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H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida, and the Department of Interdisciplinary Oncology, University of South Florida College of Medicine, Tampa, Florida (Z.C., K.N.B., J.W.); and Bristol-Myers Squibb Oncology, Princeton, New Jersey (F.Y.L.)
Received October 20, 2005; accepted January 24, 2006
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Abstract |
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tyrosine kinase activity in vitro by BMS-354825 was observed at 4 nM. Direct comparison of BMS-354825 and another PDGFR inhibitor, imatinib (Gleevec, STI571), in VSMCs indicated that BMS-354825 is 67-fold more potent than imatinib in inhibition of PDGFR activation. BMS-354825 also inhibited Src tyrosine kinase in A10 cells. At the cell level, PDGF stimulated migration and proliferation of A10 cells and human AoSMCs, both of which were inhibited by BMS-354825 in a concentration dependent manner in the low nanomolar range. These results suggest that BMS-354825 is a potent inhibitor of PDGF-stimulated VSMC activities and a potential agent for the development of a new therapy for vascular obstructive diseases such as restenosis.
; Michaels and Chatterjee, 2002). Therefore, restenosis represents a major problem limiting the long-term efficacy of these revascularization therapies. Moreover, after coronary artery bypass surgery, narrowing (stenosis) of the transplanted artery can also occur that necessitates further medical treatments.
Abnormal migration and proliferation of VSMCs are critical events in the pathogenesis of artery obstructive diseases such as restenosis (Bailey, 2002; Sanz-Gonzalez et al., 2004; Levitzki, 2005). Although several growth factors and cytokines are involved in the development of restenosis, many lines of evidence have indicated that PDGF plays a prominent role in the pathogenesis of restenosis. PDGF is the most potent mitogen and motogen for VSMCs (Heldin and Westermark, 1999). PDGF is present at sites of vascular injury from activated platelets, monocytes, and cells of the artery wall (Raines, 2004). Expression of exogenous PDGF in animal arteries can induce intimal thickening through stimulation of VSMC proliferation and migration and synthesis of extracellular matrix (Pompili et al., 1995). It is noteworthy that inhibition of PDGF as well as PDGFR by immunological, molecular biological, and pharmacological methods can suppress development of restenotic lesions in animal models (Ferns et al., 1991; Sirois et al., 1997; Myllarniemi et al., 1999; Levitzki, 2005).
Imatinib (Gleevec, STI571, CGP57148B) is the first protein tyrosine kinase inhibitor that has been successfully developed into a targeted therapy drug. It is currently used to treat chronic myeloid leukemia (CML) and gastrointestinal stromal tumor based on inhibition of Bcr-Abl and c-Kit protein tyrosine kinases, respectively (Logrono et al., 2004; Deininger et al., 2005). Besides Bcr-Abl and c-Kit, imatinib also inhibits PDGFR tyrosine kinase (Buchdunger et al., 1996; Druker et al., 1996). Experiments in animals have shown that imatinib inhibits restenosis after balloon angio-plasty and stenosis after allograft (Myllarniemi et al., 1999; Sihvola et al., 2003). Inhibition of PDGFR by imatinib, however, requires micromolar concentrations in cell-based assays (Buchdunger et al., 1996; Sanz-Gonzalez et al., 2004).
After the findings by us and others that a Src tyrosine kinase inhibitor, PD180970, and other pyrido[2,3-d]pyrimidine derivatives could cross-inhibit Bcr-Abl (Dorsey et al., 2000; Wisniewski et al., 2002; Huron et al., 2003), several dual Src/Bcr-Abl inhibitors are now being developed as new therapeutic agents for CML. One of these is the orally bio-available BMS-354825 (Lombardo et al., 2004; Shah et al., 2004; Burgess et al., 2005; O'Hare et al., 2005), which is currently in clinical trials in CML patients. Because the Bcr-Abl tyrosine kinase inhibitor imatinib was originally identified in the screening of PDGFR tyrosine kinase inhibitors, we sought to determine whether BMS-354825 could also cross-inhibit PDGFR in VSMCs and suppress PDGF-stimulated migration and proliferation of VSMCs. We provide evidence here that BMS-354825 is a potent PDGFR tyrosine kinase inhibitor in VSMCs and that it inhibits cellular responses to PDGF in VSMCs at low nanomolar concentrations. These results reveal BMS-354825 as a potential agent for development of a new therapy for restenosis and stenosis, which will help to improve the long-term success of angioplasty/stenting and coronary bypass surgery.
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Materials and Methods |
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was from Upstate Biotechnology (Lake Placid, NY). Antibodies to PDGFR and Src were from Calbiochem (San Diego, CA). Antibodies to Akt, STAT3, Erk1/2, Src (for immunoblotting), insulin-like growth factor-1 receptor (IGF-1R), and epidermal growth factor receptor (EGFR) were from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-activated Erk1/2 antibody was from Promega (Madison, WI). PDGF-BB was from PeproTech (Rocky Hill, NJ). Propidium iodide and rat tail type I collagen were from Roche (Indianapolis, IN). BMS-354825 (N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide monohydrate) was provided by Bristol-Myers Squibb. Imatinib (Gleevec, STI571) was provided by Novartis (Basel, Switzerland). Cell Culture. The A10 rat aortic smooth muscle cell line was obtained from American Type Culture Collection (Manassas, VA) and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 µg/ml streptomycin. Primary culture of human aortic smooth muscle cells (AoSMCs) was obtained from Cambrex Bio Science Walkersville, Inc. (Walkersville, MD) and grown in SmGM-2 Bulletkit medium (Cambrex) plus 100 units/ml penicillin and 100 µg/ml streptomycin. Human AoSMCs were used before the 7th passage. Cells were maintained at 37°C in a humidified 95% air and 5% CO2 incubator.
Immunoprecipitation and Immunoblotting. Cells were lysed in ice-cold lysis buffer (25 mM Tris-HCl, pH 7.2, 150 mM NaCl, 25 mM NaF, 1 mM benzamidine, 1% Triton X-100, 1 mM Na3VO4, 20 mM p-nitrophenyl phosphate, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 100 µg/ml phenylmethylsulfonyl fluoride). Cell lysate supernatants were obtained by microcentrifugation at 4°C for 15 min and the protein concentration was determined. For immunoblotting analysis of cell lysates, cell lysate supernatants were heat-denatured in SDS loading buffer. Antibodies to activated STAT3, activated Akt, activated Erk1/2, and phosphotyrosine were used to assess the activation of these molecules and receptor tyrosine kinases by immunoblotting. Immunoblotting was performed essentially as described previously (Cunnick et al., 2001, 2002; Ren and Wu, 2003) using the SuperSignal West Pico chemiluminescent reagent (Pierce, Rockford, IL). Each immunoprecipitation was performed using 600 µg of cell lysate supernatant, 2 µg of specific antibody indicated in the figure legends, and 30 µl of Protein-A or Protein-G at 4°C for 2 h. Immune complex was collected by microcentrifugation and washed three times with the lysis buffer. Quantification of immunoreactive band intensity was achieved using the ImageQuant program (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
Protein Tyrosine Kinase Assays. PDGFR was immunoprecipitated with an anti-PDGFR antibody (Calbiochem). Immunoprecipitates were washed twice with the lysis buffer and twice with kinase buffer (50 mM HEPES, pH 7.4, 10 mM MnCl2, and 0.1 mM Na3VO4). The immune complex kinase assay was performed in a 50-µl reaction mixture (kinase buffer plus 20 µCi of [
-32P]ATP, and 10 µM ATP) at 30°C for 15 min. Kinase reaction was terminated by addition of 17 µl of 4x SDS loading buffer and heat-denaturation at 95°C for 10 min. Samples were subjected to 8% SDF-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane. Phosphorylation was quantified by filmless autoradiographic analysis or autoradiography. The immune complex kinase assay for c-Src was performed as described previously (Ren et al., 2004).
Cell Migration Assay. Cell migration was measured using the Transwell cell migration assay (Ren et al., 2004). Transwell cell culture insert polycarbonate membrane (6.5 mm, 8.0-µm pore size; Costar, Corning, NY) was coated with rat tail type I collagen (10 µg/ml in PBS) at 4°C for 18 h and air-dried. VSMCs (80% confluent) were serum-starved in DMEM/0.1% BSA for 18 h, detached from plates by digestion with 1:3 diluted trypsin-EDTA (Invitrogen), washed with DMEM, and resuspended in DMEM/0.1% BSA at 5 x 104 cells/ml for A10 cells or 2.5 x 104 cells/ml for human AoSMCs. Cell suspension (0.2 ml per well) was incubated with BMS-354825 or solvent (DMSO) in 1.5-ml microcentrifuge tube for 20 min before been placed in the upper chamber of Transwell. The lower chamber contained 0.6 ml of DMEM/0.1% BSA with or without 5 ng/ml PDGF-BB (PeproTech) and BMS-354825. After incubation at 37°C/5% CO2 for 4 h, cells remaining on the upper membrane surface were mechanically removed with a cotton swab. Migrated cells on the lower side of membrane were fixed and stained with the HEMA3 reagents (Fisher Scientific, Swanee, GA) and enumerated under a microscope in eight randomly chosen fields with a 10 x 10 lens. Each field for quantification of the migrated cell number has an area of 0.8 x 0.6 mm.
Cell Proliferation Analysis. A10 cells and human AoSMC cells were seeded in triplicate at 2 x 104 per plate in 6-cm plates in DMEM/1% FBS with or without PDGF-BB (10 ng/ml). After 24 h (day 0), cells were treated with DMSO (solvent) or 10 to 50 nM BMS-354825. Medium was changed on day 3. Viable cell number was determined on day 6 as described previously (Dorsey et al., 2000). For the recovery experiment, cells were treated as above for 6 days and then cultured without BMS-354825 for another 6 days.
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, STAT3, Akt, and Erk2 by immunoblotting analyses (Fig. 1A). PDGFR is the predominant PDGFR subunit in VSMCs (Raines, 2004) and the only functional PDGFR subunit in A10 cells (Rao et al., 1997). STAT3, Akt, and Erk2 signaling pathways have been reported to mediate PDGF-induced cell migration and proliferation in VSMCs (Graf et al., 1997; Shibata et al., 2003; Zhan et al., 2003; Neeli et al., 2004; Kim et al., 2005; Vantler et al., 2005). PDGF treatment markedly increased PDGFR tyrosine phosphorylation, which was detectable at the earliest time point examined (1 min). BMS-354825 (50 nM) effectively blocked PDGF-stimulated PDGFR tyrosine phosphorylation at all time points examined. Figure 1A also shows that STAT3, Akt, and Erk2 were activated by PDGF in A10 cells as measured by activation-specific phosphorylation and these were inhibited by BMS-354825.
We next immunoprecipitated PDGFR from A10 cells that had been treated with PDGF-BB (5 ng/ml, 5 min) in the presence of 0 to 25 nM BMS-354825 and then analyzed the PDGFR tyrosine phosphorylation by immunoblotting. As illustrated in Fig. 1B, tyrosine phosphorylation of PDGFR was induced by PDGF stimulation in A10 cells; the response was inhibited by BMS-354825 with an IC50 of 3 nM in this assay.
To confirm that BMS-354825 inhibits PDGFR tyrosine kinase activity, PDGFR was immunoprecipitated from A10 cells with or without prior PDGF stimulation (5 ng/ml, 5 min). The PDGFR tyrosine kinase activity was determined in vitro in the immune complexes by autophosphorylation in the presence of various concentrations of BMS-354825. As shown in Fig. 1C, BMS-354825 inhibited PDGFR tyrosine kinase activity in a concentration-dependent manner. The 50% inhibition of PDGFR phosphorylation was observed at 4 nM BMS-354825.
Comparison of PDGFR Inhibition by BMS-354825 and Imatinib. In addition to inhibiting Bcr-Abl and c-Kit, imatinib also inhibits PDGFR tyrosine kinase. To compare the inhibition of PDGFR in VSMCs by BMS-354825 and imatinib, A10 cells were preincubated with various concentrations of BMS-354825 or imatinib, stimulated with PDGF-BB, and activation of PDGFR, STAT3, Akt, and Erk2 were analyzed. Figure 2A shows that PDGF-stimulated PDGFR tyrosine phosphorylation was completely blocked by 50 nM BMS-354825. In contrast, 5 µM imatinib was required to achieve the same level of PDGFR inhibition in parallel experiments. Likewise, complete inhibition of PDGF-stimulated Akt and Erk2 activation were observed in cells treated with 50 nM BMS-354825 but only with 5 µM imatinib, whereas decrease in STAT3 tyrosine phosphorylation appeared at lower concentrations of imatinib (Fig. 2A). It is noteworthy that inhibition of Akt by both BMS-354825 and imatinib was observed only at the highest drug concentration tested; PDGFR is a strong activator of the phosphoinositide-3 kinase signaling pathway because it binds phosphoinositide-3 kinase directly. Comparison of 50% inhibition of PDGFR tyrosine phosphorylation by BMS-354825 and imatinib in these experiments indicated that BMS-354825 was 67-fold more potent than imatinib in inhibiting PDGFR tyrosine phosphorylation (Fig. 2B).
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The primary culture of human AoSMCs seemed to have an elevated level of active STAT3 in the absence of PDGF stimulation (Fig. 3). Nevertheless, STAT3 was further activated by PDGF, which was blocked by BMS-354825. BMS-354825, however, was unable to reduce the active STAT3 to a level below the basal activation state in human AoSMCs. Complete inhibition of PDGF-stimulated Akt and Erk2 activation was achieved at 50 nM BMS-354825 in human AoSMCs (Fig. 3), which is similar to data obtained in A10 cells (Fig. 2).
Inhibition of c-Src Tyrosine Kinase by BMS-354825 in VSMCs. To determine whether BMS-354825 inhibits c-Src activity in VSMCs, c-Src was immunoprecipitated from serum-starved A10 cells treated with PDGF-BB (5 ng/ml) for 0 to 30 min in the presence or absence of 50 nM BMS-354825, and the Src tyrosine activity was determined by an immune complex kinase assay using a GST fusion protein of Gab1 fragment as an exogenous substrate (Ren et al., 2004). c-Src isolated from A10 cells without exposure to BMS-354825 and PDGF had detectable kinase activity (Fig. 4A). This kinase activity was reduced to a residual level in c-Src isolated from BMS-354825-treated cells (Fig. 4A). PDGF treatment did not lead to a detectable increase in c-Src kinase activity in A10 cells in repeated experiments. Nevertheless, as in unstimulated cells, the activity of c-Src tyrosine kinase in PDGF-stimulated A10 cells was suppressed by BMS-354825 to the residual level (Fig. 4A). This result indicated that c-Src was basally active in A10 cells and that BMS-354825 effectively inhibited the c-Src kinase activity in these cells.
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IGF-1R and EGFR in A10 Cells Are Not Sensitive to BMS-354825 Inhibition. Besides PDGFR, A10 cells also express IGF-1R and EGFR. To assess whether BMS-354825 inhibits other receptor tyrosine kinases in VSMCs, we examined the effects of BMS-354825 on these two receptor tyrosine kinases in A10 cells. As shown in Fig. 5, IGF-1 induced IGF-1R tyrosine phosphorylation, whereas EGF induced EGFR tyrosine phosphorylation in A10 cells. No detectable inhibition of IGF-1R and EGFR tyrosine phosphorylation was observed when A10 cells were treated with up to 50 nM BMS-354825. These data are consistent with a previous report that the IC50s for inhibition of IGF-1R and EGFR by BMS-354825 in the in vitro kinase assays were >5 µM and 180 nM, respectively (Lombardo et al., 2004).
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The primary human AoSMCs had a higher basal migration activity, which is 4 times that of A10 cells. In the presence of PDGF (5 ng/ml), migration of human AoSMCs was increased 3-fold. Again, PDGF-stimulated human AoSMC cell migration was inhibited by BMS-354825 (Fig. 6C).
Inhibition of VSMC Proliferation by BMS-354825. To determine the effect of BMS-354825 on PDGF-stimulated VSMC proliferation, rat A10 cells were incubated in medium containing low serum (1% FBS), PDGF (10 ng/ml), and/or 0 to 50 nM BMS-354825 for 6 days and viable cell numbers were determined. As shown in Fig. 7A, A10 cell proliferation was stimulated 5-fold by PDGF. BMS-354825 effectively inhibited PDGF-stimulated A10 cell proliferation. Complete inhibition was achieved at 50 nM BMS-354825. Similar results were obtained in human AoSMCs (Fig. 7B).
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A previous study showed that the PDGFR specific tyrosine kinase inhibitor AG-1295 could inhibit porcine SMC proliferation, and the effect was reversible and not toxic (Banai et al., 1998). To evaluate whether the inhibitory effect of BMS-354825 on A10 cell proliferation is reversible, two sets of A10 cells were treated with 0 to 50 nM BMS-354825 for 6 days. After this time, BMS-354825 was removed from one set of cells and cells were cultured for another 6 days. Viable cell numbers were determined on day 12. As shown in Fig. 7D, A10 cells proliferation resumed after BMS-354825 withdrawal. Thus, the inhibitory effect of BMS-354825 on A10 cell proliferation was reversible, suggesting that BMS-354825 does not exert a cytotoxic effect on these cells.
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; Bowman et al., 2001). However, biochemical evidence suggests that inhibition of PDGFR plays a major role in suppression of PDGF-stimulated responses in VSMCs by BMS-354825.
First, our data show that BMS-354825 is a potent inhibitor of PDGFR. The IC50 for inhibition of PDGFR tyrosine kinase in PDGF-stimulated A10 cells is 3 nM. A similar IC50 (4 nM) was obtained in the in vitro kinase assay. While this work was in progress, Lombardo et al. (2004) reported the kinase selectivity profile of BMS-354825, which showed that BMS-354825 inhibited PDGFR in vitro with an IC50 of 28 nM. Although Lombardo et al. (2004) performed no cell-based assay in their study, our data and theirs are consistent in demonstrating that BMS-354825 is a potent PDGFR inhibitor in vitro. The slight difference in IC50 for inhibition of PDGFR may reflect different assay conditions used to determine the PDGFR kinase activity between these two studies.
We found that Src is basally active in A10 cells and that BMS-354825 inhibits the basal Src activity (IC50 = 2 nM). Src kinase activity may be essential for cellular activities such as migration (Heldin and Westermark, 1999). Thus, blocking Src activity could contribute to the overall effect of BMS-354825 in inhibition of PDGF-stimulated VSMC migration. However, we did not detect measurable PDGF-induced Src activation in A10 cells, whereas marked activation of PDGFR was readily detectable. Thus, Src kinase activity alone is insufficient to account for the higher migratory activity of VSMCs observed in the presence of PDGF. Furthermore, although imatinib has no Src tyrosine kinase inhibitor activity (Druker et al., 1996), it suppressed the PDGF-stimulated activation of PDGFR, STAT3, Akt, and Erk2 in A10 cells. Therefore, inhibition of the PDGFR tyrosine kinase activity alone could have major effects on blocking PDGF-stimulated responses in VSMCs.
Several studies have shown that PDGFR inhibitors such as imatinib and AG-1295 are effective in suppressing restenosis and stenosis in experimental animals (Myllarniemi et al., 1999; Fishbein et al., 2000; Sihvola et al., 2003; Levitzki, 2005). Inhibition of PDGFR in VSMCs by imatinib and AG-1295 requires micromolar concentrations (Fishbein et al., 2000; Sanz-Gonzalez et al., 2004). In contrast, near-complete inhibition of PDGFR activation and PDGF-stimulated activities in VSMCs was observed with 50 nM BMS-354825 in this study. Direct comparison of BMS-354825 and imatinib in A10 cells indicates that BMS-354825 is 67-fold more potent that imatinib (Fig. 2). Thus, BMS-354825 is a much more potent PDGFR inhibitor than imatinib.
Inhibition of PDGF-stimulated VSMC proliferation by BMS-354825 is reversible. It has been reported that the vascular endothelial growth factor receptor is not sensitive to BMS-354825 inhibition (Lombardo et al., 2004). These observations suggest that BMS-354825 is unlikely to cause unmanageable damage to the vascular system. In support of this notion, imatinib, which also inhibits PDGFR, does not cause an adverse effect in the cardiovascular system in CML patients who have been treated with the drug for more than 4 years (Lahaye et al., 2005). The reversibility of the inhibitory effect of PDGF-stimulated VSMC proliferation by BMS-354825 also raises the question of whether BMS-354825 will be effective in suppressing restenosis. It is possible that restenosis-promoting activities peak shortly after vascular injury and lessen afterward. Therefore, one would predict that VSMC proliferation is not likely to resume when BMS-354825 is terminated after sufficient period of treatment unless there is new vascular injury.
BMS-354825 is currently being developed as an anticancer drug. We demonstrated here that it is very effective in inhibiting PDGF-stimulated VSMC migration and proliferation. VSMC migration and proliferation are critical events in the development of restenosis, which is a major problem for the long-term efficacy of the widely performed angioplasty and stenting procedures for coronary artery disease. Thus, in addition to being a promising anticancer drug, BMS-354825 is a potential novel therapeutic agent for cardiovascular diseases involving abnormal VSMC activities such as restenosis and stenosis.
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Footnotes |
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ABBREVIATIONS: VSMC, vascular smooth muscle cell; PDGF, platelet-derived growth factor; CML, chronic myeloid leukemia; PDGFR, platelet-derived growth factor receptor; STAT, signal transducer and activator of transcription; Erk, extracellular signal-regulated kinase; IGF-1R, insulin-like growth factor-1 receptor; EGFR, epidermal growth factor receptor; BMS-354825, N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide monohydrate; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; AoSMC, aortic smooth muscle cell; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; SMC, smooth muscle cells; AG-1295, 6,7-dimethyl-2-phenylquinoxaline; PD180970, 6-(2,6-dichloro-phenyl)-2-(4-fluoro-3-methylphenylamino)-8-methyl-8H-pyrido[2,3-d]pyrimidin-7-one.
Address correspondence to: Dr. Jie Wu, Molecular Oncology Program, SRB-3, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612. E-mail: wu{at}moffitt.usf.edu
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