Carboxyamidotriazole inhibits cell growth of imatinib-resistant chronic myeloid leukaemia cells...

This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

http://www.elsevier.com/copyright

Author's personal copy

Carboxyamidotriazole inhibits cell growth of imatinib-resistantchronic myeloid leukaemia cells including T315I Bcr–Abl mutantby a redox-mediated mechanism

Chiara Corrado a, Stefania Raimondo a, Anna Maria Flugy a, Simona Fontana a,Alessandra Santoro b, Giorgio Stassi c, Anna Marfia b, Flora Iovino c, Ralph Arlinghaus d,Elise C. Kohn e, Giacomo De Leo a, Riccardo Alessandro a,⇑a Dipartimento di Biopatologia e Biotecnologie Mediche e Forensi, Sezione di Biologia e Genetica, Palermo, Italyb Laboratorio di Ematologia, Dipartimento di Ricerca Clinica e Biotecnologia, A.O. ‘‘V. Cervello”, Italyc Dipartimento di Scienze Chirurgiche ed Oncologiche, Palermo, Italyd Molecular Pathology, University of Texas, M.D. Anderson Cancer Center, Houston, TX, USAe Molecular Signaling Section, Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA

a r t i c l e i n f o

Article history:Received 23 June 2010Received in revised form 8 October 2010Accepted 11 October 2010

Keywords:Chronic myelogenous leukemia (CML)Carboxyamidotriazole (CAI)Drug resistanceReactive-oxygen species (ROS)

a b s t r a c t

Mutation of the Bcr–Abl oncoprotein is one of most frequent mechanisms by which chronicmyelogenous leukemia (CML) cells become resistant to imatinib. Here, we show that treat-ment of cell lines harbouring wild type or mutant BCR–ABL with carboxyamidotriazole(CAI), a calcium influx and signal transduction inhibitor, inhibits cell growth, the expres-sion of Bcr–Abl and its downstream signalling, and induces apoptosis. Moreover, we showthat CAI acts by increasing intracellular ROS. Clinically significant, CAI has also inhibitoryeffects on T315I Bcr–Abl mutant, a mutation that causes CML cells to become insensitiveto imatinib and second generation abl kinase inhibitors.

� 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Chronic myeloid leukemia is a myeloproliferative disor-der characterized by the t(9:22) (q34:q11) reciprocaltranslocation, resulting in the expression of the chimericBcr–Abl oncoprotein with constitutive tyrosine kinaseactivity [1,2]. Deregulated Bcr–Abl induces in turn thehyperactivation of various signalling pathways, such asthe Ras/Raf/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway, thephosphoinositide 3-kinase (PI3K) pathway, Janus kinase 2pathway, and the signal transducer and activator of

transcription 5 pathway (STAT-5) [3,4] that promote cellgrowth, suppress apoptosis and alter cell adhesion [5,6].Imatinib mesylate (IM) is a selective well tolerated inhibi-tor of the Bcr–Abl tyrosine kinase that has significantly im-proved the prognosis of patients with chronic phase CML[7,8]. Whereas clinical data from imatinib treatment seempromising, the development of resistance due to primaryor acquired point mutations in BCR–ABL [9], especially inindividuals with accelerated and blast phase disease, is agrowing problem. A set of BCR–ABL mutants, especiallythe T315I mutation (located within the kinase domain ofBcr–Abl), leads to alteration in the structure of the enzymeactive site thus resulting in a constitutive kinase activityand resistance to IM [10]. To circumvent resistance, highlypotent kinase inhibitors, such as AMN107 and BMS-354825 [11], have been recently developed, however thesecompounds do not have therapeutic activity against all

0304-3835/$ - see front matter � 2010 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.canlet.2010.10.007

⇑ Corresponding author. Address: Dipartimento di Biopatologia eBiotecnologie Mediche e Forensi, Università di Palermo, Via Divisi 83,90133 Palermo, Italy. Tel.: +39 091 655 4600; fax: +39 091 655 4624.

E-mail address: [email protected] (R. Alessandro).

Cancer Letters 300 (2011) 205–214

Contents lists available at ScienceDirect

Cancer Letters

journal homepage: www.elsevier .com/locate /canlet


imatinib-resistant mutants of Bcr–Abl. Alternative strate-gies, such as Aurora Kinase inhibitors [12] or PEITC [13],as well as combination therapies have shown some successin the treatment of the T315I mutant in in vitro settingsand in mouse models but have not completed clinical trialsyet [14]. Therefore, there is an urgent need for new anti-cancer agents and combinations that could improve re-sponses and survival rates for CML.

Recent studies from our laboratory have shown thataddition of carboxyamidotriazole (CAI), an inhibitor ofcalcium-mediated signal transduction [15], to imatinib-resistant human CML cells induces a marked decrease incell viability and augmented apoptosis, events associatedwith downregulation of Bcr–Abl protein, inhibition oftyrosine phosphorylation of Bcr–Abl, STAT5, CrkL, as wellas inhibition of ERK1/2 phosphorylation [16]. Evidencehas linked calcium signalling with reactive-oxygen spe-cies (ROS) production [17,18]. ROS production and themodification of the cellular redox state play importantroles in many cellular events [19] and are known to medi-ate, in several types of cancer including leukaemia [20–22], caspase-dependent or -independent cell death bynumerous anticancer agents [23,24]. Taking these datainto account, the aims of our study were (i) to test ifCAI exerts its inhibitory effect on Bcr–Abl mutants,including the T315I mutant, and (ii) to investigate themechanism by which CAI is exerting its action. Our resultsindicate that addition of CAI to 32D murine cells transfec-ted with wild type BCR–ABL or mutated forms, and to LA-MA84R, a human model of imatinib-resistant CML,induces inhibition of cell growth and cell death due to in-crease in ROS production.

2. Materials and methods

2.1. Cell culture and reagents

LAMA84R are imatinib-resistant cells kindly providedby Dr. P. Vigneri (University of Catania, Italy). 32D BCR–ABL wild type (32D P210), 32D BCR–ABL mutated T315I(32D T315I) and 32D BCR–ABL mutated E255K (32DE255K) were kindly provided by Dr. Ralph Arlinghaus(MD Anderson Cancer Center, University of Texas, USA).Cells were cultured in RPMI 1640 medium (BioWhittaker,Verviers, Belgium) supplemented with 10% fetal bovineserum (BioWhittaker, Verviers, Belgium), 2 mM L-gluta-mine, 100 U/ml penicillin and 100 lg/ml streptomycin(Euroclone, UK). All experiments were performed usinglogarithmically growing cells (4–6 � 105 cells/ml). Imati-nib, kindly provided by Dr. R. Bertieri (Novartis, Milan,Italy), was prepared as a 5 mM stock solution in sterilephosphate-buffered saline (PBS); CAI was prepared as a40 mM stock solution in DMSO; Dasatinib, kindly providedby Dr. G. Pitrone (Bristol-Meyers Squibb, New Brunswick,NJ) was prepared as a 10 lM stock solution in ethanol. Glu-tathione-reduced ethyl ester (GSH, Sigma Chemical Co., St.Louis, MO) was prepared as a 0.5 M stock solution in PBS.All stocks were kept at �20 �C. Serial dilutions of all drugstocks were performed in culture medium the same dayof use.

2.2. Proliferation assay (MTT assay)

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide) assay was done as previously described[25]. Briefly, cells were plated in quadruplicate at 1.5 � 104

per well and exposed to escalating doses of CAI (1–5–10 lM) or dasatinib (0.5–1–5 nM) for up to 4 days. To testthe effect of GSH on CAI-induced death, cells were treatedfor 48–72–96 h with 5 and 10 lM CAI in presence or ab-sence of GSH. Means and standard deviations generatedfrom four independent experiments are reported as thepercentage of growth versus control (cells treated withDMSO 0.01%). Cell proliferation curves were derived fromthese data by using Microsoft Excel software.

2.3. Annexin V assay

Cells were treated with 5 lM CAI for 72 h; as a control,cells were also treated with DMSO vehicle (0.01%) for thesame duration (72 h). After incubation, cells were washedin PBS twice and apoptosis assay was performed as follows.Cells were resuspended in cold 1� binding buffer (10�:0.1 M Hepes pH 7.4; 1.4 M NaCl; 25 mM CaCl2) at 1 � 106

cells/ml. A 0.1 ml aliquot of the cellular suspension wastransferred in a FACS tube and 5 ll of Annexin V-FITC(BD-Biosciences, San Jose, CA) plus 10 ll of 7-AAD (7-Ami-no-actinomycin D, BD-Biosciences, San Jose, CA) wereadded. After 15 min of incubation at room temperature(RT) in the dark, 400 ll of binding buffer 1� was addedto each tube and the fluorescence was determined by flowcytometric analysis with BD FACScantoII, software BDFACSDiva (BD-Biosciences, San Jose, CA).

2.4. Cell cycle analysis

Cells were treated with 5 lM and 10 lM CAI for 72 h; asa control, cells were also treated with DMSO vehicle(0.01%) for the same duration. After treatment, 1 � 106

cells were washed in cold PBS, fixed in 1 ml of Ethanol70% and incubated overnight at 4 �C. Cells were resus-pended in 300 ll of propidium iodide solution (SigmaChemical Co., St. Louis, MO) and analysed by flow cytome-ter with BD FACScan, software CellQuest (BD-Biosciences,San Jose, CA).

2.5. Western blot analysis

Total protein cell lysates were obtained and analysedby SDS–PAGE followed by Western blotting as previouslydescribed [16]. Briefly, LAMA84R or 32D murine BCR–ABLtransfected cells were treated as indicated, then washedand lysed in lysis buffer (300 mM NaCl, 50 mM Tris HClpH 7.6, 0.1% Triton X-100, 1 mM PMSF, 10 lg/ml leupep-tin, 10 lg/ml aprotinin, 4 mM EDTA, 2 mM sodium ortho-vanadate, 10 mM NaPPi, 100 mM NaF) on ice for 1.5 h. Forcytoplasmatic and nuclear fractions, cells were washedtwice in ice PBS, resuspended in solution I (Hepes10 mM pH 7.9, KCl 10 mM, MgCl2 0.1 mM, EDTA0.1 mM, DTT 0.1 mM, PMSF 0.5 mM, 10 lg/ml leupeptin,10 lg/ml aprotinin, 2 mM sodium orthovanadate, 10 mMNaPPi, 100 mM NaF) and passed seven times through a

206 C. Corrado et al. / Cancer Letters 300 (2011) 205–214


syringe equipped with a 23 gauge needle. After centrifu-gation, the supernatant was collected as cytoplasmaticfraction. To recover the nuclear fraction, the pellet waswashed 4 times in solution I, resuspended in solution II(Hepes 10 mM pH 7.9, NaCl 400 mM, MgCl2 1.5 mM, EDTA0.1 mM, DTT 0.1 mM, PMSF 0.5 mM, 10 lg/ml leupeptin,10 lg/ml aprotinin, 2 mM sodium orthovanadate, 10 mMNaPPi, 100 mM NaF) and incubated on ice for 30 min.The total cell lysates or the nuclear fraction were clarifiedat high speed centrifugation for 15 min and an aliquot ofthe supernatant was assayed to determine protein con-centration by Coomassie plus protein assay reagent (Ther-mo Scientific, Rockford, IL).

Proteins were separated by SDS-polyacrylamide gelelectrophoresis and transferred to nitrocellulose mem-brane (Protran Schleicher & Schuell, Dassel, Germany).The membrane was incubated in blocking solution (5%non-fat dry milk, 20 mM Tris, 140 mM NaCl, 0.1% Tween-20), and probed overnight at 4 �C with specific antibodiesagainst c-Abl, phospho-Abl, phospho-Stat-5, phospho-CrkL,cleaved caspase 3, PARP, AIF, beta-actin (all from Cell Sig-nalling Technology, Beverly, MA, USA) and anti-phosphoty-rosine, anti-Stat5 and anti-CrkL (all from Santa CruzBiotechnology, SantaCruz, CA, USA). After three washeswith 20 mM Tris, 140 mM NaCl, 0.1% Tween-20 (WS), themembrane was incubated with horseradish peroxidase-conjugated secondary antibody and proteins detected bythe enhanced chemiluminescence detection system (WestPico chemiluminescent substrate, Thermo Scientific,Rockford, IL, USA).

2.6. Measurement of cellular ROS

The levels of ROS were examined using 5–6-chloro-methyl-20,70-dichlorodihydrofluorescein diacetate, acetylester (CM-H2DCFDA; Invitrogen, Carlsbad, CA, USA). Thismolecule passively diffuses into the cells; chemically re-duced and acetylated form are nonfluorescent until theacetate groups are removed by intracellular esterasesand oxidation occurs within the cell. Oxidation of theprobes can be detected by monitoring the increase influorescence by a flow cytometer, using filter appropriatefor FITC. The probe was diluted in DMSO to the concen-tration of 1 mM just immediately before the use. Cellswere treated for 72 and 96 h with 5 lM CAI in presenceor absence of GSH 5 mM; as a control, cells were treatedwith DMSO 0.01% or GSH 5 mM for the same period oftime (72 and 96 h). After treatment, cells were washedtwice in PBS, resuspended at 1 � 106 cells/ml in PBS con-taining the probe at a final concentration of 1 lM, incu-bated in the dark for 15 min and analyzed by flowcytometer, software BD FACSDiva. As a positive controlcells were treated with H2O2 0.5 mM for 3 h and analysedas previously described.

2.7. Statistical analysis

Statistically significant differences between mean val-ues (from three independent experiments at least) weredetermined using two-tailed Student’s t-test. Differenceswere considered statistically significant at p < 0.05.

Fig. 1. CAI inhibits cell proliferation of 32D cell lines. 32D p210 (A), 32D T315I (B), 32D E255K (C) cell growth was measured by MTT assay after 48, 72, 96 hof treatment with increasing doses of Dasatinib (0.5–1–5 nM), left panel, or CAI (1–5–10 lM), right panel. The values were plotted as a percentage of thecontrol (cells treated with DMSO). Each point represents the mean ± SD for three independent experiments.

C. Corrado et al. / Cancer Letters 300 (2011) 205–214 207


3. Results

3.1. CAI decreases viability of murine 32D cells expressing wild type or mutantBCR–ABL

We have previously demonstrated that CAI is able to inhibit in atime- and dose-dependent manner the growth of IM-resistant humanCML cells [16]. In order to better characterize the inhibitory activity ofCAI, parallel studies were carried out in murine 32D cells with ectopicexpression of either the unmutated BCR–ABL (32D p210) or the pointmutants BCR–ABL E255K (32D E255K) or BCR–ABL T315I (32D T315I).MTT assays were performed as described in Section 2 and data pre-sented in Fig. 1 show results of 4-day treatment. As demonstrated byother studies [26,27], treatment of IM-resistant CML cells with secondgeneration inhibitors of Bcr–Abl has remarkable effects on Bcr–Abl, ex-cept for the mutant T315I. Dasatinib, a �325-fold more potent com-pound than IM in inhibiting the kinase activity of Bcr–Abl [28,29],induced a net decrease of cell viability in 32D p210 cells while 32DE255K cells were less sensitive and 32D T315I cells were not responsive(Fig. 1, left panel). On the contrary, CAI inhibits cell growth of all 32Dcell lines including 32D T315I cells in the low micromolar range, a phys-iologically attainable concentration range [15], in a dose dependentfashion (Fig. 1, right panel). The results herein show a 40–50% growthreduction of the murine cell lines.

3.2. Effects of CAI on Bcr–Abl expression and its downstream signalling inmurine 32D cells expressing wild type or mutant Bcr–Abl

To investigate the biochemical basis of the antiproliferative effects ofCAI, the global Bcr–Abl expression and its phosphorylation, the phospho-tyrosine signature and phosphorylation of Bcr–Abl downstream targets,Stat5 and CrkL, were evaluated in whole cell extracts of the 32D p210,32D E255K and 32D T315I cells after 96 h of drug treatment. Cells wereincubated with increasing concentrations of CAI, harvested and sub-jected to immunoblotting with antibodies that recognize p210Bcr–Abl,phospho-p210Bcr–Abl, phosphotyrosine, phospho-Stat5 and Stat5, phos-pho-CrkL and CrkL. As shown in Fig. 2A upper panel, treatment of allthree BCR–ABL transfected 32D cell lines with CAI causes a reductionnot only in the levels of phosphorylated Bcr–Abl after 96 h of drug expo-sure but also of the total amount of the kinase and inhibits the phos-phorylation of two targets of Bcr–Abl, namely Stat5 and CrkL, aspreviously demonstrated by our group for IM-resistant human CML celllines [16]. In fact the tyrosine phosphorylation of Stat5 and CrkL was re-duced by CAI treatment in all cell lines. Moreover, several heavily tyro-sine-phosphorylated proteins were observed in the untreated 32D cellsand a dose-dependent inhibition of the overall pattern of tyrosine phos-phorylated protein was observed starting at 5 lM CAI (Fig. 2A, lower pa-nel). Treatment of the cells with 5 nM Dasatinib for 15 h determines adrastic inhibition of tyrosine phosphorylation of wild type Bcr–Abl and

Fig. 2. (A) CAI treatment diminishes Bcr–Abl expression, inhibits Bcr–Abl tyrosine phosphorylation and its downstream signalling on 32D cell lines. 32Dp210, 32D T315I and 32D E255K were treated with increasing doses of CAI (1–10 lM) or DMSO (Ctrl) for 96 h after which protein lysates were prepared andsubjected to western blot analysis as described in Section 2 using anti-phospho-abl, anti-abl, anti-phospho-stat5, anti-stat5, anti-phospho-crkl, anti-crkl,and anti-phospho-tyr antibodies. Blots were then stripped and subsequently reprobed with antibody against beta-actin to ensure equal loading. (B)Dasatinib treatment inhibits Bcr–Abl tyrosine phosphorylation, except on 32D T315I cell lines. 32D p210, 32D T315I and 32D E255K were treated with 5 nMDasatinib or DMSO (Ctrl) for 15 h. Protein lysates obtained were subjected to western blot analysis using anti-abl and anti-phospho-abl antibodies.



Fig. 3. CAI treatment induces apoptosis in 32D murine cells expressing wild type, E255K- and T315I-mutant Bcr–Abl. 32D p210, 32D T315I and 32D E255Kcell death was detected by Annexin V/7-AAD assay after 72 h of treatment with CAI 5 lM. Left panel: cells treated with DMSO, as a control; right panel: cellstreated with CAI 5 lM. The number shown inside panels indicates the percentage of the Annexin V positive and 7-AAD negative cells (apoptotic).

Table 1Percentage of cells in different phases of cell cycle.

LAMA84R 32D p210

Ctrl 5 lM CAI 10 lM CAI Ctrl 5 lM CAI 10 lM CAI

Sub G1 11.52 30.52 48.97 2.05 23.78 19.17G1 53 35.39 30.83 37.95 44.97 48.78S 13 11.2 7.74 21.25 11.97 12.09G2/M 19.25 14.92 9.52 29.03 18.45 18.85

32D T315I 32D E255K

Ctrl 5 lM CAI 10 lM CAI Ctrl 5 lM CAI 10 lM CAI

Sub G1 8.73 24.43 40.05 11.74 25.47 60.65G1 47.82 40.95 31.77 49.13 35.34 16.53S 14.38 11.14 11.44 15.23 13.53 7.58G2/M 23.61 20.09 13.56 21.57 24.39 14.76

LAMA84R, 32D p210, 32D T315I and 32D E255K cell cycle analysis was performed after 72 h of treatment with 5 lM and 10 lM CAI. The table shows thepercentage of cells in subG1–G1–S–G2/M phases of cell cycle.



Fig. 4. CAI treatment increases intracellular ROS in LAMA84R and in 32D cell lines. To determine the intracellular concentration of ROS, cells, after 72 h oftreatment with the drug, were incubated with dichlorodihydrofluorescein diacetate, as described in Section 2, and the fluorescence was measured by flowcytometry. Left panel: cells treated with CAI 5 lM (grey) or DMSO (light grey). Right panel: cells treated with CAI 5 lM (grey) or GSH 5 mM-CAI 5 lM(black). (A) LAMA84R; (B) 32D p210; (C) 32D T315I; (D) 32D E255K; (E) Histogram of Mean Intensity Fluorescence from cells treated as previouslydescribed. These results are representative of three independent experiments for each cell line.



of Bcr–Abl E255K while it was ineffective for the phosphorylation ofBcr–Abl T315I; in contrast of CAI, Dasatinib has no effect on Bcr–Ablexpression (Fig. 2B).

3.3. CAI induces apoptosis in 32D murine cells expressing wild type, E255K-and T315I-mutant Bcr–Abl

We have previously showed that treatment of IM-resistant humanCML cells with CAI increases apoptosis [16]. To investigate if the effectof CAI on the viability of 32D cell lines was mediated by activation ofapoptotic pathway, Annexin V–FITC/7-AAD fluorescence was measuredby flow cytometer on 32D p210, 32D T315I, 32D E255K cell lines trea-ted with CAI 5 lM for 72 h. Fig. 3 shows an increase of cell death in32D cell lines treated with CAI up to 20%, as also confirmed by acridineorange/ethidium bromide (data not shown). Furthermore the cell cycleanalysis of LAMA84R and 32D cell lines shows a dose dependentincrease of cells in subG1 phase, indicating cell death, as reported inTable 1.

3.4. CAI increases intracellular reactive-oxygen species (ROS) in humanLAMA84R cells and in murine wild type and resistant CML cells

To investigate whether CAI stimulates ROS generation in 32D celllines and in a model of human IM-resistant CML cells, LAMA84R, we usedCMH2DCFDA fluorochrome as an indicator of peroxides and superoxideaccumulation. These cell lines are also different because of the mecha-nism of resistance to imatinib: imatinib-resistance of LAMA84R is causedby BCR–ABL gene amplification while 32D mutant cell lines have beentransfected with mutated form of BCR–ABL that abrogates the capabilityof imatinib to bind and inhibit the oncoprotein kinase activity. We treatedcells with 5 lM CAI for 72–96 h and evaluated intracellular ROS accumu-lation by flow cytometric analysis. After treatment with CAI, an increaseof intracellular ROS (shift on the right of grey peak) was observed as earlyas at 72 h in IM-resistant CML cell line (LAMA84R) and in all 32D cell lines(Fig. 4, left panel). To further address if the increase of intracellular ROS incells treated with CAI was specific, cells were treated with 5 mM glutathi-one (GSH), a physiologic ROS scavenger, in combination with CAI. Thepeak of fluorescence intensity of cells treated with GSH-CAI (black peak)

is shifted on the left compared with treatment of CAI alone (Fig. 4, rightpanel); in Fig. 4E is shown the histogram of mean intensity fluorescencefrom cells treated with CAI and GSH-CAI.

3.5. Treatment of cells with GSH reverses CAI-dependent effects on Bcr–Abl,induction of apoptosis and inhibition of proliferation in CML cell lines

To investigate if the effects of CAI on Bcr–Abl expression and on sig-nalling are mediated by increase of intracellular ROS, LAMA84R and32D cells were incubated for 96 h with 5 lM CAI alone or in combinationwith GSH 5 mM, harvested and subjected to immunoblotting with anti-bodies that recognize p210Bcr–Abl and phospho-p210Bcr–Abl. As shownin Fig. 5A, GSH reverts the effect of CAI on Bcr–Abl expression and on itsphosphorylation.

We subjected the cells to CAI and GSH with CAI and investigated bio-chemical measures of apoptosis. As shown in Fig. 5B, CAI induces cleavageof caspase 3 and subsequent cleavage of PARP in 32D cell lines. GSH, asROS scavenger, partially reverts the effect of CAI on this caspase-depen-dent apoptosis. No cleavage of caspase 3 and PARP were observed in LA-MA84R cells, but there is an increase of total PARP with addition of GSH,indicating a reduction in apoptotic activity. In Fig. 5C we show that treat-ment of LAMA84R with CAI causes the translocation of Apoptosis Induc-ing Factor (AIF) in nuclear fraction of the cells while AIF increases but itremains localized in the cytoplasm in 32D cell lines treated with CAI.The detection of beta-actin exclusively in cytoplasmic fraction confirmsthe purity of cellular fractionation.

Furthermore, our findings were complemented by measures of cellsurvival in the presence of CAI with or without addition of GSH. GSH re-verts the effect of CAI on cell growth suggesting that CAI exerts its inhib-itory effects on cell growth via increase of intracellular ROS (Fig. 6).

4. Discussion

The structural basis for imatinib-resistance in chronicmyeloid leukaemia involves the emergence of point muta-tions in BCR–ABL coding region altering drug affinity. Morethan 50 different point mutations have been described [30]that predominantly cause the impairment of drug binding

Fig. 5. GSH treatment reverts the effects of CAI on Bcr–Abl and on the induction of apoptosis in LAMA84R and in 32D cell lines. All cell lines were treatedwith CAI 5 lM, GSH 5 mM or GSH 5 mM-CAI 5 lM for 96 h after which protein lysates were prepared and subjected to western blot analysis, as described inSection 2, using anti-abl and anti-phospho-abl antibodies (5A), anti-cleaved caspase 3 and anti-PARP antibodies (5B). Blots were subsequently reprobedwith antibody against beta-actin to ensure equal loading. (5C) All cell lines were treated with CAI 5 lM for 96 h after which cytoplasmic and nuclearfractions were prepared and subjected to western blot analysis using anti-AIF antibody and anti-beta-actin antibodies as described in Section 2. Ctrl: cellstreated with DMSO. C: cytoplasmic fraction. N: nuclear fraction. These results were representative of three independent experiments for each cell line.



to the enzyme active site. However, many of these muta-tions are relatively rare and the most common are affectingresidues Tyr253, Phe359, Glu255 and especially Thr315.Second generation inhibitors such as dasatinib or nilotinib,have demonstrated activity against a number of mutationsalthough none are strongly successful against Thr315[28,31]. New approaches are necessary to overcome theemergence of new mutations causing drug resistance. Wehave previously published that CAI, an inhibitor of cal-cium-mediated signal transduction [15], decreases cell via-

bility and induces apoptosis of imatinib-resistant CMLcells, reducing both total and phosphorylated Bcr–Abl[16]. We hypothesized that reactive-oxygen species arean effector for the CAI activity.

Reactive-oxygen species (ROS) play an important role incancer phenotype due to their stimulating effects on cellgrowth and proliferation [32,33]. Increased ROS generationis common in cancer cells with active metabolism under theinfluence of oncogenic signals including RAS, BCR–ABL, andc-MYC [34–36]. In addition, high levels of ROS cause cellular

Fig. 6. GSH reverts the cytotoxic effect of CAI in LAMA84R and in 32D cell lines. Cell growth was measured by MTT assay after 48, 72 and 96 h of treatmentwith increasing doses of CAI (5–10 lM), GSH 5 mM or cotreatment GSH-CAI. The values were plotted as a percentage of the control (cells treated withDMSO). (A) LAMA84R; (B) 32D p210; (C) 32D T315I; (D) 32D E255K. Each point represents the mean ± SD for three independent experiments. *p < 0.05;**p < 0.001; ***p < 0.0001.



damage [37] and cancer cell death [38], thus suggestingthat alterations of ROS might be an amenable targeting incancer therapy. Moreover, there has been some correlationbetween the Bcr–Abl oncogenic signal and ROSgeneration [39,40]. Zhang and colleagues have shown thatPEITC, a natural compound, is able to kill imatinib-resistantCML cells through an increase of intracellular ROS andfollowing redox-mediated degradation of the Bcr–Ablprotein [13].

Calcium signalling has been linked recently with gener-ation of reactive-oxygen species (ROS) production [17,18].Both are intracellular signalling molecules that cross-talkthus enhancing signalling coordination and integration ofcellular functions involving both the mitochondria andthe cytosol. Su and colleagues showed that curcumininduced apoptosis of human colon cancer cells throughproduction of reactive-oxygen species and intracellularcalcium modulation [41]. Zhang et al. demonstrated thatcombined mitochondrial ROS generation and calciuminflux blockade is required for leukaemia cell death [18].Recently, Ciarcia and colleagues showed a link betweenintracellular calcium and oxidative stress in CML disease[42].

On the basis of these observations, we posited that CAIexerts its effects on CML cells through increase of intracel-lular ROS. Our data have identified a new mechanism of ac-tion of CAI, ROS generation, and demonstrated thatmechanism as responsible for inhibition of CML throughdownregulation of the Bcr–Abl oncogenic activity and sub-sequent apoptosis.

Overactivation of PARP, instead of its cleavage, and sub-sequent translocation of AIF to the nucleus are emerging asalternative molecular mechanism of caspase-independentcell death. In fact Parreno and colleagues demonstratedthat Bobel-24, a non-steroid anti-inflammatory molecule,induces Ros-dependent cell death in leukaemia and pan-creatic cancer cells through caspase-independent mito-chondrial pathway activated by PARP and followed bynuclear translocation of AIF [23,24,43]. Our data suggestthat, in LAMA84R cells but not in BCR–ABL transfected32D cells, CAI induce a caspase-independent apoptosisthrough a AIF mediated pathway.

The different effects of CAI on the caspase pathway maybe explained taking in consideration that 32D murine cellsand LAMA84R are characterized by different modality ofimatinib-resistance.

In conclusion, the results suggest that the molecularmechanism by which CAI inhibits CML cell proliferationis through an increase of reactive-oxygen species that inturn modulate Bcr–Abl levels, tyrosine phosphorylationof oncoprotein, downstream signalling and apoptosis evenif perturbation of other pathways cannot be excluded atthis time.

CAI is already in phase II clinical trial for treatment ofother solid tumours with minor side effects, our resultspropose that it might be a possible candidate for patientswith CML that are resistant to monotherapy of imatinib.Use of signal transduction inhibitors, as CAI, targeting a dif-ferent pathway with respect to Bcr–Abl inhibitors, asimatinib or dasatinib, could achieve a significative thera-peutic result in the occurrence of drug resistance.

Conflicts of interest

None declared.

Acknowledgements

This work was supported by Italian Association for Can-cer Research (AIRC) to G.D.L. and R.A., University of Paler-mo (International Cooperation) to R.A.; ex 60% MURST toR.A., A.M.F., S.F. and to G.D.L.

Dr. Kohn is supported by the Intramural Program of theCenter for Cancer Research, National Cancer Institute, Na-tional Institutes of Health, USA.

References

[1] Y. Ben-Neriah, G. Daley, A. Mes-Masson, O. Witte, D. Baltimore, Thechronic myelogenous leukemia-specific P210 protein is the productof the bcr/abl hybrid gene, Science 233 (1986) 212–214.

[2] J. Rowley, A new consistent chromosomal abnormality in chronicmyelogenous leukaemia identified by quinacrine fluorescence andGiemsa staining, Nature 243 (1973) 290–293.

[3] M. Nieborowska-Skorska, M. Wasik, A. Slupianek, P. Salomoni, T.Kitamura, B. Calabretta, T. Skorski, Signal transducer and activator oftranscription (STAT)5 activation by BCR/ABL is dependent on intactSrc homology (SH)3 and SH2 domains of BCR/ABL and is required forleukemogenesis, J. Exp. Med. 189 (1999) 1229–4122.

[4] C. Sillaber, F. Gesbert, D. Frank, M. Sattler, J. Griffin, STAT5 activationcontributes to growth and viability in Bcr/Abl-transformed cells,Blood 95 (2000) 2118–2125.

[5] J. Sonoyama, I. Matsumura, S. Ezoe, Y. Satoh, X. Zhang, Y. Kataoka, E.Takai, I.M. Mizuk, T. Machii, H. Wakao, Y. Kanakura, Functionalcooperation among Ras, STAT5, and phosphatidylinositol 3-kinase isrequired for full oncogenic activities of BCR/ABL in K562 cells, J. Biol.Chem. 277 (2002) 8076–8082.

[6] L. Hazlehurst, N. Bewry, R. Nair, J. Pinilla-Ibarz, Signaling networksassociated with BCR–ABL-dependent transformation, Cancer Control16 (2009) 100–107.

[7] B. Druker, S. Tamura, E. Buchdunger, S. Ohno, G. Segal, S. Fanning, J.Zimmermann, N. Lydon, Effects of a selective inhibitor of the Abltyrosine kinase on the growth of Bcr–Abl positive cells, Nat. Med. 2(1996) 561–566.

[8] H. Kantarjian, J. Cortes, S. O’Brien, F. Giles, G. Garcia-Manero, S.Faderl, D. Thomas, S. Jeha, M. Rios, L. Letvak, K. Bochinski, R.Arlinghaus, M. Talpaz, Imatinib mesylate therapy in newlydiagnosed patients with Philadelphia chromosome-positive chronicmyelogenous leukemia: high incidence of early complete and majorcytogenetic responses, Blood 101 (2003) 97–100.

[9] F. Lee, A. Fandi, M. Voi, Overcoming kinase resistance in chronicmyeloid leukemia, Int. J. Biochem. Cell Biol. 40 (2008) 334–343.

[10] N. Shah, J. Nicoll, B. Nagar, M. Gorre, R. Paquette, J. Kuriyan, C.Sawyers, Multiple BCR–ABL kinase domain mutations conferpolyclonal resistance to the tyrosine kinase inhibitor imatinib(STI571) in chronic phase and blast crisis chronic myeloidleukemia, Cancer Cells 2 (2002) 117–125.

[11] S. Redaelli, R. Piazza, R. Rostagno, V. Magistroni, P. Perini, M. Marega,C. Gambacorti-Passerini, F. Boschelli, Activity of bosutinib, dasatinib,and nilotinib against 18 imatinib-resistant BCR/ABL mutants, J. Clin.Oncol. 27 (2009) 469–471.

[12] T. Carter, L. Wodicka, N. Shah, A. Velasco, M. Fabian, D. Treiber, Z.Milanov, C. Atteridge, W.R. Biggs, P. Edeen, M. Floyd, J. Ford, R.Grotzfeld, S. Herrgard, D. Insko, S. Mehta, H. Patel, W. Pao, C.Sawyers, H. Varmus, P. Zarrinkar, D. Lockhart, Inhibition of drug-resistant mutants of ABL, KIT, and EGF receptor kinases, Proc. Natl.Acad. Sci. USA 102 (2005) 11011–11016.

[13] H. Zhang, D. Trachootham, W. Lu, J. Carew, F. Giles, M. Keating, R.Arlinghaus, P. Huang, Effective killing of Gleevec-resistant CML cellswith T315I mutation by a natural compound PEITC through redox-mediated mechanism, Leukemia 22 (2008) 1191–1199.

[14] J. Zhang, F. Adria, W. Jahnke, S. Cowan-Jacob, A. Li, R. Iacob, T. Sim, J.Powers, C. Dierks, F. Sun, G. Guo, Q. Ding, B. Okram, Y. Choi, A.Wojciechowski, X. Deng, G. Liu, G. Fendrich, A. Strauss, N. Vajpai, S.Grzesiek, T. Tuntland, Y. Liu, B. Bursulaya, M. Azam, P. Manley, J.Engen, J. Daley, M. Warmuth, N. Gray, Targeting Bcr–Abl by



combining allosteric with ATP-binding-site inhibitors, Nature 463(2010) 501–506.

[15] M. Hussain, H. Kotz, L. Minasian, A. Premkumar, G. Sarosy, E. Reed, S.Zhai, S. Steinberg, M. Raggio, V.K. Oliver, W. Figg, E. Kohn, Phase IItrial of carboxyamidotriazole in patients with relapsed epithelialovarian cancer, J. Clin. Oncol. 21 (2003) 4356–4363.

[16] R. Alessandro, S. Fontana, M. Giordano, C. Corrado, P. Colomba, A.Flugy, A. Santoro, E. Kohn, G. De Leo, Effects of carboxyamidotriazoleon in vitro models of imatinib-resistant chronic myeloid leukemia, J.Cell. Physiol. 215 (2008) 111–121.

[17] C. Nathan, Specificity of a third kind: reactive oxygen andnitrogen intermediates in cell signaling, J. Clin. Invest. 111 (2003)769–778.

[18] Y. Zhang, J. Soboloff, Z. Zhu, S. Berger, Inhibition of Ca2+ influx isrequired for mitochondrial reactive oxygen species-inducedendoplasmic reticulum Ca2+ depletion and cell death in leukemiacells, Mol. Pharmacol. 70 (2006) 1424–1434.

[19] D. Trachootham, J. Alexandre, P. Huang, Targeting cancer cells byROS-mediated mechanisms: a radical therapeutic approach?, NatRev. Drug Discov. 8 (2009) 579–591.

[20] R. Feng, H. Ni, S. Wang, I. Tourkova, M. Shurin, H. Harada, X. Yin,Cyanidin-3-rutinoside, a natural polyphenol antioxidant, selectivelykills leukemic cells by induction of oxidative stress, J. Biol. Chem.282 (2007) 13468–13476.

[21] D. Xiao, A. Powolny, S. Singh, Benzyl isothiocyanate targetsmitochondrial respiratory chain to trigger reactive oxygen species-dependent apoptosis in human breast cancer cells, J. Biol. Chem. 283(2008) 30151–30163.

[22] R. Zhang, I. Humphreys, R. Sahu, Y. Shi, S. Srivastava, In vitro andin vivo induction of apoptosis by capsaicin in pancreatic cancer cellsis mediated through ROS generation and mitochondrial deathpathway, Apoptosis 13 (2008) 1465–1478.

[23] M. Parreno, I. Casanova, M. Cespedes, J. Vaque, M. Pavon, J. Leon, R.Mangues, Bobel-24 and derivatives induce caspase-independentdeath in pancreatic cancer regardless of apoptotic resistance, CancerRes. 68 (2008) 6313–6323.

[24] M. Parreno, J. Vaque, I. Casanova, P. Frade, M. Cespedes, M. Pavon, A.Molins, M. Camacho, L. Vila, J. Nomdedeu, R. Mangues, J. Leon, Noveltriiodophenol derivatives induce caspase independentmitochondrial cell death in leukemia cells inhibited by Myc, Mol.Cancer Ther. 5 (2006) 1166–1175.

[25] S. Fontana, R. Alessandro, M. Barranca, M. Giordano, C. Corrado, I.Zanella-Cleon, M. Becchi, E. Kohn, G. De Leo, Comparative proteomeprofiling and functional analysis of chronic myelogenous leukemiacell lines, J. Proteome Res. 6 (2008) 4330–4342.

[26] H. Kantarjian, F. Giles, L. Wunderle, K. Bhalla, S. O’Brien, B.Wassmann, C. Tanaka, P. Manley, P. Rae, W. Mietlowski, K.Bochinski, A. Hochhaus, J. Griffin, D. Hoelzer, M. Albitar, M. Dugan,J. Cortes, L. Alland, O. Ottmann, Nilotinib in imatinib-resistant CMLand Philadelphia chromosome-positive ALL, New Engl. J. Med. 354(2006) 2542–2551.

[27] M. Puttini, A. Coluccia, F. Boschelli, L. Cleris, E. Marchesi, A. Donella-Deana, S. Ahmed, S. Redaelli, R. Piazza, V. Magistroni, F. Andreoni, L.Scapozza, F. Formelli, C. Gambacorti-Passerini, In vitro and in vivoactivity of SKI-606, a novel Src–Abl inhibitor, against imatinib-resistant Bcr–Abl+ neoplastic cells, Cancer Res. 66 (2006) 11314–11322.

[28] T. O’Hare, D. Walters, E. Stoffregen, T. Jia, P. Manley, J. Mestan, S.Cowan-Jacob, F. Lee, M. Heinrich, M. Deininger, B. Druker, In vitroactivity of Bcr–Abl inhibitors AMN107 and BMS-354825 against

clinically relevant imatinib-resistant Abl kinase domain mutants,Cancer Res. 65 (2005) 4500–4505.

[29] A. Quintas-Cardama, H. Kantarjian, D. Jones, C. Nicaise, S. O’Brien, F.Giles, M. Talpaz, J. Cortes, Dasatinib (BMS-354825) is active inPhiladelphia chromosome-positive chronic myelogenous leukemiaafter imatinib and nilotinib (AMN107) therapy failure, Blood 109(2007) 497–499.

[30] N. von Bubnoff, F. Schneller, C. Peschel, J. Duyster, BCR–ABL genemutations in relation to clinical resistance of Philadelphia-chromosome-positive leukaemia to STI571: a prospective study,Lancet 359 (2002) 487–491.

[31] P. Ramirez, J. DiPersio, Therapy options in imatinib failures, TheOncologist 13 (2008) 424–434.

[32] Y. Hu, D. Rosen, Y. Zhou, L. Feng, G. Yang, J. Liu, P. Huang,Mitochondrial manganese-superoxide dismutase expression inovarian cancer: role in cell proliferation and response to oxidativestress, J. Biol. Chem. 280 (2005) 39485–39492.

[33] D. Radisky, D. Levy, L. Littlepage, H. Liu, C. Nelson, J. Fata, D. Leake, E.Godden, D. Albertson, M. Nieto, Z. Werb, M. Bissell, Rac1b andreactive oxygen species mediate MMP-3-induced EMT and genomicinstability, Nature 436 (2005) 123–127.

[34] K. Irani, Y. Xia, J. Zweier, S. Sollott, C. Der, E. Fearon, M. Sundaresan, T.Finkel, P. Goldschmidt-Clermont, Mitogenic signaling mediated byoxidants in Ras-transformed fibroblasts, Science 275 (1997) 1649–1652.

[35] M. Sattler, S. Verma, G. Shrikhande, C. Byrne, Y. Pride, T. Winkler, E.Greenfield, R. Salgia, J. Griffin, The BCR/ABL tyrosine kinase inducesproduction of reactive oxygen species in hematopoietic cells, J. Biol.Chem. 275 (2000) 24273–24278.

[36] O. Vafa, M. Wade, S. Kern, M. Beeche, T. Pandita, G. Hampton, G.Wahl, c-Myc can induce DNA damage, increase reactive oxygenspecies, and mitigate p53 function: a mechanism for oncogene-induced genetic instability, Mol. Cell. 9 (2002) 1031–1044.

[37] H. Pelicano, D. Carney, P. Huang, ROS stress in cancer cells andtherapeutic implications, Drug Resist. Update 7 (2004) 97–110.

[38] H. Pelicano, L. Feng, Y. Zhou, J. Carew, E. Hileman, W. Plunkett, M.Keating, P. Huang, Inhibition of mitochondrial respiration: a novelstrategy to enhance drug-induced apoptosis in human leukemiacells by a reactive oxygen species-mediated mechanism, J. Biol.Chem. 278 (2003) 37832–37839.

[39] M. Koptyra, R. Falinski, M. Nowicki, T. Stoklosa, I. Majsterek, M.Nieborowska-Skorska, J. Blasiak, T. Skorski, BCR/ABL kinase inducesself-mutagenesis via reactive oxygen species to encode imatinibresistance, Blood 108 (2006) 319–327.

[40] D. Trachootham, Y. Zhou, H. Zhang, Y. Demizu, Z. Chen, H. Pelicano, P.Chiao, G. Achanta, R. Arlinghaus, J. Liu, P. Huang, Selective killing ofoncogenically transformed cells through a ROS-mediatedmechanism by beta-phenylethyl isothiocyanate, Cancer Cells 10(2006) 241–252.

[41] C. Su, J. Lin, T. Li, J. Chung, J. Yang, S. Ip, W. Lin, G. Chen, Curcumin-induced apoptosis of human colon cancer colo 205 cells through theproduction of ROS, Ca2+ and the activation of caspase-3, AnticancerRes. 26 (2006) 4379–4389.

[42] R. Ciarcia, D. D’Angelo, C. Pacilio, D. Pagnini, M. Galdiero, F. Fiorito, S.Damiano, E. Mattioli, C. Lucchetti, S. Florio, A. Giordano,Dysregulated calcium homeostasis and oxidative stress in chronicMyeloid leukemia (CML) cells, J. Cell. Physiol. 224 (2010) 443–453.

[43] S. Hong, T. Dawson, V. Dawson, Nuclear and mitochondrialconversations in cell death: PARP-1 and AIF signaling, TrendsPharmacol. Sci. 25 (2004) 259–264.


Carboxyamidotriazole inhibits cell growth of imatinib-resistant chronic myeloid leukaemia cells...

Documents

Transcript of Carboxyamidotriazole inhibits cell growth of imatinib-resistant chronic myeloid leukaemia cells...