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Biochemical Pharmacology (v.73, #11)
CagA protein of Helicobacter pylori: A hijacker of gastric epithelial cell signaling by Osamu Handa; Yuji Naito; Toshikazu Yoshikawa (pp. 1697-1702).
Epidemiological study has shown strong correlation between the Helicobacter pylori ( H. pylori) infection and gastric carcinogenesis. However, the mechanism by which H. pylori induces gastric carcinogenesis is not known. In this review, we focused on the product of cytotoxin-associated gene A (CagA), one of the important virulence factors of H. pylori. H. pylori injects CagA protein into the host gastric epithelial cells through its needle-like structure, type IV secretion system. Injected CagA hijacks physiological signal transduction and causes pathological cellular response such as increased cell proliferation, motility, apoptosis and morphological change through different mechanisms. H. pylori has been shown to produce reactive oxygen species (ROS) in infected gastric mucosa. Although the main source of ROS production is possibly host neutrophil, we propose novel source of ROS production in this review; CagA itself can induce ROS production in gastric epithelial cell. Excessive ROS production in gastric epithelial cells can cause DNA damage and thus might involve in gastric carcinogenesis. Understanding the molecular mechanism by which H. pylori-induced carcinogenesis is important for developing new strategies against gastric cancer.
Keywords: Phosphorylation; Mitochondria; DNA damage; Signal transduction; Carcinogenesis
Down-regulation of human telomerase reverse transcriptase through specific activation of RNAi pathway quickly results in cancer cell growth impairment by Paolo Gandellini; Marco Folini; Roberto Bandiera; Michelandrea De Cesare; Mara Binda; Silvio Veronese; Maria Grazia Daidone; Franco Zunino; Nadia Zaffaroni (pp. 1703-1714).
Targeting of human telomerase reverse transcriptase (hTERT) by different small interfering RNAs (siRNAs) resulted in a variable degree of telomerase activity inhibition in PC-3 and DU145 prostate cancer cells. In addition, transfection with siRNA5 and siRNA41, which caused high levels (∼80 and ∼55%, respectively) of enzyme activity inhibition in both cell lines, led to a marked reduction of hTERT mRNA and protein expression and a significant inhibition of cell proliferation within a few days, without concomitant telomere shortening or telomeric 3′ overhang impairment. Such an antiproliferative effect was not ascribable to the activation of non-specific responses, since siRNA5 and siRNA41 did not induce the expression of 2′–5′ oligoadenylate synthetase-1 and were able to cause a significant growth impairment also in HCT 116 colon cancer cells, which have a defective interferon pathway. Cell growth inhibition was indeed associated with hTERT down-regulation, as it was almost completely rescued in siRNA-treated HCT 116 cells co-transfected with an hTERT-expressing vector. Moreover, siRNA5 and siRNA41 failed to affect the proliferation of hTERT-negative U2-OS osteosarcoma cells. Interestingly, transfection with siRNA5 significantly reduced the tumorigenic and growth potential of PC-3 cells when xenotransplanted into nude mice. Such data suggest siRNA-mediated hTERT down-regulation as an efficient strategy to impair prostate cancer cell growth.
Keywords: RNA interference; Small interfering RNA; Telomerase; hTERT; Prostate cancer; Cell growth
Inhibition of G1/S transition potentiates oxaliplatin-induced cell death in colon cancer cell lines by Tatiana V. Rakitina; Irina A. Vasilevskaya; Peter J. O’Dwyer (pp. 1715-1726).
In a series of colorectal cancer cell lines, both necrosis and apoptosis were induced upon exposure to oxaliplatin, and enhanced by co-administration of the Hsp90 inhibitor 17-AAG. We analyzed the effects of these interventions on the cell cycle, and found that oxaliplatin treatment caused G1 and G2 arrest in HCT116 cells, and S-phase accumulation in two p53-deficient cell lines (HT29 and DLD1). Addition of 17-AAG enhanced cell cycle effects of oxaliplatin in HCT116, and induced G1 arrest and decrease in S-phase population in the other cell lines. Analysis of cell cycle proteins revealed that the major difference between the cell lines was that in HCT116, 17-AAG resulted in profound inhibition of expression and phosphorylation of late G1 proteins cyclin E and cdk2, with no effect on p21/WAF1 induction. Consistent with these, an HCT116 p53−/− line, lacking p21, showed resistance to oxaliplatin, failure to enter apoptosis, and an accumulation of cells in S-phase. Introduction of p21 in these cells caused reversal of that phenotype, including restoration of the G1 block and re-sensitization to oxaliplatin. Inhibition of G1/S progression using cdk2 inhibitor also enhanced oxaliplatin cytotoxicity. We conclude that in colon cancer cells with impaired p53 function, interventions directed to cycle arrest in G1 may potentiate oxaliplatin activity.
Keywords: Oxaliplatin; 17-AAG; Cell death; p21; Cell cycle; Cdk modulators
Modulation of GSH levels in ABCC1 expressing tumor cells triggers apoptosis through oxidative stress by Rémi-Martin Laberge; Joel Karwatsky; Maximilian C. Lincoln; Mara L. Leimanis; Elias Georges (pp. 1727-1737).
The over-expression of ABCC1 transmembrane protein has been shown to cause multidrug resistance in tumor cell lines. ABCC1 is a member of the ABC transmembrane proteins that function as efflux pumps with diverse substrate specificity. Several endogenous cell metabolites, including the leukotriene C4 (LTC4) and glutathione (GSH) are substrates for ABCC1 protein. ABCC1 expression in certain tumor cells was demonstrated to confer hypersensitivity to glutathione modulating agents. In this report we have investigated the mechanism of collateral sensitivity seen in tumor cells over-expressing ABCC1 protein. The results of this study show that ABCC1 expression in tumor cells correlates with their hypersensitivity to various glutathione modulating agents, as demonstrated in H69AR-drug selected and HeLa/ABCC1-transfectant cells. This effect was triggered either through inhibition of GSH synthesis with BSO or by increasing ABCC1-mediated GSH transport with verapamil or apigenin. In addition, our results show that the hypersensitivity of ABCC1-expressing cells to BSO, verapamil or apigenin was preceded by an increase in reactive oxygen species (or ROS). A decrease in GSH level is also observed prior the increase in ROS. In addition, we show that hypersensitivity to the BSO, verapamil or apigenin leads to tumor cell death by apoptosis. Together, the results of this study demonstrate that ABCC1 potentiates oxidative stress in tumor cells through reductions in cellular GSH levels.
Keywords: Abbreviations; MRP1 or ABCC1; multidrug resistance associated protein; ROS; reactive oxygen species; GSH; glutathione; ABC; ATP binding cassette; MDR; multidrug resistance; BSO; l; -buthionine (; S; ,; R; )-sulfoximine; API; apigenin; VRP; verapamil; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideReactive oxygen species; Glutathione; ATP binding cassette; Multidrug resistance; l; -Buthionine (; S; ,; R; )-sulfoximine; Apigenin
HIV-1 protease inhibitors do not interfere with provirus transcription and host cell apoptosis induced by combined treatment TNF-α+TSA by Claire Vandergeeten; Vincent Quivy; Michel Moutschen; Carine Van Lint; Jacques Piette; Sylvie Legrand-Poels (pp. 1738-1748).
HIV-1 latency represents a major hurdle to the complete eradication of the virus from patients under highly active anti-retroviral therapy (HAART) regimens. One solution to this problem would be to eliminate the latently infected cellular reservoirs by forcing gene expression in presence of HAART to prevent spreading of the infection by the newly synthesized viruses. Many studies have reported that a combination of a histone deacetylase inhibitor (HDACi) (i.e. TSA, NaBut, Valproic acid, …) with a pro-inflammatory cytokine (i.e. TNFα, IL-1, …) reactivates in a synergistic manner HIV-1 transcription in latently infected cells. The aim of the present study was to determine whether HIV-1 protease inhibitors (PIs) used in HAART (such as Saquinavir, Indinavir, Nelfinavir, Lopinavir, Ritonavir and Amprenavir) could interfere with the potential purge of the cellular reservoirs induced by a combined treatment involving TSA and TNFα. We showed, in two HIV-1 latently infected cell lines (ACH-2 and U1) that all PIs efficiently inhibited release of mature viral particles but did neither affect cell apoptosis nor NF-κB induction and HIV-1 transcription activation following combined treatment with TNFα+TSA. This study is encouraging in the fight against HIV-1 and shows that PIs should be compatible with an inductive adjuvent therapy for latent reservoir reduction/elimination in association with efficient HAART regimens.
Keywords: HIV-1 protease inhibitor; NF-κB; ApoptosisAbbreviations; HIV-1; human immunodeficiency virus type 1; LTR; long terminal repeat; HDACi; histone deacetylase inhibitor; TNFα; tumor necrosis factor alpha; IL-1; interleukin-1; HAART; highly active anti-retroviral therapy; PI; protease inhibitor; TSA; trichostatin A; CPT; camptothecin; EMSA; electrophoretic mobility shift assay; SQV; saquinavir; IND; indinavir; NFV; nelfinavir; LPV; lopinavir; RTV; ritonavir; APV; amprenavir
Promotion of DNA strand breaks, interstrand cross-links and apoptotic cell death in A2780 human ovarian cancer cells by transplatinum planar amine complexes by Sheena M. Aris; David A. Gewirtz; John J. Ryan; Kenneth M. Knott; Nicholas P. Farrell (pp. 1749-1757).
Cisplatin is one of the primary drugs utilized in the treatment of ovarian cancer. However, despite the initial effectiveness of chemotherapy in suppressing this disease, drug resistance almost invariably develops and cures are relatively rare. While it is generally thought that only compounds of the cis geometry express antitumor activity, a number of transplatinum derivates have shown preclinical promise. The current work investigates the influence of transplanaramine (TPA) compounds of structure trans-[Pt (O2CR)2 (L) (L′)], (L=NH3, L′=pyridine, quinoline, isoquinoline; L=L′=pyridine; R=H, CH3, CH2OH, etc.) (with a focus on the contribution of the carboxylate leaving group to drug action) on growth and viability of A2780 human ovarian carcinoma cells as well as their putative mechanism(s) of cytotoxicity. The compounds, as a class, induce cell death through caspase-dependent apoptosis, with activation of both caspase 3 and caspase 9 and concomitant PARP cleavage. The trans-platinum compounds tested show induction of p53 as well as time dependent γH2AX induction, consistent with the promotion of DNA lesions. trans-[Pt(O2CH)2(NH3)(4-pic)] can be shown to promote significant DNA strand breaks and DNA interstrand cross-linking. The enhanced cytotoxicity of trans-[Pt(O2CH)2(NH3)(4-pic)] compared to its isostructural –O2CCH3 and –O2CCH2OH analogs may be a consequence of its accelerated cellular accumulation, increased hydrolytic activation, interstrand cross-linking and abortive efforts by the cell to repair the cross linked DNA.
Keywords: DNA strand breaks; Interstrand cross-links; Apoptotic cell death; A2780 human ovarian cancer cells; Transplatinum planar amine complexes
Regulatory mechanisms underlying GKR2 levels in U937 cells: Evidence for GRK3 involvement by Natalia Fernandez; Federico Monczor; Maria R. Tubio; Carina Shayo; Carlos Davio (pp. 1758-1767).
G protein-coupled receptors represent the most diverse group of proteins involved in transmembrane signalling, that participate in the regulation of a wide range of physicochemical messengers through the interaction with heterotrimeric G proteins. In addition, GPCRs stimulation also triggers a negative feedback mechanism, known as desensitization that prevents the potentially harmful effects caused by persistent receptor stimulation. In this adaptative response, G protein-coupled receptor kinases (GRKs) play a key role and alterations in their function are related to diverse pathophysiological situations. Based on the scarce knowledge about the regulation of GRK2 by other kinases of the same family, the aim of the present work was to investigate the regulation of GRK2 levels in systems where other GRKs are diminished by antisense technique. Present findings show that in U937 cells GRK2 levels are regulated by GRK3 and not by GRK6 through a mechanism involving InsP upregulation. This work reports a novel GRK3-mediated GRK2 regulatory mechanism and further suggests that GRK2 may also act as a compensatory kinase tending to counterbalance the reduction in GRK3 levels. This study provides the first evidence for the existence of GRKs cross-regulation.
Keywords: GRK2; GRK3; Inositol phosphate; GPCR; Receptor desensitizationAbbreviations; GPCR; G protein-coupled receptor; GRK; G protein-coupled receptor kinase; G-418; geneticin; IBMX; isobutylmethylxanthine; BSA; bovine serum albumin; PBS; phosphate-buffered saline; InsP; inositol phosphate; amthamine; 2-amino-4-methylthiazole-5-ethanamine; PDE; phosphodiesterase; RPMI; Roswell Park Memorial Institute
Curcumin induces electrical activity in rat pancreatic β-cells by activating the volume-regulated anion channel by Leonard Best; Austin C. Elliott; Peter D. Brown (pp. 1768-1775).
Curcumin, the principal active component of turmeric, is reported to exert a number of therapeutic actions, including a hypoglycaemic/antidiabetic action. The underlying mechanisms to this action are essentially unknown. We have investigated the hypothesis that a direct stimulatory action on the pancreatic β-cell could contribute towards the hypoglycaemic activity of this compound. Electrical and ion channel activity were recorded in rat β-cells using the patch–clamp technique. β-Cell volume was measured using a video-imaging technique. Insulin release was measured from intact islets by radioimmunoassay. Curcumin (2–10μM) activated the volume-regulated anion channel in β-cells. Single channel studies indicated that activation was the result of increased channel open probability. This effect was accompanied by depolarisation of the cell membrane potential, the generation of electrical activity and enhanced insulin release. Curcumin also decreased β-cell volume, presumably reflecting loss of Cl− (and hence water) as a result of anion channel activation. These findings are consistent with the suggestion that Cl− fluxes play an important role in regulating β-cell function. The stimulation of β-cell function by curcumin could contribute to the hypoglycaemic actions of this compound, and these findings identify a novel potential therapeutic target for the treatment of type 2 diabetes mellitus.
Keywords: Abbreviations; NPPB; 5-nitro-2-(3-phenylpropylamino) benzoic acid; VRAC; volume-regulated anion channelCurcumin; Islet; Pancreatic beta cell; Volume-regulated anion channel; Chloride channel; Electrical activity; Insulin secretion
Involvement of reactive oxygen species on the apoptotic mechanism induced by IFN-α2b in rat preneoplastic liver by Ariel D. Quiroga; María de Luján Alvarez; Juan P. Parody; María Teresa Ronco; Daniel E. Francés; Gerardo B. Pisani; Cristina E. Carnovale; María Cristina Carrillo (pp. 1776-1785).
Interferon-α2b (IFN-α2b) is an important component in the preventive treatment of patients who have severe hepatic illness such as hepatitis B or C and hepatocarcinomas. In a previous work, using a rat liver preneoplastic model, we have demonstrated that IFN-α2b reduces the number and volume of altered hepatic foci (AHF) inducing apoptosis through a mechanism mediated by TGF-β1. In this study, the implication of hepatocytes redox status of IFN-α2b-treated preneoplastic liver in the TGF-β1-induced apoptotic death was analyzed. Results indicate that IFN-α2b induces hepatocytic TGF-β1 production and secretion by induction of reactive oxygen species (ROS) formation through the activation of a membrane bound NADPH oxidase complex. TGF-β1, in turn, reduces hepatocytes antioxidant defenses and induces programmed cell death. On the other hand, it was also demonstrated that treatment of rats with IFN-α2b plus a ROS scavenger such as ascorbic acid, abolishes the apoptotic effect of IFN-α2b in rat preneoplastic livers, leading to an increase of the foci volume. In conclusion, these findings strongly suggest that ROS have a fundamental role as signaling and/or regulator molecules in the IFN-α2b-induced apoptosis in hepatic preneoplastic cells.
Keywords: Abbreviations; IFN-α2b; interferon alfa-2b; TGF-β; 1; transforming growth factor-β; 1; ROS; reactive oxygen species; SOD; superoxide dismutase; CAT; catalase; tGSH; total intracellular glutathione; GSSG; oxidize glutathione; IP; initiation-promotion; ASC; ascorbic acid; DPI; diphenyleneiodonium; RT-PCR; reverse transcription polymerase chain reaction; GCL; glutamate cysteine ligase; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; DCFH-DA; 2′,7′-dichlorofluorescin diacetate; rGST P; placental form of rat glutathione S-transferase; AHF; altered hepatic foci; PI; proliferative index; PCNA; proliferating cell nuclear antigen; TUNEL; terminal deoxynucleotidyl transferase mediated biotin-deoxyuridine triphosphate nick-end labeling; AI; apoptotic indexJEL classification; Gastrointestinal pharmacologyHepatocarcinogenesis; Apoptosis; Oxidative stress; Transforming growth factor-β; 1; NADPH oxidase
Dihydrolipoic acid inhibits skin tumor promotion through anti-inflammation and anti-oxidation by Yuan-Soon Ho; Ching-Shu Lai; Hsin-I Liu; Sheng-Yow Ho; Chein Tai; Min-Hsiung Pan; Ying-Jan Wang (pp. 1786-1795).
α-Lipoic acid (LA) has been intensely investigated as a therapeutic agent for several diseases, including hepatic disorder and diabetic polyneuropathy. However, the effects of LA or its reduced form, dihydrolipoic acid (DHLA), on cancer chemoprevention has never been reported. In the present study, we examined the effects of DHLA/LA on the production of nitric oxide (NO) by inducible NO synthase (iNOS) and the formation of prostaglandin E2 (PGE2) by cyclooxygenase-2 (COX-2), two important mediators associated with inflammation. DHLA/LA significantly inhibited lipopolysaccharide (LPS)-induced NO and PGE2 formation in RAW 264.7 cells. Meanwhile, treatment with DHLA/LA suppressed the expression of iNOS protein but, unexpectedly, did not affect or increase the expression of COX-2 protein. The in vivo anti-inflammatory and antitumor-promoting activities were evaluated by a topical 12- O-tetradecanoylphorbol 13-acetate (TPA) application to mouse skin with measurement of edema formation, epidermal thickness and hydrogen peroxide production. DHLA significantly inhibited the priming and activation stages of skin inflammation induced by a double TPA application, by decreasing the inflammatory parameters. Furthermore, DHLA inhibited DMBA (0.3μmol)/TPA (2.0nmol)-induced skin tumor formation by reducing the tumor incidence and tumor multiplicity. When applied topically onto the shaven backs of mice prior to TPA, DHLA markedly inhibited the expression of iNOS protein. DHLA also strongly and directly inhibited COX-2 activity. These results suggest that DHLA can be a possible chemopreventive agent in inflammation-associated tumorigenesis.
Keywords: α-Lipoic acid (LA); Dihydrolipoic acid (DHLA); Cancer chemoprevention; Oxidative stress; Inflammation; Tumor promotion
Inhibition of lipopolysaccharide-stimulated NO production by a novel synthetic compound CYL-4d in RAW 264.7 macrophages involving the blockade of MEK4/JNK/AP-1 pathway by Meng-Wei Lin; Lo-Ti Tsao; Ling-Chu Chang; Ye-Long Chen; Li-Jiau Huang; Sheng-Chu Kuo; Cherng-Chyi Tzeng; Miau-Rong Lee; Jih-Pyang Wang (pp. 1796-1806).
In the present study, a novel synthetic compound 4-(2-(cyclohex-2-enylidene)hydrazinyl)quinolin-2(1 H)-one (CYL-4d) was found to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production without affecting cell viability or enzyme activity of expressed inducible NO synthase (iNOS) in RAW 264.7 macrophages. CYL-4d exhibited parallel inhibition of LPS-induced expression of iNOS protein, iNOS mRNA and iNOS promoter activity in the same concentration range. LPS-induced activator protein-1 (AP-1) DNA binding, AP-1-dependent reporter gene activity and c-Jun nuclear translocation were all markedly inhibited by CYL-4d with similar efficacy, whereas CYL-4d produced a weak inhibition of nuclear factor-κB (NF-κB) DNA binding, NF-κB-dependent reporter gene activity and p65 nuclear translocation without affecting inhibitory factor-κBα (IκBα) degradation. CYL-4d had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and its upstream activator MAPK kinase (MEK) 3, whereas it significantly attenuated the phosphorylation of c-Jun, c-Jun NH2-terminal kinase (JNK) and its upstream activator MEK4 in a parallel concentration-dependent manner. Other Toll-like receptors (TLRs) ligands (peptidoglycans, double-stranded RNA, and oligonucleotide containing unmethylated CpG motifs)-induced iNOS protein expression were also inhibited by CYL-4d. Furthermore, the NO production from BV-2 microglial cells as well as rat alveolar macrophages in response to LPS was diminished by CYL-4d. These results indicate that the blockade of NO production by CYL-4d in LPS-stimulated RAW 264.7 cells is attributed mainly to interference in the MEK4-JNK-AP-1 signaling pathway. CYL-4d inhibition of NO production is not restricted to TLR4 activation and immortalized macrophage-like cells.
Keywords: Abbreviations; AP-1; activator protein-1; COX; cyclooxygenase; CYL-4d; 4-(2-(cyclohex-2-enylidene)hydrazinyl)quinolin-2(1; H; )-one; ERK; extracellular signal-regulated kinase; LPS; lipopolysaccharide; IκBα; inhibitory factor-κBα; IKK; IκB kinase; iNOS; inducible NO synthase; JNK; c-Jun NH; 2; -terminal kinase; MAPK; mitogen-activated protein kinase; MEK; MAPK kinase; NF-κB; nuclear factor-κB; NO; nitric oxide; Q-RT-PCR; quantitative real-time reverse transcription-polymerase chain reaction; TLR; Toll-like receptorCYL-4d; RAW 264.7 macrophages; Nitric oxide; AP-1; NF-κB; BV-2 microglia; Alveolar macrophages
Kinetic analysis of reactivation and aging of human acetylcholinesterase inhibited by different phosphoramidates by Franz Worek; Nadine Aurbek; Marianne Koller; Christian Becker; Peter Eyer; Horst Thiermann (pp. 1807-1817).
The high number of fatalities due to poisoning by organophosphorus compound-based (OP) pesticides and the availability of highly toxic OP-type chemical warfare agents (nerve agents) emphasize the necessity for an effective medical treatment. Acute OP toxicity is mainly caused by inhibition of acetylcholinesterase (AChE, EC 3.1.1.7). Reactivators (oximes) of inhibited AChE are a mainstay of treatment. However, human AChE inhibited by certain OP, e.g. the phosphoramidates tabun and fenamiphos, is rather resistant towards reactivation by oximes while AChE inhibited by others, e.g. the phosphoramidate methamidophos is easily reactivated by oximes. To get more insight into a potential structure-activity relationship human AChE was inhibited by 16 different tabun analogues and the time-dependent reactivation by 1mM obidoxime, TMB-4, MMB-4, HI 6 or HLö 7, the reactivation kinetics of obidoxime and the kinetics of aging and spontaneous reactivation were investigated. A clear structure-activity relationship of aging, spontaneous and oxime-induced reactivation kinetics could be determined with AChE inhibited by N-monoalkyl tabun analogues depending on the chain length of the N-alkyl residue. N, N-dialkyl analogues bearing ethyl and n-propyl residues were completely resistant towards reactivation while N, N-di-i-propyl tabun was highly susceptible towards reactivation by oximes. AChE inhibited by phosphonoamidate analogues of tabun, bearing a N, N-dimethyl and N, N-diethyl group, could be reactivated and had comparable reactivation kinetics with obidoxime. These results in conjunction with previous data with organophosphates and organophosphonates emphasizes the necessity for kinetic studies as basis for future work on structural analysis with human AChE and for the development of effective broad-spectrum oximes.
Keywords: Acetylcholinesterase; Organophosphates; Oximes; Reactivation; Aging; Structure-activity relationshipAbbreviations; AChE; acetylcholinesterase (E.C. 3.1.1.7); BChE; butyrylcholinesterase (E.C. 3.1.1.8); ATCh; acetylthiocholine iodide; BTCh; S; -butyrylthiocholine iodide; DTNB; 5,5′-dithio-bis(-2-nitrobenzoic acid); obidoxime; 1,1′-(oxybis-methylene)bis[4-(hydroxyimino)methyl] pyridinium dichloride; pralidoxime; 2-[hydroxyimino methyl]-1-methylpyridinium chloride; HI 6; 1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2-[(hydroxyimino)methyl]pyridinium dichloride monohydrate; HLö 7; 1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2,4-bis-[(hydroxyimino)methyl] pyridinium dimethanesulfonate); TMB-4; 1,3-trimethylene-bis(4-hydroxyiminomethylpyridinium) dibromide; MMB-4; 1,1′-methylene-bis[4-(hydroxyimino)methyl] pyridinium dibromide
Effects of chronic opioid exposure on guinea pig mu opioid receptor in Chinese hamster ovary cells: Comparison with human and rat receptor by Michael Wallisch; Cole S. Nelson; Julia M. Mulvaney; Heather S. Hernandez; Sue Ann Smith; George D. Olsen (pp. 1818-1828).
Chronic opioid treatment leads to agonist-specific effects at the mu opioid receptor. The molecular mechanisms resulting from chronic opioid exposure include desensitization, internalization and down-regulation of membrane-bound mu opioid receptors (MOP). The purpose of this study was to compare the cellular regulation of guinea pig, human and rat MOP expressed in Chinese hamster ovary (CHO) cells, following exposure to two clinically important opioids, morphine and methadone. MOP expressing CHO cells were treated in culture with methadone or morphine for up to 48h. Radioligand diprenorphine and [d-AIa2, N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO)-stimulated GTPγS binding assays were carried out using paired control and opioid-exposed CHO cells. Methadone induced downregulation of the mu opioid receptor, while morphine induced desensitization of the receptor for all three species. Furthermore, morphine predominantly decreased the potency of DAMGO to stimulate GTPγS binding, whereas methadone primarily reduced its efficacy. Changes in DAMGO potency and efficacy differed among species and depended on the opioid used to treat the cells. Our results showed similarities between guinea pig and human MOP for morphine-induced desensitization, but identified differences between the two for methadone-induced desensitization. In contrast, human and rat MOP differed in response to morphine treatment, but were not distinct in their response to methadone treatment. The guinea pig is an excellent and established animal model to study opioid effects, but its molecular opioid pharmacology has not been investigated thus far. These results can assist in understanding species differences in the effects of opioid ligands activating the mu opioid receptor.
Keywords: Abbreviations; B; max; maximum receptor binding; CHO; Chinese hamster ovary; DAMGO; [; d; -AIa; 2; ,; N; -Me-Phe; 4; ,Gly; 5; -ol]-enkephalin; DPDPE; [; d; -Pen; 2,5; ]-enkephalin; E; max; maximum effect; gp; guinea pig; hu; human; K; i; inhibition constant; M3G; morphine-3-β-; d; -glucuronide; M6G; morphine-6-β-; d; -glucuronide; methadone refers to rac- methadone; MOP; mu opioid receptor; MOP-CHO; mu opioid receptor expressing Chinese hamster ovary cells; U50,488; trans-(±)-3,4-dichloro-; N; -methyl-; N; -[2-(I-pyrrolidinyl)cyclohexyl]-benzeneacetamideG protein coupled receptor; Down-regulation; Desensitization; GTPγS binding; Morphine; Methadone
Human glutathione transferases catalyzing the bioactivation of anticancer thiopurine prodrugs by Birgitta I. Eklund; Sjofn Gunnarsdottir; Adnan A. Elfarra; Bengt Mannervik (pp. 1829-1841).
cis-6-(2-Acetylvinylthio)purine (cAVTP) and trans-6-(2-acetylvinylthio)guanine (tAVTG) are thiopurine prodrugs provisionally inactivated by an α,β-unsaturated substituent on the sulfur of the parental thiopurines 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). The active thiopurines are liberated intracellularly by glutathione (GSH) in reactions catalyzed by glutathione transferases (GSTs) (EC 2.5.1.18). Catalytic activities of 13 human GSTs representing seven distinct classes of soluble GSTs have been determined. The bioactivation of cAVTP and tAVTG occurs via a transient addition of GSH to the activated double bond of the S-substituent of the prodrug, followed by elimination of the thiopurine. The first of these consecutive reactions is rate-limiting for thiopurine release, but GST-activation of this first addition is shifting the rate limitation to the subsequent elimination. Highly active GSTs reveal the transient intermediate, which is detectable by UV spectroscopy and HPLC analysis. LC/MS analysis of the reaction products demonstrates that the primary GSH conjugate, 4-glutathionylbuten-2-one, can react with a second GSH molecule to form the 4-(bis-glutathionyl)butan-2-one. GST M1-1 and GST A4-4 were the most efficient enzymes with tAVTG, and GST M1-1 and GST M2-2 had highest activity with cAVTP. The highly efficient GST M1-1 is polymorphic and is absent in approximately half of the human population. GST P1-1, which is overexpressed in many cancer cells, had no detectable activity with cAVTP and only minor activity with tAVTG. Other GST-activated prodrugs have targeted GST P1-1-expressing cancer cells. Tumors expressing high levels of GST M1-1 or GST A4-4 can be predicted to be particularly vulnerable to chemotherapy with cAVTP or tAVTG.
Keywords: Thiopurine prodrugs; Glutathione transferase; cis; -6-(2-Acetylvinylthio)purine; trans; -6-(2-Acetylvinylthio)guanine; Bioactivation; Chemotherapy
Carboxyl-glucuronidation of mitiglinide by human UDP-glucuronosyltransferases by Lushan Yu; Sijie Lu; Yongjun Lin; Su Zeng (pp. 1842-1851).
Mitiglinide (MGN) is a new potassium channel antagonist for the treatment of type 2 diabetes mellitus. In the present study, a potential metabolic pathway of MGN, via carboxyl-linked glucuronic acid conjugation, was found. MGN carboxyl-glucuronide was isolated from a reaction mixture consisting of MGN and human liver microsomes fortified with UDP-glucuronic acid (UDPGA) and identified by a hydrolysis reaction with β-glucuronidase and HPLC-MS/MS. Kinetic analysis indicated that MGN from four species had the highest affinity for the rabbit liver microsomal enzyme ( Km=0.202mM) and the lowest affinity for the dog liver microsomal enzyme ( Km=1.164mM). The metabolic activity ( Vmax/ Km) of MGN to the carboxyl-glucuronidation was in the following order: rabbit>dog>rat>human. With the assessment of MGN glucuronide formation across a panel of recombinant UDP-glucuronosyltransferase (UGT) isoforms (UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7), only UGT1A3 and UGT2B7 exhibited high MGN glucuronosyltransferase activity. The Km values of MGN glucuronidation in recombinant UGT1A3 and UGT2B7 microsomes were close to those in human liver microsomes. The formation of MGN glucuronidation by human liver microsomes was effectively inhibited by quercetin (substrate for UGT1A3) and diclofenac (substrate for UGT2B7), respectively. The MGN glucuronidation activities in 15 human liver microsomes were significantly correlated with quercetin ( r2=0.806) and diclofenac glucuronidation activities ( r2=0.704), respectively. These results demonstrate that UGT1A3 and UGT2B7 are catalytic enzymes in MGN carboxyl-glucuronidation in human liver.
Keywords: Abbreviations; ESI-MS/MS; electrospray ionization-tandem mass spectrometry; 7-HFC; 7-hydroxy-4-trifluoromethylcoumarin; HPLC-PDAD; high-performance liquid chromatography with photo-diode array detector; MGN; mitiglinide; UDPGA; UDP-glucuronic acid; UGT; UDP-glucuronosyltransferaseMitiglinide; UDP-glucuronosyltransferanse; Recombinant UGTs; Human liver microsomes; UGT1A3; UGT2B7