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OSU-03012 sensitizes TIB-196 myeloma cells to imatinib mesylate via AMP-activated protein kinase and STAT3 pathways
Leukemia Research 34 (2010) 816–820
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
OSU-03012 sensitizes TIB-196 myeloma cells to imatinib mesylate via AMP-activated protein kinase and STAT3 pathways Li-Yuan Bai a,b , Jing-Ru Weng c , Chen-Hsun Tsai d , Aaron Sargeant d , Cheng-Wen Lin e , Chang-Fang Chiu a,b,∗ a
Graduate Institute of Clinical Medical Science, College of Medicine, China Medical University, Taichung, Taiwan Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan Department of Biological Science and Technology, College of Life Sciences, China Medical University, Taichung, Taiwan d Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, USA e Department of Medical Laboratory Science and Biotechnology, College of Health Care, China Medical University, Taichung, Taiwan b c
a r t i c l e
i n f o
Article history: Received 1 August 2009 Received in revised form 16 November 2009 Accepted 18 November 2009 Available online 14 December 2009 Keywords: Myeloma OSU-03012 Imatinib mesylate AMPK STAT3
a b s t r a c t Although c-Kit is expressed on the surface of myeloma cells in one-third of myeloma patients, the efficacy of imatinib mesylate for patients with myeloma is still controversial. To investigate the combinatorial effect of OSU-03012 and imatinib mesylate, we treated a c-Kit-expressing myeloma cell line, TIB-196, with DMSO, OSU-03012 alone, imatinib mesylate alone and OSU-03012 plus imatinib mesylate. OSU-03012 sensitized TIB-196 cells to imatinib mesylate cytotoxicity. p-STAT3 (Tyr705), as well as down-stream cyclin D1 and Mcl-1, was down regulated. Additionally, there was markedly increased p-AMPK (Thr172) and down-regulation of p-p70S6K (Thr386) in the combination group. Combined treatments targeting c-Kit, AMPK and STAT3 may be a potential strategy for treating patients with myeloma. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction About one-third of myeloma patients exhibit c-Kit expression on the surface of myeloma cells but not normal plasma cells [1–3]. The biological significance of c-Kit and its ligand, stem cell factor (SCF), has also been demonstrated in myeloma cells [4]. The addition of SCF resulted in a 2.4-fold increase in the number of cell colonies, a higher proportion of cells in S-phase, and enhancement of the proliferation of MT3 and U266 cells mediated by other cytokines. When tested on fresh myeloma samples, SCF significantly increased the number of S-phase plasma cells and enhanced proliferation of myeloma cells responsive to interleukin-6 [4]. Because the downstream signaling pathways of c-Kit, including mitogen-activated protein (MAP) kinase pathways, phosphatidyl-inositol-3 kinase (PI3-kinase) pathway, phospholipase C-␥ pathway, Src pathway and Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway, are essential for cellular function and
∗ Corresponding author at: Division of Hematology and Oncology, Department of Internal Medicine, China Medical University Hospital, 2 Yude Road, North District, Taichung 404, Taiwan. Tel.: +886 4 22052121ext 2019; fax: +886 4 22038883. E-mail address: [email protected] (C.-F. Chiu). 0145-2126/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2009.11.014
survival [5–7], it is rational to treat patients with multiple myeloma by drugs targeting c-Kit. Imatinib mesylate, a tyrosine kinase inhibitor targeting ABL protein, c-Kit protein and platelet-derived growth factor receptor, has been successfully used to treat chronic myeloid leukemia and gastrointestinal stromal tumor [8–11]. It has been shown to induce apoptosis in cells with BCR-ABL chimeric proteins and gastrointestinal stromal tumors expressing c-Kit protein. In addition to the already defined targets, recently imatinib mesylate was found to act on other cellular targets as well. Larmonier and his colleagues found that imatinib mesylate at concentrations achieved clinically impaired CD4+ CD25+ FoxP3+ regulatory T cell function and FoxP3 expression by suppressing the activity of STAT3 and STAT5 [12]. Dewar et al. demonstrated that the macrophage colonystimulating factor receptor c-fms, which was implicated in ovarian cancer, rheumatoid arthritis and excess osteoclast activity in multiple myeloma, was inhibited by imatinib mesylate [13,14]. Although these data suggest value in using imatinib mesylate to treat patients with multiple myeloma, its efficacy is still controversial [1,15]. OSU-03012, a novel celecoxib derivative without cyclooxygenase-2 inhibitory activity, has potent activity against myeloma cell lines and primary myeloma cells [16]. The mechanisms of OSU-03012-mediated cytotoxicity in myeloma cells include inhibition of PI3-kinase/Akt pathway, down-regulation
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of JAK/STAT3 and down-regulation of MAP kinase pathways [16]. Because of the potential interactions between targets of imatinib mesylate and targets of OSU-03012, we hypothesize that OSU-03012 can accentuate the cytotoxicity of imatinib mesylate to myeloma cells. In this report, we showed that the combination of OSU-03012 and imatinib mesylate up-regulated the AMPK pathway, down-regulated the STAT3 pathway, and increased cytotoxicity compared with either OSU-03012 or imatinib mesylate alone in c-Kit expressing myeloma cells. 2. Materials and methods 2.1. Cells TIB-196 cells, a c-Kit expressing myeloma cell line, and a c-kit non-expressing cell line HL-60 [17], were obtained from American Type Culture Collection (Manassas, VA). Both cells were cultured in RPMI 1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) and penicillin (100 U/mL)/streptomycin (100 g/mL) (Invitrogen, Carlsbad, CA) at 37 ◦ C in an atmosphere of 5% CO2 . 2.2. Reagents OSU-03012 was kindly provided by Dr. Ching-Shih Chen, The Ohio State University, Ohio. OSU-03012 was dissolved in DMSO at a concentration of 10 M as a stock solution for further use. Imatinib mesylate was purchased from Chemie Tek (Indianapolis, IN) and was dissolved in DMSO at concentration of 20 M as a stock solution.
Fig. 1. OSU-03012 accentuates the cytotoxicity of imatinib mesylate in myeloma. The data shown here represented percentages of absorbance value in OSU-03012 group that were normalized to DMSO control (n = 5 for each group). The ratio of survival between imatinib mesylate and DMSO in the presence of OSU-03012 was compared with those in the absence of OSU-03012 for each concentration of imatinib mesylate. *denotes p < 0.05, # denotes p < 0.001.
3. Results 2.3. MTS assay Measurement of cell growth was performed using CellTiter 96 Aqueous Nonradioactive Cell Proliferation Assay kit purchased from Promega (Madison, WI) and used by the manufacturer’s instructions. Briefly, 200 L cells (2.5 × 105 /mL) were placed in a 96-well microtiter with indicated reagent and incubated in 37 ◦ C. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium] solution and PMS (phenazine methoxulfate) solution were mixed in 20:1 in volume. At indicated time points, 20 L of the mixture solution was added. The color development was measured at 490-nm wavelength by a VersaMax tunable microplate reader (Molecular Devices, Sunnyvale, CA) 4 h later. The cell viability was expressed as a percentage of absorbance value in treated samples relative to that in control samples. 2.4. Western blotting Cell lysates were prepared by exposing cells to RIPA buffer (150 mM NaCl, 50 mM Tris PH 8.0, 1% NP40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate). Protease inhibitor (Sigma, Saint Louis, MO) and phosphatase inhibitor cocktail (Calbiochem, Gibbstown, NJ) were added to RIPA buffer before lysing the cells as per the manufacturer’s instructions. Protein concentrations of cell lysates were measured using Bio-Rad protein assay dye reagent (BIO-RAD Laboratories, Hercules, CA). The mixture solution of Laemmli sample buffer (BIO-RAD, 62.5 mmol/L Tris–HCl, pH 6.8, 2% sodium dodecyl sulfate, 25% glycerol, 0.01% bromphenol blue) and mercaptoethanol (19:1) was added to the lysates, and the lysates were boiled at 95 ◦ C for 10 min. Equal amounts of protein lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Hyperbond ECL, GE Healthcare, Piscataway, NJ). After blocking with TBST (TBS containing 0.1% Tween 20) containing 5% nonfat milk for 1 h, the membranes were incubated with the indicated primary antibodies at 4 ◦ C overnight. The membrane was washed 5 times with TBST then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibodies or goat anti-rabbit IgG antibodies (Jackson ImmunoResearch) for 1 h at room temperature. After 5 washes with TBST, the blots were visualized with the enhanced chemiluminescence Amersham ECL Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ). Antibodies against various proteins were obtained from the following sources: poly (adenosine diphosphate-ribose) polymerase (PARP), Akt, p-Akt (Thr308), ERK1/2, p-ERK1/2 (pThr202Tyr204), p38 mitogen-activated protein kinase (p38 MAPK), p-p38MAPK (Thr180Tyr182), AMP-activated protein kinase (AMPK), p-AMPK (Thr172), signal transducer and activator of transcription 3 (STAT3), p-STAT3 (Tyr705), p70S6K, pp70S6K (Thr386) (Cell Signaling, Danvers, MA); cyclin D1 (Calbiochem, Gibbstown, NJ); Mcl-1 (R&D, Minneapolis, MN); actin (MP Biomedicals, Solon, OH). The goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugates and goat anti-mouse IgGHRP conjugates were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). 2.5. Statistical analysis For comparisons, t-test was used for analyzing the effect of OSU-03012 on sensitizing cells to imatinib mesylate.
3.1. OSU-03012 sensitizes TIB-196 cells to imatinib mesylate To investigate the combinatorial effect of OSU-03012 and imatinib mesylate on cell survival, TIB-196 cells (2.5 × 105 cells/mL) were incubated with medium or the indicated concentrations of imatinib mesylate in the presence of DMSO or 8 M of OSU-03012. After 72 h, the cell viability was accessed using the MTS assay and the values of absorbance in 490 nm wavelength were recorded (Fig. 1). Without OSU-03012, the ratio of survival between 10 M, 20 M, 30 M, and 40 M of imatinib mesylate and DMSO were 100%, 76%, 47% and 24%, respectively. With OSU-03012, the ratio of survival between in 10 M, 20 M, 30 M, and 40 M of imatinib mesylate and DMSO were 80%, 30%, 23% and 2%, respectively. The ratio in the presence of OSU-03012 was significantly different from those without OSU-03012 in each concentration of imatinib mesylate (p < 0.05, p < 0.001, p < 0.001 and p < 0.001 for 10 M, 20 M, 30 M, and 40 M of imatinib mesylate, respectively). In summary, OSU-03012 at concentration of 8 M increased the sensitivity of TIB-196 cells to imatinib mesylate. 3.2. OSU-03012 combined with imatinib mesylate potently modulates cellular targets Western blotting of specific proteins was employed to further analyze the additive effect and potential signaling targets involved (Fig. 2A). TIB-196 cells (2.5 × 105 cells/mL) were cultured in medium or 20 M imatinib mesylate in the presence of DMSO or 8 M OSU-03012 for 24 h. Cells were lysed by RIPA buffer and 15 g of protein was separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bands were probed by primary antibodies against PARP, p-Akt (Thr308), Akt, p-ERK1/2 (Thr202/Tyr204), ERK1/2, p-p38 MAPK (Thr180/Tyr182), p38 MAPK, p-AMPK␣ (Thr172), AMPK␣, p-p70S6K (Thr386), p70S6K, p-STAT3 (Tyr705), STAT3, cyclin D1, Mcl-1 and actin. OSU-03012 combined with imatinib mesylate more potently increased the cleaved form of PARP compared with either drug alone. Although there was no obvious change in p-Akt (Thr308), p-ERK (Thr202/Tyr204), and pp38 MAPK (Thr180/Tyr182) in the combination group compared with OSU-03012 or imatinib mesylate alone, there was a significant increase of p-AMPK (Thr172). In concordance with increased
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Fig. 2. Combinatorial effect of OSU-03012 and imatinib mesylate on indicated molecules of signaling pathways. (A) On TIB-196 myeloma cells. (B) HL-60 cells, a c-Kit non-expressing cell line, were used for comparison.
p-AMPK (Thr172) level, the p-p70S6K (Thr386) expression was attenuated. While total STAT3 protein expression was unaffected, the expression of p-STAT3 (Tyr705) decreased significantly in the combination groups. Cyclin D1 and Mcl-1, both of which are downstream targets of STAT3, were also decreased. HL-60 cells, a c-Kit non-expressing cell line, were used for comparing the protein expression (Fig. 2B). HL-60 cells (2.5 × 105 cells/mL) were cultured in medium or 20 M imatinib mesylate in the presence of DMSO or 8 M OSU-03012 for 24 h. Cell lysates were collected and probed with antibodies to p-AMPK␣ (Thr172), AMPK␣, p-STAT3 (Tyr705), STAT3 and actin. There was no change of AMPK and STAT3 protein expression in the combination group.
3.3. The modulations of p-AMPK and p-STAT3 by OSU-03102 combined with imatinib mesylate are early events To further elucidate if the changes in AMPK and STAT3 are early events, western blotting in serial time-course was performed. TIB-196 cells (2.5 × 105 cells/mL) were cultured in 20 M of imatinib mesylate in combination with 8 M of OSU-03012 for 0, 3, 6, and 24 h. Cell lysates (15 g) were separated by SDS-PAGE and
probed by primary antibodies against p-AMPK␣ (Thr172), AMPK␣, p-STAT3 (Tyr705), STAT3, cyclin D1, and actin (Fig. 3). The increment in p-AMPK expression and decrease in p-STAT3 expression occurred as early as 3 h indicating that both pathways were affected soon after the drugs were added. There was also concordant change of p-STAT3 and cyclin D1 in the time-course study.
4. Discussion Here we demonstrate that OSU-03012 increases the sensitivity of a myeloma cell line to imatinib mesylate. Specifically, the combinatorial use of OSU-03012 and imatinib mesylate leads to activation of AMPK␣, down-regulation of p-p70S6K and phosphorylated STAT3, and increased cleavage of PARP. Although c-Kit expression was reported to occur on the surface of one-third of myeloma cells [1–3], a consensus regarding the use of a c-Kit inhibitor, imatinib mesylate, to treat this disease has not been reached [1,15]. The underlying cause of this discrepancy is not clearly known. Montero et al. found that different isoforms of c-Kit influence the sensitivity and modulate the response of myeloma cells to drugs [7]. The GNNK−
Fig. 3. Western blotting analysis of time-course treatment with imatinib mesylate and OSU-03012 on TIB-196 cells.
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form of c-Kit was activated faster and more pronounced by SCF; whereas activation of the GNNK+ form is of a longer duration [7]. Another possibility is BCR-ABL-independent imatinib mesylate resistance, in which cells will survive through activation of other pathways or signaling molecules. Bewry and his colleagues noticed that increased p-STAT3 (Tyr705) expression contributed resistance toward imatinib mesylate in myeloma cells cultured in bone marrow stroma-derived conditioned medium. Reducing STAT3 levels with small interfering RNA sensitized cells to imatinib mesylate-induced cell death [18]. Because the efficacy of imatinib mesylate for treating patients with multiple myeloma is not impressive, a rational treatment strategy is to increase the sensitivity of myeloma cells to imatinib mesylate by combining drugs acting at other targets. The potent activity of OSU-03012 against myeloma cell lines and primary myeloma cells has been attributed to inhibition of the PI3-kinase/Akt pathway, down-regulation of JAK/STAT3, and down-regulation of MAP kinase pathways [16]. These pathways targeted by OSU-03012 are important for universal cellular signaling and survival. Fittingly for the current study, they are the down-stream signaling pathways of c-Kit as well [5–7]. In our study, we found that combinatorial use of OSU-03012 and imatinib mesylate had additive effects on some target proteins compared with either drug alone. Although there was no change in p-ERK1/2 and p-Akt (Thr308) expression, there was marked attenuation of p-STAT3, a down-stream molecule of c-Kit as well as a target of OSU-03012, by the combined treatment. Additionally, we found that p-AMPK␣ (Thr172) expression increased and its down-stream p-p70S6K (Thr386) expression decreased significantly in the combined treatment group compared with either drug alone. This is the first report that either OSU03012 or imatinib mesylate has the ability to modulate the AMPK pathway. The mammalian target of rapamycin (mTOR) is a key regulator for protein synthesis and plays important roles in cellular metabolism, growth and proliferation. Aberrant activation of mTOR pathways has been noted in many cancers, and some drugs targeting mTOR pathway have been tested in preclinical and clinical studies. AMPK, a sensor of cellular energy status, is one of the Aktindependent mechanisms of mTOR regulation [19]. The activation of AMPK leads to glycolysis, fatty acid oxidation, inhibition of fatty acid synthesis, inhibition of gluconeogenesis, inhibition of protein synthesis, cell cycle control, and inhibition of cell growth [20–22]. Furthermore, some proteins in the AMPK signaling network, including LKB1, TSC1, TSC2 and p53, are tumor suppressor genes [21]. These characteristics of AMPK make it a logical target for treating patients with cancer, including breast cancer [23], melanoma [24], hepatocellular carcinoma [20], chronic lymphocytic leukemia [25], and multiple myeloma [26]. Our study shows that p-AMPK expression increases after either imatinib mesylate or OSU-03012 addition in TIB-196 cells. Combined use of these agents results in increased p-AMPK (Thr172) and decreased p-p70S6K (Thr386) compared with either drug alone. Based on Baumann’s finding and ours, we suggest value in AMPK-targeted agents for the treatment of multiple myeloma. Combination treatment has been used as a strategy to treat patients with cancer for decades. It has the advantage of reducing side effects of a single drug at higher concentration, overcoming drug resistance, reducing the financial burden of expensive compounds and, perhaps most importantly, to minimize the development of resistance to any single agent. In the present study, we demonstrate that OSU-03012 can potentiate the cytotoxicity of imatinib mesylate in myeloma cells. The combination of OSU03012 and imatinib mesylate up-regulates the AMPK pathway, down-regulates the STAT3 pathway, and increases cytotoxicity compared with either OSU-03012 or imatinib mesylate alone in c-Kit expressing myeloma cells. Collectively, our results sug-
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gest that combined treatments targeting c-Kit, AMPK and STAT3 may be a potential strategy for treating patients with multiple myeloma. Conflict of interest statement The authors declare no competing financial interests. Acknowledgements This work was supported in part by a grant from the China Medical University, Taichung, Taiwan; Research Grant CMU97-075. Contributions: L.-Y. B. performed the majority of the research, analyzed the data, and wrote the initial draft of the paper. C.H. T. assisted with the in vitro work and reviewed drafts of the paper. A. S. and C.-W. L. participated in the planning of experiments and reviewed drafts of the paper. C.-F. C. designed the research, reviewed all of the data, analyzing the data and reviewed the draft of the paper. References [1] Pandiella A, Carvajal-Vergara X, Tabera S, Mateo G, Gutierrez N, San Miguel JF. Imatinib mesylate (STI571) inhibits multiple myeloma cell proliferation and potentiates the effect of common antimyeloma agents. Br J Haematol 2003;123:858–68. [2] Ocqueteau M, Orfao A, Garcia-Sanz R, Almeida J, Gonzalez M, San Miguel JF. Expression of the CD117 antigen (c-Kit) on normal and myelomatous plasma cells. Br J Haematol 1996;95:489–93. [3] Pruneri G, Ponzoni M, Ferreri AJ, Freschi M, Tresoldi M, Baldini L, et al. The prevalence and clinical implications of c-Kit expression in plasma cell myeloma. Histopathology 2006;48:529–35. [4] Lemoli RM, Fortuna A, Grande A, Gamberi B, Bonsi L, Fogli M, et al. Expression and functional role of c-Kit ligand (SCF) in human multiple myeloma cells. Br J Haematol 1994;88:760–9. [5] Reber L, Da Silva CA, Frossard N. Stem cell factor and its receptor c-Kit as targets for inflammatory diseases. Eur J Pharmacol 2006;533:327–40. [6] Sattler M, Salgia R. Targeting c-Kit mutations: basic science to novel therapies. Leuk Res 2004;28(Suppl 1):S11–20. [7] Montero JC, Lopez-Perez R, San Miguel JF, Pandiella A. Expression of c-Kit isoforms in multiple myeloma: differences in signaling and drug sensitivity. Haematologica 2008;93:851–9. [8] Druker BJ, Guilhot F, O’Brien SG, Gathmann I, Kantarjian H, Gattermann N, et al. Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med 2006;355:2408–17. [9] Carroll M, Ohno-Jones S, Tamura S, Buchdunger E, Zimmermann J, Lydon NB, et al. CGP 57148, a tyrosine kinase inhibitor, inhibits the growth of cells expressing BCR-ABL, TEL-ABL, and TEL-PDGFR fusion proteins. Blood 1997;90:4947– 52. [10] Savage DG, Antman KH. Imatinib mesylate—a new oral targeted therapy. N Engl J Med 2002;346:683–93. [11] Tuveson DA, Willis NA, Jacks T, Griffin JD, Singer S, Fletcher CD, et al. STI571 inactivation of the gastrointestinal stromal tumor c-Kit oncoprotein: biological and clinical implications. Oncogene 2001;20:5054–8. [12] Larmonier N, Janikashvili N, LaCasse CJ, Larmonier CB, Cantrell J, Situ E, et al. Imatinib mesylate inhibits CD4+ CD25+ regulatory T cell activity and enhances active immunotherapy against BCR-ABL-tumors. J Immunol 2008;181:6955–63. [13] Dewar AL, Cambareri AC, Zannettino AC, Miller BL, Doherty KV, Hughes TP, et al. Macrophage colony-stimulating factor receptor c-fms is a novel target of imatinib. Blood 2005;105:3127–32. [14] Dewar AL, Zannettino AC, Hughes TP, Lyons AB. Inhibition of c-fms by imatinib: expanding the spectrum of treatment. Cell Cycle 2005;4:851–3. [15] Dispenzieri A, Gertz MA, Lacy MQ, Geyer SM, Greipp PR, Rajkumar SV, et al. A phase II trial of imatinib in patients with refractory/relapsed myeloma. Leuk Lymphoma 2006;47:39–42. [16] Zhang S, Suvannasankha A, Crean CD, White VL, Johnson A, Chen CS, et al. OSU-03012, a novel celecoxib derivative, is cytotoxic to myeloma cells and acts through multiple mechanisms. Clin Cancer Res 2007;13:4750–8. [17] Smith JS, Keller JR, Lohrey NC, McCauslin CS, Ortiz M. Redirected infection of directly biotinylated recombinant adenovirus vectors through cell surface receptors and antigens. Proc Natl Acad Sci USA 1999;96:8855–60. [18] Bewry NN, Nair RR, Emmons MF, Boulware D, Pinilla-Ibarz J, Hazlehurst LA. Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance. Mol Cancer Ther 2008;7:3169–75. [19] Memmott RM, Dennis PA. Akt-dependent and -independent mechanisms of mTOR regulation in cancer. Cell Signal 2009;21:656–64. [20] Imamura K, Ogura T, Kishimoto A, Kaminishi M, Esumi H. Cell cycle regulation via p53 phosphorylation by a 5 -AMP activated protein kinase activator,
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5-aminoimidazole-4-carboxamide-1--d-ribofuranoside, in a human hepatocellular carcinoma cell line. Biochem Biophys Res Commun 2001;287:562–7. [21] Motoshima H, Goldstein BJ, Igata M, Araki E. AMPK and cell proliferation—AMPK as a therapeutic target for atherosclerosis and cancer. J Physiol 2006;574(Pt 1):63–71. [22] Hardie DG. The AMP-activated protein kinase pathway—new players upstream and downstream. J Cell Sci 2004;117(Pt 23):5479–87. [23] Hadad SM, Fleming S, Thompson AM. Targeting AMPK: a new therapeutic opportunity in breast cancer. Crit Rev Hematol Oncol 2008;67:1–7.
[24] Zheng B, Jeong JH, Asara JM, Yuan YY, Granter SR, Chin L, et al. Oncogenic BRAF negatively regulates the tumor suppressor LKB1 to promote melanoma cell proliferation. Mol Cell 2009;33:237–47. [25] Campa‘s C, Lopez JM, Santidrián AF, Barragán M, Bellosillo B, Colomer D, et al. Acadesine activates AMPK and induces apoptosis in B-cell chronic lymphocytic leukemia cells but not in T lymphocytes. Blood 2003;101:3674–80. [26] Baumann P, Mandl-Weber S, Emmerich B, Straka C, Schmidmaier R. Activation of adenosine monophosphate activated protein kinase inhibits growth of multiple myeloma cells. Exp Cell Res 2007;313:3592–603.
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
OSU-03012 sensitizes TIB-196 myeloma cells to imatinib mesylate via AMP-activated protein kinase and STAT3 pathways Li-Yuan Bai a,b , Jing-Ru Weng c , Chen-Hsun Tsai d , Aaron Sargeant d , Cheng-Wen Lin e , Chang-Fang Chiu a,b,∗ a
Graduate Institute of Clinical Medical Science, College of Medicine, China Medical University, Taichung, Taiwan Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan Department of Biological Science and Technology, College of Life Sciences, China Medical University, Taichung, Taiwan d Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, USA e Department of Medical Laboratory Science and Biotechnology, College of Health Care, China Medical University, Taichung, Taiwan b c
a r t i c l e
i n f o
Article history: Received 1 August 2009 Received in revised form 16 November 2009 Accepted 18 November 2009 Available online 14 December 2009 Keywords: Myeloma OSU-03012 Imatinib mesylate AMPK STAT3
a b s t r a c t Although c-Kit is expressed on the surface of myeloma cells in one-third of myeloma patients, the efficacy of imatinib mesylate for patients with myeloma is still controversial. To investigate the combinatorial effect of OSU-03012 and imatinib mesylate, we treated a c-Kit-expressing myeloma cell line, TIB-196, with DMSO, OSU-03012 alone, imatinib mesylate alone and OSU-03012 plus imatinib mesylate. OSU-03012 sensitized TIB-196 cells to imatinib mesylate cytotoxicity. p-STAT3 (Tyr705), as well as down-stream cyclin D1 and Mcl-1, was down regulated. Additionally, there was markedly increased p-AMPK (Thr172) and down-regulation of p-p70S6K (Thr386) in the combination group. Combined treatments targeting c-Kit, AMPK and STAT3 may be a potential strategy for treating patients with myeloma. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction About one-third of myeloma patients exhibit c-Kit expression on the surface of myeloma cells but not normal plasma cells [1–3]. The biological significance of c-Kit and its ligand, stem cell factor (SCF), has also been demonstrated in myeloma cells [4]. The addition of SCF resulted in a 2.4-fold increase in the number of cell colonies, a higher proportion of cells in S-phase, and enhancement of the proliferation of MT3 and U266 cells mediated by other cytokines. When tested on fresh myeloma samples, SCF significantly increased the number of S-phase plasma cells and enhanced proliferation of myeloma cells responsive to interleukin-6 [4]. Because the downstream signaling pathways of c-Kit, including mitogen-activated protein (MAP) kinase pathways, phosphatidyl-inositol-3 kinase (PI3-kinase) pathway, phospholipase C-␥ pathway, Src pathway and Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway, are essential for cellular function and
∗ Corresponding author at: Division of Hematology and Oncology, Department of Internal Medicine, China Medical University Hospital, 2 Yude Road, North District, Taichung 404, Taiwan. Tel.: +886 4 22052121ext 2019; fax: +886 4 22038883. E-mail address: [email protected] (C.-F. Chiu). 0145-2126/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2009.11.014
survival [5–7], it is rational to treat patients with multiple myeloma by drugs targeting c-Kit. Imatinib mesylate, a tyrosine kinase inhibitor targeting ABL protein, c-Kit protein and platelet-derived growth factor receptor, has been successfully used to treat chronic myeloid leukemia and gastrointestinal stromal tumor [8–11]. It has been shown to induce apoptosis in cells with BCR-ABL chimeric proteins and gastrointestinal stromal tumors expressing c-Kit protein. In addition to the already defined targets, recently imatinib mesylate was found to act on other cellular targets as well. Larmonier and his colleagues found that imatinib mesylate at concentrations achieved clinically impaired CD4+ CD25+ FoxP3+ regulatory T cell function and FoxP3 expression by suppressing the activity of STAT3 and STAT5 [12]. Dewar et al. demonstrated that the macrophage colonystimulating factor receptor c-fms, which was implicated in ovarian cancer, rheumatoid arthritis and excess osteoclast activity in multiple myeloma, was inhibited by imatinib mesylate [13,14]. Although these data suggest value in using imatinib mesylate to treat patients with multiple myeloma, its efficacy is still controversial [1,15]. OSU-03012, a novel celecoxib derivative without cyclooxygenase-2 inhibitory activity, has potent activity against myeloma cell lines and primary myeloma cells [16]. The mechanisms of OSU-03012-mediated cytotoxicity in myeloma cells include inhibition of PI3-kinase/Akt pathway, down-regulation
L.-Y. Bai et al. / Leukemia Research 34 (2010) 816–820
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of JAK/STAT3 and down-regulation of MAP kinase pathways [16]. Because of the potential interactions between targets of imatinib mesylate and targets of OSU-03012, we hypothesize that OSU-03012 can accentuate the cytotoxicity of imatinib mesylate to myeloma cells. In this report, we showed that the combination of OSU-03012 and imatinib mesylate up-regulated the AMPK pathway, down-regulated the STAT3 pathway, and increased cytotoxicity compared with either OSU-03012 or imatinib mesylate alone in c-Kit expressing myeloma cells. 2. Materials and methods 2.1. Cells TIB-196 cells, a c-Kit expressing myeloma cell line, and a c-kit non-expressing cell line HL-60 [17], were obtained from American Type Culture Collection (Manassas, VA). Both cells were cultured in RPMI 1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) and penicillin (100 U/mL)/streptomycin (100 g/mL) (Invitrogen, Carlsbad, CA) at 37 ◦ C in an atmosphere of 5% CO2 . 2.2. Reagents OSU-03012 was kindly provided by Dr. Ching-Shih Chen, The Ohio State University, Ohio. OSU-03012 was dissolved in DMSO at a concentration of 10 M as a stock solution for further use. Imatinib mesylate was purchased from Chemie Tek (Indianapolis, IN) and was dissolved in DMSO at concentration of 20 M as a stock solution.
Fig. 1. OSU-03012 accentuates the cytotoxicity of imatinib mesylate in myeloma. The data shown here represented percentages of absorbance value in OSU-03012 group that were normalized to DMSO control (n = 5 for each group). The ratio of survival between imatinib mesylate and DMSO in the presence of OSU-03012 was compared with those in the absence of OSU-03012 for each concentration of imatinib mesylate. *denotes p < 0.05, # denotes p < 0.001.
3. Results 2.3. MTS assay Measurement of cell growth was performed using CellTiter 96 Aqueous Nonradioactive Cell Proliferation Assay kit purchased from Promega (Madison, WI) and used by the manufacturer’s instructions. Briefly, 200 L cells (2.5 × 105 /mL) were placed in a 96-well microtiter with indicated reagent and incubated in 37 ◦ C. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium] solution and PMS (phenazine methoxulfate) solution were mixed in 20:1 in volume. At indicated time points, 20 L of the mixture solution was added. The color development was measured at 490-nm wavelength by a VersaMax tunable microplate reader (Molecular Devices, Sunnyvale, CA) 4 h later. The cell viability was expressed as a percentage of absorbance value in treated samples relative to that in control samples. 2.4. Western blotting Cell lysates were prepared by exposing cells to RIPA buffer (150 mM NaCl, 50 mM Tris PH 8.0, 1% NP40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate). Protease inhibitor (Sigma, Saint Louis, MO) and phosphatase inhibitor cocktail (Calbiochem, Gibbstown, NJ) were added to RIPA buffer before lysing the cells as per the manufacturer’s instructions. Protein concentrations of cell lysates were measured using Bio-Rad protein assay dye reagent (BIO-RAD Laboratories, Hercules, CA). The mixture solution of Laemmli sample buffer (BIO-RAD, 62.5 mmol/L Tris–HCl, pH 6.8, 2% sodium dodecyl sulfate, 25% glycerol, 0.01% bromphenol blue) and mercaptoethanol (19:1) was added to the lysates, and the lysates were boiled at 95 ◦ C for 10 min. Equal amounts of protein lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Hyperbond ECL, GE Healthcare, Piscataway, NJ). After blocking with TBST (TBS containing 0.1% Tween 20) containing 5% nonfat milk for 1 h, the membranes were incubated with the indicated primary antibodies at 4 ◦ C overnight. The membrane was washed 5 times with TBST then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibodies or goat anti-rabbit IgG antibodies (Jackson ImmunoResearch) for 1 h at room temperature. After 5 washes with TBST, the blots were visualized with the enhanced chemiluminescence Amersham ECL Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ). Antibodies against various proteins were obtained from the following sources: poly (adenosine diphosphate-ribose) polymerase (PARP), Akt, p-Akt (Thr308), ERK1/2, p-ERK1/2 (pThr202Tyr204), p38 mitogen-activated protein kinase (p38 MAPK), p-p38MAPK (Thr180Tyr182), AMP-activated protein kinase (AMPK), p-AMPK (Thr172), signal transducer and activator of transcription 3 (STAT3), p-STAT3 (Tyr705), p70S6K, pp70S6K (Thr386) (Cell Signaling, Danvers, MA); cyclin D1 (Calbiochem, Gibbstown, NJ); Mcl-1 (R&D, Minneapolis, MN); actin (MP Biomedicals, Solon, OH). The goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugates and goat anti-mouse IgGHRP conjugates were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). 2.5. Statistical analysis For comparisons, t-test was used for analyzing the effect of OSU-03012 on sensitizing cells to imatinib mesylate.
3.1. OSU-03012 sensitizes TIB-196 cells to imatinib mesylate To investigate the combinatorial effect of OSU-03012 and imatinib mesylate on cell survival, TIB-196 cells (2.5 × 105 cells/mL) were incubated with medium or the indicated concentrations of imatinib mesylate in the presence of DMSO or 8 M of OSU-03012. After 72 h, the cell viability was accessed using the MTS assay and the values of absorbance in 490 nm wavelength were recorded (Fig. 1). Without OSU-03012, the ratio of survival between 10 M, 20 M, 30 M, and 40 M of imatinib mesylate and DMSO were 100%, 76%, 47% and 24%, respectively. With OSU-03012, the ratio of survival between in 10 M, 20 M, 30 M, and 40 M of imatinib mesylate and DMSO were 80%, 30%, 23% and 2%, respectively. The ratio in the presence of OSU-03012 was significantly different from those without OSU-03012 in each concentration of imatinib mesylate (p < 0.05, p < 0.001, p < 0.001 and p < 0.001 for 10 M, 20 M, 30 M, and 40 M of imatinib mesylate, respectively). In summary, OSU-03012 at concentration of 8 M increased the sensitivity of TIB-196 cells to imatinib mesylate. 3.2. OSU-03012 combined with imatinib mesylate potently modulates cellular targets Western blotting of specific proteins was employed to further analyze the additive effect and potential signaling targets involved (Fig. 2A). TIB-196 cells (2.5 × 105 cells/mL) were cultured in medium or 20 M imatinib mesylate in the presence of DMSO or 8 M OSU-03012 for 24 h. Cells were lysed by RIPA buffer and 15 g of protein was separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bands were probed by primary antibodies against PARP, p-Akt (Thr308), Akt, p-ERK1/2 (Thr202/Tyr204), ERK1/2, p-p38 MAPK (Thr180/Tyr182), p38 MAPK, p-AMPK␣ (Thr172), AMPK␣, p-p70S6K (Thr386), p70S6K, p-STAT3 (Tyr705), STAT3, cyclin D1, Mcl-1 and actin. OSU-03012 combined with imatinib mesylate more potently increased the cleaved form of PARP compared with either drug alone. Although there was no obvious change in p-Akt (Thr308), p-ERK (Thr202/Tyr204), and pp38 MAPK (Thr180/Tyr182) in the combination group compared with OSU-03012 or imatinib mesylate alone, there was a significant increase of p-AMPK (Thr172). In concordance with increased
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Fig. 2. Combinatorial effect of OSU-03012 and imatinib mesylate on indicated molecules of signaling pathways. (A) On TIB-196 myeloma cells. (B) HL-60 cells, a c-Kit non-expressing cell line, were used for comparison.
p-AMPK (Thr172) level, the p-p70S6K (Thr386) expression was attenuated. While total STAT3 protein expression was unaffected, the expression of p-STAT3 (Tyr705) decreased significantly in the combination groups. Cyclin D1 and Mcl-1, both of which are downstream targets of STAT3, were also decreased. HL-60 cells, a c-Kit non-expressing cell line, were used for comparing the protein expression (Fig. 2B). HL-60 cells (2.5 × 105 cells/mL) were cultured in medium or 20 M imatinib mesylate in the presence of DMSO or 8 M OSU-03012 for 24 h. Cell lysates were collected and probed with antibodies to p-AMPK␣ (Thr172), AMPK␣, p-STAT3 (Tyr705), STAT3 and actin. There was no change of AMPK and STAT3 protein expression in the combination group.
3.3. The modulations of p-AMPK and p-STAT3 by OSU-03102 combined with imatinib mesylate are early events To further elucidate if the changes in AMPK and STAT3 are early events, western blotting in serial time-course was performed. TIB-196 cells (2.5 × 105 cells/mL) were cultured in 20 M of imatinib mesylate in combination with 8 M of OSU-03012 for 0, 3, 6, and 24 h. Cell lysates (15 g) were separated by SDS-PAGE and
probed by primary antibodies against p-AMPK␣ (Thr172), AMPK␣, p-STAT3 (Tyr705), STAT3, cyclin D1, and actin (Fig. 3). The increment in p-AMPK expression and decrease in p-STAT3 expression occurred as early as 3 h indicating that both pathways were affected soon after the drugs were added. There was also concordant change of p-STAT3 and cyclin D1 in the time-course study.
4. Discussion Here we demonstrate that OSU-03012 increases the sensitivity of a myeloma cell line to imatinib mesylate. Specifically, the combinatorial use of OSU-03012 and imatinib mesylate leads to activation of AMPK␣, down-regulation of p-p70S6K and phosphorylated STAT3, and increased cleavage of PARP. Although c-Kit expression was reported to occur on the surface of one-third of myeloma cells [1–3], a consensus regarding the use of a c-Kit inhibitor, imatinib mesylate, to treat this disease has not been reached [1,15]. The underlying cause of this discrepancy is not clearly known. Montero et al. found that different isoforms of c-Kit influence the sensitivity and modulate the response of myeloma cells to drugs [7]. The GNNK−
Fig. 3. Western blotting analysis of time-course treatment with imatinib mesylate and OSU-03012 on TIB-196 cells.
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form of c-Kit was activated faster and more pronounced by SCF; whereas activation of the GNNK+ form is of a longer duration [7]. Another possibility is BCR-ABL-independent imatinib mesylate resistance, in which cells will survive through activation of other pathways or signaling molecules. Bewry and his colleagues noticed that increased p-STAT3 (Tyr705) expression contributed resistance toward imatinib mesylate in myeloma cells cultured in bone marrow stroma-derived conditioned medium. Reducing STAT3 levels with small interfering RNA sensitized cells to imatinib mesylate-induced cell death [18]. Because the efficacy of imatinib mesylate for treating patients with multiple myeloma is not impressive, a rational treatment strategy is to increase the sensitivity of myeloma cells to imatinib mesylate by combining drugs acting at other targets. The potent activity of OSU-03012 against myeloma cell lines and primary myeloma cells has been attributed to inhibition of the PI3-kinase/Akt pathway, down-regulation of JAK/STAT3, and down-regulation of MAP kinase pathways [16]. These pathways targeted by OSU-03012 are important for universal cellular signaling and survival. Fittingly for the current study, they are the down-stream signaling pathways of c-Kit as well [5–7]. In our study, we found that combinatorial use of OSU-03012 and imatinib mesylate had additive effects on some target proteins compared with either drug alone. Although there was no change in p-ERK1/2 and p-Akt (Thr308) expression, there was marked attenuation of p-STAT3, a down-stream molecule of c-Kit as well as a target of OSU-03012, by the combined treatment. Additionally, we found that p-AMPK␣ (Thr172) expression increased and its down-stream p-p70S6K (Thr386) expression decreased significantly in the combined treatment group compared with either drug alone. This is the first report that either OSU03012 or imatinib mesylate has the ability to modulate the AMPK pathway. The mammalian target of rapamycin (mTOR) is a key regulator for protein synthesis and plays important roles in cellular metabolism, growth and proliferation. Aberrant activation of mTOR pathways has been noted in many cancers, and some drugs targeting mTOR pathway have been tested in preclinical and clinical studies. AMPK, a sensor of cellular energy status, is one of the Aktindependent mechanisms of mTOR regulation [19]. The activation of AMPK leads to glycolysis, fatty acid oxidation, inhibition of fatty acid synthesis, inhibition of gluconeogenesis, inhibition of protein synthesis, cell cycle control, and inhibition of cell growth [20–22]. Furthermore, some proteins in the AMPK signaling network, including LKB1, TSC1, TSC2 and p53, are tumor suppressor genes [21]. These characteristics of AMPK make it a logical target for treating patients with cancer, including breast cancer [23], melanoma [24], hepatocellular carcinoma [20], chronic lymphocytic leukemia [25], and multiple myeloma [26]. Our study shows that p-AMPK expression increases after either imatinib mesylate or OSU-03012 addition in TIB-196 cells. Combined use of these agents results in increased p-AMPK (Thr172) and decreased p-p70S6K (Thr386) compared with either drug alone. Based on Baumann’s finding and ours, we suggest value in AMPK-targeted agents for the treatment of multiple myeloma. Combination treatment has been used as a strategy to treat patients with cancer for decades. It has the advantage of reducing side effects of a single drug at higher concentration, overcoming drug resistance, reducing the financial burden of expensive compounds and, perhaps most importantly, to minimize the development of resistance to any single agent. In the present study, we demonstrate that OSU-03012 can potentiate the cytotoxicity of imatinib mesylate in myeloma cells. The combination of OSU03012 and imatinib mesylate up-regulates the AMPK pathway, down-regulates the STAT3 pathway, and increases cytotoxicity compared with either OSU-03012 or imatinib mesylate alone in c-Kit expressing myeloma cells. Collectively, our results sug-
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gest that combined treatments targeting c-Kit, AMPK and STAT3 may be a potential strategy for treating patients with multiple myeloma. Conflict of interest statement The authors declare no competing financial interests. Acknowledgements This work was supported in part by a grant from the China Medical University, Taichung, Taiwan; Research Grant CMU97-075. Contributions: L.-Y. B. performed the majority of the research, analyzed the data, and wrote the initial draft of the paper. C.H. T. assisted with the in vitro work and reviewed drafts of the paper. A. S. and C.-W. L. participated in the planning of experiments and reviewed drafts of the paper. C.-F. C. designed the research, reviewed all of the data, analyzing the data and reviewed the draft of the paper. References [1] Pandiella A, Carvajal-Vergara X, Tabera S, Mateo G, Gutierrez N, San Miguel JF. Imatinib mesylate (STI571) inhibits multiple myeloma cell proliferation and potentiates the effect of common antimyeloma agents. Br J Haematol 2003;123:858–68. [2] Ocqueteau M, Orfao A, Garcia-Sanz R, Almeida J, Gonzalez M, San Miguel JF. Expression of the CD117 antigen (c-Kit) on normal and myelomatous plasma cells. Br J Haematol 1996;95:489–93. [3] Pruneri G, Ponzoni M, Ferreri AJ, Freschi M, Tresoldi M, Baldini L, et al. The prevalence and clinical implications of c-Kit expression in plasma cell myeloma. Histopathology 2006;48:529–35. [4] Lemoli RM, Fortuna A, Grande A, Gamberi B, Bonsi L, Fogli M, et al. Expression and functional role of c-Kit ligand (SCF) in human multiple myeloma cells. Br J Haematol 1994;88:760–9. [5] Reber L, Da Silva CA, Frossard N. Stem cell factor and its receptor c-Kit as targets for inflammatory diseases. Eur J Pharmacol 2006;533:327–40. [6] Sattler M, Salgia R. Targeting c-Kit mutations: basic science to novel therapies. Leuk Res 2004;28(Suppl 1):S11–20. [7] Montero JC, Lopez-Perez R, San Miguel JF, Pandiella A. Expression of c-Kit isoforms in multiple myeloma: differences in signaling and drug sensitivity. Haematologica 2008;93:851–9. [8] Druker BJ, Guilhot F, O’Brien SG, Gathmann I, Kantarjian H, Gattermann N, et al. Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med 2006;355:2408–17. [9] Carroll M, Ohno-Jones S, Tamura S, Buchdunger E, Zimmermann J, Lydon NB, et al. CGP 57148, a tyrosine kinase inhibitor, inhibits the growth of cells expressing BCR-ABL, TEL-ABL, and TEL-PDGFR fusion proteins. Blood 1997;90:4947– 52. [10] Savage DG, Antman KH. Imatinib mesylate—a new oral targeted therapy. N Engl J Med 2002;346:683–93. [11] Tuveson DA, Willis NA, Jacks T, Griffin JD, Singer S, Fletcher CD, et al. STI571 inactivation of the gastrointestinal stromal tumor c-Kit oncoprotein: biological and clinical implications. Oncogene 2001;20:5054–8. [12] Larmonier N, Janikashvili N, LaCasse CJ, Larmonier CB, Cantrell J, Situ E, et al. Imatinib mesylate inhibits CD4+ CD25+ regulatory T cell activity and enhances active immunotherapy against BCR-ABL-tumors. J Immunol 2008;181:6955–63. [13] Dewar AL, Cambareri AC, Zannettino AC, Miller BL, Doherty KV, Hughes TP, et al. Macrophage colony-stimulating factor receptor c-fms is a novel target of imatinib. Blood 2005;105:3127–32. [14] Dewar AL, Zannettino AC, Hughes TP, Lyons AB. Inhibition of c-fms by imatinib: expanding the spectrum of treatment. Cell Cycle 2005;4:851–3. [15] Dispenzieri A, Gertz MA, Lacy MQ, Geyer SM, Greipp PR, Rajkumar SV, et al. A phase II trial of imatinib in patients with refractory/relapsed myeloma. Leuk Lymphoma 2006;47:39–42. [16] Zhang S, Suvannasankha A, Crean CD, White VL, Johnson A, Chen CS, et al. OSU-03012, a novel celecoxib derivative, is cytotoxic to myeloma cells and acts through multiple mechanisms. Clin Cancer Res 2007;13:4750–8. [17] Smith JS, Keller JR, Lohrey NC, McCauslin CS, Ortiz M. Redirected infection of directly biotinylated recombinant adenovirus vectors through cell surface receptors and antigens. Proc Natl Acad Sci USA 1999;96:8855–60. [18] Bewry NN, Nair RR, Emmons MF, Boulware D, Pinilla-Ibarz J, Hazlehurst LA. Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance. Mol Cancer Ther 2008;7:3169–75. [19] Memmott RM, Dennis PA. Akt-dependent and -independent mechanisms of mTOR regulation in cancer. Cell Signal 2009;21:656–64. [20] Imamura K, Ogura T, Kishimoto A, Kaminishi M, Esumi H. Cell cycle regulation via p53 phosphorylation by a 5 -AMP activated protein kinase activator,
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L.-Y. Bai et al. / Leukemia Research 34 (2010) 816–820
5-aminoimidazole-4-carboxamide-1--d-ribofuranoside, in a human hepatocellular carcinoma cell line. Biochem Biophys Res Commun 2001;287:562–7. [21] Motoshima H, Goldstein BJ, Igata M, Araki E. AMPK and cell proliferation—AMPK as a therapeutic target for atherosclerosis and cancer. J Physiol 2006;574(Pt 1):63–71. [22] Hardie DG. The AMP-activated protein kinase pathway—new players upstream and downstream. J Cell Sci 2004;117(Pt 23):5479–87. [23] Hadad SM, Fleming S, Thompson AM. Targeting AMPK: a new therapeutic opportunity in breast cancer. Crit Rev Hematol Oncol 2008;67:1–7.
[24] Zheng B, Jeong JH, Asara JM, Yuan YY, Granter SR, Chin L, et al. Oncogenic BRAF negatively regulates the tumor suppressor LKB1 to promote melanoma cell proliferation. Mol Cell 2009;33:237–47. [25] Campa‘s C, Lopez JM, Santidrián AF, Barragán M, Bellosillo B, Colomer D, et al. Acadesine activates AMPK and induces apoptosis in B-cell chronic lymphocytic leukemia cells but not in T lymphocytes. Blood 2003;101:3674–80. [26] Baumann P, Mandl-Weber S, Emmerich B, Straka C, Schmidmaier R. Activation of adenosine monophosphate activated protein kinase inhibits growth of multiple myeloma cells. Exp Cell Res 2007;313:3592–603.