverse cholesterol transport; TnI; Troponin I; t-PA; tissue plasminogen activatorAcute Myocardial infarction; Alteplase; Apolipoprotein A-I; Fibrinolysis; High density lipoproteins; Tenecteplase

Interleukin-6 induces vascular endothelial growth factor expression and promotes angiogenesis through apoptosis signal-regulating kinase 1 in human osteosarcoma by Huey-En Tzeng; Chun-Hao Tsai; Zi-Ling Chang; Chen-Ming Su; Shih-Wei Wang; Wen-Li Hwang; Chih-Hsin Tang (pp. 531-540).

Osteosarcoma is characterized by a high malignant and metastatic potential. Angiogenesis is essential for the caner metastasis. Interleukin-6 (IL-6) is a multifunctional cytokine that is associated with the disease status and outcomes of cancers. However, the relationship between IL-6 and vascular endothelial growth factor (VEGF) expression in human osteosarcoma is mostly unknown. Here we found that the IL-6 and VEGF expression was correlated with tumor stage and significantly higher than that in normal bone. Incubation of osteosarcoma cells with IL-6 increased VEGF mRNA and protein expression. Pretreatment of cells with IL-6R antibody reduced IL-6-mediated VEGF production. The apoptosis signal-regulating kinase 1 (ASK1)/p38/AP-1 pathway was activated after IL-6 treatment, and IL-6-induced VEGF expression was abolished by the specific inhibitor and siRNA of ASK1, p38, and AP-1 cascades. Importantly, knockdown IL-6 reduced VEGF expression and abolished osteosarcoma conditional medium-mediated angiogenesis. Taken together, these results indicate that IL-6 occurs through ASK1 and p38, which in turn activates AP-1, resulting in the activations of VEGF expression and contributing the angiogenesis of human osteosarcoma cells.

Keywords: IL-6; VEGF; ASK1; Angiogenesis; Osteosarcoma

Analysis by substituted cysteine scanning mutagenesis of the fourth transmembrane domain of the CXCR4 receptor in its inactive and active state by Philip E. Boulais; Emanuel Escher; Richard Leduc (pp. 541-550).

The chemokine SDF-1 (CXCL12) selectively binds to CXCR4, a member of the G protein-coupled receptor (GPCR) superfamily. In this study, we used the substituted-cysteine accessibility method (SCAM) to identify specific residues of the fourth transmembrane domain (TM4) that contribute to the formation of the binding pocket of CXCR4 in its inactive and active state. We successively substituted each residue from E179^(4.68) to K154^(4.43) with cysteine and expressed the mutants in COS-7 cells. Mutant receptors were then alkylated with methanethiosulfonate-ethylammonium (MTSEA), and binding inhibition was monitored using the CXCR4 antagonist FC131 [ cyclo(-D-Tyr¹-Arg²-Arg³-Nal⁴-Gly⁵-)], which displays anti-HIV activity. MTSEA treatment resulted in a significant reduction of FC131 binding to D171C^(4.60) and P170C^(4.59). To assess TM4 accessibility in an active state of CXCR4, TM4 cysteine mutants were transposed within the constitutively active mutant N119S^(3.35). MTSEA treatment of TM4 mutants N119S-S178C^(4.67), N119S-V177C^(4.66) and N119S-I173C^(4.62) resulted in a significant reduction in FC131 binding. Protection assays using FC131 prior to MTSEA treatment significantly reduced the alkylation of all MTSEA-sensitive mutants. The accessibility of the D171C^(4.60) and P170C^(4.59) residues suggests that they are oriented towards a water-accessible area of the binding pocket of CXCR4. S178C^(4.67), V177C^(4.66) and I173C^(4.62) showed binding inhibition only in an N119S^(3.35) background. Taken together our results suggest that TM4 and ECL2 undergo conformational changes during CXCR4 activation and also demonstrate how TM4 is an important feature for the binding of anti-HIV compounds.

Keywords: CXCR4; G Protein-coupled receptor; Substituted-cysteine accessibility method; Binding pocket; Receptor activation

Reversible inhibition of vasoconstriction by thiazolidinediones related to PI3K/Akt inhibition in vascular smooth muscle cells by Tiziana Sinagra; Alessandra Tamburella; Vincenzo Urso; Ilias Siarkos; Filippo Drago; Claudio Bucolo; Salvatore Salomone (pp. 551-559).

Glitazones inhibit both receptor-dependent (PE-induced) and receptor independent (K⁺-induced) vasoconstriction; their effect might be accounted for by PI3K/Akt and Rho/ROK inhibition, reducing myosin phosphorylation.Thiazolidinediones (also referred to as glitazones), agonists for Peroxisome Proliferator-Activated Receptor gamma (PPARγ), are used for treating type 2 diabetes mellitus, where they decrease insulin resistance and cardiovascular risk. Compounds bearing the thiazolidinedione structure have also been shown to inhibit phosphoinositide 3-kinase (PI3K). Here we tried to elucidate the poorly defined role of PI3K/Akt in the physiology of vascular smooth muscle cell contraction and tested the hypothesis that thiazolidinediones, by affecting the PI3K/Akt pathway, may influence vascular physiology. Isolated rat femoral arteries segments were mounted in a wire myograph and challenged with 100mM KCl or phenylephrine (PE), in the absence or presence of troglitazone, rosiglitazone, pioglitazone, LY294002 (PI3K inhibitor) and 10-DEBC (Akt inhibitor). All these compounds dose-dependently inhibited vasoconstriction to KCl or PE; their effect was reversible (after 60–120min washout) and not affected by GW9662 (a PPARγ antagonist) or by N^G-nitro-L-arginine (LNNA, an inhibitor of NO biosynthesis). Analysis of phospho-Akt (ser 473) in lysates from rat arteries (by immunoblot) revealed that thiazolidinediones, LY294002 and 10-DEBC, at the same concentration and kinetics inhibiting vasoconstriction, produced a similar decrease of Akt phosphorylation. PI3K/Akt pathway therefore appears to be an important, fast acting, modulator of contraction of vascular smooth muscle. Thiazolidinediones decrease vasoconstriction of isolated vessels possibly by inhibiting PI3K/Akt pathway. Such an effect of glitazones, if occurring in vivo, may impact cardiovascular syndromes related to vasospasm in diabetic patients.

Keywords: Vascular tone; Anti-diabetic drugs; Thiazolidinediones; PPARγ; Phosphoinositide 3-kinase/Akt

The interactions of apamin and tetraethylammonium are differentially affected by single mutations in the pore mouth of small conductance calcium-activated potassium (SK) channels by Sébastien Dilly; Fabian Philippart; Cédric Lamy; Sylvie Poncin; Dirk Snyders; Vincent Seutin; Jean-François Liégeois (pp. 560-569).

Phenylalanine replacing valine residues in the pore region prevents a crucial interaction remotely from the pore between arginine 13 of apamin (highlighted in green) and aspartate 341 of SK2 channels.Valine residues in the pore region of SK2 (V366) and SK3 (V520) were replaced by either an alanine or a phenylalanine to evaluate the impact on the interactions with the allosteric blocker apamin. Unlike TEA which showed high sensitivity to phenylalanine mutated channels, the binding affinity of apamin to the phenylalanine mutants was strongly reduced. In addition, currents from phenylalanine mutants were largely resistant to block by apamin. On the other hand, when the valine residue was replaced by an alanine residue, an increase of the binding affinity and the amount of block by apamin was observed for alanine mutated SK2 channels, but not for mutated SK3 channels. Interestingly, the VA mutation reduced the sensitivity to TEA. In silico data confirmed these experimental results. Therefore, such mutations in the pore region of SK channels show that the three-dimensional structure of the SK tetramers can be disorganized in the outer pore region leading to reduced interaction of apamin with its target.

Keywords: Abbreviations; SK/K; _Ca; 2 channel; small conductance calcium-activated potassium channel; mAHP; medium afterhyperpolarization; CaM; calmodulin; CNS; central nervous system; TEA; tetraethylammonium; HEK; human embryonic kidney; EDTA; ethylene diamine tetraacetic acid; PEI; polyethylenimine; EGFP; enhanced green fluorescent protein; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EGTA; ethylene glycol tetraacetic acid; MD; molecular dynamicsSmall conductance calcium-activated potassium channel; Apamin; TEA; Single mutation; Site-directed mutagenesis; Docking

Urinary chemokine (C-C motif) ligand 2 (monocyte chemotactic protein-1) as a tubular injury marker for early detection of cisplatin-induced nephrotoxicity by Kumiko Nishihara; Satohiro Masuda; Haruka Shinke; Aiko Ozawa; Takaharu Ichimura; Atsushi Yonezawa; Shunsaku Nakagawa; Ken-ichi Inui; Joseph V. Bonventre; Kazuo Matsubara (pp. 570-582).

Because of the difficulty in detecting segment-specific response in the kidney, we investigated the molecular events underlying acute kidney injury in the proximal tubules of rats with cisplatin (cis-diamminedichloroplatinum II)-induced nephrotoxicity. Microarray analysis revealed that mRNA levels of several cytokines and chemokines, such as interleukin-1beta, chemokine (C-C motif) ligand (CCL) 2, CCL20, chemokine (C-X-C motif) ligand (CXCL) 1, and CXCL10 were significantly increased after cisplatin treatment in both isolated proximal tubules and whole kidney. Interestingly, tubular CCL2 mRNA levels increased soon after cisplatin administration, whereas CCL2 mRNA levels in whole kidney first decreased and then increased. Levels of both CCL2 and kidney injury molecule-1 (KIM-1) in the whole kidney increased after cisplatin administration. Immunofluorescence analysis revealed CCL2 changes in the proximal tubular cells initially and then in the medullary interstitium. Urinary CCL2 excretion significantly increased approximately 3-fold compared with controls the day after cisplatin administration (5mg/kg), when no changes were observed plasma creatinine and blood urea nitrogen levels. Urinary levels of KIM-1 also increased 3-fold after cisplatin administration. In addition, urinary CCL2 rather than KIM-1 increased in chronic renal failure rats after administration of low-dose cisplatin (2mg/kg), suggesting that urinary CCL2 was selective for cisplatin-induced nephrotoxicity in renal impairment. These results indicated that the increase in cytokine and chemokine expression in renal epithelial cells might be responsible for kidney deterioration in cisplatin-induced nephrotoxicity, and that urinary CCL2 is associated with tubular injury and serves as a sensitive and noninvasive marker for the early detection of cisplatin-induced tubular injury.

Keywords: Acute kidney injury; Microarray analysis; Renal proximal tubule cells; Monocyte chemotactic protein-1; MCP-1; Kidney injury molecule-1; KIM-1

Metformin-mediated downregulation of p38 mitogen-activated protein kinase-dependent excision repair cross-complementing 1 decreases DNA repair capacity and sensitizes human lung cancer cells to paclitaxel by Sheng-Chieh Tseng; Yu-Ching Huang; Huang-Jen Chen; Hsien-Chun Chiu; Yi-Jhen Huang; Ting-Yu Wo; Shao-Hsing Weng; Yun-Wei Lin (pp. 583-594).

Metformin, an extensively used and well-tolerated drug for treating individuals with type 2 diabetes, has recently gained significant attention as an anticancer drug. On the other hand, paclitaxel (Taxol) is a new antineoplastic drug that has shown promise in the treatment of non-small cell lung cancer (NSCLC). High expression levels of excision repair cross-complementary 1 (ERCC1) in cancers have been positively associated with the DNA repair capacity and a poor prognosis in NSCLC patients treated with platinum-containing chemotherapy. In this current study, paclitaxel was found to increase phosphorylation of mitogen-activated protein kinase (MAPK) kinase 3/6 (MKK3/6)-p38 MAPK as well as protein and mRNA levels of ERCC1 in H1650 and H1703 cells. Moreover, paclitaxel-induced ERCC1 protein and mRNA levels significantly decreased via the downregulation of p38 activity by either a p38 MAPK inhibitor SB202190 or p38 knockdown with specific small interfering RNA (siRNA). Specific inhibition of ERCC1 with siRNA was found to enhance the paclitaxel-induced cytotoxic effect and growth inhibition. Furthermore, metformin was able to not only decrease the paclitaxel-induced p38 MAPK-mediated ERCC1 expression, but also augment the cytotoxic effect induced by paclitaxel. Finally, expression of constitutive activate MKK6 or HA-p38 MAPK vectors in lung cancer cells was able to abrogate ERCC1 downregulation by metformin and paclitaxel as well as cell viability and DNA repair capacity. Overall, our results suggest that inhibition of the p38 MAPK signaling by metformin coupled with paclitaxel therapy in human NSCLC cells may be a clinically useful combination, which however will require further validation.

Keywords: Metformin; Paclitaxel; ERCC1; Non-small cell lung cancer; p38 MAPK

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Biochemical Pharmacology (v.85, #4)

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Biochemical Pharmacology (v.85, #4)