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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Natural and Synthetic Drugs Research Centre, Laboratory of Medicinal Chemistry (X.d.L., J.-M.D., B.P.), Metastasis Research Laboratory, Center for Experimental Cancer Research (T.D., D.W., A.B., V.C.), and Cellular and Molecular Therapeutic Center (V.B.), University of Liège, Liège, Belgium
Received January 10, 2006; accepted May 22, 2006.
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Abstract |
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; Gately, 2000). Therefore, COX-2 inhibition has been considered as an interesting approach for the management of tumor-associated angiogenesis (Gately and Li, 2004). Accordingly, results from preclinical investigations have indicated that both selective COX-2 inhibitors and nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) may effectively reduce or inhibit tumor angiogenesis (Gately and Li, 2004; Wei et al., 2004). Although the vast majority of studies have provided compelling and converging evidence that COX-2 inhibition may protect against tumor angiogenesis, the implementation of cancer prevention strategies, based on these findings, is hampered by the recognized toxicity of chronic use of NSAIDs (Fortun and Hawkey, 2005). Furthermore, the recent withdrawal of specific COX-2 inhibitor Vioxx due to cardiovascular toxicity (Bresalier et al., 2005) represents a new serious obstacle to the targeting of COX-2 for cancer prevention and/or treatment (Vanchieri, 2004). Therefore, the selective targeting of downstream COX-2 pathway metabolites or enzymes may appear to be a promising approach with the potential of avoiding the cardiovascular toxicity of COX-2 inhibitors while maintaining their antiangiogenic properties. One of these downstream metabolites, TXA2, a powerful aggregation mediator involved in thrombotic disorders, has been recently demonstrated to participate in cancer progression (Nie et al., 2004; Moussa et al., 2005) and cancer cell-induced platelet aggregation, a process that favors blood-borne metastasis (Jurasz et al., 2004). On the other hand, TXA2 also acts as a potent angiogenesis stimulator, both directly and by inducing platelet vascular endothelium growth factor (VEGF) and platelet-derived growth factor secretion after platelet aggregation (Arisato et al., 2003; Rhee et al., 2004). Furthermore, in endothelial cells, VEGF and basic fibroblast growth factor have been shown to stimulate the biosynthesis of TXA2, which is correlated with enhanced cell migration (Nie et al., 2000). Finally, treatment of endothelial cells with U-46,619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin F2), a stable TXA2 receptor agonist, augments the migration of human umbilical vein endothelial cells (HUVECs) in vitro and resumes the basic fibroblast growth factor-induced angiogenic response under COX-2-inhibited conditions in vivo in a model of corneal angiogenesis in mice (Daniel et al., 1999). Altogether, these studies underline the potential of TXA2 modulation for inhibiting angiogenesis.
Following the observation according to which the pyridinic sulfonylurea torasemide, a high ceiling loop diuretic, was able to induce a concentration-dependent relaxation of the canine coronary artery precontracted with carbocyclic TXA2, a stable TXA2 agonist (Uchida et al., 1992), a series of torasemide derivatives were synthesized in the Laboratory of Medicinal Chemistry (University of Liège) to obtain compounds displaying enhanced TXA2 inhibitory effects. We herein describe the in vitro pharmacologic characterization of nine torasemide derivatives that display dual TXA2 pathway inhibitory activity toward both TXA2 synthase and TXA2 receptors. We further show that these original compounds bear antiangiogenic properties.
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Materials and Methods |
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,2(Z),3,4]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid), a TXA2 receptor (TP receptor) antagonist, furegrelate [5-(3-pyridinylmethyl)-2-benzofurancarboxyclic acid, sodium salt], a TXA2 synthase (TXS) inhibitor and U-46,619, a stable TP receptor agonist, were purchased from Cayman Chemical (Ann Arbor, MI). These compounds were dissolved in ethanol (furegrelate and SQ-29,548) or methyl acetate (U-46,619) and diluted to the required final concentration with saline. Original compounds synthesized in the Laboratory of Medicinal Chemistry, University of Liège, Belgium (Fig. 1), were as follows: BM-500 (N-isopropyl-N'-[5-nitro-2-m-toluylaminobenzenesulfonyl]urea); BM-520 (N-tert-butyl-N'-[5-nitro-2-m-toluylaminobenzenesulfonyl]urea); BM-531 (N-tert-butyl-N'-[2-(cyclohexylamino)-5-nitrobenzenesulfonyl]urea); BM-567 (N-n-pentyl-N'-[2-(cyclohexylamino)-5-nitrobenzenesulfonyl]urea); BM-573 (N-tert-butyl-N'-[5-nitro-2-p-toluylaminobenzenesulfonyl]urea); BM-600 (N-n-butyl-N'-[2-(cyclohexylamino)-5-nitrobenzenesulfonyl]urea); BM-616 (N-tert-butyl-N'-[2-(4-bromo-3-methylphenylamino)-5-nitrobenzenesulfonyl]ure-a); BM-519 (N-homopiperidinocyanoiminocarboxyl-5-nitro-2-m-toluylaminobenzenesulfonamide); BM-615 (N-n-pentyl-N'-[2-(cyclohexylamino)-5-benzonitrilsulfonyl]urea); and torasemide (N-isopropyl-N'-[(4-m-toluylamino-3-pyridinyl)-sulfonyl]urea). They were dissolved in dimethylsulfoxide and phosphate-buffered saline (30:70 v/v, respectively) to obtain 1 mM stock solutions. Further dilutions were performed using phosphate-buffered saline alone.
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95% viability in each experiment performed.
Animals. Eight- to 12-week-old male Wistar rats weighing 250 to 300 g were sacrificed by decapitation after i.p. injection of chloral hydrate (400 mg/kg). The thoracic aorta was removed under a dissection microscope and immediately transferred into culture dishes containing cold serum-free DMEM (Invitrogen, Grand Island, NY). The periaortic fibroadipose tissue was removed with microdissection forceps and iridectomy scissors, paying attention not to damage the aortic wall. One-millimeter-long aortic rings (
22–25 per thoracic aorta portion) were sectioned with a surgical blade and further rinsed in five consecutive cold DMEM baths. These experimental procedures were carried out in accordance with the Declaration of Helsinki (Principles of Laboratory Animal Care, National Institutes of Health publication 85-23, revised 1985) and had been reviewed and approved by the Ethics Committee of the University of Liège (Liège, Belgium).
Human Platelet TP Receptor Affinity. The binding test was performed using washed human platelets according to a method described previously (Dogne et al., 2003). The concentration of drug that reduced the amount of specifically bound [5,6-3H]SQ-29,548 by 50% (IC50) was determined for each drug by nonlinear regression analysis (GraphPad Prism software). Results were expressed as means ± S.D.
In Vitro Human Platelet Aggregation. Blood was collected by venipuncture from healthy volunteers reported to be free from any medication for at least 10 days and diluted (9:1) with trisodium citrate (3.8% w/w) in a polypropylene tube. Platelet-rich plasma (PRP) was obtained from the supernatant fraction after centrifugation for 10 min at 90g (25°C). The remaining fraction was further centrifuged at 2000g for 10 min (25°C), and the supernatant, a platelet-poor plasma (PPP), was harvested. The platelet concentration of PRP was determined using a hemacytometer and adjusted to 3 x 105 cells/µl by dilution with PPP. An aggregation test was performed according to Born's turbidimetric method (Born and Cross, 1963) using a two-channel aggregometer (Chrono-log). PPP was used to adjust the photometric measurement to the minimal optical density. PRP (225 µl) was added in a silanized cuvette and stirred (1100 rpm). Drug solution (25 µl) was then added, and the mixture was incubated at 37°C for 3 min. Platelet aggregation was initiated by addition of 5 µl of arachidonic acid (AA) (600 µM final concentration) or 2 µl of U-46,619 (1 µM final concentration). To evaluate platelet aggregation, the maximal increase in light transmission was determined from the aggregation curves 7 min after addition of the inducer. The drug concentrations able to decrease U-46,619-induced platelet aggregation by 50% (IC50) were calculated by nonlinear regression analysis from at least three concentration-response curves. Results were expressed as means ± S.D. of at least three independent experiments.
Arachidonic Acid-Induced Platelet TXA2 Generation. After incubation of PRP with arachidonic acid in the presence or absence of the drugs being investigated, platelet TXA2 generation was stopped by addition of indomethacin (100 µM final concentration). The PRP was then centrifuged, and supernatant TXB2 levels were measured as the stable metabolite of TXA2 by a specific enzyme immunoassay (Cayman Chemical) according to the manufacturer's instructions. Results are expressed as the means ± S.D. of at least three independent experiments.
Purified Ovine COX-1 and COX-2 Enzyme Inhibition Test. Purified ovine COX-1 and COX-2 inhibition assays were performed as described previously (de Leval et al., 2001) and prostaglandin (PG) E2 production was quantified by a specific enzyme immunoassay (Cayman Chemical) according to the manufacturer's instructions.
HUVEC Chemotaxis Assay. HUVECs were harvested from 70 to 80% confluent colonies and resuspended in endothelial basal medium-2 (Biowhittaker) containing 2% fetal bovine serum (Biowhittaker, Petit-Rechain, Belgium) at a density of 5 x 105 cells/ml. HUVEC migration was assessed using modified Boyden chambers (BD Biosciences, San Jose, CA). Cells were placed in the upper chamber, and migration was initiated by placing 1 ml of endothelial basal medium-2 containing 10 ng/ml VEGF (Biowhittaker) in the bottom chamber. Drugs to be investigated were added to both the upper and bottom chambers to obtain a 10 µM or 1 µM final concentration. After 12 h of incubation at 37°C in a humidified incubator with a 5% CO2-air atmosphere, the migration was stopped by replacement of the medium by methanol followed by a 20-min incubation at –20°C. Cells were stained in a 6% Giemsa solution in water. Cells at the upper side of the membrane were removed using a cotton swab, and the membrane was cut out and fixed. Cells that had migrated through the membrane were counted. For each experimental condition, a minimum of three chambers were used.
HUVEC Intracellular Calcium Pool Mobilization. The protocol used to assess intracellular calcium pool mobilization was performed essentially as described by Kroll et al. (2005). In brief, HUVECs were harvested from 70 to 80% confluent populations, washed twice with Krebs-HEPES buffer, and incubated for 1 h at 37°C in 10 ml of Krebs-HEPES buffer supplemented with 50 µg of Fluo-4 (Invitrogen, Eugene, OR). After incubation, the cell suspension was centrifuged and resuspended in Krebs-HEPES at a density of 5 x 106 cells/ml; 150 µl of Fluo-4-treated HUVEC suspension were added to each well of a 96-well plate and further incubated in a Fluoroskan Ascent (Thermo Electron Corporation, Waltham, MA) for 15 min in the presence or absence of BM-567 or BM-573 (10 µM final concentration). HUVEC intracellular Ca2+ influxes were measured fluorimetrically after the addition of U-46,619 (1 µM final concentration) to each well (excitation at 485 nM wavelength; measurements at 538 nM wavelength). In a second step, 10 µl of Triton X-100 solution (10% in water) were added to each well to evaluate the maximal fluorimetric response measured. Finally, 10 µl of a 140 mM EGTA solution were added to each well, and the minimal fluorimetric response was measured. Ca2+ influxes were calculated using the equation: [Ca2+]i = 385 x (F – Fmin)/Fmax – F). In this equation, 385 is the Fluo-4 KD for Ca2+, F represents the absorbance measured in the presence of U-46,619, Fmax represents the absorbance measured after treatment with Triton X-100 solution, and Fmin represents the absorbance measured after the treatment with EGTA solution.
Assessment of Endothelial Cell Adhesion. HUVEC adhesion tests were performed as described previously (Bellahcene et al., 2000). In brief, microtiter 96-well plates (Greiner, Wemmel, Belgium) were coated with 10 µl of vitronectin (1 µg/ml) or fibronectin (60 µg/ml). HUVECs were incubated with various concentrations of test compound or with vehicle in endothelial growth medium-2 medium. After 20 h of incubation, cells were harvested and counted. Then, 2 x 104 living cells were incubated at 37°C for 2 h in wells precoated with vitronectin or fibronectin. Attached cells were stained with crystal violet, and the incorporated dye was measured by reading absorbance at 560 nm using a scanning multiwell spectrophotometer.
Assessment of Endothelial Cell Viability and Proliferation. HUVECs were seeded at a density of 104 cells/well in 96-well microtiter plates precoated with 0.2% (v/v) gelatin (Sigma, Bornem, Belgium). Twenty-four hours later, medium was removed and replaced by 100 µl of basal medium (MCDB 131; Invitrogen) supplemented or not with 20% fetal bovine serum (proliferation or viability, respectively) in the presence of the drugs to be investigated (10 µM final concentration) or vehicle alone. Viability of HUVECs was determined by a colorimetric assay based on formazan dye formation (WST-1; Boehringer Mannheim, Mannheim, Germany), which directly correlates with the number of metabolically active cells in the culture. After incubation of the cells for a period of 20 h, 10 µl of WST-1/well were added and further incubated for 2 h at 37°C. A decrease in the number of viable cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample with an ensuing loss in formazan dye formation. The formazan dye was quantified by measuring the optical density of the dye solution at 450 nm with a scanning multiwell spectrophotometer using 690 nm as the internal reference.
Rat Aortic Ring Model of Angiogenesis. A rat aortic ring model of angiogenesis was essentially used as described previously (Nicosia and Ottinetti, 1990). In brief, cleaned rat aortic rings were embedded into rat tail interstitial type I collagen gel (1.5 mg/ml). This final collagen solution was obtained by mixing 7.5 volumes of type I collagen (2 mg/ml, Collagen R; Serva, Heidelberg, Germany) with 1 volume of 10 times concentrated DMEM (Invitrogen, Paisley, Scotland), 1.5 volumes of sodium bicarbonate solution (15.6 mg/ml), and 0.1 volume of sodium hydroxide solution (1 M) to adjust the pH to 7.4. Collagen-embedded rat aortic rings were processed in cylindrical agarose wells and placed in triplicate in 60-mm bacteriologic polystyrene dishes containing 8 ml of serum-free MCDB-131 medium (Invitrogen) supplemented with 25 mM NaHCO3, 1% glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. These ex vivo organotypic cultures were treated with single compound concentrations ranging from 10 to 0.1 µM. After 9 days of culture at 37°C under a air-CO2 (95%:5%) atmosphere, the aortic rings were photographed under an optic microscope (25x magnification, Carl Zeiss AxioCam HR Workstation, KS100 3.0 software). Images were digitalized into 1300 x 1300 pixels with 256 gray levels and assessed infographically using Psion 1.62 software. Areas of neovascularization and maximal neoformed vessel lengths were evaluated as parameters of the observed angiogenic response.
Statistical Analysis. All results are expressed as means ± S.D. Statistical significance was determined using the Student's t test. Probability values less than 0.05 were considered to be significant.
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Results |
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Original Torasemide Derivatives Prevent U-46,619-Induced Human Platelet Aggregation. The ability of our original compounds to prevent human platelet aggregation was determined using U-46,619, a TXA2 stable agonist, as inducer. The use of U-46,619 allowed us to estimate the IC50 for each original compound (Table 1). In this test, all of the torasemide-derived compounds investigated displayed significantly enhanced antiaggregatory properties compared with torasemide or furegrelate whereas SQ-29,548 exhibited a significantly higher preventive effect with an IC50 of 0.035 ± 0.007 µM. Among the torasemide derivatives tested, the highest antiaggregatory effects were observed with BM-567 and BM-573, which prevented U-46,619-induced platelet aggregation with IC50 values of 0.298 ± 0.042 and 0.240 ± 0.013 µM, respectively.
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Original Torasemide Derivatives Inhibit TXS Activity. To determine whether our compounds displayed a dual TXA2 modulation activity, we evaluated their ability to decrease platelet TXA2 release occurring during platelet aggregation. This hypothesis was tested by measuring the levels of TXB2, a stable TXA2 metabolite, after platelet aggregation induced by AA. An important release of TXB2 was observed during AA-induced platelet aggregation (632 ± 179 versus 85,525 ± 11,481 pg/ml in PRP before and after treatment with AA, respectively). Pretreatment of platelets with our original torasemide derivatives at the final concentrations of 100 and 10 µM resulted in a significant and concentration-dependent inhibition of TXS activity (Table 1). Using the same assay, SQ-29,548, a TP receptor antagonist, failed to prevent this release whereas furegrelate, a TXS inhibitor, dose dependently inhibited TXS activity.
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Original Torasemide Derivatives Inhibit Endothelial Cell Migration. Migration of HUVECs was evaluated in Boyden chambers. The chemotactism of HUVECs was significantly enhanced by VEGF (10 ng/ml final concentration) and U-46,619 (0.3 and 0.1 µM final concentrations) (Fig. 2A). Reference drugs and our original compounds were assayed for their ability to affect the migration of VEGF-challenged HUVECs through the chambers. As shown in Fig. 2B, all the drugs tested, except torasemide and BM-500, significantly reduced the migration of VEGF-challenged HUVECs at the concentration of 10 µM. When the drugs were used at 1 µM final concentration, migration inhibition remained statistically significant for SQ-29,548 and the torasemide derivatives BM-519, BM-567, and BM-573.
Original Torasemide Derivatives Do Not Affect the Viability and Adhesion of Endothelial Cells. Incubation of HUVECs with our original compounds at final concentrations of 10, 1, and 0.1 µM in the presence or absence of fetal bovine serum did not significantly inhibit formazan dye formation compared with appropriate controls (data not shown). These data demonstrated the lack of cytotoxicity or antiproliferative effects of our original compounds on HUVECs. The impact of the torasemide-derived compounds on cellular adhesion were also evaluated in cell adhesion assays using vitronectinand fibronectin-coated microtiter plates. Neither U-46,619 (at final concentrations of 1, 0.3, and 0.1 µM) nor the original compounds altered the adhesion of HUVECs to vitronectin or fibronectin (Fig. 3, A and B).
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; Joo et al., 2002; LaMontagne et al., 2006), and it has been shown previously that activation of TXA2 signaling leads to a release of Ca2+ from intracellular stores (Kinsella, 2001). We observed that U-46,619 (1 µM final concentration) induced Ca2+ influxes in HUVECs with a measured intracellular Ca2+ mobilization of 3213 ± 300 nM. Pretreatment of HUVECs with BM-567 and BM-573 (10 µM final concentration) significantly prevented U-46,619-induced Ca2+ mobilization (1484 ± 49.5 and 1056 ± 108; P < 0.01 for each experimental condition).
Torasemide-Derived Compounds Inhibit Angiogenesis in the ex Vivo Aortic Ring Assay. Treatment of rat aortic rings with VEGF (20 ng/ml) resulted in a significant increase in vessel sprouting (Fig. 4A), which was quantified by measurement of the neovascularization area and the maximal vessel outgrowth length (0.937 ± 0.055 mm2 and 1.44 ± 0.105 mm for 20 ng/ml VEGF-treated explants versus 0.630 ± 0.036 mm2 and 0.78 ± 0.301 mm for controls, respectively). U-46,619 at final concentrations of 0.3 and 0.1 µM also significantly increased the neovascularization area of the rat aortic ring explants whereas it decreased this neovascularization at the final concentration of 1 µM (Fig. 4, B and C). SQ-29,548 and our original compounds were tested at final concentrations of 0.1, 1, and 10 µM. Because they exhibited a weak inhibitory profile in the chemotaxis assay, BM-600 and BM-615 were not assayed. Experiments carried out with SQ-29,548 and torasemide showed that these drugs did not modify the angiogenic response of the rat aortic ring explants in terms of both neovascularization area and maximal vessel outgrowth length. Results obtained with our original compounds are summarized in Fig. 5, A and B. BM-616 significantly decreased the neovascularization area (Fig. 5, C, ,D and E). The torasemide derivatives were also investigated in this model under VEGF stimulation (20 ng/ml). Under these conditions, BM-519, BM-520, and BM-616 still significantly decreased the angiogenic response when evaluated in the presence of VEGF (Fig. 6, A and B).
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Discussion |
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). Ridogrel and terbogrel belong to this family. They exhibit inhibitory activities toward TXS and/or TP receptors that are weaker than or equivalent to those observed with our torasemide-derived compounds. They have been tested in clinical trials as antithrombotic and anti-inflammatory agents (Dogne et al., 2000; Carty et al., 2001). Yet, to the best of our knowledge, none of these molecules has been tested for its capacity to inhibit angiogenesis.
In this study, we have synthesized, produced, characterized, and selected nine compounds that modulate the pathway of TXA2, a prostanoid involved in cancer development/progression. These original compounds, derived from torasemide, represent a new family of chemical molecules that act as TXA2 receptor antagonists and TXA2 synthase inhibitors and are able to inhibit endothelial cell migration in vitro and angiogenesis in an ex vivo animal model.
The present evaluation of our dual TXA2 modulators demonstrates that these compounds, when used at the final concentration of 10 µM (with the exception of BM-500), significantly inhibit the migration of VEGF-challenged HUVECs. This inhibitory activity remains significant for BM-567, BM-573, and BM-519 when they are added to the cells at the final concentration of 1 µM. It is interesting to note that these three compounds, which have been identified as the most potent ligands of human platelet TP receptors, also appear to be the most efficient for preventing human platelet aggregation induced by U-46,619. This observation suggests a direct correlation between the potency of the compounds to modulate the TXA2 pathway and their ability to inhibit VEGF-driven migration of endothelial cells.
In this study, we have further investigated the effects of our original torasemide derivatives in the rat aortic ring assay, which represents a valuable ex vivo angiogenic model because it does not require exogenous growth factors and recapitulates virtually all of the biologic steps of a complete and spontaneous angiogenic phenomenon. We have found that all torasemide-derived compounds assayed in this ex vivo angiogenesis model display significant antiangiogenic effects, with the highest inhibition obtained with BM-616. It is noteworthy that this molecule was not the most potent inhibitor of endothelial cell migration. This difference may, at least partly, be explained by the difference in stability of the different drugs tested. For example, BM-616 presents a particular substitution pattern with a bromine atom adjacent to the methyl group of R2. This substitution is known to prevent the commonly observed oxidations of methyl substituents in the ortho position.
In humans, TXA2 signals through two TP receptor isoforms named TP and TP
. These two TP receptors contain seven-transmembrane domains and can be coupled to a large number of G-proteins. Depending on the G-protein, various intracellular signaling pathways involving adenyl cyclase, phospholipase C, or AG1478-sensitive epidermal growth factor receptor, can be switched on (Sinead et al., 2002). Yet, TP receptors are thought to prominently signal through the activation of the -isoenzymes of phospholipase C, leading to phosphatidylinositol turnover and release of Ca2+ from intracellular stores (Kinsella, 2001). Interestingly, several studies have suggested the involvement of intracellular Ca2+ fluxes in endothelial cell migration (Shukla et al., 2001; Joo et al., 2002; LaMontagne et al., 2006). In this context, we have sought to determine whether some of our most potent torasemide derivatives (BM-567 and BM-573) may in fact modulate the mobilization of intracellular Ca2+ pools in HUVECs. We have observed that BM-567 and BM-573 are able to decrease U-46,619-induced intracellular Ca2+ influxes in endothelial cells. Therefore, the ability of our drugs to inhibit endothelial cell migration may be explained, at least in part, by their interference with intracellular Ca2+ mobilization in HUVECs. These findings are in agreement with the general view that 1) the TXA2 pathway signals through the activation of phospholipase C, leading to phosphatidylinositol turnover, and subsequent release of Ca2+ from intracellular stores (Kinsella, 2001) and 2) intracellular Ca2+ fluxes are involved in endothelial cell migration (Shukla et al., 2001; Joo et al., 2002; LaMontagne et al., 2006).
Accumulating evidence suggests an important role for the TXA2 pathway in tumor growth and associated angiogenesis. For example, Pradono et al. (2002) have transduced a retroviral vector carrying TXS or prostacyclin synthase cDNA to colon-26 adenocarcinoma cells and have subsequently inoculated the transformants to BALB/c mice. They have observed that tumors derived from TXS transformants grow significantly faster than null-vector controls and show more abundant vasculature, indicating that the TXA2 pathway may be a critical determinant for angiogenesis and subsequent tumor development. In the same model, tumors derived from prostacyclin synthase transformants grow significantly less than null-vector controls and show reduced vasculature. These data further illustrate the antagonistic effects of TXA2 and prostacyclin (PGI2) in tumor angiogenesis and clearly suggest that the selective targeting of COX-2 downstream mediators, such as TXA2, may be an interesting approach to inhibit tumor-associated angiogenesis (Pradono et al., 2002). In general terms, the growth of primary cancers is dependent on the angiogenic process, and this is also the case for metastatic lesions. Whether these lesions would respond similarly to anti-TXA2 modulators still remains to be answered. Clinically, it is well known that some cancer types, such as clear cell carcinoma of the kidney, appear more "angiogenic" than others. This type of malignant lesion may therefore be more susceptible to an inhibition of the TXA2 pathway. However, this theory undoubtedly remains to be tested in trials. Our findings describing the antiangiogenic effects of dual TXA2 modulators might be useful in other therapeutic areas. Indeed, angiogenesis is also a critical process and a potential therapeutic target in several nonmalignant diseases such as rheumatoid arthritis and osteoarthritis (Walsh, 1999), diabetic retinopathy (Gargiulo et al., 2004), psoriasis (Leong et al., 2005), endometriosis (Fujimoto et al., 1999), and preeclampsia (Ahmad and Ahmed, 2004).
The key role of prostanoids in cancer is unanimously recognized and has been demonstrated through epidemiologic, experimental, and clinical observations. It is also generally admitted that the toxicity associated with the administration of COX inhibitors, whether selective or not, limits their use in cancer prevention strategies as well as for the treatment of patients suffering from chronic diseases. Indeed, uptake of NSAIDs is commonly associated with gastrointestinal side effects. This toxicity is related to the ability of these drugs to inhibit the COX-1 isoform, which participates in gastric mucosa protection through the synthesis of prostaglandins (de Leval et al., 2000). This gastrointestinal toxicity is not observed with COX-2 selective inhibitor treatment. However, rofecoxib, a selective COX-2 inhibitor, was withdrawn from the market at the end of September 2004 because of an increased risk of cardiovascular events. This cardiovascular toxicity has been demonstrated in two double-blind, randomized clinical trials: the APPROVe (Adenomatous Polyp Prevention on VIOXX) and VIGOR (Vioxx Gastrointestinal Outcomes) studies, which have demonstrated a 2-fold increase in the risk of serious thromboembolic events, including stroke and myocardial infarction in the rofecoxib-treated group compared with the placebo group (Bombardier et al., 2000; Bresalier et al., 2005). This cardiovascular toxicity can be associated with the COX-2 inhibitory properties of rofecoxib. Indeed, although COX-2 is an inducible enzyme expressed in pathologic conditions such as inflammation and cancer progression, this enzyme is also expressed in physiologic conditions by several tissues such as vascular endothelium, kidney, and brain. In terms of cardiovascular physiology, COX-2 has been demonstrated to be the prominent source of PGI2 in vivo (Belton et al., 2000). This prostanoid displays powerful vasodilatative and antiaggregatory properties. On the other hand, COX-1 and TXA2 synthase are constitutively expressed within platelets. COX-1 is responsible for the enzymatic transformation of arachidonic acid into PGH2 which is subsequently transformed by the TXA2 synthase into TXA2, a potent inducer of platelet aggregation and vasoconstriction (Dogne et al., 2004). Nonspecific NSAIDs block both COX isoforms and therefore have a balanced effect on reducing the prothrombotic actions of TXA2 and the antithrombotic effects of PGI2 (Dogne et al., 2004). In contrast, rofecoxib suppresses the formation of endogenous PGI2 without affecting TXA2 generation in healthy volunteers, potentially creating an alteration of the vascular homeostasis (FitzGerald, 2003). Such results have also been demonstrated with celecoxib, another COX-2 selective inhibitor. The shift in the TXA2/PGI2 balance is a strong theoretical basis for an association between use of coxibs and the occurrence of thromboembolic disorders (Pratico and Dogne, 2005). In this context, our original dual TXA2 modulators should have a positive effect on the TXA2/PGI2 balance and may prevent thromboembolic occurrence in vivo. Our original compounds indeed combine the advantages of pure TXA2 receptor antagonist and TXS inhibitors: blocking the agonistic activities of TXA2 on its receptors and allowing the accumulation of endoperoxide PGH2 that can be metabolized into PGI2 by endothelial cell prostacyclin synthase (Marcus et al., 1981). It is also noteworthy that our original compounds lack inhibitory effects against COX-1 or COX-2 activity in vitro. Therefore, we think that no major cardiovascular adverse effect should be expected from the administration of our compounds. Yet, we acknowledge that this speculation, based on strong in vitro data, should be confirmed. Overall, we believe that our torasemide derivatives represent an exciting and potentially viable alternative to COX inhibitors for the prevention and treatment of diseases in which angiogenesis is turned on.
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Footnotes |
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A. Bellahcène and D. Waltregny are research associates and T. Dassesse is a Télévie Research Fellow at the National Fund for Scientific Research (FNRS, Belgium).
X.d.L. and T.D. contributed equally to this work. B.P. and V.C. codirected the study.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: COX, cyclooxygenase; NSAIDs, nonsteroidal anti-inflammatory drugs; TXA2, thromboxane A2; VEGF, vascular endothelial growth factor; U46,619, 9,11-dideoxy-9,11-methanoepoxy-prostaglandin F2; HUVEC, human umbilical endothelial cell; SQ-29,548, 1S-[1,2(Z),3,4]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid; TP receptor, TXA2 receptor; TXS, thromboxane synthase; BM-500, N-isopropyl-N'-[5-nitro-2-m-toluylaminobenzenesulfonyl]urea; BM-520, N-tert-butyl-N'-[5-nitro-2-m-toluylaminobenzenesulfonyl]urea; BM-531, N-tert-butyl-N'-[2-(cyclohexylamino)-5-nitrobenzenesulfonyl]urea; BM-567, N-n-pentyl-N'-[2-(cyclohexylamino)-5-nitrobenzenesulfonyl]urea; BM-573, N-tert-butyl-N'-[5-nitro-2-p-toluylaminobenzenesulfonyl]urea; BM-600, N-n-butyl-N'-[2-(cyclohexylamino)-5-nitrobenzenesulfonyl]urea; BM-616, N-tert-butyl-N'-[2-(4-bromo-3-methylphenylamino)-5-nitrobenzenesulfonyl]urea; BM-519, N-homopiperidinocyanoiminocarboxyl-5-nitro-2-m-toluylaminobenzenesulfonamide; BM-615, N-n-pentyl-N'-[2-(cyclohexylamino)-5-benzonitrilsulfonyl]urea; DMEM, Dulbecco's modified Eagle's medium; PRP, platelet-rich plasma; PPP, platelet-poor plasma; AA, arachidonic acid; PG, prostaglandin; PGI2, prostacyclin.
Address correspondence to: Dr. Xavier de Leval, Natural and Synthetic Drugs Research Center, Laboratory of Medicinal Chemistry, University of Liège, Avenue de l'hôpital, Bat. B36, B-4000 Liège, Belgium. E-mail: xdeleval{at}ulg.ac.be
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