Check out our New Publishers' Select for Free Articles

Biochemical Pharmacology (v.75, #9)

Editorial Advisory Board (pp. iii).

Physiological and pharmacokinetic roles of H⁺/organic cation antiporters (MATE/SLC47A) by Tomohiro Terada; Ken-ichi Inui (pp. 1689-1696).

Vectorial secretion of cationic compounds across tubular epithelial cells is an important function of the kidney. This uni-directed transport is mediated by two cooperative functions, which are membrane potential-dependent organic cation transporters at the basolateral membranes and H⁺/organic cation antiporters at the brush-border membranes. More than 10 years ago, the basolateral organic cation transporters (OCT1-3/SLC22A1-3) were isolated, and molecular understandings for the basolateral entry of cationic drugs have been greatly advanced. However, the molecular nature of H⁺/organic cation antiport systems remains unclear. Recently, mammalian orthologues of the multidrug and toxin extrusion (MATE) family of bacteria have been isolated and clarified to function as H⁺/organic cation antiporters. In this commentary, the molecular characteristics and pharmacokinetic roles of mammalian MATEs are critically overviewed focusing on the renal secretion of cationic drugs.

Keywords: Abbreviations; MATE; multidrug and toxin extrusion; MPP; 1-methyl-4-phenylpyridinium; NMN; N; ¹; -methylnicotinamide; OCT; organic cation transporter; OCTN; novel organic cation transporter; SLC; solute carrier; SNP; single nucleotide polymorphism; TEA; tetraethylammoniumH; ⁺; /organic cation antiporter; Organic cation transporter; Renal tubular secretion; MATE1; MATE2-K; OCT

Histone deacetylase inhibitor SAHA induces ERα degradation in breast cancer MCF-7 cells by CHIP-mediated ubiquitin pathway and inhibits survival signaling by Xin Yi; Wei Wei; Sheng-Yu Wang; Zhi-Yan Du; Yuan-Ji Xu; Xiao-Dan Yu (pp. 1697-1705).

Estrogen receptor α (ERα) plays an important role in the development and progression of breast cancer, and recent studies showed that ERα expression is associated with resistance to hormonal therapy. Therefore, a number of studies have explored ways to deplete ERα from breast cancer cells as a new therapy especially for hormone-refractory breast cancer. We reported here that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, effectively depletes ERα in breast cancer MCF-7 cells. However, the intrinsic mechanisms by which SAHA decreases ERα levels are not clear. Our present data demonstrated that both inhibition of ERα mRNA level and promotion of ERα degradation by the proteasome contribute to SAHA-induced ERα depletion, indicating that SAHA may exert its effects through transcriptional and posttranslational mechanisms. Furthermore, the decrease of ERα protein level in MCF-7 cells after SAHA treatment is mainly the result of its rapid degradation by the ubiquitin-proteasome pathway rather than transcriptional inhibition. In addition, we showed that inactivation of the heat shock protein-90 (Hsp90) is involved in SAHA-induced ERα degradation, and ubiquitin ligase CHIP (C-terminal Hsc70 interacting protein) enhances SAHA-induced ERα degradation. SAHA-induced ERα depletion is paralleled with reduction of transcriptional activity of ERα and SAHA is able to effectively inhibit cell proliferation and induce apoptosis of MCF-7 cells. Taken together, our results revealed a mechanism for SAHA-induced ERα degradation and indicated that SAHA is a suitable pharmacological agent for depletion of ERα and a potential choice for breast cancer expressing high ERα.

Keywords: HDACi; SAHA; ERα; Breast cancer; CHIP; Hsp90

Suppression of ClC-3 channel expression reduces migration of nasopharyngeal carcinoma cells by Jianwen Mao; Lixin Chen; Bin Xu; Lijing Wang; Hongzhi Li; Jiao Guo; Weidong Li; Sihuai Nie; Tim J.C. Jacob; Liwei Wang (pp. 1706-1716).

Recent studies suggest that chloride (Cl⁻) channels regulate tumor cell migration. In this report, we have used antisense oligonucleotides specific for ClC-3, the most likely molecular candidate for the volume-activated Cl⁻ channel, to investigate the role of ClC-3 in the migration of nasopharyngeal carcinoma cells (CNE-2Z) in vitro. We found that suppression of ClC-3 expression inhibited the migration of CNE-2Z cells in a concentration-dependent manner. Whole-cell patch-clamp recordings and image analysis further demonstrated that ClC-3 suppression inhibited the volume-activated Cl⁻ current ( I_Cl,vol) and regulatory volume decrease (RVD) of CNE-2Z cells. The expression of ClC-3 positively correlated with cell migration, I_Cl,vol and RVD. These results strongly suggest that ClC-3 is a component or regulator of the volume-activated Cl⁻ channel. ClC-3 may regulate CNE-2Z cell migration by modulating cell volume. ClC-3 may be a new target for cancer therapies.

Keywords: ClC-3; Chloride channels; Antisense oligonucleotide; Cell migration; Cell volume

An insight into the pharmacophores of phosphodiesterase-5 inhibitors from synthetic and crystal structural studies by Gong Chen; Huanchen Wang; Howard Robinson; Jiwen Cai; Yiqian Wan; Hengming Ke (pp. 1717-1728).

Selective inhibitors of cyclic nucleotide phosphodiesterase-5 (PDE5) have been used as drugs for treatment of male erectile dysfunction and pulmonary hypertension. An insight into the pharmacophores of PDE5 inhibitors is essential for development of second generation of PDE5 inhibitors, but has not been completely illustrated. Here we report the synthesis of a new class of the sildenafil derivatives and a crystal structure of the PDE5 catalytic domain in complex with 5-(2-ethoxy-5-(sulfamoyl)-3-thienyl)-1-methyl-3-propyl-1,6-dihydro-7 H-pyrazolo[4,3- d]pyrimidin-7-one (12). Inhibitor12 induces conformational change of the H-loop (residues 660–683), which is different from any of the known PDE5 structures. The pyrazolopyrimidinone groups of12 and sildenafil are well superimposed, but their sulfonamide groups show a positional difference of as much as 1.5Å. The structure–activity analysis suggests that a small hydrophobic pocket and the H-loop of PDE5 are important for the inhibitor affinity, in addition to two common elements for binding of almost all the PDE inhibitors: the stack against the phenylalanine and the hydrogen bond with the invariant glutamine. However, the PDE5–12 structure does not provide a full explanation to affinity changes of the inhibitors. Thus alternatives such as conformational change of the M-loop are open and further structural study is required.

Keywords: Phosphodiesterase (PDE); Sildenafil; Viagra; Crystal structure

Critical role of Bid and Bax in indirubin-3′-monoxime-induced apoptosis in human cancer cells by Jie Shi; Han-Ming Shen (pp. 1729-1742).

Indirubin-3′-monoxime (I3M) is a derivative of indirubin, an active component from a Chinese medicinal recipe with known anti-cancer function. I3M has been well established as a cyclin-dependent kinase (CDK) inhibitor, while the molecular mechanism underlying I3M-induced apoptosis has not been fully elucidated. In this study, we focused on the critical role of the pro-apoptosis Bcl-2 family members in I3M-induced apoptosis. We first observed I3M-induced apoptosis in a time- and dose-dependent manner in three different types of human cancer cells—cervical cancer HeLa, hepatoma HepG2 and colon cancer HCT116. Induction of the caspase cascade for both the extrinsic and intrinsic pathways was demonstrated, including caspase-8, -9 and -3 activation. Initiation of the death receptor pathway started with enhanced surface expression of DR4 and DR5, as well as increased total protein level, which correlated with the up-regulation of p53 and its transcriptional activity. Importantly, we found in HeLa cells that caspase-8 activation resulted in Bid cleavage, followed by Bax conformational change and hence the amplification of the apoptotic signals through the mitochondrial pathway. Consistently, stable knockdown of Bid abrogated I3M-induced Bax conformational change and cell death. Moreover, ectopic expression of a viral caspase inhibitor (CrmA) or Bcl-2 partially protected I3M-induced apoptosis. In conclusion, our results indicate that I3M mainly elicites apoptosis through extrinsic pathway with type II response mediated by the pro-apoptotic Bcl-2 family members (Bid and Bax).

Keywords: Abbreviations; I3M; indirubin-3′-monoxime; DAPI; 4′,6-diamidino-2-phenylindole; MTT; thiazolyl blue tetrazolium bromide; PARP; poly(ADP-ribose)polymerase; z-VAD-FMK; z-Val-Ala-Asp(OMe)-fluoromethylketone; ac-DEVD-CHO; N; -acetyl-Asp-Glu-Val-Asp-CHO (aldehyde); z-DEVD-FMK; z-Asp(OMe)-Glu(OMe)-Val-Asp; Ac-IETD-CHO; N; -acetyl-Ile-Glu-Thr-Asp-CHO (aldehyde); Ac-LEHD-CHO; N; -acetyl-Leu-Glu-His-Asp-CHO (aldehyde); PI; propidium iodide; CrmA; cytokine response member A; GFP; green fluorescence proteinIndirubin-3′-monoxime; Death receptor; Bid; Bax; Apoptosis

A facile method to screen inhibitors of protein–protein interactions including MDM2–p53 displayed on T7 phage by Kazutomo Ishi; Fumio Sugawara (pp. 1743-1750).

Protein–protein interactions are essential in many biological processes including cell cycle and apoptosis. It is currently of great medical interest to inhibit specific protein–protein interactions in order to treat a variety of disease states. Here, we describe a facile multiwell plate assay method using T7 phage display to screen for candidate inhibitors of protein–protein interactions. Because T7 phage display is an effective method for detecting protein–protein interactions, we aimed to utilize this technique to screen for small-molecule inhibitors that disrupt these types of interaction. We used the well-characterized interaction between p53 and MDM2 and an inhibitor of this interaction, nutlin 3, as a model system to establish a new screening method. Phage particles displaying p53 interacted with GST–MDM2 immobilized on 96-well plates, and the interaction was inhibited by nutlin 3. Multiwell plate assay was then performed using a natural product library, which identified dehydroaltenusin as a candidate inhibitor of the p53–MDM2 interaction. We discuss the potential applications of this novel T7 phage display methodology, which we propose to call ‘reverse phage display’.

Keywords: Abbreviations; PBS; phosphate-buffered saline; DMSO; dimethyl sulfoxide; SDS; sodium dodecyl sulfate; GST; glutathione; S; -transferase; p; NPP; p; -nitrophenyl phosphate; CBB; Coomassie brilliant blue R250; IPTG; isopropyl-beta-; d; -galactoside; PFU; plaque forming unitPhage display; Nutlin 3; Dehydroaltenusin; Protein–protein interaction; p53; MDM2

Implication of NAG-1 in synergistic induction of apoptosis by combined treatment of sodium salicylate and PI3K/MEK1/2 inhibitors in A549 human lung adenocarcinoma cells by Cho Hee Kim; Min Young Kim; Ji Young Moon; Ji Won Hwang; Su Yeon Lee; Young Mi Joo; Song Iy Han; Hye Gyeong Park; Ho Sung Kang (pp. 1751-1760).

Aspirin is used as chemopreventive agents in a variety of human cancer cells including those of colon, lung, breast, and leukemia. Sodium salicylate (NaSal, the natural deacetylated form of aspirin) induced cell cycle arrest and apoptosis in a dose-dependent manner in A549 cells; high dose (20mM) of NaSal-induced apoptosis, whereas low dose (2–10mM) induced cell cycle arrest. We found that NaSal-activated Akt/PKB, ERK1/2, and p38MAPK signal cascades. Twenty micromolar of NaSal-induced apoptotic response of A549 cells was enhanced by the PI3K inhibitors (LY294002 and wortmannin) and in a less extent by the MEK1/2 inhibitors (U0126 and PD98059), whereas it was suppressed by the p38MAPK inhibitor (SB203580). Furthermore, simultaneous inhibition of the Akt/PKB and ERK1/2 signal cascades could lower the dose of NaSal to induce apoptosis to 2mM in A549 lung cancer cells. Similar enhancement was observed in cells treated with 2mM NaSal and 100μM genistein, an inhibitor of receptor tyrosine kinases (RTKs) that are upstream of PI3K and MEK1/2 signaling. We further demonstrated that NAG-1 plays a key role in apoptosis by NaSal-based combined treatment. Collectively, our findings indicate that inhibition of the pro-survival Akt/PKB and ERK1/2 signaling may increase the chemopreventive effects of NaSal and combined treatment of two natural compounds (NaSal and genistein) results in a highly synergistic induction of apoptosis, thereby increasing the chemopreventive effects of NaSal against cancer.

Keywords: Abbreviations; NaSal; sodium salicylate; RTK; receptor tyrosine kinases; NSAID; non-steroidal anti-inflammatory drugs; NAG-1; non-steroidal anti-inflammatory drug-activated gene-1; NSCLC; non-small cell lung cancer; GSK-3β; glycogen synthase kinase 3β; PBS; phosphate-buffered saline; PI; propidium iodide; FACS; fluorescence-activated cell sorter; DTT; dithiothreitol; IGF-1R; insulin-like growth factor-1 receptor; EGFR; epidermal growth factor receptor; TPA; 12-; O; -tetradecanoylphorbol-13-acetate; siRNA; small interference RNA; RT-PCR; reverse transcription-polymerase chain reactionSodium salicylate; Apoptosis; PI3K; Akt/PKB; ERK1/2; NAG-1; Genistein

STI571/doxorubicin concentration-dependent switch for diverse caspase actions in CML cell line K562 by Malgorzata Czyz; Justyna Jakubowska; Malgorzata Sztiller-Sikorska (pp. 1761-1773).

We examined the response of the apoptosis-reluctant CML cell line K562 to doxorubicin alone or in combination with the tyrosine kinase inhibitor STI571. We found that at clinically relevant concentrations, doxorubicin induced differentiation and senescence, but did not induce apoptosis. Doxorubicin induced G₂/M arrest and mitochondrial transmembrane potential dissipation. Interestingly, drug-induced differentiation could be diminished by caspase inhibitors. STI571 caused a graded response characterized by differentiation at low concentrations and apoptosis at higher. STI571 was not observed to induce senescence. Combination of STI571 and caspase inhibitors protected cells from apoptosis but did not influence differentiation. The diverse mode of action of both drugs contributed to the response observed during combination treatment. An additive effect on proliferation was obtained. The mechanisms contributing to inhibition of cellular proliferation were complex and strongly dependent on the applied drug concentrations. Differentiation or apoptosis were enhanced by combined treatment only in narrow ranges of concentrations. Conclusion: DOX and STI571 along diverse mechanisms contributed to elevated levels of activated caspases which might be then responsible for a switch from differentiation to apoptosis.

Keywords: Abbreviations; Ara-C; cytarabine, cytosine arabinoside; Bcl-X; _L; (B-cell leukemia/lymphoma extra long); anti-apoptotic protein; CML; chronic myelogenous leukemia; DAPI; 4,6-diamidino-2-phenylidole; Δ; Ψ; _m; mitochondrial transmembrane potential; DMSO; dimethyl sulfoxide; DOX; doxorubicin; FITC; fluorescein isothiocyanate; GATA-1; transcription factor; GPA; glycophorine A; IC; ₅₀; concentration with 50% inhibitory effect; MAP; mitogen-activated protein; PBS; phosphate-buffered saline; PI; propidium iodide; SA-β-gal; senescence-associated β-galactosidase (activity); TMRE; tetramethylrhodamine ethyl ester; zDEVD-fmk; N; -benzyloxycarbonyl-Asp(OCH; ₃; )-Glu(OCH; ₃; )-Val-Asp(OCH; ₃; )-fluoromethylketone; zVAD-fmk; N; -benzyloxycabonyl-Val-Ala-Asp-fluoromethylketoneCML; Caspases; Differentiation; Doxorubicin; STI571

Indole-3-carbinol enhances anti-proliferative, but not anti-invasive effects of oxaliplatin in colorectal cancer cell lines by Lynne M. Howells; Christopher P. Neal; Mhairi C. Brown; David P. Berry; Margaret M. Manson (pp. 1774-1782).

The primary aim of this study was to determine whether combination of the chemopreventive agent indole-3-carbinol (I3C) with oxaliplatin would decrease proliferative index and invasive potential of human colorectal tumour cells.Combination of the agents resulted in a 170-fold decrease in proliferative capacity in SW480 and SW620 cell lines, which was approximately 6-fold greater than for oxaliplatin alone. Decreased proliferation was attributed to enhanced S-phase cell cycle arrest for SW480, and increased apoptosis for SW620 cells.The combined agents resulted in significantly increased E-cadherin levels in SW480 cells, and β-catenin levels in both cell lines (assessed by in-cell westerns). In SW480 cells confocal microscopy revealed an increase in membrane-associated β-catenin levels, with oxaliplatin treatments enhancing nuclear export and cytoplasmic localisation. In SW620 cells, all treatments increased membrane localisation of E-cadherin.Whilst both oxaliplatin and I3C decreased invasive capacity of SW480 cells, this was not further enhanced by the combined treatment.

Keywords: Colorectal cancer; Oxaliplatin; Chemoprevention; Indole-3-carbinol; E-Cadherin; β-Catenin

SP1-regulated p27/Kip1 gene expression is involved in terbinafine-induced human A431 cancer cell differentiation: An in vitro and in vivo study by Ching-Shui Huang; Wei-Lu Ho; Wen-Sen Lee; Ming-Thau Sheu; Ying-Jan Wang; Shih-Hsin Tu; Rong-Jane Chen; Jan-Show Chu; Li-Ching Chen; Chia-Hwa Lee; How Tseng; Yuan-Soon Ho; Chih-Hsiung Wu (pp. 1783-1796).

In this study, the differentiation-promoting effects of terbinafine (Lamisil^®, TB) were investigated in human epithelioid squamous carcinoma (A431) cells. The polyhydroxyethylmethacrylate (poly-HEMA)- and type-I collagen-coated culture plate models were adapted to harvest the TB-induced differentiated cells by agitation of the suspension medium. We demonstrated that p27/Kip1, p21/Cip1 and the keratinocyte differentiation marker, human involucrin (hINV), were induced (>25μM) in TB-induced differentiated A431 cells. Animal studies demonstrated that administration of TB (10mg/kg body weight) inhibited A431-xenografted tumor growth through differentiation processes as evidenced by expression of pancytokeratin in tumor tissues. Immunocytochemical staining analysis showed that p27/Kip1, but not p21/Cip1, positive-stained cells were detected in the early-differentiated cells of TB-treated tumor tissues. SP1, which regulates p27/Kip1 expression, was induced by TB (>10μM) in A431 cells. The TB-induced promoter activity and protein expression levels of p27/Kip1 were significantly attenuated by pretreatment with mithramycin A, a SP1 specific inhibitor. We also demonstrated that TB-induced differentiated A431 cells sorted from the poly-HEMA-coated culture plates were arrested in the G1 phase. TB-induced G1 arrest in the suspension-cultured cells was attenuated by mithramycin A pretreatment. Such results suggest that SP1 plays a critical role in the p27/Kip1 gene transcriptional activation that may be subsequently involved in the TB-induced A431 cancer cell differentiation process.

Keywords: Abbreviations; BM; basement membrane; ChIP; chromatin immunoprecipitation analysis; CDK; cyclin-dependent kinase; DMEM; Dulbecco's modified Eagle's medium; DMSO; dimethylsulfoxide; ECM; extracellular matrix; FACS; fluorescence-activated cell sorter; FCS; fetal calf serum; FIL; filaggrin; hINV; human involucrin; LD; lower differentiated region; LOR; loricrin; NBT; nitro blue tetrazolium; NHEKs; Normal human epidermal keratinocytes; poly-HEMA; polyhydroxyethylmethacrylate; RT-PCR; reverse transcriptase-polymerase chain reaction; SC; stratum corneum; TB; terbinafine; TGase-1; tissue transglutaminase-1; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; WD; well differentiated regionTerbinafine (Lamisil; ^®; ); G1 cell cycle arrest; Differentiation; A431 cells; p27/Kip1

14-3-3 protein regulates Ask1 signaling and protects against diabetic cardiomyopathy by Rajarajan A. Thandavarayan; Kenichi Watanabe; Meilei Ma; Punniyakoti T. Veeraveedu; Narasimman Gurusamy; Suresh S. Palaniyandi; Shaosong Zhang; Anthony J. Muslin; Makoto Kodama; Yoshifusa Aizawa (pp. 1797-1806).

Mammalian 14-3-3 proteins are dimeric phosphoserine-binding proteins that participate in signal transduction and regulate several aspects of cellular biochemistry. Diabetic cardiomyopathy is associated with increased oxidative stress and inflammation. In order to study the pathogenic changes underlying diabetic cardiomyopathy, we examined the role of 14-3-3 protein and apoptosis signal-regulating kinase 1 (Ask1) signaling by using transgenic mice with cardiac-specific expression of a dominant-negative 14-3-3η protein mutant (DN 14-3-3η) after induction of experimental diabetes. The elevation in blood glucose was comparable between wild type (WT) and DN 14-3-3η mice. However, a marked downregulation of thioredoxin reductase was apparent in DN 14-3-3η mice compared to WT mice after induction of diabetes. Significant Ask1 activation in DN 14-3-3η after diabetes induction was evidenced by pronounced de-phosphorylation at Ser-967 and intense immunofluorescence observed in left ventricular (LV) sections. Echocardiographic analysis revealed that cardiac functions were notably impaired in diabetic DN 14-3-3η mice compared to diabetic WT mice. Marked increases in myocardial apoptosis, cardiac hypertrophy, and fibrosis were observed with a corresponding up-regulation of atrial natriuretic peptide and galectin-3, as well as a downregulation of sarcoendoplasmic reticulum Ca²⁺ ATPase2 expression. Furthermore, diabetic DN 14-3-3η mice displayed significant reductions of platelet-endothelial cell adhesion molecule-1 staining as well as endothelial nitric acid synthase and vascular endothelial growth factor expression. In conclusion, our data suggests that enhancement of 14-3-3 protein could provide a novel therapeutic strategy against hyperglycemia-induced left ventricular dysfunction and can limit the progression of diabetic cardiomyopathy by regulating Ask1 signaling.

Keywords: Diabetic cardiomyopathy; 14-3-3 protein; Apoptosis signal-regulating kinase 1; Thioredoxin reductase; Hypertrophy; Fibrosis; Endothelial cells

Aspirin inhibits human bradykinin B₂ receptor ligand binding function by Joëlle Gardes; Stéphanie Michineau; Anne Pizard; François Alhenc-Gelas; Rabary M. Rajerison (pp. 1807-1816).

The bradykinin B₂ receptor, a member of the G protein-coupled receptors superfamily, is involved in a variety of physiological functions, including vasodilation, electrolyte transfer in epithelia, mediation of pain, and inflammation. The effect of aspirin on bradykinin binding to cell-surface receptor and on signal transduction were studied in CHO-K1 cells, stably expressing the human B₂ receptor. Cell-surface organization of the receptor was assessed by immunoprecipitation and Western blot analysis in CHO-K1 cells expressing N-terminally V5-tagged B₂ receptor. We found that the widely used analgesic, anti-thrombotic, and anti-inflammatory drug aspirin alters the B₂ receptor ligand binding properties. Aspirin reduces the apparent affinity of the receptor for [³H]-bradykinin by accelerating the dissociation rate of [³H]-bradykinin–receptor complexes. In addition, aspirin reduces the capacity of unlabeled bradykinin or the B₂ receptor antagonist icatibant to destabilize pre-formed [³H]-bradykinin–receptor complexes. Kinetic and reversibility studies are consistent with an allosteric type of mechanism. Aspirin effect on B₂ receptor binding properties is not accompanied by alteration of the cell-surface organization of the receptor in dimers and monomers. Aspirin does not influence the receptor ability to transduce bradykinin binding into activation of G-proteins and phospholipase C. These results suggest that aspirin is an allosteric inhibitor of the B₂ receptor, a property that may be involved in its therapeutic actions.

Keywords: Abbreviations; GPCR; G protein-coupled receptor; BK; bradykinin; B; ₂; receptor; bradykinin receptor B; ₂; subtype; B; ₁; receptor; bradykinin receptor B; ₁; subtype; CHO-K1 cells; K1 type of Chinese hamster ovary cells; IPs; inositol phosphatesBradykinin; Aspirin; B; ₂; receptor; B; ₂; antagonist; Allosteric regulation

Inhibitory effect of fluoxetine on lymphoma growth through the modulation of antitumor T-cell response by serotonin-dependent and independent mechanisms by Luciana Romina Frick; María Laura Palumbo; María Paula Zappia; Marcela Adriana Brocco; Graciela Alicia Cremaschi; Ana María Genaro (pp. 1817-1826).

Fluoxetine, a selective serotonin reuptake inhibitor, is widely used for the treatment of depressive symptoms of cancer patients. However, there are contradictory evidences about its effects on immunity and cancer. Thus, we studied the effects of fluoxetine on tumor growth and on antitumoral T-cell-mediated immunity. In vivo chronic fluoxetine treatment inhibited tumor growth, and increased latency of appearance of solid tumors and survival of mice. Fluoxetine administration also increased mitogen-induced T-cell proliferation and Tumor Necrosis Factor-α (TNF-α) and Interferon-γ (IFN-γ) expression, without altering CD4⁺/CD8⁺ ratio. In vitro, fluoxetine did not affect tumor cells proliferation, but it exerted a direct effect on T lymphocytes. Both fluoxetine and serotonin stimulated proliferation induced by a suboptimal mitogen concentration but inhibited proliferation at the optimal one. When both drugs were combined the results indicated that the effects of fluoxetine are in part independent of its ability to elevate serotonin extracellular levels. Finally, continue fluoxetine administration in nude mice – devoid of T lymphocytes – did not modify tumor progression, thus supporting the hypothesis of an immuno-modulatory effect of this drug on T cells that drives tumor growth control. These findings indicate, for the first time, that fluoxetine inhibits tumor growth through modulation of T-cell-mediated immunity by the already known serotonin-dependent pathway and by a novel independent mechanism.

Keywords: Fluoxetine; Serotonin; T-cell; T lymphoma; Tumor progression; Cellular immunity

Effects of estrogen on intracellular signaling pathways linked to activation of muscarinic acetylcholine receptors and on acetylcholinesterase activity in rat hippocampus by Renato Tavares dos Santos Pereira; Catarina Segreti Porto; Rosely Oliveira Godinho; Fernando Maurício Francis Abdalla (pp. 1827-1834).

The aim of the present study was to investigate the effects of estrogen lack and estrogen replacement on the production of total [³H]inositol phosphate ([³H]IP) induced by the activation of muscarinic acetylcholine receptors (mAChRs) and on the mechanisms for inactivation of acetylcholine. Hippocampi were obtained from rats in proestrus (PE), ovariectomized for 15 days (C15), ovariectomized for 15 days and then treated with 17β-estradiol for 7 days (E7) and ovariectomized and immediately treated with 17β-estradiol for 21 days (E21). Ovariectomy did not change the basal level of total [³H]IP in the hippocampus. 17β-Estradiol replacement (E7 and E21) reduced the basal level of total [³H]IP. In all experimental groups, carbachol (CCh) caused a concentration-dependent rise in total [³H]IP. The maximum effect was reached with 10⁻⁴M CCh. The response to 10⁻⁴M CCh in the hippocampi from C15 and E7 rats was twofold higher than in hippocampi from PE and E21 animals and was blocked by pirenzepine, but not by methoctramine. Ovariectomy or 17β-estradiol treatment for 7 days did not change neither the total acetylcholinesterase (AChE) activity nor the relative amount of mono- and dimeric G₁/G₂ and tetrameric G₄ globular forms. Conversely, hormonal treatment for 21 days induced an increase in AChE activity of G₁/G₂ and G₄ forms, indicating that 17β-estradiol stimulates both synthesis and assembly of AChE molecular forms. The present results suggest that the duration and/or a critical period with regard to the initiation of estrogen therapy are important to regulate the function of mAChRs and AChE activity in female rat hippocampus.

Keywords: Muscarinic acetylcholine receptors; Hippocampus; Estrogen; Ovariectomy; Inositol phosphate; Acetylcholinesterase

Bakuchiol analogs inhibit monoamine transporters and regulate monoaminergic functions by Gang Zhao; Shao-Yun Zang; Xiang-Wei Zheng; Xiao-Hua Zhang; Li-He Guo (pp. 1835-1847).

Monoamine transporters play key roles in controlling monoamine levels and modulating monoamine reuptake. The objective of the present study was to identify monoamine transporter inhibitors from herbal sources. We discovered that bakuchiol analogs isolated from Fructus Psoraleae inhibited monoamine transporter uptake to differing degrees. The bakuchiol analog, Δ³,2-hydroxybakuchiol was the most potent and efficacious reuptake blocker and was thus selected as the candidate target. Monoamine transporter inhibition by Δ³,2-hydroxybakuchiol was more selective for the dopamine transporter (DAT) (IC₅₀=0.58±0.1μM) and norepinephrine transporter (NET) (IC₅₀=0.69±0.12μM) than for the serotonin transporter (SERT) (IC₅₀=312.02±56.69μM). Δ³,2-Hydroxybakuchiol exhibited greater potency (pEC₅₀ for DAT and NET) than bupropion and exhibited similar efficacy ( E_max for DAT and/or NET) to bupropion and GBR12,935. Pharmacokinetically, Δ³,2-hydroxybakuchiol competitively inhibited DAT and NET with partial reversibility and occupied cocaine binding sites. Moreover, Δ³,2-hydroxybakuchiol counteracted 1-methyl-4-phenylpyridinium-induced toxicity in cells expressing DAT with similar efficacy to GBR12,935. In vivo studies showed that Δ³,2-hydroxybakuchiol increased the activity of intact mice and improved the decreased activity of reserpinized mice. In the conditioned place preference test, preference scores in intact mice were unaffected by Δ³,2-hydroxybakuchiol treatment. Bakuchiol analogs, especially Δ³,2-hydroxybakuchiol, are monoamine transporter inhibitors involved in regulating dopaminergic and noradrenergic neurotransmission and may have represented potential pharmacotherapies for disorders such as Parkinson's disease, depression, and cocaine addiction.

Keywords: Abbreviations; CHO; Chinese hamster ovary; DAT; dopamine transporter; DPM; disintegrations-per-minute; fr.; fraction; GABA; γ-aminobutyric acid; GAT-1; γ-aminobutyric acid transporter; GBR12,935; 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)homopiperazine hydrochloride; HBSS; Hank's balanced salt solution; i.p.; intraperitoneal; MPP; ⁺; 1-methyl-4-phenylpyridinium; MPTP; 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MTT; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NET; norepinephrine transporter; PBS; phosphate-buffered saline; SERT; serotonin transporter; Tr-CHO; transgenic Chinese hamster ovary; WIN35,428; (−)-2β-carbomethoxy-3β-(4-fluorophenyl) tropaneBakuchiol analogs; Monoamine transporter; Dopamine transporter; Norepinephrine transporter; Reuptake inhibitor

Metabolism of 17α-hydroxyprogesterone caproate by hepatic and placental microsomes of human and baboons by Ru Yan; Tatiana N. Nanovskaya; Olga L. Zharikova; Donald R. Mattison; Gary D.V. Hankins; Mahmoud S. Ahmed (pp. 1848-1857).

Recent data from our laboratory revealed the formation of an unknown metabolite of 17 hydroxyprogesterone caproate (17-HPC), used for treatment of preterm deliveries, during its perfusion across the dually perfused human placental lobule. Previously, we demonstrated that the drug is not hydrolyzed, neither in vivo nor in vitro, to progesterone and caproate. Therefore, the hypothesis for this investigation is that 17-HPC is actively metabolized by human and baboon ( Papio cynocephalus) hepatic and placental microsomes. Baboon hepatic and placental microsomes were investigated to validate the nonhuman primate as an animal model for drug use during pregnancy. Data presented here indicate that human and baboon hepatic microsomes formed several mono-, di-, and tri-hydroxylated derivatives of 17-HPC. However, microsomes of human and baboon placentas metabolized 17-HPC to its mono-hydroxylated derivatives only in quantities that were a fraction of those formed by their respective livers, except for two metabolites (M16′ and M17′) that are unique for placenta and contributed to 25% and 75% of the total metabolites formed by human and baboon, respectively. The amounts of metabolites formed, relative to each other, by human and baboon microsomes were different suggesting that the affinity of 17-HPC to CYP enzymes and their activity could be species-dependent.

Keywords: Preterm delivery; 17α-Hydroxyprogesterone caproate; Metabolism; Microsomes; Human placenta; Baboon placenta; Human liver; Baboon liver

3,3′-Diindolylmethane reduces levels of HIF-1α and HIF-1 activity in hypoxic cultured human cancer cells by Jacques E. Riby; Gary L. Firestone; Leonard F. Bjeldanes (pp. 1858-1867).

3,3′-Diindolylmethane (DIM) is a chemopreventive and chemotherapeutic phytochemical derived from the metabolism of indoles found at high concentrations in cruciferous vegetables. We have previously shown that DIM exhibits anti-angiogenic properties in cultured vascular endothelial cells and in Matrigel plug assays in rodents. In the present study, we demonstrate that DIM reduces the level of hypoxia-inducible factor (HIF)-1α in hypoxic tumor cell lines, as well as HIF-1 transcriptional activity as measured by a reporter assay. Moreover, DIM inhibited the expression of HIF-1-responsive endogenous genes, resulting in the reduced expression of key hypoxia responsive factors, VEGF, furin, enolase-1, glucose transporter-1 and phosphofructokinase. DIM reduced the level of HIF-1α in hypoxic cells by increasing the rate of the prolylhydroxylase- and proteasome-mediated degradation of HIF-1α, and by decreasing the rate of HIF-1α transcription. Using enzyme kinetics studies, we established that DIM interacts with the oligomycin-binding site on the F0 transmembrane component of mitochondrial F1F0-ATPase. The contributions of the resulting increases in levels of ROS and O₂ in hypoxic cells to the inhibitory effects of DIM on HIF-1α expression are discussed. These studies are the first to show that DIM can decrease the accumulation and activity of the key angiogenesis regulatory factor, HIF-1α, in hypoxic tumor cells.

Keywords: 3,3′-Diindolylmethane; Angiogenesis; Hypoxia-inducible factor; Cancer; ATPase; Prolylhydroxylase

Sections

Personal tools

Biochemical Pharmacology (v.75, #9)

Sections

Personal tools

Local resources

Biochemical Pharmacology (v.75, #9)