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Mutation Research 709–710 (2011) 60–66

Contents lists available at ScienceDirect

Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis

journa l homepage: www.e lsev ier .com/ locate /molmutCommuni ty address : www.e lsev ier .com/ locate /mutres

karugamycin induces DNA damage, intracellular calcium increase, p38 MAPinase activation and apoptosis in HL-60 human promyelocytic leukemia cells

uxandra Popescua, Elke Hannelore Heissa, Franziska Ferkb, Andrea Peschelc,iegfried Knasmuellerb, Verena Maria Dirscha, Georg Krupitzac, Brigitte Koppa,∗

Department of Pharmacognosy, University of Vienna, Althanstrasse 14, A-1090 Vienna, AustriaDepartment of Medicine I, Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a, AustriaInstitute of Clinical Pathology, Medical University of Vienna, Waeringer Guertel 18-20, A-1090 Vienna, Austria

r t i c l e i n f o

rticle history:eceived 30 June 2010eceived in revised form 28 February 2011ccepted 2 March 2011vailable online 8 March 2011

a b s t r a c t

Ikarugamycin (IKA) is an antibiotic with strong antiprotozoal and cytotoxic activity. The purpose of ourwork was to provide insight into the mechanism of action characterizing the cytotoxic effect of IKAin HL-60 leukemia cells in order to evaluate its potential as an antineoplastic agent. Cell viability wasreduced in response to IKA (IC50 of 221.3 nM), while the amount of HL-60 cells with a subdiploid DNAcontent increased significantly after 24 h. Apoptotic cell death was confirmed by the cleavage of caspase-

eywords:karugamycinL-60poptosisenotoxicityalcium

9, -8 and -3 using immunoblotting. Single cell gel electrophoresis pointed to an early genotoxic effect.Monitoring of intracellular calcium ([Ca2+]i) levels by flow cytometric analysis of Fluo-3-AM fluorescenceindicated an increase in cytosolic calcium that correlated with the cleavage of caspases. In addition, IKAtriggered the activation of p38 MAP kinase which was partly dependent on elevated [Ca2+]i concentrationsand contributed to caspase activation. The data demonstrate that IKA induced apoptosis in HL-60 cellsthrough genotoxicity and caspase activation which was in part correlated to an increase in intracellular

tion o

38 calcium levels and activa

. Introduction

Ikarugamycin (IKA) is a pentacyclic tetramic acid first isolatedrom Streptomyces phaeochromogenes subsp. ikaruganensis as anntibiotic with strong antiprotozoal activity [1,2]. IKA belongs tohe class of macrocyclic tetramic acids, which includes discoder-

ide, isolated from the Carribean deep-sea sponge Discodermiaissolute, alteramide A from the marine bacterium Altermonasp., and cylindramide, extracted from the marine sponge Hali-hondria cylindrata [3–5]. All macrocyclic tetramic acids exhibitignificant cytotoxicity on different cancer cell lines, which raisesnterest in their potential as cancer therapeutics. IKA is reportedo elicit cytotoxic effects in MCF-7 mamma carcinoma, HMO2 gas-ric adenocarcinoma, Hep G2 hepatocellular carcinoma and Huhhepatoma cells [6]. However, the data concerning the biological

ode of action of these compounds is limited to a recent study by

ramer et al. showing calcium-dependent cytotoxicity caused byylindramide [7]. Hence, the purpose of the present study was tonvestigate the mechanism of action underlying the cytotoxic effect

∗ Corresponding author at: Department of Pharmacognosy, University of Vienna,lthanstrasse 14, A-1090 Vienna, Austria. Tel.: +43 1 4277 55255;

ax: +43 1 4277 55256.E-mail address: brigitte.kopp@univie.ac.at (B. Kopp).

027-5107/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.mrfmmm.2011.03.001

f p38 MAP kinase.© 2011 Elsevier B.V. All rights reserved.

of IKA and its potential as an antineoplastic agent. IKA inhibitedcell proliferation in HL-60 cells through genotoxicity and inducedapoptosis and activation of caspases which was partially correlatedwith a rise in intracellular calcium levels and activation of p38 MAPkinase.

2. Materials and methods

2.1. Chemicals, reagents and antibodies

IKA was purchased from Biomol International, LP, (Plymouth Meeting, PA).BAPT-AM and MDL28170 were obtained from Tocris Bioscience (Bristol, UK).SB203580 was purchased from Calbiochem (Darmstadt, Germany). DMSO wasobtained from Fluka (Buchs, Switzerland). The CompleteTM protease inhibitor waspurchased from Roche Diagnostics (Basel, Switzerland). The polyvinylidene diflu-oride membrane and the Precision Plus ProteinTM standard were obtained fromBio-Rad (Hercules, CA). Antibodies: anti-cleaved caspase-3 (Asp175), anti-cleavedcaspase-8 (Asp391), anti-cleaved caspase-9 (Asp330), anti-p38 MAP kinase, anti-phospho-p38 MAP kinase (Thr180/Thr182), anti-phospho-Chk2 (Thr68) and theanti-rabbit IgG antibody were from Cell Signalling Technology, Inc. (Danvers, MA);

anti-H2AX (pSer139) was purchased from Calbiochem (Darmstadt, Germany); anti-�-tubulin was from Santa Cruz Biotechnology (Santa Cruz, CA); the anti-mouseIgG was obtained from Upstate (Billerica, MA). Propidium iodide, luminol and iso-proterenol hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO). Allother reagents were from Carl Roth (Karlsruhe, Germany) unless stated other-wise.

Research 709–710 (2011) 60–66 61

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Fig. 1. Effect of IKA on HL-60 cell viability and cell cycle distribution. (A) HL-60 cellswere seeded in 96-well plates, cultured to logarithmic growth phase for 24 h andthen treated with solvent (0.5% DMSO) or increasing concentrations (0.04–2 �M)of IKA for 24 h. The percentage of viable cells was determined by the WST-1 col-orimetric assay. (B) Cells were seeded in 24-well plates, grown for 24 h, and thenincubated with 400 nM IKA or solvent (0.1% DMSO) for 8 h and 24 h, harvested,fixed in 70% ethanol and stained with propidium iodide. The percentage of cellsin G0-G1, S and G2-M phase was quantified by FACS analysis. (C) After exposure to

R. Popescu et al. / Mutation

.2. Cell culture

Human promyelocytic leukemia HL-60, breast cancer MCF-7, and breast epithe-ial MCF-10A cells were obtained from the American Type Culture Collection (ATCC).eripheral blood mononuclear PBMC cells were isolated from human peripherallood. HL-60 and PBMC cells were maintained in RPMI 1640 medium and MCF-cells in DMEM medium, supplemented with 10% heat inactivated fetal bovine

erum, 1% l-glutamine and 1% penicillin/streptomycin. MCF-10A cells were culturedn MEGM SingleQuots medium with 100 ng/ml isoproterenol hydrochloride. Cells

ere incubated at 37 ◦C in a humidified atmosphere of 5% CO2. RPMI 1640 mediumnd its supplements were purchased from Life Technologies (Carlsbad, CA). MEGMingleQuots medium was from Lonza (Walkersville, MD, USA).

.3. Cell viability

Cells were seeded in 96-well plates (0.05 × 106 cells/well), grown for 24 h andhen incubated with IKA or solvent for 24 h. Cell viability was assessed by the WST-1olorimetric method, according to the manufacturer’s instructions (Roche Appliedcience, Mannheim, Germany). The absorbance was measured at 450 nm with aecan spectrophotometer GENios ProTM.

.4. Propidium iodide (PI) staining and flow cytometric cell cycle analysis

Cells were cultured in 24-well plates (0.5 × 106 cells/ml) and grown for 24 h.fter treatment with IKA, cells were harvested, centrifuged for 5 min at 800 rpm at◦C, washed with ice-cold PBS and fixed in cold 70% ethanol for at least 30 min at◦C. Then, samples were washed with ice-cold PBS and incubated with 50 �g/ml PIver night at 4 ◦C and analysed by flow cytometry (FACSCaliburTM, BD Biosciences,an Jose, CA).

.5. Measurement of [Ca]i levels

Cells were grown in 24-well plates (0.5 × 106 cells/ml) for 24 h and then incu-ated with IKA or solvent for the indicated periods of time. Cells were harvested,entrifuged for 5 min at 800 rpm at 4 ◦C and incubated with 2 �M Fluo-3-AM (Molec-lar Probes Inc., Eugene, OR) intracellular calcium indicator for 45 min at 37 ◦C. Thenamples were centrifuged for 5 min at 800 rpm at 4 ◦C, washed with and resuspendedn PBS and analysed by flow cytometry (FACSCaliburTM, BD Biosciences, San Jose, CA).

.6. SDS gel electrophoresis and Western blot analysis

HL-60 cells were seeded in 24-well plates (0.5 × 106 cells/ml) and grown for4 h. After treatment cells were harvested, centrifuged for 5 min at 2,000 rpm at◦C, washed with ice-cold PBS and incubated with lysis buffer (50 mM HEPES, 50 mMaCl, 50 mM NaF, 10 mM Na4P2O7·10H2O, 1 mM Na3VO4, 5 mM EDTA, 1 mM phenyl-ethylsulphonyl fluoride, CompleteTM, and 1% Triton X-100, pH 7.5) for 10 min at◦C. Then the samples were centrifuged for 15 min at 13,000 rpm at 4 ◦C. Proteinoncentration in the supernatant was assessed by the Bradford method. The lysatesere stored at −80 ◦C. Prior to use, supernatants were mixed with 3× SDS sample

uffer and heated to 95 ◦C for 5 min. Customary gel electrophoresis and blottingrotocols were employed. Primary antibodies were diluted 1:500 and secondaryntibodies 1:1000 in TBS-T. Blots were analysed using an enhanced chemolumines-ence technique, a CCD camera (LAS-3000TM, Fujifilm, Tokyo, Japan), and AIDATM

mage analyser 4.06 software (raytest, GmbH, Straubenhardt, Germany).

.7. Single cell gel electrophoresis (SCGE)/comet assay

The assay was performed as previously described by Tice et al. [8]. Cells werereated with solvent (0.5% DMSO) or 400 nM IKA for 2, 4 and 8 h. Then cells wereentrifuged at 400 × g, for 5 min at 23 ◦C, and the pellet resuspended in 200 �l PBS.ytotoxicity was measured utilizing tryptan blue exclusion assay [9] and only cul-ures with survival rates ≥80% were analysed for comet formation. 0.05 × 106 cellsere mixed with 80 �l low melting agarose (0.5%, Gibco, Paisley, Scotland) and

ransferred to agarose-coated slides. Slides were immersed in lysis solution (1% Tri-on X, 10% DMSO, 2.5 M NaCl, 10 mM Tris, 100 mM Na2EDTA, pH 10) at 4 ◦C for 1 hnd then samples were subjected to unwinding and electrophoresis (300 mA, 25 V,0 min) under alkaline solutions (pH > 13) for the determination of single and dou-le strand breaks, DNA–protein crosslinks, and apurinic sites. The DNA was stainedith 40 �l ethidium bromide (20 �g/ml, Sigma–Aldrich, Munich, Germany) and

he percentage of DNA in tail was analysed using Comet IV image analysis systemPerceptive Instruments Ltd., Haverhill, UK). Three slides were prepared for eachxperimental point and 50 cells were scored per slide.

.8. Statistical methods

For the statistical analysis and IC50 calculation GraphPad PrismTM software ver-ion 4.03 (Graphpad Software Inc., La Jolla, CA) was used. For the comparison ofwo groups Student’s t test was utilized and P values <0.05 were considered signifi-ant (*). As far as applicable, graph figures represent mean ± S.E.M. of at least threendependent experiments.

100 nM, 200 nM and 400 nM IKA for 24 h, the percentage of cells in the Sub-G1 peak,indicative of fragmented DNA, was determined by flow cytometry. Data representmean ± S.E.M. from four independent experiments performed in triplicate. *P < 0.05,**P < 0.01, ***P < 0.001 as compared to control cells.

3. Results

3.1. Effect of IKA on HL-60 cell viability and cell cycle distribution

In order to investigate the potential toxicity of IKA, HL-60 humanpromyelocytic leukemia cells were exposed to increasing concen-trations (0.04–2 �M) of IKA for 24 h. IKA led to a dose-dependentreduction of cell viability, with an IC50 of 221.3 nM (Fig. 1A).

For the comparison of the effect of IKA in tumour and normalcells, MCF-7 breast cancer, PBMC peripheral blood mononuclearand MCF-10A non-tumorigenic breast epithelial cells were alsosubjected to dose-response analysis of cell viability and prolifer-ation. The results indicated IC50 values of 712 nM, 196.8 nM and

62 R. Popescu et al. / Mutation Research 709–710 (2011) 60–66

Fig. 2. Activation of the caspase cascade upon treatment with 400 nM IKA. (A) HL-60cells were cultured for 24 h before drug treatment in 24-well plates. Cells were incu-bated with 400 nM IKA or solvent (0.1% DMSO) for the specified periods of time. CelllDra

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Fig. 3. Evaluation of DNA damage in IKA treated cells. (A) HL-60 cells were handledand treated as described in Fig. 2. Cell lysates were analysed by immunoblotting forthe levels of phosphorylated H2AX (�-H2AX) and phosphorylated Chk2 (p-Chk2).

ysates were then subjected to Western blot analyses for the indicated proteins. (B)ensitometric values of cleaved caspase-9, -8 and -3 as fold of vehicle control. Data

epresent mean ± S.E.M. from three independent experiments. *P < 0.05, **P < 0.01s compared to control samples.

.15 �M respectively, thus, implying a general cytotoxic effect ofKA (Supplementary information).

The influence of IKA on cell cycle distribution was determinedy flow cytometric analysis of PI-stained nuclei of HL-60 cells incu-ated with 400 nM IKA for 8 h and 24 h. The results show a decrease

n the proportion of cells in G0-G1 phase after 8 h of treatment,ccompanied by a slight increase in the percentage of cells in S and2-M phase (Fig. 1B). After exposure to 400 nM IKA for 24 h, the per-entage of cells in G0-G1 and S phase markedly decreased, while theumber of cells in G2-M phase did not significantly change whenompared to vehicle-treated control cells. However, treatment ofL-60 cells with 100–400 nM IKA for 24 h resulted in a significantose-dependent increase in the number of cells in Sub-G1 phase

ndicating DNA fragmentation, a hallmark of apoptosis (Fig. 1C).

.2. IKA induces apoptosis in HL-60 cells

To confirm that the increase in the Sub-G1 population of HL-0 cells was associated with apoptotic cell death, the effect of IKAn the cleavage of caspases was determined by Western blot anal-sis. Treatment with 400 nM IKA for 0.5, 2, 4, 6 and 8 h showed aime-dependent activation of the initiator caspase-9 and -8 and theffector caspase-3 with a first obvious effect after 4 h (Fig. 2A and). In addition, staining with Annexin V-FITC and PI coupled withuorescence microscopy showed that IKA induced the externaliza-ion of phosphatidylserine after 6 h, which is another hallmark ofpoptosis (Supplementary information).

.3. IKA causes DNA damage in HL-60 cells

In response to severe genotoxic agents cells can trigger theirelf-elimination and undergo apoptosis. In order to assess the asso-iation of IKA-induced apoptosis with DNA damage response wenalysed the levels of phosphorylated histone H2AX (�-H2AX), a

ost-translational modification of this histone that is specifically

ncreased upon DNA double strand breaks, as well as the expres-ion of phosphorylated kinase Chk2 that plays a role in DNA damageesponse. In addition, single cell gel electrophoresis (comet assay)or the determination of DNA strand breaks was employed. Comet

(B) Densitometric values of �-H2AX and p-Chk2 as fold of vehicle control. (C) Cometassay. The genotoxicity of IKA was investigated using solvent-treated cells as nega-tive control and H2O2 as positive control. Data represent mean ± S.E.M. from threeindependent experiments. **P < 0.01 as compared to control samples.

assay analysis detected DNA damage 2 h after treatment of cellswith 400 nM IKA, while immunoblotting showed the activation ofChk2 and H2AX after 6 h and 8 h, respectively (Fig. 3A–C). AlthoughDNA damage was seen as early as 2 h following IKA treatment(comet assay) no change in H2AX phosphorylation indicating DNAdouble strand breaks was observed. This suggests that alkalinecomet assay traced single strand breaks as primary event and latera DNA repair response when Chk2 was activated.

3.4. IKA leads to elevated levels of the intracellular calcium

IKA is an inhibitor of clathrin-dependent endocytosis [10,11].Since calcium is one of the factors that regulates endocytosis andalso apoptosis [12–17], we were interested whether IKA couldinfluence intracellular calcium ([Ca2+]i) levels. Elevated [Ca2+]i con-centrations are known to have an important role in apoptoticsignalling and cell death. The fast activation of caspases by IKAsuggested a possible involvement of rapid calcium signalling in

this process. Hence, we investigated whether IKA influenced thecytosolic calcium levels using flow cytometry and the intracellularcalcium indicator Fluo-3-AM. Exposure of HL-60 cells to 400 nMIKA for 0.5, 1 and 3 h showed a time-dependent increase in [Ca2+]i(Fig. 4A), which was quenched by the intracellular calcium chelator

R. Popescu et al. / Mutation Research 709–710 (2011) 60–66 63

Fig. 4. Effect of IKA on the intracellular Ca2+ release in HL-60 cells. (A) Cells weregrown for 24 h before drug treatment. Cells were incubated with solvent (0.1%DMSO) or with 400 nM IKA for the indicated periods of time. Intracellular Ca2+ levelswere quantified by Fluo-3-AM staining and flow cytometry. (B) Cells were pretreatedwith 500 nM intracellular calcium chelator BAPT-AM for 45 min prior to 400 nM IKAe 2+

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Fig. 5. Activation of p38 MAP kinase by IKA treatment and role of BAPT-AM andMDL28170 in IKA-induced phosphorylation of p38 MAP kinase and cleavage ofcaspase-9 and -8. (A) After 24 h of cultivation, cells were incubated with solvent(0.1% DMSO) or 400 nM IKA for the indicated periods of time. Protein extracts wereprepared and analysed for the levels of p38 and phospho-p38 MAP kinase by West-ern blotting. (B) Densitometric quantification of Western blot bands. (C) HL-60 cellswere seeded and grown in 24-well plates for 24 h prior to drug treatment. Cellswere preincubated with solvent (0.1% DMSO) or 50 nM calcium chelator BAPT-AM and 10 �M calpain inhibitor MDL28170 before exposure to 400 nM IKA for 6 h.Then, the total lysates were analysed for the cleaved form of caspase-9 and -8 andp38 MAP kinase activation by Western blotting. (D) Densitometric values of p38,phospho-p38 MAP kinase and cleaved caspase-9 and -8 as fold of control. Values aremean ± S.E.M. of three independent experiments. *P < 0.05, **P < 0.01 as compared

xposure. After 3 h the cytosolic Ca was determined by flow cytometry. The valuesre mean ± S.E.M. of four independent experiments performed in triplicate. *P < 0.05,*P < 0.01 as compared to control cells (A) and IKA-only treated cells, respectivelyB).

APT-AM (Fig. 4B). Thus, treatment with IKA results in activationf intracellular calcium signalling.

.5. Effect of IKA on the phosphorylation of p38 MAP kinase andole of calcium in IKA-induced p38 MAP kinase activation andaspase-cleavage

Elevation of [Ca2+]i levels has been shown to lead to the acti-ation of p38 MAP kinase [18–20], one of the effectors of the MAPinase stress-activated signalling pathway. Since activation of p38AP kinase has furthermore been linked to induction of apoptosis

21–24], we were prompted to test the effect of IKA on the phos-horylation of p38 MAP kinase. After treatment with 400 nM IKAor 0.5, 2, 4, 6 and 8 h, Western blot analysis revealed that the phos-horylation of p38 MAP kinase at Thr180/Thr182 occurred as earlys 2 h and increased gradually until 8 h after exposure to IKA (Fig. 5And B). Thus, IKA is a strong inducer of the p38 MAP kinase stress-ctivated signalling pathway, whereas ERK1/2 remained unaffectednd JNK was induced only weakly (Supplementary information).

To prove whether increased calcium levels are causally con-ected to p38 MAP kinase activation and apoptosis in IKA-treatedL-60 cells, we made use of the calcium chelator BAPT-AM and

he calpain inhibitor MDL28170. Calpains are calcium-dependentroteases with the potential to initiate cell death [25–28]. West-

rn blot analysis showed that IKA (400 nM)-triggered activation of38 MAP kinase and caspases-9 and -8 was significantly decreasedpon chelation of calcium with BAPT-AM and inhibition of calpainith MDL28170 (Fig. 5C and D). Thus, the observed rise in intra-

ellular calcium and subsequent activation of calpain upon IKA

to control samples.

64 R. Popescu et al. / Mutation Resea

Fig. 6. Effect of SB203580 on the IKA-induced caspase activation. (A) Cells weregrown for 24 h and then preincubated with solvent (0.1% DMSO) or 5 �M p38 MAPkinase inhibitor SB203580 for 45 min prior to administration of 400 nM IKA. After 6 hthe cells were harvested, lysed and the cellular proteins were analysed by Western

rch 709–710 (2011) 60–66

treatment contribute in part to the activation of p38 MAP kinaseand the cleavage of caspases-9 and -8.

3.6. Role of p38 MAP kinase in IKA-induced caspase activation

In order to determine whether the observed p38 MAP kinaseactivation was causally involved in IKA-induced apoptosis and nota mere coincidental event due to elevated calcium, we employedthe p38-selective inhibitor SB203580 and investigated its effect onIKA-induced cleavage of caspase-9, -8 and -3. Western blot analy-sis showed a marked decrease in the levels of activated p38 MAPkinase (showing functionality of the used inhibitor) and caspases-8 and -3 as well as a moderate reduction of activated caspase-9when cells were incubated with SB203580 prior to the exposureof 400 nM IKA for 6 h (Fig. 6A–E). Therefore, IKA-triggered caspaseactivation is, at least partly, mediated by p38 MAP kinase. Notably,neither cotreatment with SB203580 nor BAPT-AM improved cellviability 24 h upon IKA treatment (data not shown). We thereforeassume that the genotoxic effect of IKA is dominant and indepen-dent of [Ca2+]i increase, and represents the major trigger for celldeath. Moreover, IKA may also be able to trigger, next to the justdescribed apoptosis, a form of cell death that is independent ofcaspase-activation.

Hence, we could show that IKA activates caspase-3 via genotox-icity and calcium-dependent activation of calpain, p38 MAP kinaseand finally caspase-9 and -8. A proposed scheme of the mechanismof action of IKA on HL-60 cells is shown in Fig. 7.

4. Discussion

In this study we showed that nanomolar concentrations of IKA(IC50 of 221.3 nM) decreased the viability of p53-null HL-60 humanpromyelocytic leukemia cells. As Bertasso et al. reported before [6],IKA was also active against p53-positive MCF-7 breast cancer cellssuggesting a cell-line- and p53-independent pro-apoptotic effect.However, the substance failed to show a selective antineoplasticeffect and indicated similar cytotoxicity in PBMC peripheral bloodmononuclear and MCF-10A non-tumorigenic breast epithelial cells.IKA led to only small changes in the S- and G2-M phase after 8 hand to an accumulation of HL-60 cells with a subdiploid DNA con-tent after 24 h, thus, implying that IKA may induce HL-60 cell deathwithout an apparent preceding and specific cell cycle arrest. How-ever, without extensive cell analyses over time, it cannot be ruledout that inhibition of DNA replication (as seen for most genotoxicagents in appropriate doses) occurs prior to the observed inductionof apoptosis.

Checkpoint kinase Chk2 plays a role in the DNA damageresponse of the cell. Chk2 is inactive in normal cells and becomesphosphorylated, mainly by the ATM kinase, in response to DNAdouble-stranded breaks and related lesions [29,30]. DNA damagecan generate modifications in the structure of chromatin, of whichone of the best-characterized is the phosphorylation of histoneH2AX (�-H2AX). This post-translational modification is essential

for the protection of genome integrity as it prevents cells to re-enterthe cell cycle and provides time for DNA repair [31,32]. The geno-toxic effect, either direct or through intermediates, was confirmedby comet assay which identified DNA stranded breaks 2 h aftertreatment with IKA. This was followed by Chk2 and caspase-9 and

blotting using antibodies against p38 and phospho-p38 MAP kinase and cleavedcaspase-9, -8 and -3. One representative blot of at least three individual experimentsis shown. (B) Densitometric values of p38 and phospho-p38 MAP kinase (C–E) andcleaved caspase-9, -8 and -3 as fold of control. Values are mean ± S.E.M. of at leastthree experiments. *P < 0.05 as compared to IKA-only treated cells.

R. Popescu et al. / Mutation Resea

Fig. 7. Schematic representation of the proposed mechanism of action of IKA onHL-60 cells. IKA induced DNA damage and intracellular calcium ([Ca2+]i) increasefkIi

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ollowed by p38 MAP kinase and caspase activation leading to apoptosis; p38 MAPinase was phosphorylated downstream of calpain activation (continuous line).n addition to apoptosis, IKA may be able to trigger a form of cell death that isndependent of caspase-activation (dotted line).

3 activation and cell death. It is likely that DNA double-strandedreaks are a trigger for these events, which were detected by �-2AX Western blotting. The increase in the level of �-H2AX andhk2 did not follow exactly the same time course which might bexplained by the different sensitivity of the assays.

In addition, IKA induced caspase activation through an increasef intracellular calcium levels which could be diminished byntracellular calcium chelation. Multiple reports have identifiedntracellular calcium overload or redistribution as trigger for apo-tosis via the endoplasmic reticulum, mitochondria or throughifferent Ca2+-mediated effector mechanisms involving enzymesuch as calcineurin, endonucleases, phospholipases and calpain33–38]. [Ca2+]i release initiates downstream processes that canesult in caspase activation and apoptosis [39–41]. Interestingly,KA is an inhibitor of endocytosis which is co-regulated by cal-ium [12–17] that underlines the role of calcium for modulationf cellular signalling by IKA. How exactly IKA exerts its effectn cellular calcium levels needs to be addressed by future stud-es. Cylindramide, another cytotoxic macrocyclic tetramic acid,as been shown to exert its effect through a short-term reduc-ion of free [Ca2+]i and a long-term interaction with the ER [7].he authors hypothesize that cylindramide exercises its cytotox-city through complexation of [Ca2+]i. IKA leads to the elevation ofhe levels of cytosolic Ca2+, thus indicating different mechanismsnderlying the cytotoxicity triggered by macrocyclic tetramic

cids.

Employing MDL28170, we could furthermore reveal an involve-ent of calpain as Ca2+-dependent mediator of IKA-induced

aspase activation. Calpains are proteases that translate calcium-nto pro-apoptotic signals [25–28]. The calpain inhibitor MDL28170

rch 709–710 (2011) 60–66 65

decreased the levels of IKA-triggered caspase-9 and -8 activation,consistent with our proposed Ca2+/calpain/caspase axis of caspase-3 induced by IKA.

MAP kinases are involved in several important intracellularsignalling cascades regulating cell differentiation, motility, prolif-eration and death. The p38 MAP kinase stress-activated signallingpathway is one of the three major MAP kinase pathways, togetherwith the extracellular signal-regulated kinase (ERK)-, and c-Jun-N-terminal kinase pathways (JNK) [42,43]. The role of p38 MAPkinase in apoptosis can be dualistic; it has been shown that p38can either activate or block apoptosis [44–47]. One of the factorsleading to p38 MAP kinase activation is the release of intracellu-lar calcium [18–20] and here we demonstrate that p38 MAP kinasewas involved in the calcium-mediated caspase activation inducedby IKA, while ERK was not influenced and JNK showed a lesserlevel of transient activation. The p38 inhibitor SB203580 exerted astronger attenuating effect on IKA-activated caspase -8 and -3 thanon caspase-9. This is in accordance with earlier reports evidencingstrong regulation of caspase-8 activity by p38 MAP kinase [48–52].In addition to its impact on the cleavage of caspase-9 and -8, thecalpain inhibitor MDL28170 decreased the levels of activated p38MAP kinase placing calpain activation upstream of p38 MAP kinasephosphorylation as has been previously reported by Lizama et al.[53]. The inhibition of p38 MAP kinase and chelation of calciumhowever, did not significantly improve HL-60 cell viability in thepresence of IKA indicating that the major pro-apoptotic signal wastriggered directly by the DNA damage. Furthermore, the possibilitythat IKA-induced genotoxic stress is able to trigger an additionalcaspase-independent form of cell death should raise attention infurther investigations.

In conclusion, the present study contributes to the understand-ing of the cytotoxicity of macrocyclic tetramic acid lactams byproviding an insight into the mechanism of action underlying thecytotoxic effect of IKA. IKA exhibited a general toxic effect andinduced apoptosis in HL-60 cells by DNA damage and activationof caspases-9, -8 and -3. Caspase cleavage was in part causally con-nected also to intracellular calcium increase and the activation ofp38 MAP kinase.

Conflict of interest statement

There is no conflict of interests.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.mrfmmm.2011.03.001.

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