Biochemical Pharmacology (v.75, #2)
Editorial Board (iii).
Bridging the gap between protein carboxyl methylation and phospholipid methylation to understand glucose-stimulated insulin secretion from the pancreatic β cell by Anjaneyulu Kowluru (335-345).
Recent findings have implicated post-translational modifications at C-terminal cysteines [e.g., methylation] of specific proteins [e.g., G-proteins] in glucose-stimulated insulin secretion [GSIS]. Furthermore, methylation at the C-terminal leucine of the catalytic subunit of protein phosphatase 2A [PP2Ac] has also been shown to be relevant for GSIS. In addition to these two classes of protein methyl transferases, a novel class of glucose-activated phospholipid methyl transferases have also been identified in the β cell. These enzymes catalyze three successive methylations of phosphatidylethanolamine to yield phosphatidylcholine. The “newly formed” phosphatidylcholine is felt to induce alterations in the membrane fluidity, which might favor vesicular fusion with the plasma membrane for the exocytosis of insulin. The objectives of this commentary are to: (i) review the existing evidence on the regulation, by glucose and other insulin secretagogues, of post-translational carboxylmethylation [CML] of specific proteins in the β cell; (ii) discuss the experimental evidence, which implicates regulation, by glucose and other insulin secretagogues, of phosphatidylethanolamine methylation in the islet β cell; (iii) propose a model for potential cross-talk between the protein and lipid methylation pathways in the regulation of GSIS and (iv) highlight potential avenues for future research, including the development of specific pharmacological inhibitors to further decipher regulatory roles for these methylation reactions in islet β cell function.
Keywords: Pancreatic β cells; Protein phosphatase 2A; Glucose-stimulated insulin secretion; G-proteins; Phospholipid methylation; Protein carboxylmethylation;
Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease by Joseph D. Raffetto; Raouf A. Khalil (346-359).
Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade various components of the extracellular matrix (ECM). Members of the MMP family include collagenases, gelatinases, stromelysins, matrilysins and membrane-type MMPs. ProMMPs are cleaved into active forms that promote degradation of ECM proteins. Also, recent evidence suggests direct or indirect effects of MMPs on ion channels in the endothelium and vascular smooth muscle, and on other mechanisms of vascular relaxation/contraction. Endogenous tissue inhibitors of metalloproteinases (TIMPs) reduce excessive proteolytic ECM degradation by MMPs. The balance between MMPs and TIMPs plays a major role in vascular remodeling, angiogenesis, and the uterine and systemic vasodilation during normal pregnancy. An imbalance in the MMPs/TIMPs activity ratio may underlie the pathogenesis of vascular diseases such as abdominal aortic aneurysm, varicose veins, hypertension and preeclampsia. Downregulation of MMPs using genetic manipulations of endogenous TIMPs, or synthetic pharmacological inhibitors such as BB-94 (Batimastat) and doxycycline, and Ro-28-2653, a more specific inhibitor of gelatinases and membrane type 1-MMP, could be beneficial in reducing the MMP-mediated vascular dysfunction and the progressive vessel wall damage associated with vascular disease.
Keywords: MMP; TIMP; Blood vessels; Extracellular matrix; Aneurysm; Varicose veins;
Anticancer mechanisms of YC-1 in human lung cancer cell line, NCI-H226 by Chun-Jen Chen; Mei-Hua Hsu; Li-Jiau Huang; Takao Yamori; Jing-Gung Chung; Fang-Yu Lee; Che-Ming Teng; Sheng-Chu Kuo (360-368).
As part of a continuing search for potential anticancer drug candidates, 1-benzyl-3-(5-hydroxymethyl-2-furyl)indazole (YC-1) was evaluated in the Japanese Cancer Institute's (JCI) in vitro disease-oriented anticancer screen. The results indicated that YC-1 showed impressive selective toxicity against the NCI-H226 cell line. Therefore, the molecular mechanism by which YC-1 affects NCI-H226 cell growth was studied. YC-1 inhibited NCI-H226 cell growth in a time- and a concentration-dependent manner. YC-1 suppressed the protein levels of cyclin D1, CDK2 and cdc25A, up-regulated p16, p21 and p53, increased the number of NCI-H226 cells in the G0/G1 phase of the cell cycle. Long exposure to YC-1 induced apoptosis by mitochondrial-dependent pathway. In addition, YC-1 inhibited MMP-2 and MMP-9 protein activities to abolish tumor cells metastasis. These findings suggest a mechanism of cytotoxic action of YC-1 and indicate that YC-1 may be a promising chemotherapy agent against lung cancer.
Keywords: YC-1; NCI-H226; Cell cycle arrest; Apoptosis; Anti-metastasis;
Induction of G2/M arrest and apoptosis by sesquiterpene lactones in human melanoma cell lines by Sharon Rozenblat; Shlomo Grossman; Margalit Bergman; Hugo Gottlieb; Yigal Cohen; Sara Dovrat (369-382).
Malignant melanoma is a highly aggressive tumor which frequently resists chemotherapy, therefore, the search for new agents for its treatment is of great importance. In this study, we purified the sesquiterpene lactones (SLs), Tomentosin and Inuviscolide from Inula viscosa (Compositae) leaves and studied their anti-cancer potency against human melanoma cell lines in order to develop new agents for melanoma treatment. SLs inhibited the proliferation of three human melanoma cell lines: SK-28, 624 mel and 1363 mel in a dose-dependent manner. We further investigated SLs mechanism of action using SK-28 as a representative cell line model. SLs caused cell-cycle arrest at G2/M, accompanied by the appearance of a sub-G0 fraction, indicative of apoptotic cell death. Induction of apoptosis was further confirmed by changes in membrane phospholipids, changes in mitochondrial membrane potential (ΔΨ) and by detection of Caspase-3 activity. Rapid inhibitory phosphorylation of Cdc2 (Thr14 and Tyr15) was seen early after treatment, followed by a later decrease in the expression level of both Cyclin b1 and Cdc2. Induction of p53 and p21waf1 proteins and phosphorylation of p53 at Ser15 were also detected early after treatment. The anti-apoptotic proteins, p65 subunit of nuclear factor κB (NF-κB), and Survivin were reduced in a dose-dependent manner. Taken together, these changes partially explain the ability of the SLs to induce G2/M arrest and apoptosis. Induction of apoptosis by Tomentosin and Inuviscolide in human aggressive melanoma cell lines has high pharmacological value and implies that SLs might be developed as new agents for melanoma treatment.
Keywords: Sequiterpene lactones; Melanoma; Chemoresistance; G2/M arrest; Apoptosis;
KRIBB3, a novel microtubule inhibitor, induces mitotic arrest and apoptosis in human cancer cells by Ki Deok Shin; Young Ju Yoon; Yeong-Rim Kang; Kwang-Hee Son; Hwan Mook Kim; Byoung-Mog Kwon; Dong Cho Han (383-394).
KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) inhibited cancer cell growth in vitro and in vivo. Flow cytometry studies showed that KRIBB3 caused cell cycle arrest at the G2/M phase and subsequent apoptosis. This was confirmed as accumulation of Cyclin B1 and cleavage of poly(ADP-ribose) polymerase (PARP) were detected. While transient inhibition by KRIBB3 led to reversible mitotic arrest, prolonged exposure to KRIBB3-induced apoptosis. Co-immunoprecipitation experiments showed that KRIBB3 initially induced association of inhibitory Mad2 with p55CDC (mammalian homologue of CDC20), an activator of APC/C (anaphase-promoting complex/cyclosome), suggesting that the mitotic spindle checkpoint was activated by KRIBB3. However, the level of this inhibitory complex of Mad2 with p55CDC was gradually decreased 24 h after KRIBB3 treatment, and was hardly detectable after 48 h, indicating some slipping of the mitotic checkpoint. Consistent with these observations, KRIBB3 activated the mitotic spindle checkpoint by disrupting the microtubule cytoskeleton. KRIBB3 was proven to be a tubulin inhibitor using in vitro polymerization assays and in vivo indirect immunofluorescence staining. The temporal pattern of Bax activation by KRIBB3 was similar to PARP cleavage, suggesting that Bax is a mediator of KRIBB3-dependent apoptosis. Furthermore, when KRIBB3 was administered intraperitoneally into nude mice at 50 mg/kg or 100 mg/kg, it inhibited 49.5 or 70.3% of tumor growth, respectively. These results suggest that KRIBB3 is a good drug candidate for cancer therapy.
Keywords: Isoxazole; Microtubule inhibitor; Mitotic arrest; Apoptosis; Bax activation; Cancer therapy;
Celecoxib transiently inhibits cellular protein synthesis by Peter Pyrko; Adel Kardosh; Axel H. Schönthal (395-404).
To uncover the full spectrum of its pharmacological activities, the selective COX-2 inhibitor celecoxib is routinely being used at concentrations of up to 100 μM in cell culture. At these elevated concentrations, several COX-2-independent effects were identified, although many details of these events have remained unclear. Here, we report a COX-2-independent effect of celecoxib that might have profound consequences for the interpretation of previous results obtained at elevated concentrations of this drug in vitro. We found that celecoxib rapidly inhibits general protein translation at concentrations as low as 30 μM. This appears to be a consequence of endoplasmic reticulum (ER) stress and entails the phosphorylation and inactivation of eukaryotic translation initiation factor 2 alpha (eIF2α). These effects were not achieved by other coxibs (rofecoxib, valdecoxib) or traditional NSAIDs (indomethacin, flurbiprofen), but were mimicked by the COX-2-inactive celecoxib analog, 2,5-dimethyl-celecoxib (DMC), indicating COX-2 independence. Considering the obvious impact of blocked translation on cellular function, we provide evidence that this severe inhibition of protein synthesis might suffice to explain some of the previously reported COX-2-independent effects of celecoxib, such as the down-regulation of the essential cell cycle regulatory protein cyclin D, which is a short-lived protein that rapidly disappears in response to the inhibition of protein synthesis. Taken together, our findings establish ER stress-induced inhibition of general translation as a critical outcome of celecoxib treatment in vitro, and suggest that this effect needs to be considered when interpreting observations from the use of this drug in cell culture.
Keywords: Endoplasmic reticulum stress; Translation initiation factor 2 alpha; Dimethyl-celecoxib; NSAIDs; Coxibs; Cyclin D;
Effects of lovastatin on Rho isoform expression, activity, and association with guanine nucleotide dissociation inhibitors by Stephanie J. Turner; Shunhui Zhuang; Tong Zhang; Gerry R. Boss; Renate B. Pilz (405-413).
3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (EC184.108.40.206) inhibitors (statins) reduce cholesterol synthesis and prevent cardiovascular disease; they can also inhibit prenylation of Ras and Rho proteins, and have anti-neoplastic effects. Rho proteins cycle between an active, GTP-bound, and an inactive, GDP-bound form, and Rho prenylation is important for Rho's interaction with upstream regulators and downstream effectors, but the effects of statins on Rho signaling are incompletely understood. We found that the HMG-CoA reductase inhibitor lovastatin markedly induced the expression of RhoA, B, and C in human erythroleukemia (HEL) cells. The drug increased RhoA and C only in their unprenylated forms, but it increased both prenylated and unprenylated RhoB and did not significantly affect N- and K-Ras prenylation, suggesting that it inhibited geranyl-geranylation more efficiently than farnesylation. Quantitative analysis of nucleotides bound to Rho demonstrated a 3.7-fold increase in Rho-GTP and a similar increase in Rho-GDP in lovastatin-treated cells, leaving the fraction of Rho in the active, GTP-bound form constant at 5.8%. Lovastatin reduced Rho association with Rho guanine dissociation inhibitor (RhoGDI)-α and -β, and prenylation-deficient Rho mutants did not associate with RhoGDI. siRNA inhibition of RhoGDIα expression increased Rho-GTP, suggesting that decreased Rho/RhoGDIα association explained an increase in unprenylated Rho-GTP in lovastatin-treated cells. Unprenylated Rho A, B, and C were partly functional in activating serum response element-dependent transcription. In conclusion, we quantified effects of lovastatin on RhoA, B, and C isoforms, and provide a molecular mechanism whereby statins cause accumulation of unprenylated Rho-GTP.
Keywords: HMG-CoA reductase inhibitor; Prenylation; Rho isoforms; Rho activity; Rho guanine nucleotide dissociation inhibitor; Leukemia cells;
Transcriptional regulation of aldo-keto reductase 1C1 in HT29 human colon cancer cells resistant to methotrexate: Role in the cell cycle and apoptosis by Elisabet Selga; Véronique Noé; Carlos J. Ciudad (414-426).
While studying differentially expressed genes between sensitive and 10−5 M Methotrexate (MTX) resistant HT29 human colon cancer cells, we identified some members of the aldo-keto reductase (AKR) superfamily. The study was followed with the member AKR1C1 (EC 220.127.116.11), validating its increase in mRNA and protein levels in MTX resistant cells. The genomic content for AKR1C1 remained unchanged between sensitive and resistant cells, thereby excluding a mechanism of AKR1C1 gene amplification. Thus, we cloned the AKR1C1 human promoter and performed luciferase experiments that revealed a transcriptional regulation of the gene in the resistant cells. Computational studies showed a putative binding site for the transcription factor Sp1. The co-transfection of Sp1 or Sp3 with different constructs of AKR1C1 promoter deletions, including and excluding the proximal GC-box, demonstrated a key role for these factors in regulating AKR1C1 transcriptional activity. Gel-shift assays revealed an increase in Sp1 and Sp3 binding in resistant compared to sensitive cells, without differences in Sp1 protein levels. Dephosphorylation of the extracts coincided with a decrease in Sp1 binding, which is consistent with a process of regulation of Sp1 by phosphorylation. We also investigated the possible relationship between AKR1C1 expression and MTX action. Overexpression of AKR1C1 counteracted the S-phase accumulation of cells and apoptosis caused by MTX treatment. This suggests a role of AKR1C1 in cell proliferation. Finally, overexpression of AKR1C1 in MTX sensitive HT29 cells conferred resistance to the chemotherapeutic agent and silencing of AKR1C1 by means of iRNA technology sensitized the cells to MTX.
Keywords: Methotrexate; AKR1C1; Transcriptional regulation; Sp1 binding; DHFR; Drug-resistance;
Interaction between celecoxib and docetaxel or cisplatin in human cell lines of ovarian cancer and colon cancer is independent of COX-2 expression levels by Marcel N.A. Bijman; Cristian A. Hermelink; Maria P.A. van Berkel; Adrie C. Laan; Maarten L. Janmaat; Godefridus J. Peters; Epie Boven (427-437).
Celecoxib, an inhibitor of cyclooxygenase-2 (COX-2), is being investigated for enhancement of chemotherapy efficacy in cancer clinical trials. We determined whether continuous exposure to celecoxib would increase the antiproliferative effects of a 1-h treatment with docetaxel or cisplatin in four human ovarian cancer cell lines. COX-2 protein could not be detected in these cell lines, because of which three COX-2 positive human colon cancer cell lines were included. Multiple drug effect analysis demonstrated additive to borderline antagonistic effects of celecoxib combined with docetaxel. Combination indices with values of 1.4–2.5 in all cancer cell lines indicated antagonism between celecoxib and cisplatin regardless whether celecoxib preceded cisplatin for 3 h, was added simultaneously or immediately after cisplatin. Apoptotic features measured in COX-2-negative H134 ovarian cancer cells and COX-2-positive WiDr colon cancer cells, such as the activation of caspase-3 and the number of cells in sub-G0 of the cell cycle, induced by docetaxel were increased in the presence of celecoxib, but were abrogated upon addition of celecoxib to cisplatin. Moreover, the G2/M accumulation in cisplatin-treated cells was less pronounced when celecoxib was present. Drugs did not affect p-Akt. Celecoxib upregulated p-ERK1/2 in H134 cells, but not in WiDr cells. Platinum–DNA adduct formation measured in WiDr cells, however, was reduced when celecoxib was combined with cisplatin. Taken together, our data demonstrate clear antagonistic effects when celecoxib is given concurrently with cisplatin, which is independent of COX-2 expression levels.
Keywords: Cyclooxygenase; Combination index; Cisplatin; Docetaxel; Celecoxib;
Characterization of a new peptide agonist of the protease-activated receptor-1 by Yingying Mao; Jianguo Jin; Satya P. Kunapuli (438-447).
A new peptide (TFRRRLSRATR), derived from the c-terminal of human platelet P2Y1 receptor, was synthesized and its biological function was evaluated. This peptide activated platelets in a concentration-dependent manner, causing shape change, aggregation, secretion and calcium mobilization. Of the several receptor antagonists tested, only BMS200261, a protease activated receptor 1 (PAR-1) specific antagonist, totally abolished the peptide-induced platelet aggregation, secretion and calcium mobilization. The TFRRR-peptide-pretreated washed platelets failed to aggregate in response to SFLLRN (10 μM) but not to AYPGKF (500 μM). In addition, in mouse platelets, peptide concentrations up to 600 μM failed to cause platelet activation, indicating that the TFRRR-peptide activated platelets through the PAR-1 receptor, rather than through the PAR-4 receptor. The shape change induced by 10 μM peptide was totally abolished by Y-27632, an inhibitor of p160ROCK which is a downstream mediator of G12/13 pathways. The TFRRR-peptide, YFLLRNP, and the physiological agonist thrombin selectively activated G12/13 pathways at low concentrations and began to activate both Gq and G12/13 pathways with increasing concentrations. Similar to SFLLRN, the TFRRR-peptide caused phosphorylation of Akt and Erk in a P2Y12 receptor-dependent manner, and p-38 MAP kinase activation in a P2Y12-independent manner. The effects of this peptide are elicited by the first six amino acids (TFRRRL) whereas the remaining peptide (LSRATR), TFERRN, or TFEERN had no effects on platelets. We conclude that TFRRRL activates human platelets through PAR-1 receptors.
Keywords: PAR agonists; P2Y1 receptor; Gq; G12/13; MAP kinases; Calcium mobilization;
Neuroprotective antioxidant STAZN protects against myocardial ischemia/reperfusion injury by James J. Ley; Ricardo Prado; Jian Qin Wei; Nanette H. Bishopric; David A. Becker; Myron D. Ginsberg (448-456).
Protecting the myocardium from ischemia-reperfusion injury has significant potential to reduce the complications of myocardial infarction and interventional revascularization procedures. Reperfusion damage is thought to result, in part, from oxidative stress. Here we use a novel method of percutaneous coronary occlusion to show that the potent antioxidant and neuroprotective free-radical scavenger, stilbazulenyl nitrone (STAZN), confers marked cardioprotection when given immediately prior to reperfusion.Physiologically controlled male Sprague-Dawley rats were anesthetized with isoflurane, paralyzed with pancuronium and mechanically ventilated. A guide wire was introduced via the femoral artery and advanced retrogradely via the aorta into the left coronary artery under fluoroscopic guidance. Rats with established coronary ischemia (85 min after occlusion) were given STAZN 3.5 mg/kg or its vehicle 5 min before and 2 h after reperfusion, and were subjected to functional and histopathologic studies at 3 days. Ischemia-associated Q wave amplitude was reduced by 73% in STAZN-treated rats (P = 0.01), while infarct-related ejection fraction, fractional shortening and severe regional wall-motion impairments were improved by 48%, 54% and 37%, respectively, relative to vehicle-treated controls (P = 0.05). Total myocardial infarct volume in STAZN-treated rats was correspondingly reduced by 43% (P < 0.05), representing a sparing of 14% of the total left ventricular myocardium.STAZN, a second-generation azulenyl nitrone with potent neuroprotective efficacy in brain ischemia, is also a rapidly acting and highly effective cardioprotective agent in acute coronary ischemia. Our results suggest the potential for clinical benefit in the setting of acute coronary syndromes.
Keywords: Myocardial infarction; Antioxidants; Free radicals; Ischemia; Reperfusion;
AICAR positively regulate glycogen synthase activity and LDL receptor expression through Raf-1/MEK/p42/44MAPK/p90RSK/GSK-3 signaling cascade by Hsiang-Ming Wang; Sonya Mehta; Rishipal Bansode; Wei Huang; Kamal D. Mehta (457-467).
5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) is a commonly used pharmacological agent to study physiological effects which are similar to those of exercise. However, signal transduction pathways by which AICAR elicits downstream effects in liver are poorly understood. We report here that AICAR not only activated AMPK but also phosphorylated/deactivated glycogen synthase kinase-3 alpha/beta (GSK-3α/β) and dephophorylated/activated glycogen synthase (GS) in a time-dependent manner in human hepatoma HepG2 cells. The signal connection between AICAR and GSK-3 is indirect and involves activation of Raf-1/MEK/p42/44MAPK/p90RSK signaling cascade as pharmacologic inhibition of MEK significantly reduced phosphorylation/deactivation of GSK-3 and consequent dephosphorylation/activation of GS. Moreover, silencing the expression of p90RSK, a substrate of p42/44MAPK, attenuated AICAR-dependent GSK-3 phosphorylation, implicating this kinase as a key mediator of AICAR signaling to GSK-3. Furthermore, consistent with the involvement of Raf-1 kinase cascade, AICAR-induced low-density lipoprotein (LDL) receptor expression in a p42/44MAPK-dependent manner. Finally, AICAR requires AMPK-α2-dependent and -independent pathways to activate Raf-1 kinase cascade as suppression of AMPKα2 activity, and not of AMPKα1, partially blocked AICAR-dependent p42/44MAPK activation and GSK-3 phosphorylation/deactivation. Collectively, these results highlight Raf-1 signaling cascade as the critical mediator of AICAR action on glucose and lipid metabolism in HepG2 cells.
Keywords: AICAR; Raf-1; p90RSK; AMPK; Glycogen synthase; LDL receptor;
KATP channel openers: Tissue selectivity of original 3-alkylaminopyrido- and 3-alkylaminobenzothiadiazine 1,1-dioxides by Philippe Lebrun; Bénédicte Becker; Nicole Morel; Philippe Ghisdal; Marie-Hélène Antoine; Pascal de Tullio; Bernard Pirotte (468-475).
The present study was designed to further evaluate the biological effects and tissue selectivity of new 3-alkylaminobenzo- and 3-alkylaminopyridothiadiazine 1,1-dioxides bearing identical branched alkylamino chains at the 3-position. These original compounds were compared with their parent molecules; namely the KATP channel openers diazoxide and pinacidil.All tested 3-alkylamino-substituted derivatives provoked a marked, concentration-dependent inhibition of the glucose-induced insulin secretion from rat pancreatic islets. The 3-alkylaminopyridothiadiazine 1,1-dioxides evoked a weak vasorelaxant activity whilst their 7-halo-substituted benzothiadiazine counterparts elicited pronounced, concentration-dependent, relaxations of rat aortic rings. The myorelaxant effect of the different drugs was tightly correlated with their capacity to increase 86Rb outflow (42K substitute) from prelabelled and perifused rat aortic rings. EC50/IC50 ratios (vascular/pancreatic) revealed a pronounced selectivity of the 3-alkylaminopyridothiadiazine 1,1-dioxides towards the pancreatic endocrine tissue. Structure–activity relationships suggest that, besides the requirement of an electronegative pole at the 7-position of the heterocycle, a minimal steric hindrance confers an optimal insulin-secreting cell selectivity. Lastly, radioisotopic, electrophysiological and pharmacological investigations indicate that the marked vasorelaxant properties of the 3-alkylaminobenzothiadiazine 1,1-dioxides are related to the activation of smooth muscle KATP channels.
Keywords: ATP-sensitive K+ channel; KATP channel openers; Pyridothiadiazine dioxides; Benzothiadiazine dioxides; Insulin-secreting cell; Vascular smooth muscle;
Transient receptor potential vanilloid 1 (TRPV1) channels in cultured rat Sertoli cells regulate an acid sensing chloride channel by Céline Auzanneau; Caroline Norez; Fabrice Antigny; Vincent Thoreau; Chantal Jougla; Anne Cantereau; Frédéric Becq; Clarisse Vandebrouck (476-483).
Sertoli cells provide a controlled microenvironment for regulation and maintenance of spermatogenesis for which an acidic milieu is crucial for male fertility. Sertoli cells also contribute to protection of spermatogenetic cells. Here, we showed that TRPV1 is expressed in rat Sertoli cells and regulates an acid sensing Cl− channel (ASCC). The expression of TRPV1 in rat Sertoli cells was demonstrated by RT-PCR, immunostaining and calcium measurement experiments. ASCC activity was inhibited by capsaicin (IC50 = 214.3 ± 1.6 nM), olvanil (IC50 = 400 ± 1.7 pM) and resiniferatoxin (IC50 = 9.3 ± 1.5 nM) but potentiated by capsazepine (EC50 = 5.3 ± 1.3 μM) and ruthenium red (EC50 = 2.3 ± 1.5 μM). In the human airway epithelial cell line Calu-3 in which ASCC can be detected but not TRPV1, capsaicin and capsazepine were without any effect. Finally the application of the non-steroidal anti-inflammatory drug ibuprofen prevented the control of ASCC by TRPV1. Our study provides the first evidence for a regulation by TRPV1 of an acid sensing chloride channel in rat Sertoli cells. TRPV1 and ASCC may thus be considered as new potential physiological regulators of spermatogenesis and targets for pharmacological treatments of reproductive disorders as cryptorchidism, Sertoli cell tumors or torsion of the spermatic cord.
Keywords: Vanilloid receptor 1; Pharmacology; Sertoli cells; Acid sensing chloride channel; Calcium; Ibuprofen;
An indomethacin analogue, N-(4-chlorobenzoyl)-melatonin, is a selective inhibitor of aldo-keto reductase 1C3 (type 2 3α-HSD, type 5 17β-HSD, and prostaglandin F synthase), a potential target for the treatment of hormone dependent and hormone independent malignancies by Michael C. Byrns; Stephan Steckelbroeck; Trevor M. Penning (484-493).
Aldo-keto reductase (AKR) 1C3 (type 2 3α-HSD, type 5 17β-HSD, and prostaglandin F synthase) regulates ligand access to steroid hormone and prostaglandin receptors and may stimulate proliferation of prostate and breast cancer cells. NSAIDs are known inhibitors of AKR1C enzymes. An NSAID analogue that inhibits AKR1C3 but is inactive against the cyclooxygenases and the other AKR1C family members would provide an important tool to examine the role of AKR1C3 in proliferative signaling. We tested NSAIDs and NSAID analogues for inhibition of the reduction of 9,10-phenanthrenequinone (PQ) catalyzed by AKR1C3 and the closely related isoforms AKR1C1 and AKR1C2. Two of the compounds initially screened, indomethacin and its methyl ester, were specific for AKR1C3 versus the other AKR1C isoforms. Based on these results and the crystal structure of AKR1C3, we predicted that N-(4-chlorobenzoyl)-melatonin (CBM), an indomethacin analogue that does not inhibit the cyclooxygenases, would selectively inhibit AKR1C3. CBM inhibited the reduction of PQ by AKR1C3, but did not significantly inhibit AKR1C1 or AKR1C2. Indomethacin and CBM also inhibited the AKR1C3-catalyzed reduction of Δ4-androstene-3,17-dione but did not significantly inhibit the reduction of steroid hormones catalyzed by AKR1C1 or AKR1C2. The pattern of inhibition of AKR1C3 by indomethacin and CBM was uncompetitive versus PQ, but competitive versus Δ4-androstene-3,17-dione, indicating that two different inhibitory complexes form during the ordered bi bi reactions. The identification of CBM as a specific inhibitor of AKR1C3 will aid the investigation of its roles in steroid hormone and prostaglandin signaling and the resultant effects on cancer development.
Keywords: NSAIDs; Steroid hormone metabolism; Prostaglandin metabolism; Inhibitor design; Cancer treatment; Chemoprevention;
Cinnamaldehyde suppresses toll-like receptor 4 activation mediated through the inhibition of receptor oligomerization by Hyung S. Youn; Jun K. Lee; Yong J. Choi; Shin I. Saitoh; Kensuke Miyake; Daniel H. Hwang; Joo Y. Lee (494-502).
Toll-like receptors (TLRs) play a critical role in induction of innate immune and inflammatory responses by recognizing invading pathogens or non-microbial endogenous molecules. TLRs have two major downstream signaling pathways, MyD88- and TRIF-dependent pathways leading to the activation of NFκB and IRF3 and the expression of inflammatory mediators. Deregulation of TLR activation is known to be closely linked to the increased risk of many chronic diseases. Cinnamaldehyde (3-phenyl-2-propenal) has been reported to inhibit NFκB activation induced by pro-inflammatory stimuli and to exert anti-inflammatory and anti-bacterial effects. However, the underlying mechanism has not been clearly identified. Our results showed that cinnamaldehyde suppressed the activation of NFκB and IRF3 induced by LPS, a TLR4 agonist, leading to the decreased expression of target genes such as COX-2 and IFNβ in macrophages (RAW264.7). Cinnamaldehyde did not inhibit the activation of NFκB or IRF3 induced by MyD88-dependent (MyD88, IKKβ) or TRIF-dependent (TRIF, TBK1) downstream signaling components. However, oligomerization of TLR4 induced by LPS was suppressed by cinnamaldehyde resulting in the downregulation of NFκB activation. Further, cinnamaldehyde inhibited ligand-independent NFκB activation induced by constitutively active TLR4 or wild-type TLR4. Our results demonstrated that the molecular target of cinnamaldehyde in TLR4 signaling is oligomerization process of receptor, but not downstream signaling molecules suggesting a novel mechanism for anti-inflammatory activity of cinnamaldehyde.
Keywords: Cinnamaldehyde; Toll-like receptor; Oligomerization; MyD88; TRIF; Inflammation;
Identification and functional analysis of cyclooxygenase-1 as a molecular target of boswellic acids by Ulf Siemoneit; Bettina Hofmann; Nicole Kather; Tobias Lamkemeyer; Johannes Madlung; Lutz Franke; Gisbert Schneider; Johann Jauch; Daniel Poeckel; Oliver Werz (503-513).
Boswellic acids (BAs) are assumed as the anti-inflammatory principles of Boswellia species. Initially, it was found that BAs inhibit leukotriene biosynthesis and 5-lipoxygenase (EC number 18.104.22.168), whereas suppression of prostaglandin formation and inhibition of cyclooxygenases (COX, EC number 22.214.171.124) has been excluded. Recently, we demonstrated that BAs also interfere with platelet-type 12-lipoxygenase. Here, we show that BAs, preferably 3-O-acetyl-11-keto-β-BA (AKBA), concentration-dependently inhibit COX-1 product formation in intact human platelets (IC50 = 6 μM) as well as the activity of isolated COX-1 enzyme in cell-free assays (IC50 = 32 μM). The inhibitory effect of AKBA is reversible, and increased levels of arachidonic acid (AA) as substrate for COX-1 impair the efficacy. COX-1 in platelet lysates or isolated COX-1 selectively bound to an affinity matrix composed of immobilized BAs linked via glutaric acid to sepharose and this binding was reversed by ibuprofen or AA. Automated molecular docking of BAs into X-ray structures of COX-1 yielded positive Chemscore values for BAs, indicating favorable binding to the active site of the enzyme. In contrast, COX-2 was less efficiently inhibited by BAs as compared to COX-1, and pull-down experiments as well as docking studies exclude strong affinities of BAs towards COX-2. In conclusion, BAs, in particular AKBA, directly interfere with COX-1 and may mediate their anti-inflammatory actions not only by suppression of lipoxygenases, but also by inhibiting cyclooxygenases, preferentially COX-1.
Keywords: Boswellic acids; Cyclooxygenase; Lipoxygenase; Inflammation; Platelets; Prostaglandin;
A novel formulation of inhaled doxycycline reduces allergen-induced inflammation, hyperresponsiveness and remodeling by matrix metalloproteinases and cytokines modulation in a mouse model of asthma by Maud M. Gueders; Pascal Bertholet; Fabienne Perin; Natacha Rocks; Raphaël Maree; Vincent Botta; Renaud Louis; Jean-Michel Foidart; Agnès Noel; Brigitte Evrard; Didier D. Cataldo (514-526).
In this study, we assess the effectiveness of inhaled doxycycline, a tetracycline antibiotic displaying matrix metalloproteinases (MMP) inhibitory effects to prevent allergen-induced inflammation, hyperresponsiveness and remodeling. MMPs play key roles in the complex cascade of events leading to asthmatic phenotype.Doxycycline was administered by aerosols by the mean of a novel formulation as a complex with hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) used as an excipient. BALB/c mice (n = 16–24 in each group) were sensitized and exposed to aerosolized ovalbumin (OVA) from day 21 to 27 (short-term exposure protocol) or 5 days/odd weeks from day 22 to 96 (long-term exposure protocol).In the short-term exposure model, inhaled doxycycline decreased allergen-induced eosinophilic inflammation in bronchoalveolar lavage (BAL) and in peribronchial areas, as well as airway hyperresponsiveness. In lung tissue, exposure to doxycycline via inhaled route induced a fourfold increase in IL-10 levels, a twofold decrease in IL-5, IL-13 levels and diminished MMP-related proteolysis and the proportion of activated MMP-9 as compared to placebo.In the long-term exposure model, inhaled doxycycline significantly decreased the extent of glandular hyperplasia, airway wall thickening, smooth muscle hyperplasia and subepithelial collagen deposition which are well recognized features of airway remodeling.Doxycycline administered by aerosols decreases the allergen-induced airway inflammation and hyperresponsiveness and inhibits the development of bronchial remodeling in a mouse model of asthma by modulation of cytokines production and MMP activity.
Keywords: Asthma; Inflammation; Remodeling; Bronchial resistance; Matrix metalloproteinases; Doxycycline;
Efficient oxidation of promutagenic hydroxymethylpyrenes by cDNA-expressed human alcohol dehydrogenase ADH2 and its inhibition by various agents by Ronny Kollock; Walter Meinl; Heiko Schneider; Monika Batke; Heinz Frank; Albrecht Seidel; Hansruedi Glatt (527-537).
Alkylated polycyclic aromatic hydrocarbons can be metabolically activated via benzylic hydroxylation and sulphation to electrophilically reactive esters. However, we previously found that the predominant biotransformation route for the hepatocarcinogen 1-hydroxymethylpyrene (1-HMP) in the rat in vivo is the oxidation of the side chain by alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases to the carboxylic acid. Inhibition of this pathway by ethanol (competing ADH substrate) or 4-methylpyrazole (ADH inhibitor) led to a dramatic increase in the 1-HMP-induced DNA adduct formation in rat tissues in the preceding study. In order to elucidate the role of individual ADHs in the metabolism of alkylated polycyclic aromatic hydrocarbons, we expressed the various members of the human ADH family in bacteria. Cytosolic preparations from bacteria expressing ADH2 clearly oxidized hydroxymethylpyrene isomers (1-, 2- and 4-HMP) with the highest rate. This form was purified to near homogeneity to perform detailed kinetic analyses. High catalytic efficiencies (V max/K m) were observed with HMPs. Thus, this value was 10,000-fold higher for 2-HMP than for the reference substrate, ethanol. The corresponding aldehydes were also efficiently reduced by ADH2. 4-Methylpyrazole inhibited the oxidation of the HMP isomers as well as the reverse reaction. Daidzein, cimetidine and the competing substrate ethanol were further compounds that inhibited the ADH2-mediated oxidative detoxification of 1-HMP.
Keywords: ADH2; Alkylated polycyclic aromatic hydrocarbons; Benzylic alcohols; Detoxification; Sulphotransferase;
Microarray analysis of hepatic gene expression in pyrazole-mediated hepatotoxicity: Identification of potential stimuli of Cyp2a5 induction by Kathleen D. Nichols; Gordon M. Kirby (538-551).
Cytochrome P450 2a5 (Cyp2a5) expression is induced during liver damage caused by hepatotoxins such as pyrazole, however, the mechanism underlying this overexpression is unclear. In order to identify pathophysiological and cellular responses to pyrazole that might alter Cyp2a5 expression, we examined the effect of pyrazole on mouse hepatic gene expression in C57BL/6 mice using Affymetrix 430 2.0 microarrays. Over 3000 differentially expressed genes were identified 24-h after pyrazole treatment that were associated with a variety of cellular pathways. Upregulated genes were primarily involved in the splicing and processing of RNA and the unfolded protein response pathway, while downregulated genes were associated with amino acid and lipid metabolism, and generation of precursor metabolites for energy production. We also examined the effects of pyrazole on cellular pathways linked to metabolic and histopathological changes observed with pyrazole toxicity. Increased mRNA levels were observed for genes involved in bilirubin production, whereas the major genes of the urea cycle were strongly decreased. Changes in genes involved in carbohydrate metabolism were also observed which could explain pyrazole-induced glycogen depletion and decreased serum glucose. In addition, over 100 genes involved in the cellular stress response were upregulated by pyrazole treatment, including genes involved in the unfolded protein response and redox status. Based on these results and previous evidence concerning the regulation of Cyp2a5, we have identified several pathophysiological changes including altered energy homeostasis, hyperbilirubinemia, ER stress, and altered redox status that are associated with CYP2A5 overexpression and may represent potential stimuli for the induction of Cyp2a5.
Keywords: CYP2A5; Pyrazole; Microarray; Gene expression; Hepatotoxicity; Regulation;
Critical role of hydrogen peroxide in the differential susceptibility of Th1 and Th2 cells to tributyltin-induced apoptosis by Saeko Tada-Oikawa; Takuma Kato; Kagemasa Kuribayashi; Kohsuke Nishino; Mariko Murata; Shosuke Kawanishi (552-561).
Tributyltin (TBT), an environmental pollutant, debilitates immune responses via induction of apoptosis in CD4+ T cells through an undefined mechanism of action. Accumulating evidence indicates that the susceptibility of Th1 and Th2 cells to TBT-induced apoptosis differs. In this study, by using HL-60 cell model, we show that hydrogen peroxide (H2O2) plays a critical role in TBT-induced apoptosis. Generation of H2O2 induced by TBT resulted in a change in mitochondrial membrane potential that proceed apoptotic pathway where, at least in part, involved activation of caspase-3. We also demonstrated that Th1 clones appear to be more vulnerable to apoptosis induction than Th2 clones following exposure to TBT, which was well correlated with increased H2O2 generation in Th1 clones than Th2 clones. There was an inverse correlation between TBT-induced apoptosis and the basal levels of intracellular GSH, a major cellular antioxidant. Furthermore, the addition of NAC that replenish intracellular GSH levels inhibited generation of H2O2 and apoptosis in Th1 clones. These results suggest that TBT selectively induces apoptosis via generation of H2O2 in Th1 cells because of their low GSH levels, which may contribute to the Th2 predominance induced by TBT.
Keywords: Tributyltin; Apoptosis; Th1/Th2; GSH; H2O2; Immunotoxicity;
Thermodynamics of A2B adenosine receptor binding discriminates agonistic from antagonistic behaviour by Stefania Gessi; Eleonora Fogli; Valeria Sacchetto; Katia Varani; Stefania Merighi; Edward Leung; Stephen Mac Lennan; Pier Andrea Borea (562-569).
Thermodynamic parameters ΔG°, ΔH° and ΔS° of the binding equilibrium of 12 ligands (six agonists and six antagonists) to the A2B adenosine receptor subtype have been determined by affinity measurements carried out on HEK 293 cells stably transfected with human A2B adenosine receptors at six different temperatures (4, 10, 15, 20, 25, 30 °C) and van’t Hoff plot analysis have been performed. Affinity constants were obtained from saturation experiments of [3H]MRE 2029-F20 or by its displacement in inhibition assays for the other compounds. van’t Hoff plots were essentially linear in the temperature range investigated, showing that the Δ C p ° of the binding equilibrium is nearly zero. Thermodynamic parameters are in the range 7 ≤ ΔH° ≤ 23 kJ mol−1and 123 ≤ ΔS° ≤ 219 J K−1 mol−1 for agonists and −40 ≤ ΔH° ≤ −20 kJ mol−1 and 10 ≤ ΔS° ≤ 91 J K−1 mol−1 for antagonists indicating that agonistic binding is always totally entropy-driven while antagonistic binding is enthalpy and entropy-driven. In the −TΔS° versus ΔH° plot the thermodynamic data are clearly arranged in separate clusters for agonists and antagonists, which, therefore, turn out to be thermodynamically discriminated.
Keywords: Binding thermodynamics; Adenosine A2B receptor; Enthalpy–entropy compensation; Agonist–antagonist discrimination; Binding mechanisms; Drug development;
Characterization of mouse flavin-containing monooxygenase transcript levels in lung and liver, and activity of expressed isoforms by Lisbeth K. Siddens; Marilyn C. Henderson; Jonathan E. VanDyke; David E. Williams; Sharon K. Krueger (570-579).
The significance of active versus inactive flavin-containing monooxygenase 2 (FMO2) for human drug and xenobiotic metabolism and sensitivity is unknown, but the underlying ethnic polymorphism is well documented. We used quantitative real-time PCR to measure message levels of Fmo1, Fmo2, Fmo3 and Fmo5 in lung and liver from eight strains of 8 week old female mice to determine if a strain could be identified that predominately expressed Fmo2 in lung, recapitulating the human FMO expression profile and being the ideal strain for Fmo2 knockout studies. We also characterized enzyme activity of baculovirus expressed mouse Fmo1, Fmo2 and Fmo3 to identify a substrate or incubation conditions capable of discriminating Fmo2 from Fmo mixtures. Fmo transcript expression patterns were similar for all strains. In lung, 59% of total FMO message was Fmo2, but Fmo1 levels were also high, averaging 34%, whereas Fmo3 and Fmo5 levels were 2 and 5%, respectively. In liver, Fmo1, Fmo2, Fmo3 and Fmo5 contributed 16, 1, 7 and 76% respectively, of detected message. Peak activity varied by isoform and was pH- and substrate-dependent. Fmo3 oxidation of methyl p-tolyl sulfide was negligible at pH 9.5, but Fmo3 oxidation of methimazole was comparable to Fmo1 and Fmo2. Heating microsomes at 50 °C for 10 min eliminated most Fmo1 and Fmo3 activity, while 94% of Fmo2 activity remained. Measurement of activity in heated and unheated lung and liver microsomes verified relative transcript abundance. Our results show that dual Fmo1/2 knockouts will be required to model the human lung FMO profile.
Keywords: Quantitative real-time PCR; In vitro expression; Flavin-containing monooxygenase; Mouse; Pulmonary expression; Hepatic expression;
JNK inhibitor SP600125 is a partial agonist of human aryl hydrocarbon receptor and induces CYP1A1 and CYP1A2 genes in primary human hepatocytes by Zdenek Dvorak; Radim Vrzal; Pavla Henklova; Petra Jancova; Eva Anzenbacherova; Patrick Maurel; Lucie Svecova; Petr Pavek; Jiri Ehrmann; Roman Havlik; Petr Bednar; Karel Lemr; Jitka Ulrichova (580-588).
SP600125, a specific inhibitor of c-Jun-N-Terminal kinase (JNK), was reported as a ligand and antagonist of aryl hydrocarbon receptor (AhR) [Joiakim A, Mathieu PA, Palermo C, Gasiewicz TA, Reiners Jr JJ. The Jun N terminal kinase inhibitor SP600125 is a ligand and antagonist of the aryl hydrocarbon receptor. Drug Metab Dispos 2003;31(11):1279–82]. Here we show that SP600125 is not an antagonist but a partial agonist of human AhR.SP600125 significantly induced CYP1A1 and CYP1A2 mRNAs in primary human hepatocytes and CYP1A1 mRNA in human hepatoma cells HepG2. This effect was abolished by resveratrol, an antagonist of AhR. Consistent with the recent report, SP600125 dose-dependently inhibited CYP1A1 and CYP1A2 genes induction by a prototype AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in human hepatocytes. Moreover, SP600125 displayed typical behavior of a partial agonist in HepG2 cells transiently transfected with a reporter plasmid containing two inverted repeats of the dioxin responsive element or with a plasmid containing 5′-flanking region of human CYP1A1 gene. SP600125 transactivated the reporter plasmids with EC50 of 0.005 and 1.89 μM, respectively. On the other hand, TCDD-dependent transactivation of the reporter plasmids was inhibited by SP600125 with IC50 values of 1.54 and 2.63 μM, respectively. We also tested, whether the effects of SP600125 are due to metabolism. Using liquid chromatography/mass spectrometry approach, we observed formation of two minor monohydroxylated metabolites of SP600125 in human hepatocytes, human liver microsomes but not in HepG2 cells. These data imply that biotransformation is not responsible for the effects of SP600125 on AhR signaling.In conclusion, we demonstrate that SP600125 is a partial agonist of human AhR, which induces CYP1A genes.
Keywords: Aryl hydrocarbon receptor; Human hepatocytes; c-Jun-N-terminal kinase; SP600125;