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Biochemical Pharmacology (v.82, #11)

Editorial Advisory Board (pp. iii).

What we have learned from crystal structures of proteins to receptor function by J.-L. Reymond; Ruud van Deursen; D. Bertrand (pp. 1521-1527).

The activity of ligand gated channels is crucial for proper brain function and dysfunction of a single receptor subtype have led to neurological impairments ranging from benign to major diseases such as epilepsy, startle diseases, etc. Molecular biology and crystallography allowed the characterization at the atomic scale of the first four transmembrane ligand gated channels and of proteins sharing a high degree of homology with the neurotransmitter-binding domain.Gaining an adequate knowledge of the structural features of the ligand binding pocket led to the possibilities of developing virtual screening based approaches and probing in silico the docking of very large numbers of molecules. Development of new computing tools further extended such possibilities and rendered possible the screening of the chemical universe database GDB-11, which contains all possible organic molecules up to 11 atoms of C, N, O and F. In the case of the nicotinic acetylcholine receptors molecules identified using such screening methods were synthesized and characterized in binding assays and their pose determined in crystal structure with the acetylcholine binding protein. However, in spite of these thorough approaches, functional studies revealed that these molecules had a greater affinity for the pore domain of the channel and acted as open channel blocker rather than binding site antagonist.In this work, we discuss the potential and current limitations of how progresses made with the crystal structures of ligand gated channels, or ligand binding proteins, can be used in combination with virtual screening and functional assays, to identify novel compounds.

Keywords: Abbreviations; P; ₂; X; ligand gated purinergic receptors; GluR; glutamate receptors comprising AMPA, kainate and NMDA receptors; nAChR; nicotinic acetylcholine receptors; GABA; _A; γ-aminobutyric acid receptors; 5HT; ₃; 5-hydroxy triptamine (serotonin) receptors; GlyR; glycine receptors; GluR-Cl; invertebrate glutamate sensitive chloride gated channels; AChBP; acetylcholine binding protein; VS; virtual screening; GOLD; genetic optimization for ligand docking; SAR; structure activity relationship; PDB; protein data bank (http://www.pdb.org/pdb/home/home.do); GDB; global data baseLigand gated channel; Structure; Function; Pharmacology

New antimicrobial agents on the horizon by Karen Bush; Michael J. Pucci (pp. 1528-1539).

Antibiotic resistance issues necessitate the continued discovery and development of new antibacterial agents. Efforts are ongoing in two approaches to find new compounds that are effective against antibiotic-resistant pathogens. These efforts involve modification of existing classes including fluoroquinolones, tetracyclines, aminoglycosides, and β-lactams and identification of inhibitors against previously unexploited antibacterial targets. Examples of both approaches are described here with emphasis on compounds in late pre-clinical or clinical stages of development.

Keywords: Antibiotic; Antibiotic resistance; Drug discovery; Clinical trial; Bacterial target

Intracellular ATP levels determine cell death fate of cancer cells exposed to both standard and redox chemotherapeutic agents by Julien Verrax; Nicolas Dejeans; Brice Sid; Christophe Glorieux; Pedro Buc Calderon (pp. 1540-1548).

Cancer cells generally exhibit high levels of reactive oxygen species (ROS) that stimulate cell proliferation and promote genetic instability. Since this biochemical difference between normal and cancer cells represents a specific vulnerability that can be selectively targeted for cancer therapy, various ROS-generating agents are currently in clinical trials, either as single agents or in combination with standard therapy. However, little is known about the potential consequences of an increased oxidative stress for the efficacy of standard chemotherapeutic agents. In this context, we have assessed the influence of an oxidative stress generated by the combination of ascorbate and the redox-active quinone menadione on the capacity of melphalan, a common alkylating agent, to induce apoptosis in a chronic myelogenous leukemia cell line. Our data show that oxidative stress did not inhibit but rather promoted cancer cell killing by melphalan. Interestingly, we observed that, in the presence of oxidative stress, the type of cell death shifted from a caspase-3 dependent apoptosis to necrosis because of an ATP depletion which prevented caspase activation. Taken together, these data suggest that ROS-generating agents could be useful in combination with standard chemotherapy, even if all the molecular consequences of such an addition remain to be determined.

Keywords: Abbreviations; ROS; reactive oxygen species; NAC; N-acetyl-cysteine; CAT; catalase; LDH; lactate dehydrogenase; ANOVA; analysis of variance; PARP; poly(ADP-ribose) polymerase; AICAR; 5-aminoimidazole-4-carboxamide-1-β-; d; -ribofuranosideAscorbate; Cancer; Chemotherapy; Menadione; Oxidative stress

Biochemical disorders associated with antiproliferative effect of dehydroepiandrosterone in hepatoma cells as revealed by LC-based metabolomics by Mei-Ling Cheng; Ming-Shi Shiao; Daniel Tsun-Yee Chiu; Shiue-Fen Weng; Hsiang-Yu Tang; Hung-Yao Ho (pp. 1549-1561).

DHEA is known to have chemopreventive and antiproliferative activities, and was initially thought to be mediated by inhibition of G6PD. Our previous study has shown that DHEA may act through interference with energy metabolism. To study the effect of pharmacological dose of DHEA on cellular metabolism, and to further delineate the mechanism underlying its antiproliferative effect, we applied a metabolomic approach to globally profile the changes in metabolites in SK-Hep1 cells underexpressing G6PD (Sk-Gi) and control cells (Sk-Sc) after DHEA treatment. RRLC-TOF-MS was used to identify metabolites, and tandem mass spectrometry was used to confirm their identity. DHEA induced changes in glutathione metabolism, lipid metabolism, s-adenosylmethionine (SAM) metabolism, as well as lysine metabolism. Elevation in level of glutathione disulfide, together with a concomitant decrease in level of reduced glutathione, was indicative of increased oxidative stress. Depletion of carnitine and its acyl derivatives reflected decline in fatty acid catabolism. These changes were associated with mitochondrial malfunction and reduction in cellular ATP content. Cardiolipin (CL) and phosphatidylcholine (PC) levels decreased significantly, suggesting that alterations in lipid composition are causally related to decline in mitochondrial function after DHEA treatment. The decline in cellular SAM content was accompanied by decreased expression of methionine adenosyltransferase genes MAT2A and MAT2B. SAM supplementation partially rescued cells from DHEA-induced growth stagnation. Our findings suggest that DHEA causes perturbation of multiple pathways in cellular metabolism. Decreased SAM production, and cardiolipin depletion and the resulting mitochondrial dysfunction underlie the antiproliferative effect of DHEA.

Keywords: Abbreviations; BPC; base peak chromatogram; DHEA; dehydroepiandrosterone; ESI; electrospray ionization; G6PD; glucose 6-phosphate dehydrogenase; GSH; glutathione; GSSG; glutathione disulfide; H; ₂; DCF-DA; 2′; ^*; 7′-dihydrodichlorofluorescein diacetate; PCA; principal component analysis; ROS; reactive oxygen species; RRLC-TOF-MS; rapid resolution liquid chromatography coupled with electrospray time-of-flight mass spectrometry; SAM; s; -adenosylmethionine; Sk-Gi; SK-Hep1 cells expressing G6PD shRNA; Sk-Sc; SK-Hep1 cells expressing scrambled control shRNAMetabolomics; Dehydroepiandrosterone; Mitochondria; s; -Adenosylmethionine; Oxidative stress

In vitro and in vivo modulation of ABCG2 by functionalized aurones and structurally related analogs by Hong-May Sim; Chung-Pu Wu; Suresh V. Ambudkar; Mei-Lin Go (pp. 1562-1571).

Over-expression of ABCG2 is linked to multidrug resistance in cancer chemotherapy. We have previously shown that functionalized aurones effectively reduced the efflux of pheophorbide A (an ABCG2 substrate) from ABCG2 over-expressing MDA-MB-231/R (“R”) cells. In the present report, we investigated the functional relevance of this observation and the mechanisms by which it occurs. Aurones and related analogs were investigated for re-sensitization of R cells to mitoxantrone (MX, a chemotherapeutic substrate of ABCG2) in cell-based assays, accumulation of intracellular MX by cell cytometry, interaction with ABCG2 by biochemical assays and in vivo efficacy in MX resistant nude mice xenografts. We found that methoxylated aurones interacted directly with ABCG2 to inhibit efflux activity, possibly by competing for occupancy of one of the substrate binding sites on ABCG2. The present evidence suggests that they are not transported by ABCG2 although they stimulate ABCG2-ATPase activity. Alteration of ABCG2 protein expression was also discounted. One member was found to re-sensitize R cells to MX in both in vitro and in vivo settings. Our study identified methoxylated aurones as promising compounds associated with low toxicities and potent modulatory effects on the ABCG2 efflux protein. Thus, they warrant further scrutiny as lead templates for development as reversal agents of multidrug resistance.

Keywords: ABCG2; ABC transporter; Multidrug resistance; Functionalized aurones; Reversing agents

Underexpression of miR-224 in methotrexate resistant human colon cancer cells by Núria Mencia; Elisabet Selga; Véronique Noé; Carlos J. Ciudad (pp. 1572-1582).

MicroRNAs (miRNAs) are small non-coding RNAs involved in RNA silencing that play a role in many biological processes. They are involved in the development of many diseases, including cancer. Extensive experimental data show that they play a role in the pathogenesis of cancer as well as the development of drug resistance during treatments. The aim of this work was to detect differentially expressed miRNAs in MTX-resistant cells. Thus, miRNA microarrays of sensitive and MTX-resistant HT29 colon cancer cells were performed. The results were analyzed using the GeneSpring GX11.5 software. Differentially expressed miRNAs in resistant cells were identified and miR-224, which was one of the most differentially expressed miRNAs and with high raw signal values, was selected for further studies. The underexpression of miR-224 was also observed in CaCo-2 and K562 cells resistant to MTX. Putative targets were predicted using TargetScan 5.1 software and integrated with the data from expression microarrays previously performed. This approach allowed us to identify miR-224 targets that were differentially expressed more than 2-fold in resistant cells. Among them CDS2, DCP2, HSPC159, MYST3 and SLC4A4 were validated at the mRNA level by qRT-PCR. Functional assays using an anti-miR against miR-224 desensitized the cells towards MTX, mimicking the resistant phenotype. On the other hand, siRNA treatment against SLC4A4 or incubation of Poly Purine Reverse Hoogsteen (PPRH) hairpins against CDS2 or HSPC159 increased sensitivity to MTX. These results revealed a role for miR-224 and its targets in MTX resistance in HT29 colon cancer cells.

Keywords: Abbreviations; MTX; methotrexate; DHFR; dihydrofolate reductase; miR and miRNA; microRNA; CRC; colorectal carcinoma; PDAC; pancreatic ductal adenocarcinoma; APRT; adenine phosphoribosyltransferase; CDS2; CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) 2; DCP2; DCP2 decapping enzyme homolog; HSPC159; galectin-related protein; MYST3; MYST histone acetyltransferase (monocytic leukemia) 3; SLC4A4; Na/bicarbonate cotransporter 1; ENO2; enolase 2; CLU; clusterin; PDCD4; programmed cell death 4; S100A4; S100 calcium binding protein A4; TOP1; topoisomerase1; TERT; telomerase reverse transcriptase; XRCC4; X-ray repair complementing defective repair in Chinese hamster cells 4; HAPLN1; hyaluronan and proteoglycan link protein 1Methotrexate; miR-224; Drug resistance; DHFR

The tyrphostin AG1478 inhibits proliferation and induces death of liver tumor cells through EGF receptor-dependent and independent mechanisms by Laia Caja; Patricia Sancho; Esther Bertran; Conrad Ortiz; Jean S. Campbell; Nelson Fausto; Isabel Fabregat (pp. 1583-1592).

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related death. Different signaling pathways are de-regulated in this pathogenesis, among them the epidermal growth factor receptor one (EGFR/Erb1). Here we show that blockage of this pathway by the tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) in different liver tumor cell lines promotes both inhibition of cell proliferation and induction of cell death, which are coincident with arrest in the G1 phase of the cell cycle, caspase-3 activation and DNA fragmentation. AG1478 up-regulates the expression of the pro-apoptotic member of the BCL-2 family BIM and down-regulates the expression of the anti-apoptotic BCL-XL and MCL1. Furthermore, it also decreases the levels of the caspase inhibitors HIAP2 and XIAP. The treatment of HCC cells with AG1478 enhanced the apoptosis induced by other pro-apoptotic stimuli, such as the physiological cytokine, TGF-β, highly expressed in liver tumors, or the chemotherapeutic drug doxorubicin. The effects observed by AG1478 were broader than the ones seen by silencing of the EGFR with siRNA, which indicates that this drug might act on other targets different from the EGFR. In this same line of evidence, AG1478 retained some cytotoxic effects in cells where EGFR has been targeted knock-down with shRNA. Interestingly, AG1478 preferentially acts on liver tumor cells, being untransformed cells much less responsive to its cytotoxic effects. In conclusion, AG1478 could be a potential therapeutic drug to be used in HCC.

Keywords: Abbreviations; TGF-β; transforming growth factor-beta; EGFR; epidermal growth factor receptor; HCC; hepatocellular carcinoma; TGF-α; transforming growth factor-alpha; HB-EGF; heparin binding-epidermal growth factor-like growth factor; AR; amphiregulin; HIAP; human inhibitors of apoptosis protein; TACE; TNF-alpha converting enzyme; HFH; human fetal hepatocytes; HBV; hepatitis B virus; HCV; hepatitis C virusHCC; Hepatocytes; TGF-beta; Doxorubicin; Apoptosis; Liver

H1, a derivative of Tetrandrine, exerts anti-MDR activity by initiating intrinsic apoptosis pathway and inhibiting the activation of Erk1/2 and Akt1/2 by Ning Wei; Geng-Tao Liu; Xiao-Guang Chen; Qian Liu; Feng-Peng Wang; Hua Sun (pp. 1593-1603).

Currently, multi-drug resistance (MDR) to anticancer drugs is a major obstacle to successful treatment of cancer. Looking for novel compounds with anti-MDR activity is an effectively way to overcome cancer drug resistance. Here, we found that H1, a novel derivate of Tetrandrine, displayed anti-MDR activity in vitro and in vivo. Average resistant factor of H1 is only 1.6. In KB and KBv200 cancer cells xenograft mice, H1 also displayed favorable anti-MDR activity. It could induce typical apoptosis as indicated by morphologic changes, DNA fragmentation in sensitive and resistant cancer cells. Further studies showed that H1 treatment resulted in the increase of ROS generation, elevation of the Bax/Bcl-2 ratio, loss of mitochondrial transmembrane potential (Δ Ψ_m), release of cytochrome c and AIF from mitochondria into cytosol, and activation of caspase-9 and caspase-3, but had no effect on activation of caspase-8 and the expression of Fas/FasL. On the other hand, H1 also inhibited survival pathways such as the activation of Erk1/2 and Akt1/2. In conclusion, H1 exerts good anti-MDR activity in vitro and in vivo, its mechanisms may be associated with initiating intrinsic apoptosis pathway and inhibiting the activation of Erk1/2 and Akt1/2. These findings further support the potential of H1 to be used in clinical trial of MDR cancer treatment.

Keywords: Drug resistance; Tetrandrine; Anti-cancer; Apoptosis; Survival pathway

M2, a novel anthracenedione, elicits a potent DNA damage response that can be subverted through checkpoint kinase inhibition to generate mitotic catastrophe by Benny J. Evison; Mile Pastuovic; Rebecca A. Bilardi; Robert A. Forrest; Paul P. Pumuye; Brad E. Sleebs; Keith G. Watson; Don R. Phillips; Suzanne M. Cutts (pp. 1604-1618).

Pixantrone is a promising anti-cancer aza-anthracenedione that has prompted the development of new anthracenediones incorporating symmetrical side-chains of increasing length varying from two to five methylene units in each pair of drug side-chains. A striking relationship has emerged in which anthracenedione-induced growth inhibition and apoptosis was inversely associated with side-chain length, a relationship that was attributable to a differential ability to stabilise the topoisomerase II (TOP2) cleavage complex. Processing of the complex to a DNA double strand break (DSB) flanked by γH2AX in nuclear foci is likely to occur, as the generation of the primary lesion was antecedent to γH2AX induction. M2, bearing the shortest pair of side-chains, induced TOP2-mediated DSBs efficiently and activated cell cycle checkpoints via Chk1 and Chk2 phosphorylation, implicating the involvement of ATM and ATR, and induced a protracted S phase and subsequent G2/M arrest. The inactive analogue M5, containing the longest pair of side-chains, only weakly stimulated any of these responses, suggesting that efficient stabilisation of the TOP2 cleavage complex was crucial for eliciting a strong DNA damage response (DDR). An M2 induced DDR in p53-defective MDA-MB-231 cells was abrogated by UCN-01, a ubiquitous inhibitor of kinases including Chk1, in a response associated with substantial mitotic catastrophe and strong synergy. The rational selection of checkpoint kinase inhibitors may significantly enhance the therapeutic benefit of anthracenediones that efficiently stabilise the TOP2 cleavage complex.

Keywords: Abbreviations; AN-9; pivaloyloxymethyl butyrate; ATM; ataxia telangiectasia mutated; ATR; ataxia telangiectasia mutated-related; CC; closed; Chk1; checkpoint kinase 1; Chk2; checkpoint kinase 2; CI; combination index; DDR; DNA damage response; DNA-PK; DNA protein kinase; DSB; double strand break; γH2AX; phosphorylated H2AX; IC; ₅₀; 50% inhibitory concentration; K; _app; apparent binding constant; kDNA; kinetoplast DNA; MTT; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; MX; mitoxantrone; NAC; N-acetyl-cysteine; NC; decatenated nicked; OTM; olive tail moment; PIX; pixantrone; PMSF; phenylmethylsulfonyl fluoride; ROS; reactive oxygen species; Sur; suramin; TOP2; topoisomerase IITopoisomerase II; Anthracenedione; Double strand break; Checkpoint kinase; Mitotic catastrophe

Increased apoptotic efficacy of lonidamine plus arsenic trioxide combination in human leukemia cells. Reactive oxygen species generation and defensive protein kinase (MEK/ERK, Akt/mTOR) modulation by Eva Calviño; María Cristina Estañ; Gloria P. Simón; Pilar Sancho; María del Carmen Boyano-Adánez; Elena de Blas; Jacqueline Bréard; Patricio Aller (pp. 1619-1629).

Lonidamine is a safe, clinically useful anti-tumor drug, but its efficacy is generally low when used in monotherapy. We here demonstrate that lonidamine efficaciously cooperates with the anti-leukemic agent arsenic trioxide (ATO, Trisenox™) to induce apoptosis in HL-60 and other human leukemia cell lines, with low toxicity in non-tumor peripheral blood lymphocytes. Apoptosis induction by lonidamine/ATO involves mitochondrial dysfunction, as indicated by early mitochondrial permeability transition pore opening and late mitochondrial transmembrane potential dissipation, as well as activation of the intrinsic apoptotic pathway, as indicated by Bcl-X_L and Mcl-1 down-regulation, Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release to the cytosol, XIAP down-regulation, and caspase-9 and -3 cleavage/activation, with secondary (Bcl-2-inhibitable) activation of the caspase-8/Bid axis. Lonidamine stimulates reactive oxygen species production, and lonidamine/ATO toxicity is attenuated by antioxidants. Lonidamine/ATO stimulates JNK phosphorylation/activation, and apoptosis is attenuated by the JNK inhibitor SP600125. In addition, lonidamine elicits ERK and Akt/mTOR pathway activation, as indicated by increased ERK, Akt, p70S6K and rpS6 phosphorylation, and these effects are reduced by co-treatment with ATO. Importantly, co-treatment with MEK/ERK inhibitor (U0126) and PI3K/Akt (LY294002) or mTOR (rapamycin) inhibitors, instead of ATO, also potentiates lonidamine-provoked apoptosis. These results indicate that: (i) lonidamine efficacy is restrained by drug-provoked activation of MEK/ERK and Akt/mTOR defensive pathways, which therefore represent potential therapeutic targets. (ii) Co-treatment with ATO efficaciously potentiates lonidamine toxicity via defensive pathway inhibition and JNK activation. And (iii) conversely, the pro-oxidant action of lonidamine potentiates the apoptotic efficacy of ATO as an anti-leukemic agent.

Keywords: Abbreviations; Akt/mTOR; protein kinase B/mammalian target of rapamycin; ANT; adenine nucleotide translocator; AML; acute myeloid leukemia; APL; acute promyelocytic leukemia; ATO; arsenic trioxide; CsA; cyclosporin A; DAPI; 4,6-diamino-2-phenylindole; DHE; dihydroethidium; DHR; dihydrorhodamine-123; H; ₂; DCFDA; dichlorodihydrofluorescein diacetate; IL2; interleukin-2; IMP; inner mitochondrial membrane permeabilization; JNK; c-Jun NH; ₂; -terminal kinase; LY294002; 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; mAb; monoclonal antibody; MAPK; mitogen-activated protein kinase; MEK/ERK; mitogen-induced extracellular kinase/extracellular signal-regulated kinase; mPTP; mitochondrial transition pore; MTT; 3[4,5-dimethyl-2-thiazolyl]-2,5diphenyl-2H-tetrazolium bromide; NAC; N; -acetyl-; l; -cysteine; pAb; polyclonal antibody; PBS; phosphate buffered saline; PBLs; peripheral blood lymphocytes; PEG–catalase; catalase–polyethylene glycol; PI3K; phosphatidylinositol 3-kinase; PHA; phytohemagglutinin; PI; propidium iodide; R123; rhodamine 123; ROS; reactive oxygen species; SP600125; anthra[1,9-c; d; ]pyrazol-6(2; H; )-one; U0126; 1,4-diamino-2,3-dicyano-1,4-; bis; (2-aminophenylthio)butadiene; z-VAD-fmk; Z-Val–Ala–Asp(OMe)–CH; ₂; FLonidamine; Arsenic trioxide; Apoptosis; ROS; Protein kinases

Silencing of the SNARE protein NAPA sensitizes cancer cells to cisplatin by inducing ERK1/2 signaling, synoviolin ubiquitination and p53 accumulation by Zchong-Zcho Wu; Nian-Kang Sun; Kun-Yi Chien; Chuck C.-K. Chao (pp. 1630-1640).

Working model of cisplatin stress and related signal pathways implicated in the regulation of SYVN1 degradation, impaired ERAD complex formation, accumulation of p53, and enhancement of cisplatin-induced apoptosis following NAPA knockdown.We found earlier that NAPA represents an anti-apoptotic protein that promotes resistance to cisplatin in cancer cells by inducing the degradation of the tumor suppressor p53. In the present study, we investigated the cellular mechanism underlying the degradation of p53 by NAPA. Knockdown of NAPA using short-hairpin RNA was shown to induce p53 accumulation and to sensitize HEK293 cells to cisplatin. On the other hand, this sensitization effect was not found in H1299 lung carcinoma cells which lack p53. Expression of exogenous p53 in H1299 cells was increased following knockdown of NAPA and these cells showed increased sensitivity to cisplatin-induced apoptosis. Notably, knockdown of NAPA induced the ubiquitination and degradation of the E3 ubiquitin ligase synoviolin and the accumulation of p53 in unstressed HEK293 cells. Conversely, NAPA overexpression decreased the ubiquitination and degradation of synoviolin, and reduced p53 protein level. Knockdown of NAPA disrupted the interaction between synoviolin and proteins that form the endoplasmic reticulum-associated degradation (ERAD) complex and in turn decreased the ability of this complex to ubiquitinate p53. In addition, knockdown of NAPA induced the activation of the MAPK kinases ERK, JNK and p38, but only inhibition of ERK reduced synoviolin ubiquitination and p53 accumulation. These results indicate that NAPA promotes resistance to cisplatin through synoviolin and the ERAD complex which together induce the degradation of p53 and thus prevent apoptosis. Based on these findings, we propose that the combination of cisplatin and knockdown of NAPA represents a novel and attractive strategy to eradicate p53-sensitive cancer cells.

Keywords: Abbreviations; BiP; binding immunoglobulin protein; CHX; cycloheximide; DMEM; Dulbecco's modified Eagle's medium; EDTA; ethylenediaminetetraacetic acid; ER; endoplasmic reticulum; ERAD; endoplasmic reticulum-associated degradation; ERK; extracellular signal-regulated kinases; FBS; fetal bovine serum; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; IP; immunoprecipitation; JNK; c-Jun N-terminal protein kinase; Luc; luciferase; MAPK; mitogen-activated protein kinase; NAPA; NSF attachment protein α; ORF; open-reading frame; PARP; poly-ADP ribose polymerase; PCR; polymerase chain reaction; PVDF; polyvinylidene fluoride; RT; reverse transcription; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; shRNA; short-hairpin RNA; SYVN1; synoviolin/Der3p/Hrd1p; UPR; unfolding protein responseApoptosis; Chemotherapy; Cisplatin resistance; NAPA; Synoviolin

Guggulsterone sensitizes hepatoma cells to TRAIL-induced apoptosis through the induction of CHOP-dependent DR5: Involvement of ROS-dependent ER-stress by Dong-Oh Moon; Sung-Yong Park; Yung Hyun Choi; Jong Seog Ahn; Gi-Young Kim (pp. 1641-1650).

Guggulsterone (GGS) has anti-tumor and anti-angiogenesis potential by suppressing nuclear factor-κB and STAT3 activity. Although GGS has been suggested as a potential therapeutic agent for treating various cancers, the underlying molecular mechanisms are unknown. Therefore, we investigated whether GGS sensitizes hepatocellular carcinoma cells (HCC) to apoptosis mediated by tumor necrosis factor-related apoptosis inducing ligand (TRAIL). The apoptotic mechanism induced by treatment with a GGS/TRAIL combination involved the loss of mitochondrial transmembrane potential and consequent activation of caspases. GGS also induced upregulation of the death receptor DR5 for TRAIL. The effects seemed to be associated with eIF2α and CHOP activation, which are related to the endoplasmic reticulum (ER) stress response and apoptosis. This relationship was suggested by the observation that CHOP downregulation by specific siRNA attenuated both GGS-mediated DR5 upregulation and the cytotoxicity induced by GGS/TRAIL co-treatment. Moreover, salubrinal, a specific eIF-2α phosphorylation-inducing agent, enhanced the expression of CHOP and DR5 induced by GGS and sensitized cells to GGS/TRAIL-induced apoptosis. Thus, GGS-induced eIF2α phosphorylation seems to be important for CHOP and DR5 upregulation. Furthermore, these events were accompanied by an increase in the generation of reactive oxygen species. Pretreatment with N-acetyl-l-cysteine and glutathione inhibited GGS-induced ER-stress, and CHOP and DR5 upregulation and almost completely blocked GGS/TRAIL-induced apoptosis. These results collectively indicate that DR5 induction via eIF-2α and CHOP is crucial for the marked synergistic effects induced by TRAIL and GGS. Taken together, these results indicate that a GGS/TRAIL combination could represent a novel important tool for cancer therapy.

Keywords: Abbreviations; GGS; guggulsterone; HCC; hepatocellular carcinoma cells; TRAIL; tumor necrosis factor-related apoptosis inducing ligand; ER; endoplasmic reticulum; CHOP; C/EBP homologous transcription factor; DR4; TRAIL-R1; DR5; TRAIL-R2; NF-κB; nuclear factor-κB; eIF2α; initiation factor-2α; ROS; reactive oxygen species; NAC; N; -acetyl-; l; -cysteine; GSH; glutathione; DiOC; ₆; 3,3′-dihexyloxacarbocyanine iodide; HE; hydroethidine; DCFDA; 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; siRNA; small interfering RNAGuggulsterone; TRAIL; CHOP; ER stress

A novel steroidal antiandrogen targeting wild type and mutant androgen receptors by Thomas Andrieu; Reto Bertolini; Sara E. Nichols; Raschid Setoud; Felix J. Frey; Michael E. Baker; Brigitte M. Frey (pp. 1651-1662).

The dihydrotestosterone derivative, RB346, acts as a new highly potent androgen receptor (AR) antagonist targeting wild type and mutant ARs.Prostate cancer (PCa) progression is enhanced by androgen and treatment with antiandrogens represents an alternative to castration. While patients initially respond favorably to androgen ablation therapy, most experience a relapse of the disease within 1–2 years by expressing androgen receptor (AR) mutants. Such mutations, indeed, promote unfavorable agonistic behavior from classical antagonists. Here, we have synthesized and screened 37 novel compounds derived from dihydrotestosterone (DHT), cyanolutamide and hydroxyflutamide. These derivatives were tested for their potential antagonistic activity using a luciferase reporter gene assay and binding properties were determined for wild type (WT) and mutant ARs (T877A, W741C, W741L, H874Y). In the absence and presence of antiandrogens, androgen dependent cellular proliferation and prostate specific antigen (PSA) expression were assayed in the prostate cancer cell line LNCaP by crystal violet, real time PCR and by Western blots. Also, cellular proliferation and PSA expression were assayed in 22Rv1. A novel compound RB346, derived from DHT, was found to be an antagonist for all tested AR forms, preventing DHT induced proliferation and PSA expression in LNCaP and 22Rv1 cells. RB346 displayed no agonistic activity, in contrast to the non-steroidal antiandrogen bicalutamide (Casodex^®) with unfavorable agonistic activity for W741L-AR. Additionally, RB346 has a slightly higher binding affinity for WT-AR, T877A-AR and H874Y-AR than bicalutamide. Thus, RB346 is the first potent steroidal antiandrogen with efficacy for WT and various AR mutants.

Keywords: Prostate cancer; Androgen receptor; Steroidal antiandrogen; Anti-androgen; LNCaP; 22Rv1

Lovastatin inhibits proliferation of anaplastic thyroid cancer cells through up-regulation of p27 by interfering with the Rho/ROCK-mediated pathway by Wen-Bin Zhong; Sung-Po Hsu; Pei-Yin Ho; Yu-Chih Liang; Tien-Chun Chang; Wen-Sen Lee (pp. 1663-1672).

Previously, we demonstrated that lovastatin, a HMG-CoA reductase inhibitor, induced apoptosis, differentiation, and inhibition of invasiveness of human anaplastic thyroid carcinoma cells (ATCs). Here, we further examined the effect of lovastatin on the growth of ARO cells. Lovastatin (0–20μM) concentration-dependently decreased cell number in cultured ATC and arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that lovastatin caused an increase of the protein level of p27 and cyclin-dependent kinase (CDK)4 and a decrease of the protein level of cyclin A2, cyclin D3, and phosphorylated Rb (pRb), but did not significantly change the protein levels of p21, cyclins D1 and E, and CDK2, in ARO cells. The formation of the CDK2–p27 complex was increased and the CDK2 activity was decreased in the lovastatin-treated ARO cells. Pretreatment of ARO cells with a p27, but not p21, antisense oligonucleotide prevented the lovastatin-induced G0/G1 arrest in ARO cells. The lovastatin-induced growth inhibition and translocation of RhoA and Rac1 in ARO cells were completely prevented by mevalonate and partially by geranylgeranyl pyrophosphate. Treatment of ARO cells with Y27632, an inhibitor of Rho-associated kinase, abolished the GGPP-mediated prevention of lovastatin-induced anti-proliferation and up-regulation and prolonged degradation of p27. Taken together, these data suggest that lovastatin treatment caused a reduction of Rho geranylgeranylation, which in turn increased the expression and stability of p27, and then inhibited ARO cell proliferation. These data suggest that lovastatin merits further investigation as multipotent therapy for treatment ATC.

Keywords: Anaplastic thyroid cancer; HMG-CoA reductase; Lovastatin; p27; Rho

Impact of divalent metal ions on regulation of adenylyl cyclase isoforms by forskolin analogs by Miriam Erdorf; Tung-Chung Mou; Roland Seifert (pp. 1673-1681).

We show that the interactions of diterpenes of mammalian membranous adenylyl cyclases are determined by Mg²⁺ and Mn²⁺. Moreover, we report that AC2 exhibits a unique regulation by diterpenes.Mammalian membranous adenylyl cyclases (mACs) play an important role in transmembrane signalling events in almost every cell and represent an interesting drug target. Forskolin (FS) is an invaluable research tool, activating AC isoforms 1–8. However, there is a paucity of AC isoform-selective FS analogs. Therefore, we examined the effects of FS and six FS derivatives on recombinant ACs 1, 2 and 5, representing members of different mAC families. Correlations of the pharmacological properties of the different AC isoforms revealed pronounced differences between ACs 1, 2 and 5. Additionally, potencies and efficacies of FS derivatives changed for any given AC isoform, depending on the metal ion, Mg²⁺ or Mn²⁺. The most striking effects of Mg²⁺ and Mn²⁺ on the diterpene profile were observed for AC2 where the large inhibitory effect of BODIPY-FS in the presence of Mg²⁺ was considerably reduced in the presence of Mn²⁺. Sequence alignment and docking experiments confirmed an exceptional position of AC2 compared to ACs 1 and 5 with respect to the structural environment of the catalytic core and cation-dependent diterpene effects. In conclusion, mAC isoforms 1, 2 and 5 exhibit a distinct pharmacological diterpene profile, depending on the divalent cation present. mAC crystal structures and modelling/docking studies provided an explanation for the pharmacological differences between the AC isoforms. Our study constitutes an important step towards the development of isoform-specific diterpenes exhibiting stimulatory or inhibitory effects.

Keywords: Abbreviations; mAC; mammalian membranous adenylyl cyclase; cAMP; cyclic adenosine 3′,5′-monophosphate; FS; forskolin; 1d-FS; 1-deoxy-forskolin; 9d-FS; 9-deoxy-forskolin; 7DA-FS; 7-deacetyl-forskolin; 6A7DA-FS; 6-acetyl-7-deacetyl-forskolin; DMB-FS; 7-deacetyl-7-[; O; -(; N; -methylpiperazino)-γ-butyryl]-forskolin; BODIPY-FS; boron-dipyrro-methene-forskolin; GTPγS; guanosine 5′-[γ-thio]triphosphate; RMSD; root mean square deviations; Sf9; Spodoptera frugiperdaAdenylyl cyclase; Diterpenes; Divalent cations; Forskolin; Molecular modelling

Interferon-α2b and transforming growth factor-β1 treatments on HCC cell lines: Are Wnt/β-catenin pathway and Smads signaling connected in hepatocellular carcinoma? by María Paula Ceballos; Juan Pablo Parody; María de Luján Alvarez; Paola Inés Ingaramo; Cristina Ester Carnovale; María Cristina Carrillo (pp. 1682-1691).

Wnt/β-catenin pathway is often dysregulated in hepatocellular carcinoma (HCC). Activated β-catenin accumulates in the cytosol and nucleus and forms a nuclear complex with TCF/LEF factors like TCF4. Interferon-α (IFN-α) has recently been recognized to harbor therapeutic potential in prevention and treatment of HCC. Transforming Growth Factor-β1 (TGF-β1) is a mediator of apoptosis, exerting its effects via Smads proteins. One mode of interaction between Wnt/β-catenin and TGF-β1/Smads pathways is the association of Smads with β-catenin/TCF4. In this study we analyzed the effects of IFN-α2b and TGF-β1 treatments on Wnt/β-catenin pathway, Smads proteins levels, β-catenin/TCF4/Smads interaction and proliferation and apoptotic death in HepG2/C3A and Huh7 cell lines. IFN-α2b and TGF-β1 attenuated Wnt/β-catenin signal by decreasing β-catenin and Frizzled7 receptor proteins contents and the interaction of β-catenin with TCF4. Truncated β-catenin form present in C3A cell line also diminished after treatments. Both cytokines declined Smads proteins and their interaction with TCF4. The overall cellular response to cytokines was the decrease in proliferation and increase in apoptotic death. Treatment with Wnt3a, which elevates β-catenin protein levels, also generated the increment of Smads proteins contents when comparing with untreated cells. In conclusion, IFN-α2b and TGF-β1 proved to be effective as modulators of Wnt/β-catenin pathway in HCC cell lines holding both wild-type and truncated β-catenin. Since the inhibition of β-catenin/TCF4/Smads complexes formation may have a critical role in slowing down oncogenesis, IFN-α2b and TGF-β1 could be useful as potential treatments in patients with HCC.

Keywords: β-catenin; Transforming growth factor-β1; Interferon-α; Smad proteins; Hepatocellular carcinoma

Functional analysis of pharmacogenetic variants of human organic cation/carnitine transporter 2 (hOCTN2) identified in Singaporean populations by Dorothy Su Lin Toh; Michael Murray; Kuan Pern Tan; Vishwaroop Mulay; Thomas Grewal; Edmund Jon Deoon Lee; Fanfan Zhou (pp. 1692-1699).

The human organic cation/carnitine transporter-2 (hOCTN2; SLC22A5) mediates the cellular influx of organic cations such as carnitine, which is essential for fatty acid oxidation. Primary carnitine deficiency has been associated with a wide range of hOCTN2 gene mutations. Six novel nonsynonymous single nucleotide polymorphisms in the hOCTN2 gene were identified recently in Chinese and Indian populations of Singapore. The present study evaluated the impact of these polymorphisms on hOCTN2 function and expression in HEK-293 cells. Transport function was markedly impaired in variants that encoded amino acid substitutions D122Y (<20% of wild-type control) and K302E (∼45% of wild-type) in the large extracellular loop and large intracellular loop of hOCTN2, respectively. The function of the other four variants was unimpaired (E109K, V175M, K191N and A214V). From biotinylation and immunofluorescence experiments, the expression of the D122Y and K302E-hOCTN2 variants at the plasma membrane of HEK-293 cells was decreased relative to the wild-type hOCTN2 but total cellular expression was unchanged. Transporter kinetic studies indicated a decrease in the V_max forl-carnitine influx by K302E-hOCTN2 to 49% of wild-type control, while K_m remained unchanged; kinetic evaluation of D122Y-hOCTN2 was not possible due to its low transport function. The K302E-hOCTN2 variant was also more susceptible than the wild-type transporter to inhibition by the drugs cimetidine, pyrilamine and verapamil. These findings indicate that impaired plasma membrane targeting of the D122Y and K302E-hOCTN2 variants that occur in Singaporean populations contributes to decreased carnitine influx.

Keywords: Abbreviations; GlpT; glycerol 3-phosphate transporter; hOCTN2; human organic cation/carnitine transporter 2; OPM; Orientations of Proteins in Membranes; PBS; phosphate-buffered saline; PDB; protein data bank; SNPs; single nucleotide polymorphisms; TEA; tetraethylammonium; TMD; transmembrane domainHuman organic cation/carnitine transporter 2; SLC22A5; Single nucleotide polymorphisms; Transporter function; l; -Carnitine

Emodin, a naturally occurring anthraquinone derivative, suppresses IgE-mediated anaphylactic reaction and mast cell activation by Yue Lu; Ju Hye Yang; Xian Li; Kyoung Hwangbo; Seung-Lark Hwang; Yoshitaka Taketomi; Makoto Murakami; Young-Chae Chang; Cheorl-Ho Kim; Jong-Keun Son; Hyeun Wook Chang (pp. 1700-1708).

The high-affinity receptor for IgE (FcɛRI)-mediated activation of mast cells plays an important role in allergic diseases such as asthma, allergic rhinitis and atopic dermatitis. Emodin, a naturally occurring anthraquinone derivative in oriental herbal medicines, has several beneficial pharmacologic effects, such as anti-cancer and anti-diabetic activities. However, the anti-allergic effect of emodin has not yet been investigated. To assess the anti-allergic activity of emodin, in vivo passive anaphylaxis animal model and in vitro mouse bone marrow-derived mast cells were used to investigate the mechanism of its action on mast cells. Our results showed that emodin inhibited degranulation, generation of eicosanoids (prostaglandin D₂ and leukotriene C₄), and secretion of cytokines (TNF-α and IL-6) in a dose-dependent manner in IgE/Ag-stimulated mast cells. Biochemical analysis of the FcɛRI-mediated signaling pathways demonstrated that emodin inhibited the phosphorylation of Syk and multiple downstream signaling processes including mobilization of intracellular Ca²⁺ and activation of the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and NF-κB pathways. When administered orally, emodin attenuated the mast cell-dependent passive anaphylactic reaction in IgE-sensitized mice. Thus, emodin inhibits mast cell activation and thereby the anaphylactic reaction through suppression of the receptor-proximal Syk-dependent signaling pathways. Therefore, emodin might provide a basis for development of a novel anti-allergic drug.

Keywords: Abbreviations; BMMC; bone marrow-derived mast cell; Syk; spleen tyrosine kinase; LAT; linker of activated T cells; PLCγ1; phospholipase Cγ1; cPLA; ₂; α; cytosolic phospholpase A; ₂; α; COX-2; cyclooxygenase-2; 5-LO; 5-lipoxygenase; PCA; passive cutaneous anaphylaxisEmodin; Passive anaphylactic reaction; Mast cells; IgE; Syk kinase

Discovery of a novel mechanism of steroid receptor antagonism: WAY-255348 modulates progesterone receptor cellular localization and promoter interactions by Matthew R. Yudt; Louise A. Russo; Thomas J. Berrodin; Scott A. Jelinsky; Debra Ellis; Jeff C. Cohen; Neil Cooch; Elizabeth Haglund; Raymond J. Unwalla; Andrew Fensome; Jay Wrobel; Zhiming Zhang; Sunil Nagpal; Richard C. Winneker (pp. 1709-1719).

WAY-255348 is a potent nonsteroidal progesterone receptor (PR) antagonist previously characterized in rodents and nonhuman primates. This report describes the novel mechanism by which WAY-255348 inhibits the activity of progesterone. Most PR antagonists bind to and block PR action by inducing a unique “antagonist” conformation of the PR. However, WAY-255348 lacks the bulky side chains or chemical groups that have been associated with the conformation changes of helix 12 that lead to functional antagonism. We show that WAY-255348 achieves antagonist activity by binding to and subsequently preventing progesterone-induced nuclear accumulation, phosphorylation and promoter interactions of the PR. This effect was concentration dependent, as high concentrations of WAY-255348 alone are able to induce nuclear translocation, phosphorylation and subsequent promoter interactions resulting in partial agonist activity at these concentrations. However, at lower concentrations where nuclear accumulation and phosphorylation are prevented, the progesterone-induced DNA binding is blocked along with PR-dependent gene expression. Analysis of the PR conformation induced by WAY-255348 using a limited protease digestion assay, suggested that the WAY-255348 bound PR conformation was similar to that of a progesterone agonist-bound PR and distinct from steroidal antagonist-bound PR conformations. Furthermore, the recruitment and binding of peptides derived from nuclear receptor co-activators is consistent with WAY-255348 inducing an agonist-like conformation. Taken together, these data suggest that WAY-255348 inhibits PR action through a novel molecular mechanism that is distinct from previously studied PR modulators and may be a useful tool to further understanding of PR signaling pathways. Development of therapeutic molecules with this ‘passive’ antagonism mechanism may provide distinct advantages for patients with reproductive disorders or PR positive breast cancers.

Keywords: Transcriptional regulation; Nuclear localization; Peptide profiling; Chromatin immunoprecipitation; Nonsteroidal selective progesterone receptor modulator (SPRM)

Hepatocyte growth factor suppresses the anticancer effect of irinotecan by decreasing the level of active metabolite in HepG2 cells by Manabu Okumura; Tomomi Iwakiri; Akinori Takagi; Yasutoshi Hirabara; Yohei Kawano; Kazuhiko Arimori (pp. 1720-1730).

In the liver, carboxylesterase (CES) converts irinotecan (CPT-11) to its active metabolite SN-38, which exerts anticancer effects. SN-38 is metabolized to an inactive metabolite SN-38 glucuronide by uridine 5′-diphospho-glucuronosyltransferase 1A1 (UGT1A1). Therefore, single nucleotide polymorphisms (SNPs) of the UGT1A1 gene are responsible for the severe adverse effects associated with the disruption of SN-38 metabolism. However, despite having SNPs of the UGT1A1 gene, many patients metabolize SN-38 sufficiently to avoid severe adverse effects. Among these patients, we found individuals with elevated serum concentrations of hepatocyte growth factor (HGF). The aim of this study was to evaluate whether HGF alters the metabolism of CPT-11, resulting in a reduction in the anticancer effect of CPT11. The cytotoxicity of CPT-11 and SN-38 was evaluated in HepG2 cells pretreated with HGF. Furthermore, we explored the level of expression and mechanisms of activity of CES and UGT1A1. HGF suppressed the cytotoxicity of CPT-11 by decreasing intracellular SN-38 levels that resulted from a decrease in CES2 and an increase in UGT1A1. Furthermore, this HGF-induced suppression was improved by pretreatment with an inhibitor of HGF receptor c-Met, and the improvement was synergistically potentiated by epidermal growth factor receptor (EGFR) inhibitors. Moreover, HGF induced phosphorylation of signal transducer and activator of transcription 3 and transactivated EGFR. These results suggest that HGF is a possible causative agent of acquired clinical resistance in chemotherapy with CPT-11 and could be useful as a predictor of clinical resistance. Additional treatment using c-Met and/or EGFR inhibitors could be a novel strategy to overcome resistance.

Keywords: Abbreviations; Akt; anti-apoptotic serine–threonine kinase; CES; carboxylesterase; CYP; cytochrome P450; EGF; epidermal growth factor; EGFR; epidermal growth factor receptor; Erk; extracellular signal-regulated kinase; HCC; hepatocellular carcinoma; HGF; hepatocyte growth factor; Jak; Janus kinase; NPA; p; -nitrophenylacetate; RTK; receptor tyrosine kinase; SNP; single nucleotide polymorphism; STAT3; signal transducer and activator of transcription 3; UGT; uridine 5′-diphospho-glucuronosyltransferaseHepatocyte growth factor; CPT-11; SN-38; CES; UGT1A1

Fisetin disposition and metabolism in mice: Identification of geraldol as an active metabolite by Yasmine S. Touil; Nicolas Auzeil; François Boulinguez; Hanane Saighi; Anne Regazzetti; Daniel Scherman; Guy G. Chabot (pp. 1731-1739).

Although the natural flavonoid fisetin (3,3′,4′,7-tetrahydroxyflavone) has been recently identified as an anticancer agent with antiangiogenic properties in mice, its in vivo pharmacokinetics and metabolism are presently not characterized. Our purpose was to determine the pharmacokinetics and metabolism of fisetin in mice and determine the biological activity of a detected fisetin metabolite. After fisetin administration of an efficacious dose of 223mg/kg i.p. in mice, the maximum fisetin concentration reached 2.5μg/ml at 15min and the plasma concentration declined biphasically with a rapid half-life of 0.09h and a terminal half-life of 3.1h. Three metabolites were detected, one of which was a glucuronide of fisetin (M1), whereas another glucuronide (M2) was a glucuronide of a previously unknown fisetin metabolite (M3). HPLC–MS/MS analysis indicated that M3 was a methoxylated metabolite of fisetin (MW=300Da). The UV spectrum of M3 was identical to that of fisetin and standard 3,4′,7-trihydroxy-3′-methoxyflavone (geraldol). In addition, because M3 co-eluted with standard geraldol in 4 different chromatographic ternary gradient conditions, M3 was therefore assigned to geraldol. Of interest, this metabolite was shown to achieve higher concentrations than fisetin in Lewis lung tumors. We also compared the cytotoxic and antiangiogenic activities of fisetin and geraldol in vitro and it was found that the latter was more cytotoxic than the parent compound toward tumor cells, and that it could also inhibit endothelial cells migration and proliferation. In conclusion, these results suggest that fisetin metabolism plays an important role in its in vivo anticancer activities.

Keywords: Abbreviations; bFGF; basic fibroblast growth factor; DMEM; Dulbecco's modified Eagle's medium; DMSO; dimethylsulfoxide; EAhy 926; immortalized human umbilical vein endothelial cell line; HPLC; high-performance liquid chromatography; IC; ₅₀; inhibitory concentration for 50% of cells; LLC; Lewis lung carcinoma cells; MS/MS; tandem mass spectrometry; MTT; 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium; PBS; phosphate buffered salineFlavonoid; Fisetin; Pharmacokinetics; Metabolism; Geraldol; Cytotoxicity

Valproic acid utilizes the isoleucine breakdown pathway for its complete β-oxidation by Paula B.M. Luís; Jos P. Ruiter; Rob Ofman; Lodewijk IJlst; Marco Moedas; Luísa Diogo; Paula Garcia; Isabel Tavares de Almeida; Marinus Duran; Ronald J. Wanders; Margarida F.B. Silva (pp. 1740-1746).

The mitochondrial β-oxidation of valproic acid incorporating two novel metabolizing enzymes, short-chain enoyl-CoA hydratase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase. The potential interference with isoleucine oxidative pathway is also shown.Valproic acid (VPA) is a simple branched medium-chain fatty acid with expanding therapeutic applications beyond its prime anticonvulsant properties.(1) To resolve the underlying basis for the interference of valproate with the isoleucine degradative pathway and (2) to shed new light on the enzymology of the β-oxidation pathway of valproate.Urine organic acids were analyzed by gas chromatography/mass spectrometry. In vitro studies were performed with heterologously expressed human 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) and fibroblasts from controls and a patient with MHBD deficiency using 2-methyl-3-hydroxybutyryl-CoA and 3-hydroxyvalproyl-CoA as substrates. The respective enzymatic activities were measured using optimized HPLC procedures. Short-chain enoyl-CoA hydratase (ECHS1) immunoprecipitation in a human liver homogenate was performed and hydratase activity was measured in the supernatants by HPLC, using crotonyl-CoA and Δ^2(E)-valproyl-CoA as substrates.Patients on valproate therapy had a moderately increased urinary excretion of the isoleucine metabolite 2-methyl-3-hydroxybutyric acid. MHBD was found to convert 3-hydroxyvalproyl-CoA into 3-ketovalproyl-CoA. MHBD activity in control fibroblasts was comparable using both 2-methyl-3-hydroxybutyryl-CoA and 3-hydroxyvalproyl-CoA as substrates. In fibroblasts of a patient with MHBD deficiency, there was no detectable MHBD activity when 3-hydroxyvalproyl-CoA was used as substrate. Samples with immunoprecipitated crotonase had no detectable hydratase activity using both crotonyl-CoA and Δ^2(E)-valproyl-CoA as substrates.This work demonstrates for the first time, that MHBD is the unique enzyme responsible for the dehydrogenation of 3-hydroxyvalproyl-CoA. Furthermore, we show that crotonase is the major, if not the single hydratase involved in VPA β-oxidation, next to its role in isoleucine catabolism.

Keywords: Abbreviations; VPA; 2-; n; -propylpentanoic acid or valproic acid; Δ; ^2(E); -VPA; 2-; n; -propyl-2-pentenoic acid; CoA; coenzyme A; MHBA; 2-methyl-3-hydroxybutyric acid; 3-OHVP-CoA; 3-hydroxyvalproyl-CoA; 2-Me-3-OHBut-CoA; 2-methyl-3-hydroxybutyryl-CoA; MHBD; 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.178); ECHS1, s; hort-chain enoyl-CoA hydratase or crotonase (EC 4.2.1.17); SBCAD, s; hort branched-chain acyl-CoA dehydrogenase (EC 1.3.99.12)Valproic acid; 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase; Crotonase; Isoleucine; Mitochondrial dysfunction; Drug metabolism

Human arylacetamide deacetylase is responsible for deacetylation of rifamycins: Rifampicin, rifabutin, and rifapentine by Akinori Nakajima; Tatsuki Fukami; Yuki Kobayashi; Akinobu Watanabe; Miki Nakajima; Tsuyoshi Yokoi (pp. 1747-1756).

Rifamycins such as rifampicin, rifabutin, and rifapentine are used for the treatment of tuberculosis and induce various drug-metabolizing enzymes. Rifamycins have been reported to be mainly deacetylated by esterase(s) expressed in human liver microsomes (HLM) to 25-deacetylrifamycins, but the responsible enzyme remained to be determined. In this study, we found that recombinant human arylacetamide deacetylase (AADAC) could efficiently deacetylate rifamycins, whereas human carboxylesterases, which are enzymes responsible for the hydrolysis of many prodrugs, showed no activity. The involvement of AADAC in the deacetylation of rifamycins in HLM was verified by the similarities of the K_m and K_i values and the inhibitory characteristics between recombinant AADAC and HLM. Rifamycins exhibited potent cytotoxicity to HepG2 cells, but their 25-deacetylated metabolites did not. Luciferase assay using a reporter plasmid containing CYP3A4 direct repeat 3 and everted repeat 6 motifs revealed that 25-deacetylrifamycins have lesser potency to transactivate CYP3A4 compared with the parent drugs. Supporting these results, HepG2 cells infected with a recombinant adenovirus expressing human AADAC showed low cytotoxicity and induction potency of CYP3A4 by rifamycins. In addition, CYP3A4 induction in human hepatocytes by rifamycins was increased by transfecting siRNA for human AADAC. Thus, we found that human AADAC was the enzyme responsible for the deacetylation of rifamycins and would affect the induction rate of drug-metabolizing enzymes by rifamycins and their induced hepatotoxicity.

Keywords: Abbreviations; AADAC; arylacetamide deacetylase; AdAADAC; a recombinant adenovirus expressing human AADAC; AdGFP; a recombinant adenovirus expressing a green fluorescence protein; BNPP; bis-; p; -nitrophenylphosphate; CES; carboxylesterase; ChE; cholinesterase; DTNB; 5,5′-dithiobis(2-nitrobenzoic acid); DFP; diisopropyl fluorophosphate; HLM; human liver microsomes; HPLC; high performance-liquid chromatography; NaF; sodium fluoride; PCR; polymerase chain reaction; PMSF; phenylmethylsulfonyl fluoride; PON; paraoxonaseArylacetamide deacetylase; Rifamycins; Gene induction; Cytotoxicity

Characterizing the effect of UDP-glucuronosyltransferase (UGT) 2B7 and UGT1A9 genetic polymorphisms on enantioselective glucuronidation of flurbiprofen by Haina Wang; Lingmin Yuan; Su Zeng (pp. 1757-1763).

Flurbiprofen (FPF), available commercially as a racemic mixture, is a propionic acid derivative of non-steroidal anti-inflammatory drugs (NSAIDs) with known stereoselective glucuronidation. The major enzyme catalyzing this conjugation reaction is UDP-glucuronosyltransferase (UGT) 2B7, with minor contributions by UGT1A9. This study examines the role of the genetic variants of UGT2B7 and 1A9 enzymes involved in the formation of acyl glucuronides (FPFGs). Variants caused by three single nucleotide polymorphisms (SNPs) (A71S, 211G>T; H268Y, 802C>T; and D398N, 1192G>A) in UGT2B7 and three SNPs (C3Y, 8G>A; M33T, 98T>C; D256N, 766G>A) in UGT1A9 showed activity changes toward different substrates. However the functional impacts of these SNPs on chiral substrates were not examined. Upon stable expression in Bac-to-Bac system, UGT2B7*71S (A⁷¹S), UGT2B7*2 (H²⁶⁸Y) and UGT2B7*5 (D³⁹⁸N) were all associated with a decrease in the formation of FPFGs. Compared with UGT2B7*1 (wild-type), UGT2B7*71S exhibited a >2-fold lower intrinsic clearance mainly by altered capacities ( V_max). Furthermore, a >14-fold decreased intrinsic clearance of the *1 protein was produced by UGT2B7*2 and UGT2B7*5. However, no significantly stereoselective difference for the formation of (R)- and (S)-FPFG was found among these UGT2B7 allozymes. UGT1A9*2 (C³Y) exhibited a higher V_max (3.2-fold), K_m (2.1-fold) and intrinsic clearance (1.6-fold) toward (S)-FPF than UGT1A9*1 (wild-type). UGT1A9*3 (M³³T) almost lost the catalytic activity to FPF. A significantly stereoselective difference on the glucuronidation of rac-FPF was seen between the two variants compared with the wild type of UGT1A9.

Keywords: Abbreviations; RP-HPLC; reverse phase high performance liquid chromatography; UGT; uridine 5-diphosphoglucuronosyl transferases; UDPGA; uridine 5-diphosphoglucuronic acid; FBS; fetal bovine serum; FPF; flurbiprofen; FPFG; flurbiprofen glucuronide; ZPF; zaltoprofenFlurbiprofen; UGT2B7; UGT1A9; Single nucleotide polymorphism; Stereoselectivity

Regioselectivity of human UDP-glucuronosyltransferase isozymes in flavonoid biotransformation by metal complexation and tandem mass spectrometry by Scott A. Robotham; Jennifer S. Brodbelt (pp. 1764-1770).

Based on reactions with five flavonoids, the regioselectivities of twelve human UDP-glucuronosyltransferase (UGT) isozymes were elucidated. The various flavonoid glucuronides were differentiated based on LC–MS/MS fragmentation patterns of [Co(II)(flavonoid-H)(4,7-diphenyl-1,10-phenanthroline)₂]⁺ complexes generated upon post-column complexation. Glucuronide distributions were evaluated to allow a systematic assessment of the regioselectivity of each isozyme. The various UGT enzymes, including eight UGT1A and four UGT2B, displayed a remarkable range of selectivities, both in terms of the positions of glucuronidation and relative reactivity with flavanones versus flavonols.

Keywords: Human UDP-glucuronosyltransferase; Flavonoid; Regioselectivity; Mass spectrometry; Metal complexation; Glucuronidation

Metformin suppresses pregnane X receptor (PXR)-regulated transactivation of CYP3A4 gene by Lucie Krausova; Lucie Stejskalova; Hongwei Wang; Radim Vrzal; Zdenek Dvorak; Sridhar Mani; Petr Pavek (pp. 1771-1780).

Metformin is widely used in the treatment of type-2 diabetes. The pleotropic effects of metformin on glucose and lipid metabolism have been proposed to be mediated by the activation of AMP-activated protein kinase (AMPK) and the subsequent up-regulation of small heterodimer partner (SHP). SHP suppresses the functions of several nuclear receptors involved in the regulation of hepatic metabolism, including pregnane X receptor (PXR), which is referred to as a “master regulator” of drug/xenobiotic metabolism.In this study, we hypothesize that metformin suppresses the expression of CYP3A4, a main detoxification enzyme and a target gene of PXR, due to SHP up-regulation.We employed various gene reporter assays in cell lines and qRT-PCR in human hepatocytes and in Pxr^−/− mice.We show that metformin dramatically suppresses PXR-mediated expression of CYP3A4 in hepatocytes. Consistently, metformin significantly suppressed the up-regulation of Cyp3a11 mRNA in the liver and intestine of wild-type mice, but not in Pxr^−/− mice. A mechanistic investigation of the phenomenon showed that metformin does not significantly up-regulate SHP in human hepatocytes. We further demonstrate that AMPK activation is not involved in this process. We show that metformin disrupts PXR's interaction with steroid receptor coactivator-1 (SRC1) in a two-hybrid assay independently of the PXR ligand binding pocket. Metformin also inhibited vitamin D receptor-, glucocorticoid receptor- and constitutive androstane receptor (CAR)-mediated induction of CYP3A4 mRNA in human hepatocytes.We show, therefore, a suppressive effect of metformin on PXR and other ligand-activated nuclear receptors in transactivation of the main detoxification enzyme CYP3A4 in human hepatocytes.

Keywords: Metformin; Cytochrome P450; Induction; PXR; AMPK

Indomethacin induces cellular morphological change and migration via epithelial-mesenchymal transition in A549 human lung cancer cells: A novel cyclooxygenase-inhibition-independent effect by Tomoko Kato; Hiromichi Fujino; Satomi Oyama; Tatsuo Kawashima; Toshihiko Murayama (pp. 1781-1791).

Levels of cyclooxygenase (COX)-2 and its metabolite prostaglandin E₂ (PGE₂) are frequently increased in colon cancer and other cancers including lung cancer. Non-steroidal anti-inflammatory drugs are considered to have chemo-preventive effects on these diseases by reducing the biosynthesis of PGE₂ via their inhibition of COX-2. Although the COX-2/PGE₂ pathway may directly impact on lung carcinogenesis, some population-based cohort studies of NSAIDs showed no significant protective effects. In this study, using human non-small-cell lung cancer A549 cells, we examined the effects of indomethacin, a potent NSAID, on the growth and motility of lung cancer cells. Besides inhibiting PGE₂ production and cellular growth, indomethacin caused drastic morphological changes with a loss of stress fibers in a time- and dose-dependent manner. Interestingly, the change in cellular shape caused by indomethacin was not seen when the cells were treated with aspirin or diclofenac, two other NSAIDs, despite the concentrations used being sufficient to inhibit PGE₂ production. The indomethacin-induced morphological changes in A549 cells were accompanied by a reduction in levels of the adhesion molecule E-cadherin and a component of basal lamina, collagen IV, as well as an increase in the activity of a collagenase, matrix metalloprotease-9. Furthermore, indomethacin-induced shape changes resulted in enhanced motility via regulation of peroxisome proliferator-activated receptor γ. The dual effects of indomethacin, inhibition of cellular growth and enhancement of migration, would explain, to some extent, the difficulty in using this NSAID for lung cancer therapy.

Keywords: Abbreviations; NSAIDs; non-steroidal anti-inflammatory drugs; COX; cyclooxygenase; PGE; ₂; prostaglandin E; ₂; EMT; epithelial-mesenchymal transition; MMP; matrix metalloproteinase; NSCLC; non-small-cell lung cancer; EGFR; epidermal growth factor receptor; PPAR; peroxisome proliferator-activated receptor; MET; mesenchymal-epithelial transitionIndomethacin; EMT; E-cadherin; MMP-9; NSCLC

Ethanol impairs muscarinic receptor-induced neuritogenesis in rat hippocampal slices: Role of astrocytes and extracellular matrix proteins by Gennaro Giordano; Marina Guizzetti; Khoi Dao; Hayley A. Mattison; Lucio G. Costa (pp. 1792-1799).

In an in vitro co-culture system of astrocytes and neurons, stimulation of cholinergic muscarinic receptors in astrocytes had been shown to cause neuritogenesis in hippocampal neurons, and this effect was inhibited by ethanol. The present study sought to confirm these earlier findings in a more complex system, in vitro rat hippocampal slices in culture. Exposure of hippocampal slices to the cholinergic agonist carbachol (1mM for 24h) induced neurite outgrowth in hippocampal pyramidal neurons, which was mediated by activation of muscarinic M3 receptors. Specifically, carbachol induced a >4-fold increase in the length of the longest neurite, and a 4-fold increase in the length of minor neurites and in the number of branches. Co-incubation of carbachol with ethanol (50mM) resulted in significant inhibition of the effects induced by carbachol on all parameters measured. Neurite outgrowth in CNS neurons is dependent on various permissive factors that are produced and released by glial cells. In hippocampal slices carbachol increased the levels of two extracellular matrix protein, fibronectin and laminin-1, by 1.6-fold, as measured by Western blot. Co-incubation of carbachol with ethanol significantly inhibited these increases. Carbachol-induced increases in levels of extracellular matrix proteins were antagonized by a M3 muscarinic receptor antagonist. Furthermore, function-blocking fibronectin or laminin-1 antibodies antagonized the effect of carbachol on neurite outgrowth. These results indicate that in hippocampal slices stimulation of muscarinic M3 receptors induces neurite outgrowth, which is mediated by fibronectin and laminin-1, two extracellular matrix proteins released by astrocytes. By decreasing fibronectin and laminin levels ethanol prevents carbachol-induced neuritogenesis. These findings highlight the importance of glial–neuronal interactions as important targets in the developmental neurotoxicity of alcohol.

Keywords: Ethanol; Neuritogenesis; Muscarinic receptors; Hippocampal slice; Extracellular matrix; Fibronectin

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Biochemical Pharmacology (v.82, #11)