Fluvastatin Synergistically Improves the Antiproliferative Effect of Everolimus on Rat Smooth Muscle Cells by Altering p27/ Cyclin E Expression

Multiple intracellular signaling pathways stimulate quiescent smooth muscle cells (SMCs) to exit from G0 and re-enter the cell cycle. Thus, a combination of two drugs with different mechanisms of action may represent a suitable approach to control SMC proliferation, a prominent feature of in-stent restenosis. In the present study, we investigated the effect of everolimus, a mammalian target of rapamycin inhibitor, in combination with fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on proliferation of rat SMCs. The antiproliferative action of everolimus was amplified by 2.5-fold by the addition of subliminal concentrations of fluvastatin (5 10 7 M), lowering the IC50 value from 2.5 10 9 to 1.0 10 9 M. The increased antiproliferative effect of everolimus by fluvastatin was prevented in the presence of mevalonate, farnesol, or geranylgeraniol, suggesting the involvement of prenylated proteins. Cell cycle analysis and [H]thymidine incorporation assay demonstrated that the two drugs synergistically interfered with the progression of G1 phase. In particular, the drug combination significantly up-regulated p27 levels by 47.0%, suppressed cyclin E by 43.0%, and it reduced retinoblastoma (Rb) hyperphosphorylation by 79.0%, compared with everolimus alone. Retroviral overexpression of cyclin E conferred a significant resistance of rat SMCs to the antiproliferative action of the drug combination, measured by cell counting, [H]thymidine incorporation, and cell cycle analysis, with higher levels of hyperphosphorylated form of Rb. Taken together, these results demonstrated that everolimus acts synergistically with fluvastatin to inhibit SMC proliferation by altering the expression of cyclin E and p27, which affects Rb phosphorylation and leads to G1 phase arrest. Smooth muscle cell (SMC) proliferation in the arterial wall is the major determinant of restenosis after balloon angioplasty and stent coronary implantation (Ross, 1999; Hansson, 2005). The introduction of drug-eluting stent has significantly improved the restenosis process and the patient outcome after revascularization; but recently, the safety and the efficacy of this approach have been reevaluated (Boden et al., 2007; Stone et al., 2007). Thus, single and/or combined oral therapy has been proposed as promising approach to achieve a better clinical outcome after percutaneous coronary intervention (Mody et al., 2001; Boden et al., 2007). In particular, a combination of two different pharmacological inhibitors capable of antagonizing different intracellular signaling pathways involved in cell cycle reentry may lead to better control of SMC proliferation. The 40-O-(2-hydroxyethyl)-derivative of rapamycin, everolimus, is a proliferation signal inhibitor that affects growth factor-induced proliferation of hematopoietic and nonhematopoietic cells via cell cycle arrest at the late G1 phase (Price et al., 1992; Brown et al., 1995; Decker et al., 2003; Hafizi et al., 2004). The antiproliferative action of everolimus is elicited through binding to the mammalian target of rapamycin complex (mTORC) 1 composed of mTOR, a common regulatory subunit called LST8, and the raptor subunit that specifies the downstream substrates (Schuler et al., 1997; Sarbassov et al., 2004; Shaw and Cantley, 2006). The binding of everolimus to mTORC1 complex strongly inhibits its catalytic activity and the activation of two well characterized mTORC1 complex substrates that control translation and cell growth, This research was supported by Novartis-Pharma AG (Basel, Switzerland). 1 Current affiliation: Nicox S. A., Sophia Antiplois, France. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.046045. ABBREVIATIONS: SMC, smooth muscle cell; mTOR, mammalian target of rapamycin; mTORC, mammalian target of rapamycin complex; 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1; TEMED, N,N,N ,N -tetramethylethylenediamine; FOH, farnesol; GGOH, geranylgeraniol; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; MVA, mevalonate; Rb, retinoblastoma; IRES, internal ribosomal entry site; cdk, cyclin-dependent kinase; SCH66336, lonafarnib. 0026-895X/08/7401-144–153$20.00 MOLECULAR PHARMACOLOGY Vol. 74, No. 1 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 46045/3352965 Mol Pharmacol 74:144–153, 2008 Printed in U.S.A. 144 http://molpharm.aspetjournals.org/content/suppl/2008/07/25/mol.108.046045.DC1 Supplemental material to this article can be found at: at A PE T Jornals on July 5, 2017 m oharm .aspeurnals.org D ow nladed from the p70S6 protein kinase (p70S6) and the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) family of proteins (Brown et al., 1995; Brunn et al., 1997). More recently, everolimus has been shown to directly interfere with the assembly of the rapamycin-insensitive rictor/mTOR protein complex, mTORC2, and to block AKT signaling (Zeng et al., 2007). Thus, the inhibition of both mTORC1 and mTORC2 is considered the pivotal molecular mechanism for the antiproliferative effect of everolimus. The inhibition of cell proliferation is thought to be the basic molecular mechanism for the multiple actions of everolimus, such as immunosuppression, prevention of renal and heart transplant rejection, and retardation of cardiac allograft vasculopathy (Schuler et al., 1997; Nashan, 2002). In an experimental model of in-stent restenosis, oral administration of everolimus inhibited SMC proliferation at similar degree to that seen with rapamycin-eluting stents, suggesting a potential oral use of this drug for restenosis (Farb et al., 2002). This feature has made rapamycin and everolimus an attractive pharmacological tool for the development of drug-eluting stents. Indeed, everolimus-eluting stents as rapamycin-eluting stents, have been reported to inhibit in-stent neointimal growth in patients with coronary artery disease (Grube et al., 2004). A second class of drugs that strongly affects cell proliferation is represented by the HMG-CoA reductase inhibitors, also called statins. We have previously shown that fluvastatin interferes with SMC proliferation in vitro at therapeutic concentrations (0.1–1 10 6 M), and more importantly, sera from patients treated with fluvastatin can significantly reduce SMC proliferation in an ex vivo assay (Corsini et al., 1996). The ability of statins to inhibit SMC proliferation seems to be independent from their cholesterol-reducing properties, and more likely to be related to the depletion of intracellular nonsteroidal isoprenoid compounds, such as farnesol (FOH) and geranylgeraniol (GGOH), which inhibits intracellular protein prenylation process (Corsini et al., 1993; Raiteri et al., 1997; Bellosta et al., 2000). Several prenylated proteins belonging to different intracellular signaling pathways have been documented to be indispensable for cell proliferation, including the small GTP-binding protein Ras, and Ras-like proteins, such as Rho, Rac, and Rap (Corsini et al., 1999; Brown et al., 2006). Interestingly, the combination fluvastatin everolimus has been shown previously to have a beneficial effect on graft vascular disease in a rat model of chronic heart rejection, measured as arterial intimal thickness, suggesting a potential positive effect between the two drugs on SMC proliferation. The basic molecular mechanisms, however, have not been elucidated (Gregory et al., 2001). On this basis, in the present study we explored the potential synergistic inhibitory effect of the combination everolimus fluvastatin on SMC proliferation and the underling molecular mechanisms. Materials and Methods Reagents and Antibodies. DMEM, trypsin ethylenediaminetetraacetate, penicillin (10,000 U ml ), streptomycin (10 mg ml ), 1 M Tricine buffer, pH 7.4, nonessential amino acid solution (100 ), and fetal calf serum (FCS) were purchased from Invitrogen (Carlsbad, CA). Disposable culture flasks and Petri dishes were from Corning Life Sciences (Acton, MA), and filters were from Millipore Corporation (Billerica, MA). [6-H]Thymidine, sodium salt (2 Ci/mmol) was from GE Healthcare (Milan, Italy), and molecular weight protein standards were from Bio-Rad Laboratories (Hercules, CA). Isoton II was purchased from Instrumentation Laboratories (Milan, Italy). SDS, TEMED, ammonium persulfate, glycine, and acrylamide solution (30% T, 2.6% C) were obtained from Bio-Rad Laboratories. Cytox-Dye was purchased from Invitrogen. Fluvastatin (Corsini et al., 1995) and everolimus (SDZ RAD) (Schuler et al., 1997) were provided by Novartis-Pharma AG (Basel, Switzerland). FOH, GGOH, and mevalonate (MVA) were from Sigma (Milan, Italy). For Western blot analysis, the following antibodies were used: anti-cyclin D1, anti-cyclin E, anti-cdk2, anti-p70S6 kinase, and anti-phosphop70S6 kinase Thr 412 (Millipore, Vimodrone, Italy); anti-p27 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-Rb protein (Millipore); anti 4E-BP1 (Cell Signaling Technology Inc., Danvers, MA); anti-p21 (Abcam plc, Cambridge, UK); and anti-mouse and anti-rabbit peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Cell Proliferation and DNA Synthesis. SMCs were cultured from the intimal-medial layers of aorta of male Sprague-Dawley rats as described previously (Corsini et al., 1995). Cells were seeded at a density of 1 10 SMC/35-mm Petri dish, and then they were incubated with DMEM supplemented with 10% FCS. Twenty-four hours later, the medium was changed to medium containing 0.4% FCS to stop cell growth, and the cultures were incubated for 72 h. At this time (time 0), the medium was replaced with medium containing 10% FCS in the presence or absence of known concentrations of the drugs, and the incubation was continued for further 72 h at 37°C. Cell proliferation was evaluated by cell counting with a Coulter Counter model ZM (Beckman Coulter, Fullerton, CA) after trypsinization of the monolayers. At time 0, just before the addition of the substances to be tested, three Petri dishes were used for cell counting. The total cell number determined at time 0 was subtracted from cell number found in each triplicate after 72 h of cell growth. For DNA synthesis, synchronization of SMCs to the G0/G1 phase of the cell cycle was accomplished by incubating logarithmically growing cultures (3 10 myocytes/Petri dish) for 5 days in a medium containing 0.4% FCS. Quiescent cells were then incubated for 16 h in fresh medium containing 10% FCS in the presence or absence of drugs. DNA synthesis was estimated by nuclear incorporation of [H]thymidine (Ferri et al., 2003). HMG-CoA Reductase Assay. The experimental conditions were the same than those used for cell proliferation assay. HMG-CoA reductase activity was determined by measuring the rate of conversion of radioactive HMG-CoA into MVA in detergent-solubilized cellfree extract (Corsini et al., 1995). Aliquots of the cell-free extracts (30–40 g) were assayed in a buffer containing 0.25 M K2HPO4, pH 7.4, 100 mM glucose 6-phosphate, 15 mM NADP, 50 mM dithiothreitol, and 110 M HMG-CoA (90,000 dpm/sample [C]HMG-CoA) in a total volume of 200 l. Microsomes were preincubated in the reaction buffer at 37°C for 10 min before the addition of HMG-CoA, and then they were incubated for 120 min at 37°C with moderate shaking. The reaction was stopped by the addition of 20 l of 5 M HCl, and 90,000 dpm [H]mevalonolactone standard was added to measure recovery. The reaction solution was then incubated at 37°C for 30 min to allow lactonization of the mevalonate. The mixture was extracted twice with 10 ml (20 ml total) of diethyl ether. The top phase was transferred to a 50-ml conical tube, and the combined upper phases were dried; the residue was resuspended in acetone, spotted on a thin layer chromatography plate, and chromatographed in acetone/benzene (1:1). The activity of HMG-CoA reductase was expressed as cpm incorporated in mevalonate per microgram of detergent-solubilized protein. Cell Cycle Analysis. The experimental conditions used were the same as those used for DNA synthesis assay. Flow cytometry was used to analyze cell cycle distribution. Cells were trypsinized and centrifuged for 5 min at 1000 rpm. Pellets were resuspended in 0.5 Combination Everolimus Fluvastatin and Cell Cycle Progression 145 at A PE T Jornals on July 5, 2017 m oharm .aspeurnals.org D ow nladed from ml of permeabilizing buffer of Cytox Dye (0.5 M in 100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, and 0.1% Nonidet P-40). Samples were placed in the dark for 30 min, and the fluorescence of individual nuclei was measured. Nuclear Cytox Dye fluorescence signal was recorded on the FL2 channel of a FACScan flow cytometer (BD Biosciences, San Jose, CA) and analyzed with ModFit LT software (Verity Software House, Topsham, ME). The number of cells in G0/G1, S, and G2/M phases was expressed as percentages of total events (10,000 cells) (Ferri et al., 2003). Western Blot Analysis. Cells were washed twice with phosphate-buffered saline and lysed by incubation with a solution of 50 mM Tris, pH 7.5, 150 mM NaCl, and 0.5% Nonidet P-40, containing protease and phosphatase inhibitor cocktails (Sigma) for 30 min on ice. Cell lysates were cleared by centrifugation at 14,000g for 10 min, and protein concentrations were determined using the bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL). Lysates were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions, transferred to Immobilon polyvinylidene difluoride (Millipore Corporation), and subsequently immunoblotted with primary antibody following appropriate secondary antibody, before visualization by enhanced chemiluminescence (GE Healthcare). Quantitative densitometric analyses were performed using GelDoc acquisition system and Quantity One software (Bio-Rad Laboratories). Generation of Cyclin E Expression Construct and Retroviral Infection. Full-length rat cyclin E (accession no. D14015) was generated by polymerase chain reaction using the following primers: 5 -ATGAAAGAAGAAGGTGGTTCCG-3 and 5 -TCATTCTGTCTCCTGCTCACTGC-3 . The sequence of the polymerase chain reactiongenerated construct was confirmed by sequencing. Retroviral expression plasmid was then constructed using the pBM-IRES-PURO (Garton et al., 2002) expressing the puromycin resistance gene as a selectable second cistron gene, generated from the original pBMIRES-EGFP, generously provided by Garry P. Nolan (Stanford University, Stanford, CA). Retroviral infections of human SMC were performed as described previously (Garton et al., 2002). Analysis of Drug Synergism. According to the method of Kern et al. (1988), the expected value of cell number (CNexp, defined as the product of the percentage versus control of cell number observed after incubation with drug A alone and the percentage of cell number observed for drug B alone divided by 100) and the actual cell number observed (CNobs) for the combination of A and B were used to construct a synergistic ratio as follows: R CNexp/CNobs. Synergy was defined as any value of R greater than unity. An R value of 1.0 (additive effect) or less indicated an absence of synergy (Kern et al.,