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Biochemical Pharmacology (v.85, #8)

Editorial Board (pp. iii).

Kynurenic acid as an antagonist of α7 nicotinic acetylcholine receptors in the brain: Facts and challenges by Edson X. Albuquerque; Robert Schwarcz (pp. 1027-1032).

Kynurenic acid (KYNA), a major tryptophan metabolite, is a glutamate receptor antagonist, which is also reported to inhibit α7 nicotinic acetylcholine receptors (α7nAChRs). Due to variations in experimental approaches, controversy has arisen regarding the ability of KYNA to directly influence α7nAChR function. Here we summarize current concepts of KYNA neurobiology and review evidence pertaining to the proposed role of KYNA as an endogenous modulator of α7nAChRs and synaptic transmission. As dysfunction of α7nAChRs plays a major role in the pathophysiology of central nervous system disorders, elucidation of KYNA's action on this receptor subtype has significant therapeutic implications.

Keywords: Kynurenic acid; α7 Nicotinic receptors; Neurotransmission; Glutamate receptor antagonists

Fibroblast-mediated drug resistance in cancer by Kim H.T. Paraiso; Keiran S.M. Smalley (pp. 1033-1041).

Tumor progression relies upon the dynamic interaction of cancer cells with host fibroblasts, endothelial cells, immune cells and components of the extracellular matrix, collectively known as the tumor microenvironment. Despite this, relatively little is known about how normal host cells dictate the response of tumors to anti-cancer therapies. Emerging data suggests that host factors play a critical role in determining risks for tumor progression and decreased therapeutic responses. In particular, recent findings have provided evidence that the tumor microenvironment provides a protective niche that allows minor populations of cancer cells to escape from the cytotoxic effects of radiation, chemotherapy and targeted therapies. In this review we will outline the mechanisms by which tumor cells and host fibroblasts co-operate to drive tumor initiation and progression. In particular, we will focus upon the mechanisms by which tumor cells exposed to targeted therapies co-opt the host leading to therapeutic escape and resistance. We will end by discussing the idea that long-term responses to targeted anticancer therapies will only be achieved through strategies that target both the tumor and host.

Keywords: Melanoma; Fibroblasts; BRAF; Stroma; ECM

Involvement of Src and the actin cytoskeleton in the antitumorigenic action of adenosine dialdehyde by Ji Hye Kim; Yong Gyu Lee; Seungwan Yoo; Jueun Oh; Deok Jeong; Woo Keun Song; Byong Chul Yoo; Man Hee Rhee; Jongsun Park; Sang-hoon Cha; Sungyoul Hong; Jae Youl Cho (pp. 1042-1056).

Transmethylation is an important reaction that transfers a methyl group in S-adenosylmethionine (SAM) to substrates such as DNA, RNA, and proteins. It is known that transmethylation plays critical roles in various cellular responses. In this study, we examined the effects of transmethylation on tumorigenic responses and its regulatory mechanism using an upregulation strategy of adenosylhomocysteine (SAH) acting as a negative feedback inhibitor. Treatment with adenosine dialdehyde (AdOx), an inhibitor of transmethylation-suppressive adenosylhomocysteine (SAH) hydrolase (SAHH), enhanced the level of SAH and effectively blocked the proliferation, migration, and invasion of cancer cells; the treatment also induced the differentiation of C6 glioma cells and suppressed the neovascular genesis of eggs in a dose-dependent manner. Through immunoblotting analysis, it was found that AdOx was capable of indirectly diminishing the phosphorylation of oncogenic Src and its kinase activity. Interestingly, AdOx disrupted actin cytoskeleton structures, leading to morphological changes, and suppressed the formation of a signaling complex composed of Src and p85/PI3K, which is linked to various tumorigenic responses. In agreement with these data, the exogenous treatment of SAH or inhibition of SAHH by specific siRNA or another type of inhibitor, 3-deazaadenosine (DAZA), similarly resulted in antitumorigenic responses, suppressive activity on Src, the alteration of actin cytoskeleton, and a change of the colocalization pattern between actin and Src. Taken together, these results suggest that SAH/SAHH-mediated transmethylation could be linked to the tumorigenic processes through cross-regulation between the actin cytoskeleton and Src kinase activity.

Keywords: Transmethylation; Tumorigenic response; Adenosine dialdehyde; Src; Actin cytoskeleton; S; -adenosylhomocysteine hydrolase

Tamoxifen magnifies therapeutic impact of ceramide in human colorectal cancer cells independent of p53 by Samy A.F. Morad; James P. Madigan; Jonathan C. Levin; Noha Abdelmageed; Ramin Karimi; Daniel W. Rosenberg; Mark Kester; Sriram S. Shanmugavelandy; Myles C. Cabot (pp. 1057-1065).

Poor prognosis in patients with later stage colorectal cancer (CRC) necessitates the search for new treatment strategies. Ceramide, because of its role in orchestrating death cascades in cancer cells, is a versatile alternative. Ceramide can be generated by exposure to chemotherapy or ionizing radiation, or it can be administered in the form of short-chain analogs (C6-ceramide). Because intracellular P-glycoprotein (P-gp) plays a role in catalyzing the conversion of ceramide to higher sphingolipids, we hypothesized that administration of P-gp antagonists with C6-ceramide would magnify cell death cascades. Human CRC cell lines were employed, HCT-15, HT-29, and LoVo. The addition of either tamoxifen, VX-710, verapamil, or cyclosporin A, antagonists of P-gp, enhanced C6-ceramide cytotoxicity in all cell lines. In depth studies with C6-ceramide and tamoxifen in LoVo cells showed the regimen induced PARP cleavage, caspase-dependent apoptosis, mitochondrial membrane permeabilization (MMP), and cell cycle arrest at G1 and G2. At the molecular level, the regimen, but not single agents, induced time-dependent upregulation of tumor suppressor protein p53; however, introduction of a p53 inhibitor staved neither MMP nor apoptosis. Nanoliposomal formulations of C6-ceramide and tamoxifen were also effective, yielding synergistic cell kill. We conclude that tamoxifen is a favorable adjuvant for enhancing C6-ceramide cytotoxicity in CRC, and demonstrates uniquely integrated effects. The high frequency of expression of P-gp in CRC presents an adventitious target for complementing ceramide-based therapies, a strategy that could hold promise for treatment of resistant disease.

Keywords: Colorectal cancer; Ceramide; C6-ceramide; Tamoxifen; P-glycoprotein; p53Abbreviations; CRC; colorectal cancer; P-gp; P-glycoprotein; GCS; glucosylceramide synthase; GC; glucosylceramide; PARP; poly ADP ribose polymerase; MMP; mitochondrial membrane permeabilization; FBS; fetal bovine serum; ATCC; American type culture collection; HRP; IgG-horseradish peroxidase; TLC; thin layer chromatography; LSC; liquid scintillation counting; LC; C6-lactosylceramide; SM; C6-sphingomyelin; Δψm; mitochondrial depolarization; C6-GC; C6-glucosylceramide; C6-SM; C6-sphingomyelin

Silymarin inhibits ultraviolet radiation-induced immune suppression through DNA repair-dependent activation of dendritic cells and stimulation of effector T cells by Mudit Vaid; Ram Prasad; Tripti Singh; Craig A. Elmets; Hui Xu; Santosh K. Katiyar (pp. 1066-1076).

Silymarin repairs UV-induced DNA damage in antigen presenting dendritic cells (DCs), and that result in increase in CD8⁺ effector T cells and decrease in CD4⁺ Th2 cells in UV-irradiated mice.Silymarin inhibits UVB-induced immunosuppression in mouse skin. To identify the molecular mechanisms underlying this effect, we used an adoptive transfer approach in which dendritic cells (DCs) from the draining lymph nodes of donor mice that had been UVB-exposed and sensitized to 2,4,-dinitrofluorobenzene (DNFB) were transferred into naïve recipient mice. The contact hypersensitivity (CHS) response of the recipient mice to DNFB was then measured. When DCs were obtained from UVB-exposed donor mice that were not treated with silymarin, the CHS response was suppressed confirming the role of DCs in the UVB-induced immunosuppression. Silymarin treatment of UVB-exposed donor mice relieved this suppression of the CHS response in the recipients. Silymarin treatment was associated with rapid repair of UVB-induced cyclobutane pyrimidine dimers (CPDs) in DCs and silymarin treatment did not prevent UV-induced immunosuppression in XPA-deficient mice which are unable to repair UV-induced DNA damage. The CHS response in mice receiving DCs from silymarin-treated UV-exposed donor mice also was associated with enhanced secretion of Th1-type cytokines and stimulation of T cells. Adoptive transfer of T cells revealed that transfer of either CD8⁺ or CD4⁺ cells from silymarin-treated, UVB-exposed donors resulted in enhancement of the CHS response. Cell culture study showed enhanced secretion of IL-2 and IFNγ by CD8⁺ T cells, and reduced secretion of Th2 cytokines by CD4⁺ T cells, obtained from silymarin-treated UVB-exposed mice. These data suggest that DNA repair-dependent functional activation of DCs, a reduction in CD4⁺ regulatory T-cell activity, and stimulation of CD8⁺ effector T cells contribute to silymarin-mediated inhibition of UVB-induced immunosuppression.

Keywords: DNA repair; Contact hypersensitivity; Cyclobutane pyrimidine dimer; Nucleotide excision repair; Silymarin; Ultraviolet radiation

Overcoming chemotherapy resistance of ovarian cancer cells by liposomal cisplatin: Molecular mechanisms unveiled by gene expression profiling by Martin Koch; Michaela L. Krieger; Daniel Stölting; Norbert Brenner; Manfred Beier; Ulrich Jaehde; Michael Wiese; Hans-Dieter Royer; Gerd Bendas (pp. 1077-1090).

Liposomal CDDP activates, in contrast to the free drug, the extrinsic pathway of apoptosis and thus overcomes CDDP resistance in A2780cis cells.Previously we reported that liposomal cisplatin (CDDP) overcomes CDDP resistance of ovarian A2780cis cancer cells (Krieger et al., Int. J. Pharm. 389, 2010, 10–17). Here we find that the cytotoxic activity of liposomal CDDP is not associated with detectable DNA platination in resistant ovarian cancer cells. This suggests that the mode of action of liposomal CDDP is different from the free drug. To gain insight into mechanisms of liposomal CDDP activity, we performed a transcriptome analysis of untreated A2780cis cells, and A2780cis cells in response to exposure with IC₅₀ values of free or liposomal CDDP. A process network analysis of upregulated genes showed that liposomal CDDP induced a highly different gene expression profile in comparison to the free drug. p53 was identified as a key player directing transcriptional responses to free or liposomal CDDP. The free drug induced expression of essential genes of the intrinsic (mitochondrial) apoptosis pathway ( BAX, BID, CASP9) most likely through p38MAPK activation. In contrast, liposomal CDDP induced expression of genes from DNA damage pathways and several genes of the extrinsic pathway of apoptosis ( TNFRSF10B–DR5, CD70-TNFSF7). It thus appears that liposomal CDDP overcomes CDDP resistance by inducing DNA damage and in consequence programmed cell death by the extrinsic pathway. Predictions from gene expression data with respect to apoptosis activation were confirmed at the protein level by an apoptosis antibody array. This sheds new light on liposomal drug carrier approaches in cancer and suggests liposomal CDDP as promising strategy for the treatment of CDDP resistant ovarian carcinomas.

Keywords: CDDP-resistance; Ovarian cancer; Liposomal CDDP; Gene expression profiling; Apoptosis

Creation and screening of a multi-family bacterial oxidoreductase library to discover novel nitroreductases that efficiently activate the bioreductive prodrugs CB1954 and PR-104A by Gareth A. Prosser; Janine N. Copp; Alexandra M. Mowday; Christopher P. Guise; Sophie P. Syddall; Elsie M. Williams; Claire N. Horvat; Pearl M. Swe; Amir Ashoorzadeh; William A. Denny; Jeff B. Smaill; Adam V. Patterson; David F. Ackerley (pp. 1091-1103).

Two potentially complementary approaches to improve the anti-cancer strategy gene-directed enzyme prodrug therapy (GDEPT) are discovery of more efficient prodrug-activating enzymes, and development of more effective prodrugs. Here we demonstrate the utility of a flexible screening system based on the Escherichia coli SOS response to evaluate novel nitroreductase enzymes and prodrugs in concert. To achieve this, a library of 47 candidate genes representing 11 different oxidoreductase families was created and screened to identify the most efficient activators of two different nitroaromatic prodrugs, CB1954 and PR-104A. The most catalytically efficient nitroreductases were found in the NfsA and NfsB enzyme families, with NfsA homologues generally more active than NfsB. Some members of the AzoR, NemA and MdaB families also exhibited low-level activity with one or both prodrugs. The results of SOS screening in our optimised E. coli reporter strain SOS-R2 were generally predictive of the ability of nitroreductase candidates to sensitise E. coli to CB1954, and of the k_cat/ K_m for each prodrug substrate at a purified protein level. However, we also found that not all nitroreductases express stably in human (HCT-116 colon carcinoma) cells, and that activity at a purified protein level did not necessarily predict activity in stably transfected HCT-116. These results highlight a need for all enzyme-prodrug partners for GDEPT to be assessed in the specific context of the vector and cell line that they are intended to target. Nonetheless, our oxidoreductase library and optimised screens provide valuable tools to identify preferred nitroreductase-prodrug combinations to advance to preclinical evaluation.

Keywords: CB1954; PR-104; Nitroreductase; Gene therapy; Nitroaromatic prodrug; SOS chromotest

Chemically diverse microtubule stabilizing agents initiate distinct mitotic defects and dysregulated expression of key mitotic kinases by Cristina C. Rohena; Jiangnan Peng; Tyler A. Johnson; Phillip Crews; Susan L. Mooberry (pp. 1104-1114).

Microtubule stabilizers are some of the most successful drugs used in the treatment of adult solid tumors and yet the molecular events responsible for their antimitotic actions are not well defined. The mitotic events initiated by three structurally and biologically diverse microtubule stabilizers; taccalonolide AJ, laulimalide/fijianolide B and paclitaxel were studied. These microtubule stabilizers cause the formation of aberrant, but structurally distinct mitotic spindles leading to the hypothesis that they differentially affect mitotic signaling. Each microtubule stabilizer initiated different patterns of expression of key mitotic signaling proteins. Taccalonolide AJ causes centrosome separation and disjunction failure to a much greater extent than paclitaxel or laulimalide, which is consistent with the distinct defects in expression and activation of Plk1 and Eg5 caused by each stabilizer. Localization studies revealed that TPX2 and Aurora A are associated with each spindle aster formed by each stabilizer. This suggests a common mechanism of aster formation. However, taccalonolide AJ also causes pericentrin accumulation on every spindle aster. The presence of pericentrin at every spindle aster initiated by taccalonolide AJ might facilitate the maintenance and stability of the highly focused asters formed by this stabilizer. Laulimalide and paclitaxel cause completely different patterns of expression and activation of these proteins, as well as phenotypically different spindle phenotypes. Delineating how diverse microtubule stabilizers affect mitotic signaling pathways could identify key proteins involved in modulating sensitivity and resistance to the antimitotic actions of these compounds.

Keywords: Microtubules; Microtubule stabilizers; Cell cycle; Mitosis; Centrosome

c-Src-dependent MAPKs/AP-1 activation is involved in TNF-α-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells by Chuen-Mao Yang; I-Ta Lee; Chih-Chung Lin; Chao-Hung Wang; Wen-Jin Cherng; Li-Der Hsiao (pp. 1115-1123).

TNF-α plays a critical mediator in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-α in inflammatory responses has been shown to be mediated through up-regulation of inflammatory genes, including matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-α-induced MMP-9 expression are largely unclear in the heart cells. Here, we demonstrated that in rat embryonic-heart derived H9c2 cells, TNF-α could induce MMP-9 mRNA expression associated with an increase in the secretion of MMP-9, determined by real-time PCR, zymography, and promoter activity assays. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of c-Src, EGFR, PDGFR, p110, Akt, or c-Jun. TNF-α stimulated c-Src, PDGFR, and EGFR phosphorylation, which were reduced by PP1. In addition, TNF-α-stimulated Akt phosphorylation was inhibited by PP1, AG1478, AG1296, or LY294002. We further demonstrated that TNF-α markedly stimulated p38 MAPK, p42/p44 MAPK, and JNK1/2 phosphorylation via a c-Src/EGFR, PDGFR/PI3K/Akt pathway. Finally, we showed that, in H9c2 cells, TNF-α-stimulated AP-1 promoter activity, c-Jun mRNA expression, and c-Jun phosphorylation were attenuated by PP1, AG1478, AG1296, LY294002, SB202190, SP600125, or U0126. These results suggested that TNF-α-induced MMP-9 expression is mediated through a c-Src/EGFR, PDGFR/PI3K/Akt/MAPKs/AP-1 cascade in H9c2 cells. Consequently, MMP-9 induction may contribute to cell migration and cardiovascular inflammation.

Keywords: Cytokine; Matrix metalloproteinase-9; Transcription factor; Tyrosine kinase

Gastrodin attenuation of the inflammatory response in H9c2 cardiomyocytes involves inhibition of NF-κB and MAPKs activation via the phosphatidylinositol 3-kinase signaling by Ping Yang; Yi Han; Li Gui; Jun Sun; Yuan-li Chen; Rui Song; Jia-zhi Guo; Ya-nan Xie; Di Lu; Lin Sun (pp. 1124-1133).

The phenolic glucoside gastrodin, a main constituent of a Chinese traditional herbal medicine, has been known to display several biological and pharmacological properties. However, the role and precise molecular mechanisms explaining how gastrodin suppresses the inflammatory response in septic cardiac dysfunction are unknown. To study this, rat H9c2 cardiomyocytes were treated with gastrodin and/or lipopolysaccharide (LPS). Our results showed that gastrodin treatment strongly suppressed nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) family activation and upregulation of the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in LPS-stimulated H9c2 cardiomyocytes. Simultaneously, gastrodin obviously upregulated the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling in a dose-dependent manner. However, wortmannin, a specific PI3-K inhibitor, blocked the inhibitory effects of gastrodin on LPS-stimulated H9c2 cardiomyocytes. Furthermore, PI3-K/Akt inhibition partially abolished the inhibitory effects of gastrodin on the phosphorylation of inhibitor κB-α (IκB-α), extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK), and activity of NF-κB. Here we report activation of the PI3-K/Akt signaling by gastrodin and that inhibition of this pathway reverses the inhibitory effects of gastrodin on NF-κB and MAPKs activation in H9c2 cardiomyocytes.

Keywords: Abbreviations; LPS; lipopolysaccharide; iNOS; inducible nitric oxide synthase; COX-2; cyclooxygenase-2; TNF-α; tumor necrosis factor-α; IL-6; interleukin-6; PI3-K; phosphatidylinositol 3-kinase; IκB-α; inhibitor κB-α; NF-κB; nuclear factor-κB; MAPK; mitogen-activated protein kinase; ERK1/2; extracellular signal regulated kinase1/2; JNK; c-Jun N-terminal protein kinase; p38 MAPK; p38 mitogen-activated protein kinaseGastrodin; Inflammatory response; NF-κB; PI3-K/Akt; H9c2 cardiomyocytes

Kurarinone regulates immune responses through regulation of the JAK/STAT and TCR-mediated signaling pathways by Byung-Hak Kim; Kwang-Min Na; Ikhoon Oh; Inn-Hye Song; Yun Sang Lee; Jongheon Shin; Tae-Yoon Kim (pp. 1134-1144).

Sophora flavescens is a medicinal herb that contains flavonoids and quinolizidine alkaloids and has a wide range of biological activities due to its anti-inflammatory, anti-bacterial and anti-cancer properties. We isolated a series of flavonoids from the roots of Sophora flavescens and examined their ability to inhibit immune responses. Among the flavonoids, kurarinone exhibited the strongest inhibitory effect on immune responses. Kurarinone suppressed the differentiation of CD4⁺ T cells by inhibiting the expression and production of T-cell lineage-specific master regulators and cytokines. Our results also demonstrated that kurarinone directly suppressed the cytokine-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling and T-cell receptor (TCR) pathways. In two established animal models of chronic inflammatory skin disease, one in which psoriasis-like skin disease was induced by an interleukin 23 (IL-23) injection into mouse ears and another in which 2,4,6-trinitrochlorobenzene (TNCB) application on the abdomens of mice was used to induce contact dermatitis, kurarinone repressed disease development by inhibiting the expression of pro-inflammatory mediators, including cytokines, chemokines and enzyme in murine ear skin. This study provides new evidence that kurarinone may ameliorate chronic inflammatory skin diseases through the suppression of pathogenic CD4⁺ T-cell differentiation and the overall immune response.

Keywords: Kurarinone; CD4; ⁺; T cell; JAK/STAT; T-cell receptor; Immune response; Autoimmune disease

Carbon monoxide protects against ovariectomy-induced bone loss by inhibiting osteoclastogenesis by Tien Van Phan; Ok-Joo Sul; Ke Ke; Mi-Hyun Lee; Woon-Ki Kim; Yeon-Soo Cho; Hyun-Ju Kim; Shin-Yoon Kim; Hun-Taeg Chung; Hye-Seon Choi (pp. 1145-1152).

Carbon monoxide (CO) has been shown to have remarkable therapeutic value at low dosage by suppressing inflammation via inhibitory effects on macrophages, which are also precursors of osteoclasts (OC). The objective of the present study was to determine whether CO limits bone loss through its effects on osteoclastogenesis. Intraperitoneal injection of CO-releasing molecule 2 (CORM2) into mice with reduced bone mass due to ovariectomy (OVX) resulted in significantly elevated bone mass. Increased serum levels of collagen-type I fragments, tartrate-resistant acid phosphatase 5b, and reactive oxygen species (ROS) due to OVX were also decreased when treated with CORM2. In vitro, CORM2 inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced OC formation without affecting bone resorption. CORM2 reduced long-lasting ROS levels and nuclear factor-κB (NF-κB) activation in response to RANKL. Inhibition of NADPH oxidase partially reduced the inhibitory effect of CO. CO induced increase of peroxiredoxin 1 (PRX1) in BMM. Down-regulation of PRX1 reduced the inhibitory effect of CO on OC formation and sustained the ROS levels induced by RANKL, suggesting that CO reduces generation of ROS and scavenges ROS to inhibit osteoclastogenesis. These data suggest that the inhibitory effect of CO on osteoclastogenesis is caused by impaired RANKL signaling through defective NF-κB activation and reduced levels of long-lasting ROS. These changes result in decreased bone loss. Our data highlight the potential utility of CO for ameliorating bone loss induced by loss of ovarian function.

Keywords: Bone loss; Carbon monoxide; Osteoclast; Ovariectomy; RANKL

Autophagy takes place in mutated p53 neuroblastoma cells in response to hypoxia mimetic CoCl₂ by Thomas Naves; Soha Jawhari; Marie-Odile Jauberteau; Marie-Hélène Ratinaud; Mireille Verdier (pp. 1153-1161).

Solid tumors like neuroblastoma exhibit hypoxic areas, which can lead both to cell death or aggressiveness increase. Hypoxia is a known stress able to induce stabilization of p53, implicated in cell fate regulation. Recently, p53 appeared to be involved in autophagy in an opposite manner, depending on its location: when nuclear, it enhanced transcription of pro-autophagic genes whereas when cytoplasmic, it inhibited the autophagic process. Today, we used cobalt chloride, a hypoxia mimetic that inhibits proteasomal HIF-1 degradation and generates reactive oxygen species (ROS). We focused on CoCl₂-induced cell death in a DNA-binding mutated p53 neuroblastoma cell line ( SKNBE(2c)). An autophagic signaling was evidenced by an increase of Beclin-1, ATG 5–12, and LC3-II expression whereas the p53^mut presence decreased with CoCl₂ time exposure. Activation of the pathway seemed to protect cells from ROS production and, at least in part, from death. The autophagic inhibitors activated the apoptotic signaling and the death was enhanced. To delineate the eventual implication of the p53^mut in the autophagic process in response to hypoxia, we monitored signaling in p53^WT SHSY5Y cells, after either shRNA-p53 down-regulation or transcriptional activity inhibition by pifithrin alpha. We did not detect autophagy neither with p53^wt nor when p53 was lacking whereas such a response was effective with a mutated or inactivated p53. To conclude, mutated p53 in neuroblastoma cells could be linked with the switch between apoptotic response and cell death by autophagy in response to hypoxic mimetic stress.

Keywords: Autophagy; Mutated p53; CoCl; ₂; Neuroblastoma; Hypoxia; Cell death

Signaling of an allosteric, nanomolar potent, low molecular weight agonist for the follicle-stimulating hormone receptor by Chris J. van Koppen; Pieter M. Verbost; Ruud van de Lagemaat; Willem-Jan F. Karstens; Huub J.J. Loozen; Tanja A.E. van Achterberg; Monique G.A. van Amstel; Jolanda H.G.M. Brands; Els J.P. van Doornmalen; Jesse Wat; Saskia J. Mulder; Bianca C. Raafs; Saskia Verkaik; Rob G.J.M. Hanssen; C. Marco Timmers (pp. 1162-1170).

Follicle-stimulating hormone (FSH) activates FSH receptors (FSHR) in granulosa cells to induce follicle differentiation, growth and estradiol production. FSH is used clinically to treat female infertility and is administered by injection. To increase patient convenience and compliance, compound homogeneity and composition, low molecular weight (LMW), orally bioavailable, FSHR agonists are now being developed to replace FSH. In this study, we present the signaling mechanisms of a newly developed LMW dihydropyridine agonist of the FSHR, Org 214444-0. Org 214444-0 is shown to be a stereoselective, nanomolar potent FSHR agonist and selective over the structurally related LHR and TSHR. Org 214444-0 is an allosteric agonist interacting with the transmembrane region of the FSHR. When co-incubated with FSH, Org 214444-0 augments FSH's potency in binding (6.5-fold) and adenylyl cyclase/cAMP activation (3.5-fold) in a concentration-dependent manner. Like FSH, Org 214444-0 induces FSHR internalization and is only marginally effective in stimulating phospholipase C. Moreover, Org 214444-0 stimulates cAMP and estradiol production in human granulosa cells in culture and supports the follicular phase in mature female rats. We conclude that Org 214444-0 is a bonafide FSHR agonist.

Keywords: Abbreviations; CRE; cyclic AMP-responsive element; E2; estradiol; FSH; follicle-stimulating hormone; FSHR; follicle-stimulating hormone receptor; GPCR; G protein-coupled receptor; G; _s; stimulatory G protein; LH; luteinizing hormone; LHR; luteinizing hormone receptor; TSH; thyroid-stimulating hormone; TSHR; thyroid-stimulating hormone receptorFSH receptor; Ovary; Infertility; Allosterism; Agonist

Characterization by flow cytometry of fluorescent, selective agonist probes of the A₃ adenosine receptor by Eszter Kozma; Elizabeth T. Gizewski; Dilip K. Tosh; Lucia Squarcialupi; John A. Auchampach; Kenneth A. Jacobson (pp. 1171-1181).

Various fluorescent nucleoside agonists of the A₃ adenosine receptor (AR) were compared as high affinity probes using radioligands and flow cytometry (FCM). They contained a fluorophore linked through the C2 or N⁶ position and rigid A₃AR-enhancing (N)-methanocarba modification. A hydrophobic C2-(1-pyrenyl) derivative MRS5704 bound nonselectively. C2-Tethered cyanine5-dye labeled MRS5218 bound selectively to hA₃AR expressed in whole CHO cells and membranes. By FCM, binding was A₃AR-mediated (blocked by A₃AR antagonist, at least half through internalization), with t_1/2 for association 38min in mA₃AR-HEK293 cells; 26.4min in sucrose-treated hA₃AR-CHO cells ( K_d 31nM). Membrane binding indicated moderate mA₃AR affinity, but not selectivity. Specific accumulation of fluorescence (50nM MRS5218) occurred in cells expressing mA₃AR, but not other mouse ARs. Evidence was provided suggesting that MRS5218 detects endogenous expression of the A₃AR in the human promyelocytic leukemic HL-60 cell line. Therefore, MRS5218 promises to be a useful tool for characterizing the A₃AR.

Keywords: Abbreviations; cAMP; 3′,5′-cyclic adenosine monophosphate; CHO; Chinese hamster ovary; Cl-IB-MECA; 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9; H; -purin-9-yl]-1-deoxy-; N; -methyl-β-; d; -ribofuranuronamide; DMEM; Dulbecco's Modified Eagle Medium; DMSO; dimethyl sulfoxide; FBS; fetal bovine serum; PBS; phosphate buffered saline; FCM; flow cytometry; GPCR; G protein-coupled receptor; HEK; human embryonic kidney; [; ¹²⁵; I]I-AB-MECA; [; ¹²⁵; I]4-amino-3-iodobenzyl-5′-; N; -methylcarboxamidoadenosine; IB-MECA; 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9; H; -purin-9-yl]-; N; -methyl-β-; d; -ribofuranuronamide; MESF; molecules of equivalent soluble fluorochrome; MFI; measured fluorescent intensity; MRS1220; N; -[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-; c; ]quinazolin-5-yl]benzene acetamide; MRS5218; (1′S,2′R,3′S,4′S,5′S)-4′-[6-(3-chlorobenzylamino)-2-(N-cyanine(β-aminoethylaminocarbonyl)-1-butynyl)-9-yl]-2′,3′-dihydroxybicyclo[3.1.0]hexane-1′-carboxylic acid; N; -methylamide; MRS5449; 2-(6-amino-3-iminio-4,5-disulfonato-3; H; -xanthen-9-yl)-5-((6-(4-(4-((9-chloro-2-(furan-2-yl)-[1,2,4]triazolo[1,5-c]quinazolin-5-yl)amino)-4-oxobutyl)-1; H; -1,2,3-triazol-1-yl)hexyl)carbamoyl)benzoate; MRS5704; (1S,2R,3S,4R,5S)-4-(6-(3-chlorobenzylamino)-2-(pyren-1-ylethynyl)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-carboxamide; NECA; 5′-; N; -ethylcarboxamidoadenosine; XAC; xanthine amine congener,; N; -(2-aminoethyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1; H; -purin-8-yl)phenoxy]-acetamide hydrochloridePurines; Fluorescence; G protein-coupled receptor; A; ₃; adenosine receptor; Flow cytometry

Characterization of marmoset CYP2B6: cDNA cloning, protein expression and enzymatic functions by Kei Mayumi; Nobumitsu Hanioka; Kazufumi Masuda; Akiko Koeda; Shinsaku Naito; Atsuro Miyata; Shizuo Narimatsu (pp. 1182-1194).

The common marmoset is a promising species for evaluating the safety of drug candidates. To further understand the capacity for drug metabolism in marmosets, a cDNA encoding a CYP2B enzyme was cloned from the total RNA fraction of marmoset liver by 3′- and 5′-RACE methods. Nucleotide and deduced amino acid sequences showed 90.8 and 86.2% identity, respectively, with human CYP2B6. The marmoset CYP2B6 (marCYP2B6) protein was expressed in insect cells, and its enzymatic properties were compared with those of human (humCYP2B6) and cynomolgus monkey (cynCYP2B6) orthologs in liver and insect cell microsomes. Enzymatic functions were examined for the oxidation of 7-ethoxy-4-(trifluoromethyl)coumarin (7-ETC), bupropion (BUP) and efavirenz (EFV). The kinetic profiles for the oxidation of the three substrates by liver microsomal fractions were similar between humans and cynomolgus monkeys (biphasic for 7-ETC and monophasic for BUP and EFV), but that of marmosets was unique (monophasic for 7-ETC and biphasic for BUP and EFV). Recombinant enzymes, humCYP2B6 and cynCYP2B6, also yielded similar kinetic profiles for the oxidation of the three substrates, whereas marCYP2B6 showed activity only for 7-ETC hydroxylation. In silico docking simulations suggested that two amino acid residues, Val-114 and Leu-367, affect the activity of marCYP2B6. In fact, a marCYP2B6 mutant with substitutions V114I and L367V exhibited BUP hydroxylase activity that was 4-fold higher than that of humCYP2B6, while its EFV 8-hydroxylase activity was only 10% that of the human enzyme. These results indicate that the amino acids at positions 114 and 367 affect the enzymatic capacity of marmoset CYP2B6.

Keywords: Abbreviations; CYP; cytochrome P450; marCYP2B6; marmoset CYP2B6; humCYP2B6; human CYP2B6; cynCYP2B6; cynomolgus monkey CYP2B6; 7-ETC; 7-ethoxy-4-(trifluoromethyl)coumarin; BUP; bupropion; EFV; efavirenz; RACE; rapid amplification of cDNA ends; ss-cDNA; single strand cDNA; fp; ₂; NADPH-cytochrome P450 reductaseMarmoset CYP2B6; cDNA cloning; Bupropion; Efavirenz; Docking simulation

Comparative in vitro metabolism of phospho-tyrosol-indomethacin by mice, rats and humans by Gang Xie; Dingying Zhou; Ka-Wing Cheng; Chi C. Wong; Basil Rigas (pp. 1195-1202).

Phospho-tyrosol-indomethacin (PTI; MPI 621), a novel anti-cancer agent, is more potent and safer than conventional indomethacin. Here, we show that PTI was extensively metabolized in vitro and in vivo. PTI was rapidly hydrolyzed by carboxylesterases to generate indomethacin as its major metabolite in the liver microsomes and rats. PTI additionally undergoes cytochromes P450 (CYP)-mediated hydroxylation at its tyrosol moiety and O-demethylation at its indomethacin moiety. Of the five major human CYPs, CYP3A4 and CYP2D6 catalyze the hydroxylation and O-demethylation reactions of PTI, respectively; whereas CYP1A2, 2C9 and 2C19 are inactive towards PTI. In contrast to PTI, indomethacin is primarily O-demethylated by CYP2C9, which prefers acidic substrates. The hydrolyzed and O-demethylated metabolites of PTI are further glucuronidated and sulfated, facilitating drug elimination and detoxification. We observed substantial inter-species differences in the metabolic rates of PTI. Among the liver microsomes from various species, PTI was the most rapidly hydrolyzed, hydroxylated and O-demethylated in mouse, human and rat liver microsomes, respectively. These results reflect the differential expression patterns of carboxylesterase and CYP isoforms among these species. Of the human microsomes from various tissues, PTI underwent more rapid carboxylesterase- and CYP-catalyzed reactions in liver and intestine microsomes than in kidney and lung microsomes. Together, our results establish the metabolic pathways of PTI, reveal significant inter-species differences in its metabolism, and provide insights into the underlying biochemical mechanisms.

Keywords: Abbreviations; CES; carboxylesterase; CYP; cytochrome P450; DFP; diisopropyl fluorophosphates; HLMs; human liver microsomes; IND; indomethacin; PTI; phospho-tyrosol-indomethacin; UGT; UDP-glucuronosyltransferasesPhospho-tyrosol-indomethacin; Cytochrome P450; Liver microsomes; Glucuronidation

P300 regulates the human RLIP76 promoter activity and gene expression by Archana Sehrawat; Sushma Yadav; Yogesh C. Awasthi; Alakananda Basu; Charles Warden; Sanjay Awasthi (pp. 1203-1211).

A 76-kDa Ral-interacting protein (RLIP76) has been implicated in the pathogenesis of cancer and diabetes. It is often over expressed in human malignant cell lines and human tumor samples and has been associated with metastasis and chemoresistance. RLIP76 homozygous knockout mice exhibit increased insulin sensitivity, hypoglycemia, and hypolipidemia, and resist cancer development. Little is known about the mechanism by which the expression of RLIP76 is regulated. In the present study, we functionally characterized the RLIP76 promoter using deletion mapping and mutational analysis to investigate the regulation of RLIP76 transcription. We have identified the promoter regions important for RLIP76 transcription, including a strong cis-activating element in the proximal promoter containing overlapping consensus cMYB and cETS binding sites. Transcription factor cMYB and the coactivator p300 associated with RLIP76 gene promoter as shown by CHIP assay. Knockdown of p300 in HEK293 cells reduced the activity of the promoter fragment containing wild type cMYB/cETS binding site in comparison to that with deleted or mutated cMYB/cETS binding site. Knockdown of p300 also decreased the RLIP76 expression as indicated by immunoblotting, immunocytochemistry and flow cytometry analysis. Thus, we report for the first time that p300 associates with the RLIP76 promoter via an overlapping cMYB and cETS binding site and regulates RLIP76 promoter activity and its expression.

Keywords: RLIP76; Transcription; Promoter activity; p300; cMYB; cETSAbbreviations; RalBP1; Ral-binding protein-1; RLIP76; 76; KDa Ral-interacting protein; p300; E1A binding protein; TFBS; transcription factor binding site; DNP-SG; dinitrophenyl glutathione; CHIP; chromatin immunoprecipitation; LRF; leukemia-related factor; TTF1; thyroid transcription factor-1; EMSA; electrophoretic mobility shift assay

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Biochemical Pharmacology (v.85, #8)

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Biochemical Pharmacology (v.85, #8)