Synergistic killing effect of imatinib and simvastatin on imatinib-resistant chronic myelogenous leukemia cells
Oh, Boraa,b; Kim, Tae Y.a,b; Min, Hyun J.b; Kim, Miyoungc; Kang, Myung S.e; Huh, Ji Y.f; Kim, Youngsood; Lee, Dong S.b,c
aDepartment of Molecular and Clinical Oncology
bCancer Research Institute
cDepartment of Laboratory Medicine
dDepartment of Molecular Genomic Medicine, Seoul National University College of Medicine
eDepartment of Laboratory Medicine, CHA Gangnam Medical Center, Seoul
fDepartment of Laboratory Medicine, CHA Bundang Medical Center, Sungnam, Republic of Korea
All supplementary digital content is available directly from the corresponding author.
Correspondence to Dong S. Lee, MD, Department of Laboratory Medicine, Seoul National University College of Medicine, 28 Yongon-dong Chongno-gu, 110 744 Seoul, Republic of Korea Tel: +82 2 2072 3986; fax: +82 2 766 0827; e-mail: email@example.com
Received April 18, 2012
Accepted September 4, 2012
The antiproliferative effect of simvastatin on tumor cells has been speculated to be by intracellular signal inhibition through 3-hydroxy-3-methylglutaryl acetyl coenzyme A reductase. We examined the killing effect of simvastatin on imatinib-sensitive and resistant chronic myelogenous leukemia (CML) cells (three kinds of CML cell lines representative of each hematopoietic lineage: K562, KCL22, and LAMA84) and T315I and E255K site-directed mutant cells (Ba/F3). The in-vivo effect of simvastatin was determined in K562-xenografted nude mice. Cotreatment with imatinib and simvastatin in imatinib-resistant CML cells showed a synergistic killing effect in K562-R, KCL22-R, LAMA84-R, and E255K mutant cells, but only an additive effect in the T315I mutant cell, although a single treatment of simvastatin strongly inhibited T315I mutant cells. Mechanisms of killing were an induction of apoptosis and cell cycle arrest, through inhibition of tyrosine phosphorylation, and activated STAT5 and STAT3. Simvastatin suppressed the growth of K562-transplanted tumors, and cotreatment with imatinib was more effective in reducing tumor size. Simvastatin also killed primary CD34+ cells from patients with CML more efficiently, compared with CD34− CML cells. Our study shows a synergic effect of imatinib and simvastatin both in imatinib-sensitive and imatinib-resistant cells, but more effective synergism in resistant cells. On the basis of these findings, we suggest that a combination of simvastatin and imatinib may be a potential candidate for the treatment of imatinib-resistant CML.
Chronic myelogenous leukemia (CML) is a stem cell proliferative disorder whose hallmark is a BCR–ABL fusion gene encoding abnormal tyrosine kinase 1. The constitutively active kinase activity of Bcr–Abl in the cytosol contributes towards its transforming function 2 and towards drug resistance through the activation of several key survival pathways, including the mitogen-activated protein kinase (MEK)/extracellular signal-regulating kinase (ERK) cascade, Akt, signal transducers and activators of transcription (STATs), and nuclear factor κB 3–5. These pathways promote cellular proliferation, survival, and resistance to apoptotic stimuli induced by conventional cytotoxic drugs. It is proposed that CML, similar to many other neoplasms, may be the result of a multistep pathogenetic process 6. As the disease progresses through the accelerated phase and into the blast crisis, additional cytogenetic abnormalities become evident 7.
The introduction of imatinib mesylate has revolutionized the treatment of CML because it is a selective inhibitor of the Bcr–Abl tyrosine kinase. Imatinib mesylate has been proven to be highly active in patients with chronic-phase CML and, to a lesser extent, in patients with accelerated and blastic-phase disease 8. Despite its effectiveness against chronic-phase disease, patients with CML eventually become refractory to imatinib through various mechanisms, including reduced drug uptake, mutation of the ATP-binding site, amplification of BCR–ABL, activation of Lyn 9, binding of imatinib to serum α-1 acid glycoprotein 10, or MDR overexpression 11. The development/pre-existence of kinase domain mutants that prevent drug binding is most commonly encountered in imatinib-refractory leukemia 12.
Various strategies to overcome this resistance have been adopted: developing novel drugs that can act on the mutated ATP-binding sites of the Bcr–Abl fusion protein, increasing the dose of imatinib, and combining imatinib with other drugs to block the downstream signal of the Bcr–Abl protein 13. Recently, second-generation compounds with a higher affinity for Abl, such as nilotinib (AMN107), or a high affinity for both Abl and Src kinases, such as dasatinib (BMS-354825), have been tested. However, these agents do not affect the kinase activity of the T315I mutants, which have a sterically reduced drug/kinase affinity that prevents the direct contact of these agents with the Bcr–Abl protein. Thus, the development of new strategies to eradicate such cells that are not affected by mutations in the kinase domain represents a high priority.
Statins, widely used as a cholesterol lowering agents, are competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase 14. HMG-CoA reductase is involved in the synthesis of various molecules such as ubiquinone, dolichol, farnesyl isoprenoid, and geranylgeranyl isoprenoid. These intermediates are essential for the post-translational modification of intracellular G-proteins, such as Rho, Rac, and Ras, which regulate endothelial, platelet, and leukocyte function 15–17. Inhibition of this hydrophobic modification of signaling proteins by statins has significant effects on cell growth in vitro. Furthermore, statins have pleiotropic, or seemingly unrelated, distinct effect on processes such as angiogenesis and inflammation, and also affect a number of novel molecular targets and complex signaling pathways 18. Because of these pleiotropic effects, statins potentially have activity in a number of chronic human diseases. Statins are believed to inhibit downstream signals of Bcr–Abl, contributing toward the growth of CML cells, through inhibition of Ras, Rho, and JAK/STAT.
In this study, we used simvastatin, one of the most pharmacologically potent inhibitors of HMG-CoA reductase. Simvastatin is similar to other relatively lipophilic statins: lovastatin, atrovastatin, fluvastatin, and pitavastatin. We found an antiproliferative activity of simvastatin in CML cells and its killing mechanism. Second, we established imatinib-resistant cell lines through mutation or progressive exposure to increasing concentrations of imatinib and examined the effectiveness of simvastatin alone and in combination with imatinib to overcome imatinib resistance. Simvastatin strongly inhibited the growth of cell lines expressing either the T315I or the E255K mutant form of Bcr–Abl as well as the wild type (WT). However, the synergistic killing interactions between simvastatin and imatinib were not observed in Ba/F3 cells expressing T315I mutant Bcr–Abl. However, cotreatment of both drugs showed synergistic killing interactions in imatinib-resistant K562, KCL22, LAMA84, and in primary CD34+ CML cells. Finally, simvastatin also enhanced imatinib-induced growth arrest in an in-vivo mouse model. Together, these results indicate that simvastatin may be useful in the treatment of imatinib-resistant CML.
Materials and methods
K562 cells were obtained from the KCLB (Seoul, Korea). KCL22 and LAMA84 cells were obtained from DSMZ (Braunschweig, Germany). All CML cell lines used in this study were grown in RPMI-1640 supplemented with 10% FBS, 2 mmol/l L-glutamine, 100 μg/ml streptomycin sulfate, and 100 U/ml penicillin G (all from GIBCO, Carlsbad, California, USA). Imatinib-resistant CML cell lines (K562-R, KCL22-R, and LAMA84-R) were derived from the parental K562, KCL22, and LAMA84 cell line after exposure to progressively higher concentrations of imatinib mesylate. K562, KCL22, and LAMA84 cells were cultured with continuous exposure to imatinib mesylate in stepwise increments of 100 nmol/l from 0.1 to 1 μmol/l after every 10 days of culture. Viable imatinib-resistant CML cells were maintained under a selected pressure in a medium containing 1 μmol/l imatinib mesylate. Mouse pro-B Ba/F3 and WEHI3 cell line were obtained from RIKEN (Tokyo, Japan). Ba/F3 cells and variants of Ba/F3 stably expressing Bcr–Abl or the T315I or the E255K mutation of Bcr–Abl were cultured in complete RPMI-1640 medium with or without filtered 10% WEHI medium supplementation as the source of IL-3. They were maintained in a 37°C, 5% CO2, fully humidified incubator and prepared for experimental procedures when in log-phase growth.
Generation of stable Ba/F3 Bcr–Abl wild-type or mutant-expressing cells
RNA was extracted from K562 cells and used to synthesize cDNA. First, Bcr–Abl fragments were amplified by a PCR with K562 cDNA as a template and Bcr–Abl full genes were amplified by an overlapping extension PCR (OE-PCR) and subcloned into the pGEM-T easy vector (Promega, Madison, Wisconsin, USA). Abl mutants (E255K and T315I) were constructed by site-directed mutagenesis using an OE-PCR. The OE-PCR DNA fragments were ligated into TA easy vectors. The presence of the mutations was confirmed by sequencing. The enhanced green fluorescent protein-expressing retroviral vector, MigR1, was a kind gift from Dr Warren S. Pear. The WT and mutated kinase domains in the TA easy vectors were transferred to the MigR1 by EcoRI digestion. Transient transfection of Mig constructs was carried out using the Neon transfection system (Invitrogen, Carlsbad, California, USA) following the manufacturer’s protocol. Cells transfected by electroporation were immediately transferred to Ba/F3 cell cultured medium. Cells were selected by FACS sorting using a FACSVantage equipped with a TurboSort option (Becton Dickinson Immunocytometry Systems, San Jose, California, USA).
Normal and patient samples
Normal peripheral blood mononuclear cells were isolated from four healthy donors using Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation. Normal hematopoietic cells were collected from cord blood that had been conserved for research, because of the lack of a lymphocyte subset. Bone marrow specimens were obtained from CML patients under a protocol approved by the Seoul National University Hospital institutional review board for the use of samples for research. Mononuclear cells were prepared using Ficoll-Hypaque and CD34+ or CD34− cells isolated to more than 98% purity using MACS columns (Miltenyi Biotec, Auburn, California, USA) and CD34 microbeads (Miltenyi Biotec) according to the manufacturer’s recommendations. They were cultured in RPMI-1640 medium containing 10% FBS, 100 U/l of an antibiotic–antimycotic agent, 10 mmol/l HEPES, 1 mmol/l sodium pyruvate, 4.5 g/l glucose, and 0.05 mmol/l 2-mercaptoethanol.
Reagents and antibodies
Simvastatin was obtained as a sodium salt from Merck Chemical Ltd (Rahway, New Jersy, USA) and dissolved in DMSO. Imatinib was provided by Novartis (Seoul, Korea) and dissolved in distilled water. The primary antibodies used were antiphosphotyrosine (clone 4G10) and anti-β-actin (Upstate Biotechnology, Lake Placid, New York, USA); anti-STAT5, anti-pSTAT5 (Tyr694/699), anti-STAT3, anti-pSTAT3 (Ser727), anti-Bcr, anti-pBcr (Tyr177), anti-Bcl-2, anti-cytochrome c, anti-XIAP, anti-Mcl-1, anti-caspase 9, anti-caspase 3, and anti-PARP (Cell Signaling Technologies, Danvers, Massachusetts, USA); anti-p16, anti-p18, anti-p27, anti-Cdk2, and anti-Cdk4 (Santa Cruz Biotechnology, Santa Cruz, California, USA); and anti-Abl (8E-9; PharMingen, San Diego, California, USA). Secondary antibodies were peroxidase-conjugated affiniPure goat anti-mouse and anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, USA).
Cell viability assay
For the cell viability assay, a CellTiter-Glo luminescent cell viability assay (Promega, Madison, Wisconsin, USA) was used in accordance with the instruction manual. Briefly, 5×103 cells (CML cell lines) or 1×104 cells (patient primary cells) were plated in triplicate into microtiter-plate wells containing 80 μl of medium. The next day, the cells were incubated with the desired concentrations of simvastatin or/and imatinib to a final volume of 100 μl. After 24, 48, or 72 h, 100 μl of CellTiter-Glo reagent was added and the cells were then incubated for 10 min at room temperature. The luminescence was measured using Wallac 1420 (PerkinElmer, Boston, Massachusetts, USA). The synergistic effect of simvastatin and imatinib was determined by analysis of the combination index (CI) using Calcusyn Software (Biosoft, Ferguson, Missouri, USA and Cambridge, UK).
Two-dimensional gel electrophoresis
Two hundred micrograms of proteins were solubilized in the rehydration buffer (9 mol/l urea, 2% Chaps, 60 mmol/l DTT, and 0.5% pharmalyte (pH 3–10). Subsequently, isoelectric focusing was carried out using a pre-cast immobilized pH gradient strips (13 cm, pH 3–10, linear; Amersham Biosciences, Piscataway, New Jersey, USA). After rehydration for 12 h at 20°C, the electrophoretic conditions during isoelectric focusing were as follows: 1 h at 500 V, 1 h at 1000 V, and 3 h at 8000 V. The strips were equilibrated for 15 m in an alkylating solution that was identical to the aforementioned buffer, except that 2.5% (w/v) iodoacetamide replaced DTT. The second gel electrophoresis was carried out according to a standard sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) protocol, using the Ettan DALT6 System (Amersham Biosciences).
Western blot analysis
The cells were resuspended in a modified RIPA lysis buffer (150 mmol/l NaCl, 1 mmol/l EDTA, 1% Triton X-100, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mmol/l Tris-HCl, pH 7.4) with a protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany) and a phosphatase inhibitor (1 mmol/l sodium fluoride and 2 mmol/l sodium orthovanadate) in ice for 30 min and centrifuged at 15 000g for 30 min to collect the whole-cell lysate. The proteins (10–20 μg) were separated on an 8–12% SDS-PAGE and transferred onto a PVDF membrane (Millipore, Bedford, Massachusetts, USA). Western blotting was carried out with specific primary antibodies and peroxidase-conjugated affinipure anti-mouse or anti-rabbit secondary antibodies. Proteins were visualized with ECL plus enhanced chemiluminescence reagents (Amersham Biosciences). Western blotting for antiphosphotyrosine was carried out after two-dimensional polyacrylamide gel electrophoresis (2-DE) using the same method. Subsequently, the pattern of immunoreactive spots was matched to the pattern of spots in the silver-stained gel. The spots on the gel were in gel digested for identification by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).
Protein spots were excised from the stained gel, and the gel spots were then destained. Enzymatic in-gel digestion with trypsin (Promega, Southhampton, UK), extraction of the peptides from the gel, desalting, and concentration analysis were carried out according to the methods of Thiede et al. 19. The in-gel digested peptides extracted were identified by peptide mass fingerprinting using the matrix-assisted laser desorption/ionization time of flight/time of flight (MALDI-TOF/TOF) 4700 Proteomics Analyzer (Applied Biosystems Inc., Foster City, California, USA) at the Human Genome Research Institute (Seoul, Korea). The peptide mass fingerprinting produced by MALDI-TOF/TOF was searched using the programs MASCOT, developed by Matrix Science Ltd (available at http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=PMF), and the ExPASy molecular biology server at SWISS-PROT (http://www.expasy.ch).
Apoptosis assessment by annexin V staining
K562, KCL22, and LAMA84 cells (2×106 cells) were seeded in 60-mm dishes for 12 h before being treated with simvastatin. Cells were harvested at 48 h after treatment and washed twice with PBS. Both the untreated and the treated cells were stained with annexin V and propidium iodide according to the manufacturer’s protocol (Roche Applied Science). The percentage of apoptotic cells was determined by FACSCalibur flow cytometry (Becton Dickinson Immunocytometry Systems) using Cell Quest Software (BD Biosciences, San Jose, California, USA).
Analysis of cytosolic cytochrome c
K562, KCL22, and LAMA84 cells (2×107 cells) were seeded in 100-mm dishes for 12 h before being treated with simvastatin. Cells were harvested at 24 h after treatment and isolated between cytosol and mitochondrial fractions according to the instructions of the mitochondria isolation kit for cultured cells (Pierce, Rockford, Illinois, USA). The protein samples were quantified, separated by 15% SDS-PAGE, and subjected to western blotting.
Cell cycle assay
K562, KCL22, and LAMA84 cells were exposed to 25 or 50 μmol/l simvastatin for 24 h and washed twice with a cold PBS solution. After fixing with cold 75% ethanol overnight, cells were stained with 1 μg/ml propidium iodide and exposed to 5 μg/ml RNase I at room temperature for 1 h. Cell cycle distribution was assessed by FACSCalibur flow cytometry (Becton Dickinson Immunocytometry Systems).
Six-week-old Balb/c-nu/nu female mice were purchased from Central Lab Animal Inc. (Seoul, Korea). K562 cells were suspended at 5×106 cells/ml in Matrigel (Becton Dickinson Immunocytometry Systems). The K562 xenograft was established by a subcutaneous injection of 1×106 cells into the right flanks of mice. Ten days after inoculation, the mice were randomized into groups of five, and imatinib, simvastatin, imatinib, and simvastatin, or vehicle was administered 5 days/week for up to 30 days in total. Tumor sizes were assessed at least three times a week by caliper measurement and their volumes were calculated from the formula, tumor size (mm3)=(d2×D)/2, where d and D are the shortest and the longest diameters of the tumor, respectively. Animal procedures were approved by the institutional review board at Seoul National University Hospital.
Student’s t-test was used to calculate the difference between cell line samples; Friedman’s and Mann–Whitney’s nonparametric tests were carried out to calculate significant differences. Nonparametric tests were carried out to calculate significant differences between CML samples. Significant differences versus the control group in animal study were determined by analysis of variance.
Influence of simvastatin on the proliferation of the imatinib-sensitive chronic myelogenous leukemia cell lines
To examine the effects of simvastatin on three different CML cell lines (K562, KCL22, and LAMA84), cells were treated for 48 h with several simvastatin concentrations (5, 10, 20, 25, 40, and 50 μmol/l). Incubation of the CML cells with simvastatin or imatinib induced a dose-dependent inhibition of proliferation (Fig. 1a and b). Notably, LAMA84 cells were rather resistant to simvastatin, but more sensitive to imatinib, compared with K562 and KCL22 cells (Fig. 1a and b). The viability of K562, KCL22, and LAMA84 cells after exposure to 20 μmol/l simvastatin was significantly reduced to 49, 56, and 67%, respectively (P<0.001, K562, KCL; P<0.01, LAMA84), compared with 80% for normal human peripheral blood mononuclear cells (Fig. 1b).
Influence of simvastatin on the proliferation of wild-type or imatinib-resistant Ba/F3 cells
To determine whether simvastatin was effective against mutation-induced imatinib-resistant cells, we treated Ba/F3 cells expressing either WT Bcr–Abl or the T315I or E255K kinase mutants with simvastatin for 48 h. Ba/F3 cells expressing WT Bcr–Abl were sensitive to imatinib, but Ba/F3 cells expressing Bcr–Abl kinase mutations were resistant to imatinib mesylate (Fig. 1c). Ba/F3 cells expressing either WT or mutated Bcr–Abl were equally sensitive to simvastatin, including cells harboring the T315I mutation (Fig. 1d).
Synergistic interaction between imatinib and simvastatin
The cotreatment of imatinib and simvastatin induced synergistically enhanced antiproliferative activity in the imatinib-sensitive cell line K562 (CI<1) compared with single-agent treatment (Table 1). In KCL22 and LAMA84 cells, combination treatment showed slightly synergistic or additive effects, as indicated by CI values (Table 1). In contrast, cotreatment of imatinib and simvastatin induced a synergistic effect on antiproliferative activity in all imatinib-resistant cell lines (K562-R, KCL22-R, and LAMA84-R) (Table 1). Combination treatment of K562-R, KCL22-R, and LAMA84-R cells increased sensitivity towards both drugs as combination IC50 values were reduced by a factor of 2 in K562-R, a factor of 3 in KCL22-R, and a factor of 4 in LAMA84-R as compared with imatinib alone (Table 1). The cotreatment of simvastatin and imatinib induced synergistically enhanced antiproliferative activity in Ba/F3 cells expressing WT or E255K mutant Bcr/Abl compared with single-agent treatment (Table 1). In T315I mutant Bcr/Abl cells, combination treatment showed additive or slightly antagonistic effects, as indicated by CI values ranging between 1 and 1.5 at 50–90% growth inhibition levels (Table 1).
Effect of simvastatin and imatinib on primary CD34+ chronic myelogenous leukemia cells from patients with chronic myelogenous leukemia
Simvastatin was evaluated in CD34+ and CD34− mononuclear cells sorted by immunobead separation of bone marrow aspirates of five patients with CML in the chronic phase. The viability of CD34+ CML cells with 10, 25, and 50 μmol/l simvastatin was reduced to 51, 33, and 17%, respectively (Fig. 1e). CD34+ CML cells appeared to be more susceptible to cell death after treatment with simvastatin compared with CD34− CML cells (P<0.05; Fig. 1e). The results were compared with the effects also observed in normal CD34+ and CD34− (n=5). When the CML CD34+ cells were exposed to 10 or 25 μmol/l simvastatin, a significant enhancement in cell death was observed compared with normal CD34+ cells (P<0.05; Fig. 1f). In contrast, cell viability was not significantly different in normal CD34− and CML CD34− cells (Fig. 1f). Next, we studied whether the suppressive effects of simvastatin on CML CD34+ cells could further be promoted by cotreatment with imatinib. A synergistic effect between simvastatin and imatinib was observed in primary CML cells from patients compared with simvastatin alone (CI<1.0; Table 2).
Simvastatin enhanced imatinib-induced growth arrest in an in-vivo mouse model
Thirty Balb/c-nu/nu mice were injected subcutaneously with K562 cells. After 10 days, the tumor mean volume reached 0.13±0.04 cm3. Then, simvastatin (10 or 20 mg/kg), imatinib (5 mg/kg), or cotreatment (10 mg/kg simvastatin+5 mg/kg imatinib) was provided once daily, 5 days, weekly for 4 weeks by an intraperitoneal injection. After 10 days, the tumor volume of the 20 mg/kg simvastatin-treated group (n=5) began to decrease, and the tumor volume decreased by as much as 30% after 30 days after treatment compared with the control group (placebo-treated group, n=5) (Fig. 2a).
In the 10 mg/kg simvastatin-treated group, no significant changes in tumor volume were observed, but in the cotreated group with 5 mg/kg imatinib (n=5), a greater than 50% decrease in tumor volume was observed, indicating that cotreatment combination treatment effectively induced cell death compared with imatinib alone (Fig. 2b).
Profile changes in the tyrosine-phosphorylated proteome of K562 cells after treatment with simvastatin
To determine the differential tyrosine phosphorylation between the 50 μmol/l simvastatin-treated and the simvastatin-untreated K562 cells, the protein samples from these cell lines were separated by 2-DE and immunoblotted with the phosphotyrosine antibody, mAb 4G10. The 39 spots were identified by MALDI-TOF/TOF (Supplementary Fig. 1, http://molecular-cytogenetics.org/). As shown in Table 3, 14 proteins that have scores over 100, including values, were identified in this experiment. These proteins showed a marked decrease in tyrosine phosphorylation in response to treatment with simvastatin. Simvastatin induced a decrease in tyrosine-phosphorylated proteins, including proteases (26S proteasome non-ATPase regulatory subunit 13), DNA/RNA processing proteins (RuvB-like 2, nucleolar RNA helicase 2), oxidoreductase proteins (retinal dehydrogenase 2), chaperones (T-complex protein 1 subunits α, θ, 78 kDa glucose-regulated protein precursor, stress-induced-phosphoprotein 1, T-complex protein 1 subunit γ), glycolysis proteins (α-enolase), cytoskeleton proteins (vimentin, WD repeat-containing protein 1), and microtubule proteins (tubulin α-1B chain) (Table 3).
Simvastatin induced apoptosis in chronic myelogenous leukemia cells
The annexin V assay showed that 48 h of exposure to 25 μmol/l simvastatin induced apoptotic rates in K562, KCL22, and LAMA84 cells of 21, 16, and 21%, respectively, and 27, 44, and 39%, respectively, after 48 h of exposure to 50 μmol/l simvastatin (Fig. 3a). When exposed to 25 or 50 μmol/l simvastatin for 24 h, western blot analysis of proapoptotic and antiapoptotic proteins showed that 25 μmol/l simvastatin induced the increased expression of proapoptosis proteins [cytochrome c (cytosol), cleaved caspase 9, caspase 3, PARP), and the decreased expression of Bcl-2, Mcl-1, and XIAP (Fig. 3b).
Simvastatin induced cell cycle arrest in the G1 phase in chronic myelogenous leukemia cells
To determine the effect of simvastatin on the cell cycle, the CML cells were incubated for 24 h with 25 and 50 μmol/l simvastatin. Treatment with simvastatin induced a dose-dependent accumulation of cells in the G1 phase and a decrease in the total S-phase population of K562, KCL22, and LAMA84 cells (Table 4). Simvastatin induced upregulation of p27kip, but not of p16Ink4A or p18Ink4c in K562, KCL22, and LAMA84 cells, whereas imatinib induced upregulation of p16Ink4A, p18Ink4c, and p27kip in LAMA84 cells. Both simvastatin and imatinib induced downregulation of Cdk2 and Cdk4 (Fig. 4).
Simvastatin inhibited the Bcr–Abl downstream pathway
Treatment with simvastatin attenuated the levels of pSTAT5, which was increased in all three imatinib-resistant CML cells (Fig. 5). In imatinib-resistant cell lines, simvastatin markedly reduced the increased pSTAT5 levels. Treatment with simvastatin also inhibited the levels of pSTAT3 (Fig. 5). The expression of phospho-Lyn was also markedly elevated in K562-R and KCL22-R cells but was inhibited following exposure to simvastatin. Moreover, treatment with simvastatin inhibited increased phospho-ERK1/2 in all three imatinib-sensitive and imatinib-resistant CML cells (Fig. 5).
Statins have been shown to inhibit tumor growth and induce apoptosis in a variety of tumor cells, including prostate cancer 20, colon cancer 21,22 breast cancer 23–25, melanoma 26,27, multiple myeloma 28, and leukemia cell lines 29–31. Recently, several clinical trials have assessed the antitumor activity of statins, indicating the potential role of statins in both cancer prevention and treatment 32,33. In this study, we hypothesized that simvastatin would exert anti-CML effects independent of Bcr–Abl and sensitize imatinib-resistant cells to the residual apoptotic effects of imatinib, thereby overcoming drug resistance. To examine this hypothesis, we evaluated the effect of simvastatin on imatinib resistance in two clinically relevant cell lines, Ba/F3p210E255K and Ba/F3p210T315I, in comparison with Ba/F3p210WT and untransfected Ba/F3 cells.
Despite the presence of Bcr–Abl mutations, Ba/F3p210WT, Ba/F3p210E255K, and Ba/F3p210T315I cells were equally susceptible to the antiproliferative effects of simvastatin, yielding IC50 values of 5.0±0.1, 4.6±0.5, and 4.5±0.1 μmol/l, respectively (Table 1). In addition, cotreatment with simvastatin and imatinib exerted synergistic effects on imatinib-resistant K562, KCL22, and LAMA84 cells, as well as on Ba/F3 cells expressing E255K mutant Bcr–Abl. Interestingly, three imatinib-resistant CML cell lines (K562-R, LAMA84-R, and KCLR cells) derived from human erythrocytic (K562), erythroid-megakaryocytic (LAMA84), and myelocytic (KCL22) hematopoietic cells showed different levels of resistance to simvastatin (Fig. 1b), indicating that simvastatin can kill CML cells through a Bcr–Abl independent mechanism. Thus, the combination of simvastatin and imatinib treatment might overcome drug resistance to imatinib caused by point mutation or Bcr–Abl protein overexpression.
The resistance of Bcr–Abl-positive cells to imatinib may occur through multiple mechanisms, including amplification of the Bcr–Abl gene, mutations in the Bcr–Abl kinase ATP-binding site, or a reduction in intracellular drug uptake 34–36. Although gene amplification is not universally encountered in resistant cells, an increase in Bcr–Abl protein expression commonly occurs at least in cultured cell lines 35,36. In our study, three imatinib-resistant cell lines (K562-R, KCLR, and LAMA84-R cells) showed increased Bcr–Abl protein expression. In addition, imatinib-resistant K562-R cells showed an elevated level of Lyn protein and phosphorylation, whereas parental K562 cells suppressed Lyn expression (Supplementary Fig. 2, http://molecular-cytogenetics.org/).
Src kinases are generally considered as important but not crucial downstream targets of Bcr–Abl kinase in chronic-phase CML. However, Lyn kinase, a member of the Src family, may play an important role in certain situations. It has been reported that Lyn kinase is overexpressed in some imatinib-resistant CML cell lines 37 and imatinib-resistant patient specimens 38; moreover, its activation does not depend on Bcr–Abl kinase 9,39–42. Therefore, abnormal regulation of Lyn kinase may interfere in the Bcr–Abl signaling complex, including CrkL, STAT5, and MAPK 39–41,43. We found that simvastatin strongly inhibited Lyn phosphorylation and Bcr–Abl downstream signaling complexes such as STAT5, STAT3, and ERK1/2 phosphorylation (Fig. 5), indicating that simvastatin suppresses protein expression involved in imatinib resistance.
To examine the underlying mechanism, we carried out 2-DE protein analysis and immunoblotting with a phosphotyrosine antibody. Simvastatin induced a decrease in the levels of tyrosine-phosphorylated proteins, which play a key role in tumor development by promoting cell growth. These proteins included proteases, DNA/RNA processing proteins, oxidoreductase proteins, and chaperones. Thus, these findings support the antiproliferative and proapoptotic activities of simvastatin.
In chronic-phase CML, the stem/progenitor cells are intrinsically less sensitive to imatinib than the more mature CML cells, which compose the bulk of the leukemic clone. However, van der Weide et al. 44 have reported that simvastatin and tipifarnib exert an enhanced suppressive effect in primary sorted CD34+ acute myeloid leukemia cells. In addition, Chen et al. 45 showed that the combination of imatinib and simvastatin sensitized CD34+ K562 cells to cell death. We examined the effect of simvastatin on CML patient’s primitive CD34+ and CD34− cells. The combination of imatinib and simvastatin induced significant cell death of imatinib-resistant CD34+ cells. We also found that simvastatin was more effective in CML CD34+ cells than in CML CD34− or normal CD34+ cells. Although the detailed mechanism of how simvastatin sensitizes imatinib-resistant CD34+ cells to imatinib is still unknown, the data from the present study suggest that simvastatin may block the resistance of imatinib through downregulation of Bcr–Abl transcription, which is regulated by multiple factors in cancer cells.
Collectively, this study showed the synergistic effect of imatinib and simvastatin, as well as the effects of simvastatin alone, on CML cells. Simvastatin induces cell cycle arrest in the G1 phase through p27kip and apoptosis in CML by blocking the downstream signals of the Bcr–Abl pathway, such as STAT5 and STAT3. Simvastatin also reduces tyrosine kinase phosphorylation, which plays a key role in tumor development by promoting cell growth. Thus, simvastatin may be useful in the treatment of imatinib-resistant CML through a Bcr–Abl independent mechanism.
This work was supported in part by the Basic Science Research Program through the National Research Foundation of Korea (NRF) Funded by the Ministry of Education, Science and Technology (2012-0002257), a grant of the SNUH Research Fund (04-2007-082-0), a grant of the Korea Foundation for Cancer Research (KFCR-2009-003), a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A120216), a grant of the Korea Food & Drug Administration in 2012 (10172KFDA993), and Cancer Research Institute, Seoul National University Grant (2009).
Conflicts of interest
There are no conflicts of interest.
1. Faderl S, Talpaz M, Estrov Z, O’Brien S, Kurzrock R, Kantarjian HM. The biology of chronic myeloid leukemia. N Engl J Med. 1999;341:164–172
2. Lugo TG, Pendergast AM, Muller AJ, Witte ON. Tyrosine kinase activity and transformation potency of bcr–abl oncogene products. Science. 1990;247:1079–1082
3. Voss J, Posern G, Hannemann JR, Wiedemann LM, Turhan AG, Poirel H, et al. The leukaemic oncoproteins Bcr–Abl and Tel-Abl (ETV6/Abl) have altered substrate preferences and activate similar intracellular signalling pathways. Oncogene. 2000;19:1684–1690
4. Danial NN, Rothman P. JAK-STAT signaling activated by Abl oncogenes. Oncogene. 2000;19:2523–2531
5. Kirchner D, Duyster J, Ottmann O, Schmid RM, Bergmann L, Munzert G. Mechanisms of Bcr–Abl-mediated NF-kappaB/Rel activation. Exp Hematol. 2003;31:504–511
6. Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, et al. Efficacy and safety of a specific inhibitor of the BCR–ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med. 2001;344:1031–1037
7. Frazer R, Irvine AE, McMullin MF. Chronic myeloid leukaemia in the 21st century. Ulster Med J. 2007;76:8–17
8. O’Brien SG, Guilhot F, Larson RA, Gathmann I, Baccarani M, Cervantes F, et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med. 2003;348:994–1004
9. Donato NJ, Wu JY, Stapley J, Gallick G, Lin H, Arlinghaus R, et al. BCR–ABL independence and LYN kinase overexpression in chronic myelogenous leukemia cells selected for resistance to STI571. Blood. 2003;101:690–698
10. Gambacorti-Passerini C, Barni R, le Coutre P, Zucchetti M, Cabrita G, Cleris L, et al. Role of alpha1 acid glycoprotein in the in vivo resistance of human BCR–ABL(+) leukemic cells to the abl inhibitor STI571. J Natl Cancer Inst. 2000;92:1641–1650
11. Hochhaus A, Kreil S, Corbin AS, La Rosee P, Muller MC, Lahaye T, et al. Molecular and chromosomal mechanisms of resistance to imatinib (STI571) therapy. Leukemia. 2002;16:2190–2196
12. Walz C, Sattler M. Novel targeted therapies to overcome imatinib mesylate resistance in chronic myeloid leukemia (CML). Crit Rev Oncol Hematol. 2006;57:145–164
13. Tauchi T, Ohyashiki K. Molecular mechanisms of resistance of leukemia to imatinib mesylate. Leuk Res. 2004;28(Suppl 1):S39–S45
14. Lennernas H, Fager G. Pharmacodynamics and pharmacokinetics of the HMG-CoA reductase inhibitors. Similarities and differences. Clin Pharmacokinet. 1997;32:403–425
15. Cipollone F, Fazia M, Iezzi A, Zucchelli M, Pini B, De Cesare D, et al. Suppression of the functionally coupled cyclooxygenase-2/prostaglandin E synthase as a basis of simvastatin-dependent plaque stabilization in humans. Circulation. 2003;107:1479–1485
16. Ridker PM, Cannon CP, Morrow D, Rifai N, Rose LM, McCabe CH, et al. C-reactive protein levels and outcomes after statin therapy. N Engl J Med. 2005;352:20–28
17. Krupski WC, Layug EL, Reilly LM, Rapp JH, Mangano DT. Comparison of cardiac morbidity between aortic and infrainguinal operations. Study of Perioperative Ischemia (SPI) Research Group. J Vasc Surg. 1992;15:354–363 discussion 364–355
18. Demierre MF, Higgins PD, Gruber SB, Hawk E, Lippman SM. Statins and cancer prevention. Nat Rev Cancer. 2005;5:930–942
19. Thiede B, Otto A, Zimny-Arndt U, Muller EC, Jungblut P. Identification of human myocardial proteins separated by two-dimensional electrophoresis with matrix-assisted laser desorption/ionization mass spectrometry. Electrophoresis. 1996;17:588–599
20. He Z, Mangala LS, Theriot CA, Rohde LH, Wu H, Zhang Y. Cell killing and radiosensitizing effects of atorvastatin in PC3 prostate cancer cells. J Radiat Res. 2012;53:225–233
21. Bardou M, Barkun A, Martel M. Effect of statin therapy on colorectal cancer. Gut. 2010;59:1572–1585
22. Cho SJ, Kim JS, Kim JM, Lee JY, Jung HC, Song IS. Simvastatin induces apoptosis in human colon cancer cells and in tumor xenografts, and attenuates colitis-associated colon cancer in mice. Int J Cancer. 2008;123:951–957
23. Ghosh-Choudhury N, Mandal CC, Ghosh Choudhury G. Simvastatin induces derepression of PTEN expression via NFkappaB to inhibit breast cancer cell growth. Cell Signal. 2010;22:749–758
24. Garwood ER, Kumar AS, Baehner FL, Moore DH, Au A, Hylton N, et al. Fluvastatin reduces proliferation and increases apoptosis in women with high grade breast cancer. Breast Cancer Res Treat. 2010;119:137–144
25. Koyuturk M, Ersozc M, Altiok N. Simvastatin induces apoptosis in human breast cancer cells: p53 and estrogen receptor independent pathway requiring signalling through JNK. Cancer Lett. 2007;250:220–228
26. Zhang S, Doudican NA, Quay E, Orlow SJ. Fluvastatin enhances sorafenib cytotoxicity in melanoma cells via modulation of AKT and JNK signaling pathways. Anticancer Res. 2011;31:3259–3265
27. Shellman YG, Ribble D, Miller L, Gendall J, Vanbuskirk K, Kelly D, et al. Lovastatin-induced apoptosis in human melanoma cell lines. Melanoma Res. 2005;15:83–89
28. Tu YS, Kang XL, Zhou JG, Lv XF, Tang YB, Guan YY. Involvement of Chk1-Cdc25A-cyclin A/CDK2 pathway in simvastatin induced S-phase cell cycle arrest and apoptosis in multiple myeloma cells. Eur J Pharmacol. 2011;670:356–364
29. Lewis KA, Holstein SA, Hohl RJ. Lovastatin alters the isoprenoid biosynthetic pathway in acute myelogenous leukemia cells in vivo. Leuk Res. 2005;29:527–533
30. Yang YC, Huang WF, Chuan LM, Xiao DW, Zeng YL, Zhou DA, et al. In vitro and in vivo study of cell growth inhibition of simvastatin on chronic myelogenous leukemia cells. Chemotherapy. 2008;54:438–446
31. Li YF, Zhang R, Zhang XH, Chen GH, Cen JN, Zhu ZL. Effects of simvastatin on proliferation and apoptosis of acute monocytic leukemia cell line SHI-1. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2011;19:612–616
32. Kawata S, Yamasaki E, Nagase T, Inui Y, Ito N, Matsuda Y, et al. Effect of pravastatin on survival in patients with advanced hepatocellular carcinoma. A randomized controlled trial. Br J Cancer. 2001;84:886–891
33. Knox JJ, Siu LL, Chen E, Dimitroulakos J, Kamel-Reid S, Moore MJ, et al. A Phase I trial of prolonged administration of lovastatin in patients with recurrent or metastatic squamous cell carcinoma of the head and neck or of the cervix. Eur J Cancer. 2005;41:523–530
34. Mahon FX, Deininger MW, Schultheis B, Chabrol J, Reiffers J, Goldman JM, et al. Selection and characterization of BCR–ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance. Blood. 2000;96:1070–1079
35. le Coutre P, Tassi E, Varella-Garcia M, Barni R, Mologni L, Cabrita G, et al. Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification. Blood. 2000;95:1758–1766
36. Weisberg E, Griffin JD. Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL-transformed hematopoietic cell lines. Blood. 2000;95:3498–3505
37. Goldman JM, Melo JV. Chronic myeloid leukemia – advances in biology and new approaches to treatment. N Engl J Med. 2003;349:1451–1464
38. Kantarjian H, Sawyers C, Hochhaus A, Guilhot F, Schiffer C, Gambacorti-Passerini C, et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med. 2002;346:645–652
39. Warmuth M, Bergmann M, Priess A, Hauslmann K, Emmerich B, Hallek M. The Src family kinase Hck interacts with Bcr–Abl by a kinase-independent mechanism and phosphorylates the Grb2-binding site of Bcr. J Biol Chem. 1997;272:33260–33270
40. Stanglmaier M, Warmuth M, Kleinlein I, Reis S, Hallek M. The interaction of the Bcr–Abl tyrosine kinase with the Src kinase Hck is mediated by multiple binding domains. Leukemia. 2003;17:283–289
41. Meyn MA III, Wilson MB, Abdi FA, Fahey N, Schiavone AP, Wu J, et al. Src family kinases phosphorylate the Bcr–Abl SH3-SH2 region and modulate Bcr–Abl transforming activity. J Biol Chem. 2006;281:30907–30916
42. Donato NJ, Wu JY, Stapley J, Lin H, Arlinghaus R, Aggarwal BB, et al. Imatinib mesylate resistance through BCR–ABL independence in chronic myelogenous leukemia. Cancer Res. 2004;64:672–677
43. Miething C, Mugler C, Grundler R, Hoepfl J, Bai RY, Peschel C, et al. Phosphorylation of tyrosine 393 in the kinase domain of Bcr–Abl influences the sensitivity towards imatinib in vivo. Leukemia. 2003;17:1695–1699
44. Van der Weide K, de Jonge-Peeters SD, Kuipers F, de Vries EG, Vellenga E. Combining simvastatin with the farnesyltransferase inhibitor tipifarnib results in an enhanced cytotoxic effect in a subset of primary CD34+ acute myeloid leukemia samples. Clin Cancer Res. 2009;15:3076–3083
45. Chen R, Xiao W, Li D, Mu S. Combination of simvastatin and imatinib sensitizes the CD34+ cells in K562 to cell death. Med Oncol. 2011;28:528–531
chronic myelogenous leukemia; imatinib resistance; simvastatin
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