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Biochemical Pharmacology (v.82, #1)
Insights from the structure of estrogen receptor into the evolution of estrogens: Implications for endocrine disruption by Michael E. Baker (pp. 1-8).
Human ERα has conformational flexibility that permits binding of chemicals with diverse structures.In the last decade, there has been important progress in understanding the origins and evolution of receptors for adrenal steroids (aldosterone, cortisol) and sex steroids (estradiol, progesterone, testosterone) due to the sequencing of genomes from animals that are at key sites in vertebrate evolution. Although the estrogen receptor [ER] appears to be the ancestral vertebrate steroid receptor and estradiol [E2] is the physiological ligand for vertebrate ERs, the identity of the ancestral ligand(s) for the ER remains unknown. Here, using an analysis of crystal structures of human ERα with E2 and other chemicals and 3D models of human ERα with 27-hydroxycholesterol and 5-androsten-3β,17β-diol, I propose that one or more Δ5 steroids were the ancestral ligands for the ER, with E2 evolving later as the canonical estrogen. The evidence that chemicals with a β-hydroxy at C3 in a saturated A ring can act as estrogens and the conformational flexibility of the vertebrate ER can explain the diversity of synthetic chemicals that disrupt estrogen responses by binding to vertebrate ERs.
Keywords: Evolution; Ancestral estrogen; Endocrine disruptors; Estrogen receptor; Hydroxy-cholesterol
From differential induction of UDP-glucuronosyltransferases in rat liver to characterization of responsible ligand-activated transcription factors, and their multilevel crosstalk in humans by Karl Walter Bock (pp. 9-16).
UDP-glucuronosyltransferases (UGTs) catalyze a major Phase II reaction in the endo- and xenobiotic-metabolizing enzyme (XME) system consisting of Phases I–III proteins and ligand-activated transcription factors. Differential induction of liver microsomal CYP activities following treatment of rats with aryl hydrocarbons or phenobarbital, discovered over 50 years ago, initiated studies to characterize multiple CYPs and the transcription factors Ah receptor (AhR) and CAR, respectively. Similar studies of UGT activities initiated studies of multiple UGTs. However, inducible human UGTs differed from those in rats. In addition, induction of UGTs is complicated, for example, by coordinate regulation of some XMEs by AhR and the antioxidant Nrf2 transcription factor. Functions of UGTs in the XME system are discussed using the following examples: (i) Tight coupling between Phase I and II enzymes in benzo[a]pyrene detoxification. In particular, AhR- and Nrf2-controlled quinone reductases and UGTs may prevent quinone–quinol redox cycling with generation of oxidative stress. (ii) CAR-mediated induction of UGT1A1 may be involved in perinatal detoxification of bilirubin neurotoxicity. (iii) PPARα-mediated glucuronidation of eicosanoids may contribute to their detoxification and homeostasis. Identification of the role of UGTs is challenged by intense crosstalk of transcription factors at the genetic level, the level of protein–protein interaction and control by signaling networks. Nevertheless, as drug targets ligand-activated transcription factors provide promising therapeutic possibilities.
Keywords: Abbreviations; AhR; Ah receptor; BaP; benzo[a]pyrene; CAR; constitutive androstane receptor; CYP; cytochrome P450; GR; glucocorticoid receptor; GST; glutathione S-transferase; gtPBREM; glucuronyltransferase Phenobarbital-Responsive Enhancer Module; HETE; hydroxyeicosatetraenoic acid; LATFs; ligand-activated transcription factors; LTB4; leucotriene B4; Nrf2; nuclear erythroid-related factor 2; PB; phenobarbital; PXR; pregnane X receptor; PPARα; peroxisome proliferator-activated receptor α; ROS; reactive oxygen species; UGT; UDP-glucuronosyltransferase; XME; xenobiotic-metabolizing enzymeHepatic UGTs; Induction; Ligand-activated transcription factors; Ah receptor; CAR/PXR; Crosstalk
Dendrimers-delivered short hairpin RNA targeting hTERT inhibits oral cancer cell growth in vitro and in vivo by Xiqiang Liu; Hongzhang Huang; Jianguang Wang; Cheng Wang; Miao Wang; Bin Zhang; Chaobin Pan (pp. 17-23).
Promising therapeutic application of RNA interference (RNAi) depends on the availability of safe and efficient intracellular delivery systems. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase complex, is an attractive therapeutic target for oral cancer. Here we investigated the characteristics and anticancer effect of polyamidoamine (PAMAM) dendrimer-mediated short hairpin RNA (shRNA) against hTERT in oral cancer. Dendrimer-mediated shRNA efficiently silenced the hTERT gene in vitro, resulted in cell growth inhibition and apoptosis. Treatment with the shRNA dendriplex attenuated tumor growth in a xenograft model. These studies suggest that RNAi-mediated hTERT gene silencing, coupled with dendrimer delivery, may provide a promising approach for the treatment of oral cancer, in which hTERT is abundantly expressed.
Keywords: Human telomerase reverse transcriptase; Dendrimer; Gene delivery; Oral cancer; RNA interference
MDM2 antagonist nutlin-3a reverses mitoxantrone resistance by inhibiting breast cancer resistance protein mediated drug transport by Fan Zhang; Stacy L. Throm; Laura L. Murley; Laura A. Miller; D. Steven Zatechka Jr.; R. Kiplin Guy; Rachel Kennedy; Clinton F. Stewart (pp. 24-34).
Nutlin-3a increases the intracellular accumulation of anti-cancer drugs (D) that are BCRP substrates and enhances cell death.Breast cancer resistance protein (BCRP; ABCG2), a clinical marker for identifying the side population (SP) cancer stem cell subgroup, affects intestinal absorption, brain penetration, hepatobiliary excretion, and multidrug resistance of many anti-cancer drugs. Nutlin-3a is currently under pre-clinical investigation in a variety of solid tumor and leukemia models as a p53 reactivation agent, and has been recently demonstrated to also have p53 independent actions in cancer cells. In the present study, we first report that nutlin-3a can inhibit the efflux function of BCRP. We observed that although the nutlin-3a IC50 did not differ between BCRP over-expressing and vector control cells, nutlin-3a treatment significantly potentiated the cells to treatment with the BCRP substrate mitoxantrone. Combination index calculations suggested synergism between nutlin-3a and mitoxantrone in cell lines over-expressing BCRP. Upon further investigation, it was confirmed that nutlin-3a increased the intracellular accumulation of BCRP substrates such as mitoxantrone and Hoechst 33342 in cells expressing functional BCRP without altering the expression level or localization of BCRP. Interestingly, nutlin-3b, considered virtually “inactive” in disrupting the MDM2/p53 interaction, reversed Hoechst 33342 efflux with the same potency as nutlin-3a. Intracellular accumulation and bi-directional transport studies using MDCKII cells suggested that nutlin-3a is not a substrate of BCRP. Additionally, an ATPase assay using Sf9 insect cell membranes over-expressing wild-type BCRP indicated that nutlin-3a inhibits BCRP ATPase activity in a dose-dependent fashion. In conclusion, our studies demonstrate that nutlin-3a inhibits BCRP efflux function, which consequently reverses BCRP-related drug resistance.
Keywords: Nutlin-3a; Breast cancer resistance protein (BCRP); ABC transporter; Multi-drug resistance
UBE2M-mediated p27Kip1 degradation in gemcitabine cytotoxicity by A-Mei Huang; Yu-Ting Kao; Shinzao Toh; Pin-Yi Lin; Chi-Hsien Chou; Huei-Ting Hu; Chi-Yu Lu; Jieh-Yuan Liou; Shih-Yi Chao; Tzyh-Chyuan Hour; Yeong-Shiau Pu (pp. 35-42).
Gemcitabine (2′-deoxy-2′, 2′-difluorocytidine; Gem) is a nucleoside anti-metabolite and is commonly used for treating various human cancers including human bladder carcinoma. Gemcitabine not only functions as a suicide nucleoside analog but also inhibits DNA polymerase activity and results in the termination of chain elongation. Using 2-dimensional gel electrophoresis analysis, a Gem-induced protein was identified as UBE2M (a.k.a. UBC12), a NEDD8 conjugation E2 enzyme which contributes to protein degradation. Gem induced UBE2M expression at both RNA and protein levels in several human cancer cell lines. The induction of UBE2M by Gem was accompanied by a reduction in p27Kip1 protein levels, which could be restored by silencing UBE2M expression with siRNA or by treating cells with the proteasome inhibitor MG132, indicating that UBE2M mediates Gem-induced p27Kip1 protein degradation. The induction of UBE2M and reduction of p27Kip1 by Gem were prevented by the PI3K inhibitor LY294002. These results indicate that PI3K activity is necessary for Gem-induced UBE2M expression and that UBE2M facilitates degradation of p27Kip1. Notably, silencing of UBE2M expression reduced Gem sensitivity in NTUB1 cells, suggesting that UBE2M mediates in part cell sensitivity to Gem, possibly by degradation of p27Kip1. Analysis of Gem-resistant sub lines also showed that loss of UBE2M and increased p27Kip1 expression were associated with the acquisition of drug resistance. In conclusion, our results demonstrate a role for UBE2M in mediating cytotoxicity of gemcitabine in human urothelial carcinoma cells while also suggesting a potential function of p27Kip1 in drug resistance.
Keywords: Abbreviations; UBE2M; ubiquitin-conjugating enzyme E2M; Gemcitabine (Gem); 2′-deoxy-2′, 2′-difluorocytidine; NEDD8; neural precursor cell-expressed developmentally down-regulated 8; SRM; spermidine synthase; PMVK; phosphomevalonate kinase; DLST; dihydrolipoamide S-succinyltransferase; UROD; uroporphyrinogen decarboxylase; FASN; fatty acid synthase; PSMB6; proteasome (prosome, macropain) subunit, beta type, 6Protein degradation; Anti-metabolite drug; Cell cycle inhibitor; Drug resistance; Urothelial carcinoma
Development of screening assays and discovery of initial inhibitors of pneumococcal peptidoglycan deacetylase PgdA by Nhat Khai Bui; Samo Turk; Stephan Buckenmaier; Flint Stevenson-Jones; Benjamin Zeuch; Stanislav Gobec; Waldemar Vollmer (pp. 43-52).
Inhibitory compounds of peptidoglycan deacetylase PgdA identified by virtual HTS and confirmed by novel enzyme assay.The essential cell wall peptidoglycan is the target of several components of the innate immune system and its disruption results in lysis of invading bacteria. The pathogen Streptococcus pneumoniae produces a peptidoglycan N-acetylglucosamine deacetylase, PgdA, to modify the peptidoglycan structure. The activity of PgdA contributes to the bacteria's resistance to lysozyme, which is an important antimicrobial factor of the human innate immune system. In this study we report on the activity of PgdA against natural and artificial substrates. We have also established a virtual high-throughput screening and a new enzyme assay to search for compounds inhibiting PgdA. Two compounds with IC50 values in the micromolar range have been identified and they could serve as leads for the search of inhibitors of PgdA, an important pneumococcal virulence factor.
Keywords: Abbreviations; PG; peptidoglycan; GlcNAc; N-acetylglucosamine; MurNAc; N-acetylmuramic acid; PgdA; peptidoglycan N-acetylglucosamine deacetylase A; IC; 50; half maximal inhibitory concentration; IPTG; isopropyl-β-; d; -thiogalactopyranoside; CTAB; cetyltrimethylammonium bromide; GlcNAc; 5; N,N,N,N,N-pentaacetylchitopentaose; pNPA; p-nitrophenyl acetate; αNAα; -napthyl acetate; 4MUA; 4-methylumbelliferyl acetate; DMSO; dimethylsulfoxidePeptidoglycan deacetylase; PgdA; Lysozyme resistance; Virtual screening
Cytochrome P4504f, a potential therapeutic target limiting neuroinflammation by Neha Sehgal; Varsha Agarwal; Rupanagudi Khader Valli; Shanker Dutt Joshi; Leposava Antonovic; Henry W. Strobel; Vijayalakshmi Ravindranath (pp. 53-64).
LPS binds to pattern recognition receptors triggering inflammatory cascade, LTB4 extruded from microglia through BLT receptors enters neurons amplifying the response. Brain cytochromes P4504f resolve neuroinflammation through LTB4 hydroxylation.Inflammatory processes are involved in the pathogenesis and/or progression of acute central nervous system (CNS) infection, traumatic brain injury and neurodegenerative disorders among others indicating the need for novel strategies to limit neuroinflammation. Eicosanoids including leukotrienes, particularly leukotriene B4 (LTB4) are principle mediator(s) of inflammatory response, initiating and amplifying the generation of cytokines and chemokines. Cytochrome P450 (Cyp), a family of heme proteins mediate metabolism of xenobiotics and endogenous compounds, such as eicosanoids and leukotrienes. Cytochrome P4504F (Cyp4f) subfamily includes five functional enzymes in mouse. We cloned and expressed the mouse Cyp4f enzymes, assayed their relative expression in brain and examined their ability to hydroxylate the inflammatory cascade prompt LTB4 to its inactive 20-hydroxylated product. We then examined the role of Cyp4fs in regulating inflammatory response in vitro, in microglial cells and in vivo, in mouse brain using lipopolysacharide (LPS), as a model compound to generate inflammatory response. We demonstrate that mouse brain Cyp4fs are expressed ubiquitously in several cell types in the brain, including neurons and microglia, and modulate inflammatory response triggered by LPS, in vivo and in microglial cells, in vitro through metabolism of LTB4 to the inactive 20-hydroxy LTB4. Chemical inhibitor or shRNA to Cyp4fs enhance and inducer of Cyp4fs attenuates inflammatory response. Further, induction of Cyp4f expression lowers LTB4 levels and affords neuroprotection in microglial cells or mice exposed to LPS. Thus, catalytic activity of Cyp4fs is a novel target for modulating neuroinflammation through hydroxylation of LTB4.
Keywords: Neuroinflammation; Microglia; Cytokines; Cytochrome P450; Leukotriene
Role of Ca2+-activated K+ channels and Na+,K+-ATPase in prostaglandin E1- and E2-induced inhibition of the adrenergic response in human vas deferens by Pascual Medina; Gloria Segarra; María Dolores Mauricio; José M. Vila; Pascual Chuan; Salvador Lluch (pp. 65-71).
We studied the role of K+ channels and Na+,K+-ATPase in the presynaptic inhibitory effects of prostaglandin E1 (PGE1) and PGE2 on the adrenergic responses of human vas deferens. Furthermore, we determined the effects of increasing extracellular K+ concentrations ([K+]o) and inhibition of Na+,K+-ATPase on neurogenic and norepinephrine-induced contractile responses. Ring segments of the epididymal part of the vas deferens were taken from 45 elective vasectomies and mounted in organ baths for isometric recording of tension. The neuromodulatory effects of PGEs were tested in the presence of K+ channel blockers. PGE1 and PGE2 (10−8 to 10−6M) induced inhibition of adrenergic contractions. The presence of tetraethylammonium (10−3M), charybdotoxin (10−7M), or iberiotoxin (10−7M), prevented the inhibitory effects of PGE1 and PGE2 on the adrenergic contraction. Both glibenclamide (10−5M) and apamin (10−6M) failed to antagonize PGE1 and PGE2 effects. Raising the [K+]o from 15.8mM to 25.8mM caused inhibition of the neurogenic contractions. Ouabain at a concentration insufficient to alter the resting tension (10−6M) increased contractions induced by electrical stimulation but did not alter the contractions to norepinephrine. The inhibition of neurogenic responses induced PGE1, PGE2 and increased extracellular concentration of K+ was almost completely prevented by ouabain (10−6M). The results demonstrate that PGE1 and PGE2 inhibit adrenergic responses by a prejunctional mechanism that involves the activation of large-conductance Ca2+-activated K+ channels and Na+,K+-ATPase.
Keywords: Electrical field stimulation; Human vas deferens; Norepinephrine; Potassium channels; Prostaglandins; Smooth muscle
Neurotoxicity of methotrexate to hippocampal cells in vivo and in vitro by Miyoung Yang; Joong-Sun Kim; Juhwan Kim; Sung-Ho Kim; Jong-Choon Kim; Jinwook Kim; Hongbing Wang; Taekyun Shin; Changjong Moon (pp. 72-80).
This study investigated whether methotrexate (MTX) is neurotoxic to neural progenitor cells in the hippocampus of adult mice and whether it affects hippocampus-dependent behaviors. In addition, the cytotoxicity of MTX was elucidated in rat immature and mature hippocampal cultured cells. The number of Ki-67 (proliferating cell marker)- and doublecortin (immature progenitor neuron marker)-positive cells were significantly time- and dose-dependently changed in the dentate gyrus of adult hippocampi after MTX treatment. A learning and memory task (object recognition memory test) and depression-like behavior test (tail-suspension test) were performed after MTX treatment to assess hippocampal neurogenesis-related behavioral dysfunction. MTX-treated mice showed significant depression-like behaviors and memory defects. The cytotoxicity of MTX in immature hippocampal cells varied in a dose-dependent pattern, but was not changed in the mature cells. MTX induced marked apoptotic changes in immature hippocampal cells, with increase in active caspase-3 and cleaved poly (ADP-ribose) polymerase expressions. Results of this study suggest that the neurotoxic effect of MTX inhibits the proliferation of hippocampal progenitor cells and can cause hippocampal dysfunction, such as depression and cognitive impairment. Additionally, the significantly greater caspase-dependent MTX sensitivity of immature hippocampal cells suggests that the susceptibility of such hippocampal cells depends on their maturation.
Keywords: Methotrexate; Neurotoxicity; Neural progenitor cell; Hippocampal cell; Behavior
A bifunctional sea anemone peptide with Kunitz type protease and potassium channel inhibiting properties by Steve Peigneur; Bert Billen; Rita Derua; Etienne Waelkens; Sarah Debaveye; László Béress; Jan Tytgat (pp. 81-90).
Sea anemone venom is a known source of interesting bioactive compounds, including peptide toxins which are invaluable tools for studying structure and function of voltage-gated potassium channels. APEKTx1 is a novel peptide isolated from the sea anemone Anthopleura elegantissima, containing 63 amino acids cross-linked by 3 disulfide bridges. Sequence alignment reveals that APEKTx1 is a new member of the type 2 sea anemone peptides targeting voltage-gated potassium channels (KVs), which also include the kalicludines from Anemonia sulcata. Similar to the kalicludines, APEKTx1 shares structural homology with both the basic pancreatic trypsin inhibitor (BPTI), a very potent Kunitz-type protease inhibitor, and dendrotoxins which are powerful blockers of voltage-gated potassium channels. In this study, APEKTx1 has been subjected to a screening on a wide range of 23 ion channels expressed in Xenopus laevis oocytes: 13 cloned voltage-gated potassium channels (KV1.1–KV1.6, KV1.1 triple mutant, KV2.1, KV3.1, KV4.2, KV4.3, hERG, the insect channel Shaker IR), 2 cloned hyperpolarization-activated cyclic nucleotide-sensitive cation non-selective channels (HCN1 and HCN2) and 8 cloned voltage-gated sodium channels (NaV1.2–NaV1.8 and the insect channel DmNaV1). Our data show that APEKTx1 selectively blocks KV1.1 channels in a very potent manner with an IC50 value of 0.9nM. Furthermore, we compared the trypsin inhibitory activity of this toxin with BPTI. APEKTx1 inhibits trypsin with a dissociation constant of 124nM. In conclusion, this study demonstrates that APEKTx1 has the unique feature to combine the dual functionality of a potent and selective blocker of KV1.1 channels with that of a competitive inhibitor of trypsin.
Keywords: Abbreviations; APEKTx1; Anthopleura elegantissima; potassium channel toxin 1; MALDI TOF; matrix-assisted laser desorption–ionization time of flight; RP-HPLC; reversed-phase high performance liquid chromatography; TFA; trifluoroacetic acid; K; V; channel; voltage-gated potassium channel; DTX; dendrotoxin; BPTI; bovine pancreatic trypsin inhibitor Anthopleura elegantissima; K; V; channel inhibitor; Sea anemone toxin; Protease inhibitor