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Experimental and Toxicologic Pathology 65 (2013) 1101– 1108

Contents lists available at ScienceDirect

Experimental and Toxicologic Pathology

jo ur nal homepa ge: www.elsev ier .de /e tp

poptosis induced by paclitaxel via Bcl-2, Bax and caspases 3 and 9 activation inB4 human leukaemia cells is not modulated by ERK inhibition

aniel Morales-Cano, Eva Calvino, Virginia Rubio, Angel Herráez, Pilar Sancho, M. Cristina Tejedor, José. Diez ∗

ioquímica y Biología Molecular, Facultad de Medicina, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares (Madrid), Spain

r t i c l e i n f o

rticle history:eceived 5 September 2012ccepted 25 April 2013

eywords:poptosisaxcl-2aspaseseukaemiainases

a b s t r a c t

We have studied the role of pivotal bio-molecules involved in signalling of cytotoxic effects induced bypaclitaxel (Ptx) on acute promyelocytic human leukaemia NB4 cells. A time-dependent increase in celldeath and DNA cleavage was observed after 30 �M Ptx treatment. Cell death induction by Ptx proceedsmainly as programmed cell death as shown by annexin V-FITC, reaching up to 30% of apoptotic cells after24 h. Significant reductions of p53, changes in Bax and Bcl-2 and activation of caspases 3 and 9 wereobserved as the treatment was applied for long times. Ptx treatments produced NFkB depletion withexpression levels abolished at 19 h what could be involved in reduction of survival signals. Phosphor-ylation of intracellular kinases showed that pERK1/2 decreased significantly at 19 h of Ptx treatment.When these cells were preincubated for 90 min with 20 �M PD98059, 2′-amino-3′-methoxyflavone, aninhibitor of ERK phosphorylation, a slight reduction of cell viability was observed in comparison to that

53 produced by Ptx alone. Pretreatment with PD98059 neither activated caspases nor significantly increasedthe apoptotic effect of Ptx. Taken together, our data reveal that the inhibition of ERK phosphorylation doesnot seem to be an essential pathway for bursting an increased induction of apoptosis by Ptx. Decrease ofp53 and Bcl-2, fragmentation of DNA, increase of Bax and, finally, activation of caspases 3 and 9 in NB4leukaemia cells make the apoptotic process induced by Ptx irreversible. Application of Ptx in leukaemia

romis

cells shows therefore a p

. Introduction

Acute promyelocytic leukaemia (APL) is a subtype of acuteyeloid leukaemia (AML) producing a growth of immature cells

alled promyelocytes and their accumulation in peripheral bloodnd bone marrow, causing the displacement of normal blood cells.t is caracterized by a specific translocation between chromosomes5 and 17 (q22; q11-12) involving genes that code for PML andetinoic acid receptor � (RAR�) resulting in the generation of aML–RAR� fusion protein (Rowley et al., 1977; Roussel and Lanotte,001; Melnick and Licht, 1999). NB4 human leukaemia cell line is

unique model because it expresses different levels of some enzy-atic markers and different unpaired differentiation behaviour as

ompared to other APL-derived cell lines, such as HL-60 (Drexlert al., 1995). In addition, this NB4 cell line shows resistance toeveral chemotherapeutic drugs (Lanotte et al., 1991; Maksumova

t al., 2000). This makes this line a useful model to study the induc-ion of cell death in leukaemia cells by antitumour agents eitherlone or in combination.

∗ Corresponding author. Tel.: +34 91 8854582; fax: +34 91 8854585.E-mail address: josecarlos.diez@uah.es (J.C. Diez).

940-2993/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.etp.2013.04.006

ing potential with particular effects on different leukaemia cell types.© 2013 Elsevier GmbH. All rights reserved.

Efficiency of the antitumour action of a drug may be dependenton the mechanism of the cell death induction that could elim-inate specifically malignant cells. Leukaemia cells are sensitiveto camptothecin analogues (Song et al., 2005), arsenic trioxide(Ramos et al., 2005), retinoid molecules (Cincinelli et al., 2005)or prooxidant agents (Sen et al., 2005) that induce cell death byapoptosis. However, a rapid resistance to these compounds aftertreatment has also been reported in some patients (Lo-Coco et al.,2008).

Paclitaxel (Ptx) is a chemotherapeutic drug, a member of thetaxanes family. These compounds show antitumour activity againstseveral haematological diseases in different cell lines (Liao and Lieu,2005; Wan et al., 2004) and solid tumours such as ovary, breastand lung (Wang et al., 2000), Ptx acts by promoting microtubuleassembly (Pellegrini and Budman, 2005; Bergstralh and Ting, 2006)and interferes in several signal transduction pathways inducingsubsequent apoptosis (Ganansia-Leymarie et al., 2003; Xu et al.,2009a,b).

In several cell lines such as MCF7 or U937 the signal trans-

duction MEK/ERK pathway is involved in the cytotoxic action ofPtx (Xu et al., 2009a,b; Bacus et al., 2001; Yu et al., 2001). TheRas/Raf/MEK/ERK (MAPK) signalling pathway plays a central roleregulating activity of many proteins involved in cell progression,

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roliferation, migration, differentiation and cell death (McCubreyt al., 2007). Activation occurs by tandem phosphorylation on thre-nine and tyrosine residues. Upon activation, ERK dimerizes ands translocated to the nucleus (Yu et al., 2001; Lee and McCubrey,002). Once in the nucleus ERK phosphorylates and activates a vari-ty of transcription factors and proteins involved in cell growth bytimulating protein and nucleotide biosynthesis.

Since the ERK pathway is constitutively activated in severaluman leukaemias (Steelman et al., 2008) it is possible to actn this pathway for disabling it arresting cell growth and lead-ng to apoptosis. Concomitant with the activation of the apoptoticathway, the survival pathway can also be activated. Althoughhe ERK pathway is attributed to survival for many cell typesZhuang and Schnellmann, 2006; Zhang et al., 2012), there are suf-cient in vitro studies that support a significant role for ERK inediating apoptosis. Apoptosis culminates in the overexpression

f proapoptotic factors such as Bax, modulation of antiapoptoticroteins such as Bcl-2 and in the eventual activation of cas-ases 3, 8 and 9 among other cysteine proteases (Wang et al.,000).

Due to the implication of ERK in cell survival and to the facthat in many tumours a hyperactivation of this pathway has beeneported, several inhibitors have been developed (Stadheim et al.,001). Among them, PD98059 is a noncompetitive inhibitor that ispecific for Mek1 and 2 signalling pathway and inhibits the acti-ation of p44 (ERK1) and p42 (ERK2) (Santos-Silva et al., 2006;hrysomali et al., 2003). Some authors have claimed for a therapeu-ic efficacy of combinations of signal transduction inhibitors andhemotherapy (Abrams et al., 2010). Thus, the use of Ptx togetherith inhibitors of signal transduction kinases could be interesting

or novel antitumor therapies in leukaemia cells (Xu et al., 2009a,b,012; Shimizu and Tsujimoto, 2000).

On the basis of the correlation between of ERK signalling path-ay and cell survival and proliferation, we have studied the effects

n induction of apoptosis of Ptx combined with PD98059 inhibitorf ERK activation.

The results of this study indicate that pretreatment withD98059 plus treatment with Ptx leads to a slight reduction ofiability of NB4 cells, near disappearance of p-ERK, increase ofax/Bcl-2 ratio but no significant increases in p53 expression or inctivation of caspase-3 and caspase-9. These results might supporthat inhibition of prosurvival signalling pathway does not produce

significant sensitization of APL cells to apoptosis promoted by Ptxn acute promyelocytic disorders.

In this work, we have studied the cytotoxic and apoptotic effectsf Ptx in a useful model of human acute promyelocytic leukaemia.he involvement of apoptosis factors and MAP-kinases is consid-red and discussed.

. Materials and methods

.1. Human acute promyelocytic leukaemia (NB4) cells

Human NB4 leukaemia cells used in the experiments wereaintained at density of 3 × 105 cell/ml in RPMI – Glutamax 1640edium (Gibco-Life Technologies), supplemented with 10% heat-

nactivated foetal bovine serum (FBS), 1% penicillin/streptomycin.ell culture was carried out in an environment of 5% CO2 at 37 ◦C.

.2. Reagents and antibodies

Ptx was obtained from Sigma–Aldrich (Steinheim, Germany)nd prepared as a 58 �M in DMSO, PD98059 (2′-amino-3′-ethoxyflavone) was obtained from Calbiochem and prepared at

7 mM in DMSO. Both reagents were stored at −20 ◦C.

cologic Pathology 65 (2013) 1101– 1108

Anti-p53 mouse monoclonal IgG (Ab-1, OP03; 100 �g/ml) waspurchased from Calbiochem (Oncogene Research Products, MA,USA) and diluted 1:50 in TTBS containing 5% powder milk.

Anti-Bax mouse monoclonal IgG2b (B-9; sc-7480; 200 �g/ml)was obtained from Santa Cruz Biotechnology, CA, USA and diluted1:100 in TTBS containing 5% powder milk.

Anti-Bcl-2 mouse monoclonal IgG1 antibody (C-2; sc7382;200 �g/ml) was purchased from Santa Cruz Biotechnology, CA, USAand used after dilution at 1:200 in TTBS with 5% powder milk.

Anti Erk1/2 rabbit polyclonal antibodies diluted 1:3000 fromSanta Cruz Biotechnology sc-154.

Anti p-Erk1/2 (Tyr204) mouse monoclonal antibodies diluted1:200 from Santa Cruz Biotechnology sc-7383.

Anti-NFkB p50 and p105 (EA0) antibodies diluted 1:200 fromSanta Cruz Biotechnology sc-8414.

Anti-NFkB p65 (F-6) antibodies diluted 1:200 from Santa CruzBiotechnology from Santa Cruz Biotechnology sc-8008.

Anti-caspase-3 (H-277) rabbit polyclonal antibodies diluted1:200 from Santa Cruz Biotechnology (sc-7148).

Anti-caspase-9 (9CSP03) mouse monoclonal antibodies diluted1:200 from Santa Cruz Biotechnology (sc-73548) were also used.

Anti human �-tubulin mouse monoclonal antibodies were pur-chased from Santa Cruz Biotechnology and diluted at 1:10,000.

2.3. Treatments

For all experiments, cells were seeded in culture medium at adensity of 5 × 105 cells/ml in 12 wells culture plates. Control cellsreceived 0.1% DMSO in culture medium. Stock solutions of PD andPtx were diluted, on the day of use, with culture to the desiredconcentration with a final DMSO concentration of 0.1%.

Cells were treated with (10, 20, 40 or 60 �M) PD98059 for 90 minin culture medium and then were washed and counted. Afterwardsthey were treated with Ptx (30 �M) in the same medium for 19 h.

2.4. Cell viability: permeability to propidium iodide and flowcytometry analyses

Cell viability was determined by permeability to propidiumiodide (PI). This method was chosen because membrane perme-ability alterations can be correlated with cytofluorescence profilesshowing living and dead cells. After treatments with chemicals,cells were collected and washed with 500 �l phosphate bufferedsaline (PBS) at 1200 rpm for 5 min. The cells were resuspended in300 �l PBS and stained with 15 �l (0.1 mg/ml) PI solution (Cal-biochem) and the fluorescence of each well was measured (upto 104 cells) using a FACScalibur flow cytometer (Becton Dick-inson, San José, CA). Data analysis was done using the programWin-MDI (Windows Multiple Document Interface for Flow Cytom-etry, version 2.8, available from Scripps Research Institute, FACSCore Facility, http://facs.scripps.edu/software.html). Cell fragmentswere discriminated from the nonviable cells using dot plots of FSCvs. fluorescence associated to PI. In experiments of cell permeabilityto PI, the number of cells was plotted versus fluorescence of PI.

2.5. Analysis of cell cycle: cytometric analysis of cell populationswith subdiploid DNA and cell cycle

Apoptotic cells were counted on the basis of DNA content afterpermeabilization of the cells with NP40. After treatments with Ptx,2.5 × 105 NB4 cells were collected and washed with 500 �l PBS.In order to extract low molecular weight DNA from cell nuclei,

the pellet was resuspended in 475 �l of a solution of PBS contain-ing 0.5 mg/ml RNase (Sigma Chemical) and 0.1% NP-40 in PBS andincubated for 30 min. The remnant DNA in cells was stained with0.05 mg/ml PI immediately before measuring the fluorescence in

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he flow cytometer. The fluorescence associated to subdiploid DNAas measured by flow cytometry using a Becton Dickinson FACScal-

bur flow cytometer and the results were analized using Win-MDI.8 software.

Cells with hypodiploid DNA (apoptotic cells) were distinguishedrom those containing diploid DNA (nonapoptotic cells) on the basisf their fluorescence intensity of PI. Cell cycle progress was studiedn the same samples. The untreated cells histograms were used toefine the positions of the peaks for the G1, S and G2/M phases ofell cycle.

.6. Annexin V-FITC assays: phosphatidylserine exposure on theuter membrane side

To quantify the level of apoptosis through the presence of phos-hatidylserine in the outer membrane side the annexin V-FITCpoptosis Detection Kit from Calbiochem was used. For this assay,

× 106 treated cells were collected at 1500 rpm for 3 min. After-ards, they were incubated with 500 �l of PBS, 10 �l of binding

eagent and 1.25 �l Anexin V-FITC for 15 min at room temperaturen the dark. Subsequently, the cells were centrifuged at 2300 rpmor 5 min and the pellet resuspended in 500 �l of binding bufferiluted in PBS. 10 �l PI were added and the samples were analyzedy flow cytometry. Total apoptosis was calculated from annexin-ositive cells.

.7. Western-blot analyis

Cells were grown at a concentration of 2 × 105/ml. Cells wereollected by centrifugation at 1200 rpm for 5 min and the pel-ets resuspended in 200 �l of lysis buffer solution containing0 mM Tris/HCl pH 8.0, 150 mM NaCl; 5 mM EDTA; 0.5% NP-40;

mM PMSF and incubated for 20 min at 4 ◦C. Cells were soni-ated (duty cycle 100%, output control 50%) for 20 s. The lysateas centrifuged (14,000 rpm, 5 min, 4 ◦C) and the supernatantas frozen and stored. 40 �g of total proteins plus 5% sample

uffer (300 �M Tris–HCl, pH 6.8, 50% glycerol, 50% sodium doce-ylsulfate (SDS) and 10% bromophenol blue) were boiled at 100 ◦Cor 5 min. Then, the lysates were loaded and separated by elec-rophoresis in 12.5% or 15% polyacrylamide gel (SDS-PAGE) withrecision Plus Protein Standards (Bio-Rad). Separated proteinsere then transferred to nitrocellulose membranes (Pure Nitro-

ellulose Membrane; 0.45 �m; Biorad) which were blocked for0 min with TBS-Tween buffer (50 mM Tris–HCl, pH 7.2, 140 mMaCl, 0.06–0.1% Tween 20), containing 5% (w/v) nonfat dry milk,ashed two times in TBT-Tween and incubated overnight at +4 ◦Cith specific primary antibodies in TBS-Tween + nonfat milk. Sub-

equently, membranes were washed four times for 10 min eachn TBS-Tween buffer and then incubated with polyclonal, HRP-onjugated, goat anti-mouse antibodies (1:2000) from PromegaMadison, WI). After incubation, membranes were washed fourimes for 10 min each and bands were visualized using chemi-uminescent detection (Amersham ECL, from GE Healthcare Lifeciences).

The intensities of the bands were corrected with respect to thentensity of a constitutively expressed protein in the same blot �-ubulin, and then quantified relative to control untreated cells. Allhe experiments were repeated three or four times.

.8. Statistical analysis

Values are expressed as the mean ± standard error of the

ean (SEM) of at least three independent experiments. The dif-

erences between the control group and treated groups wereetermined. Significance of differences between treatments wasetermined by one-way analysis of variance (ANOVA) followed

cologic Pathology 65 (2013) 1101– 1108 1103

by the Bonferroni post hoc test. *, ** and *** represent significantdifferences of p < 0.05, p < 0.01 and p < 0.001, respectively, in theANOVA–Bonferroni comparison between treated cells and controls.#, ## and ### represent significant differences of p < 0.05, p < 0.01and p < 0.001, respectively, in the ANOVA–Bonferroni comparisonbetween cells pretreated with PD98059 and treated with Ptx withrespect to only with Ptx.

3. Results

3.1. NB4 cell viability and DNA fragmentation

We have determined the effects on cell viability of Ptx at 0.5,30 and 100 �M studied by permeability to PI. We observed thatonly 20% of the cells remain viable after treatment with 100 �MPtx for 19 h (data not shown). In contrast, treatment with 30 �M Ptxproduced reductions in cell viability up to 30%. Thus, we carried outtime-course studies of the action of 30 �M Ptx on cellular survivaland DNA fragmentation of these leukaemia human cells. As it canbe observed (Fig. 1A), cell viability measured as permeability to PIis clearly reduced after 14 h of treatment and is further reducedcontinuously at longer times, reaching 55% of cell viability at 24 h.

To gain further insight into the mechanism responsible for suchdecrease in cell viability, we next examined the effect of Ptx oncell cycle through the presence of subdiploid DNA (Fig. 1B). Theappearance of a peak corresponding to subdiploid DNA, is clearlyvisible for treatment times of 14 h (28%) and longer reaching valuesup to 50% after 24 h of treatment (Fig. 1B).

3.2. Paclitaxel-induced apoptosis of NB4 cells

To further study the apoptosis induction of Ptx on NB4 acutepromyelocityc leukaemia cells, we analyzed membrane alterationsby flow cytometry assay using double staining with annexin V-FITCand PI. As it can be observed (Fig. 2), a progressive increase in apop-totic cells (upper plus lower right quadrants in Fig. 2) demonstratestime-dependent cytotoxicity.

Total apoptosis showed a significant increase in Ptx-treated cellswhen compared with sham-treated cells with values of 20% apop-totic cells at 14 h up to 30% at 24 h (Fig. 2). These results supportedthe investigation of protein expression involved in signalling apo-ptosis pathways.

3.3. Expression of Bcl-2, Bax and p53

Based on the above results, we investigated the relationshipbetween apoptosis factors expression and apoptosis induction ofPtx on NB4 cells. We studied the expression of several apoptosis-related factors. Fig. 3A shows a clear reduction in p53 after 19 htreatment with 30 �M Ptx. This reduction is dependent on the dura-tion of treatment (results not shown). p53 levels remained constantand lower (70%) than those obtained in sham-treated cells until 14 hof Ptx treatment. At longer times the decrease was more relevantreaching values of around 30% and 15% of the initial level after Ptxtreatments for 19 and 24 h, respectively.

Bax and Bcl2 levels also showed variations along time (data notshown). At 19 h of Ptx treatment, Bax levels increased (180%) sig-nificantly over the control while Bcl-2 showed a clear reduction(Fig. 3A).

3.4. NF-�B levels

The levels of NF-�B factor (p105) and its components (p65and p50) in NB4 cells dramatically disminished to 46%, 37% and30%, respectively, after 5 h with 30 �M Ptx (data not shown). Con-sequently, NF-�B p105 and p50 showed reduced levels (7% and

1104 D. Morales-Cano et al. / Experimental and Toxicologic Pathology 65 (2013) 1101– 1108

Fig. 1. Cytotoxic effects of Paclitaxel on human leukaemia NB4 cells. (A) Cell viability profiles of NB4 cells treated with 30 �M Ptx for indicated times. Viability was measuredb butionh PI stad

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y flow cytometry as impermeability to PI propidium iodide. (B) Cell cycle distriypodiploid DNA was measured by flow cytometry after cell permeabililization andifferent experiments.

0%, respectively) at 19 h (Fig. 3B). NF-�B p65 also showed aower expression (21%) after 19 h in comparison to untreated cellsFig. 3B), although lower than that suffered by NF-kB p105 and p50.

.5. Intracellular kinases levels

We also considered the influence of Ptx treatment on signallingntracellular kinases such as ERK and Akt. After treatment with0 �M Ptx, the levels of both Akt and its phosphorylated formsemained constant (data not shown). A similar behaviour was dis-layed by ERK1/2. However, p-ERK1/2 decreased (about 12%) after9 h treatment (Fig. 4).

To determine whether changes in expression of pERK1/2ependent on apoptosis induction by Ptx may be significant in thisrocess, we pretreated NB4 cells with PD98059, an inhibitor of acti-ation of ERK1/2, for 90 min followed by treatment with 30 �M Ptxor 19 h.

Treatment with PD98059 (20 �M) for 90 min produced a cleareduction of ERK phosphorylation (Fig. 4) in a much more markeday that the decrease caused by either PD or Ptx alone.

.6. Effects on activation of caspases

To evaluate the underlying mechanism of apoptosis inductiony Ptx, we assessed the effect of treatment with PD and Ptx, eitherlone or in combination, on activation of caspases 3 and 9 in NB4ells.

ig. 2. Induction of apoptosis in paclitaxel-treated human leukaemia NB4 cells. Dot plo-FITC of cells treated with 30 �M Ptx for the indicated times. Upper and lower right quxperiment of at least three different experiments.

profiles of cells treated with 30 �M Ptx for the indicated times. Subdiploid andining. The figure shows, in both cases, a representative experiment of at least three

Incubation of NB-4 cells with 30 �M Ptx for 19 h induced acti-vation of caspases 3 and 9 (Figs. 5 and 6). Caspase 3 increased itsactivity up to 250% (Fig. 5) and caspase 9 increased in a lowerproportion up to 130% (Fig. 6). Ptx produced a slight decrease ofprocaspase 3 (Fig. 5) and a more relevant decrement in procaspase9 (Figs. 5 and 6). No significant changes were observed, in contrast,in cells exposed to PD98059 alone with respect to untreated cells(Fig. 5), mainly in procaspase 3.

Pretreatment with PD98059 followed by incubation with Ptxrenders slightly lower expression levels of procaspase 9 in com-parison to treatments with Ptx alone (Fig. 6). Anyhow, treatmentwith PD previous to Ptx incubation seemed to reduce slightly theactivation of both caspases measured as the proportion of the frag-mented polypeptides although these reductions are not statisticallysignificant (Figs. 5 and 6).

3.7. Cellular viability and apoptosis induction in PD98059 plusPtx treatments

Cell viability of NB4 leukaemia cells was also studied after pre-treatment with different concentrations of PD98059 (10, 20 and60 �M) for 90 min and subsequent treatment with Ptx (30 �M) for

19 h (Fig. 7). PD98059 alone just produced slight reductions on cellviability. On the other hand, PD98059 pretreatment plus additionof Ptx produced cell viability reductions higher to that producedby Ptx alone, although they were only significant at the highest

t diagrams obtained by flow cytometry measuring fluorescence of PI and annexinadrants were used to measure apoptotic cells. The figure shows a representative

D. Morales-Cano et al. / Experimental and Toxicologic Pathology 65 (2013) 1101– 1108 1105

Fig. 3. Apoptotic factors level in human leukaemia NB4 cells treated with Pacli-taxel. Western-blot analyses of NB4 cells treated with 30 �M taxol for 19 h. NB4-cellextracts (40 �g of extract proteins) were obtained and subjected to SDS-PAGE andWestern-blot for p53, Bax and Bcl2 (A) and for NFkB p105, NFkB p65, NFkB p50 (B).TAt

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Fig. 4. Levels of intracellular kinases ERK 1/2 and p-ERK 1/2 in human leukaemiaNB4 cells. (A) Representative Western-blot of cells treated with PD98059 for 90 minand subsequently with Ptx for 19 h. Anti-human �-tubulin monoclonal antibodywas used to demonstrate uniform protein load. (B) Relative intensity expressionobtained from the corresponding Western-blots. Data are expressed as mean ± SEMof at least three different experiments. Significant differences between treated cellswith respect to control (untreated) cells (used as a negative control) are indicatedas **p < 0.01 and ***p < 0.001. Significant differences between cells pretreated with

he figure represents one experiment representative of three different experiments.nti-human �-tubulin monoclonal antibody was used to demonstrate uniform pro-

ein load.

D98059 concentration used (60 �M), which decreased cell viabil-ty down to 47% (# in Fig. 7).

To further investigate tentative apoptosis induction by the samereatments, we used double staining with annexin V-FITC and PIy flow cytometry (Fig. 8). As shown in Fig. 8, all treatments withD98059 alone for 90 min did not increase the amount of total apo-tosis cells in comparison with sham-treated cells. Ptx treatmentshowed around 30% of total apoptosis (Fig. 8), which increasedlightly when combined with PD pretreatment (see Fig. 8). Ashown in Fig. 8B, the same results can be observed in one repre-entative experiment presented in a dot plot demonstrating theiscrimination between apoptotic, necrotic and viable cells.

.8. DNA fragmentation in combined treatments with PD98059lus Ptx

To gain further insights into the mechanism responsible forhe decrease of cell viability, we next examined the effect of PDlus Ptx on cell cycle and subdiploid DNA. The apoptotic effects ofoxic compounds can be related to changes in cell cycle. Cells werexposed to 10, 20 and 60 �M PD98059 for 90 min alone or withubsequent treatment with Ptx (30 �M) for 19 h. Treatments withD98059 seem to render slight but not significant effect on the levelf subdiploid DNA (Fig. 9), while Ptx increased DNA fragmentationignificantly (Figs. 1B and 9). The combined treatment with PD andtx did not diminish that action significantly (see Fig. 9).

. Discussion

Antimitotic agents interfering with the activity of micro-ubules may produce apoptotic or necrotic cell death and

PD98059 and treated with Ptx with respect to Ptx treated cells (positive control) areindicated as ###p < 0.001.

alterations in cell signalling pathways involved in cell survivaland proliferation (Ganansia-Leymarie et al., 2003; Bacus et al.,2001).

In addition, many tumour cell lines show an activation ofMEK/ERK pathway with an overexpressed activity of the corre-sponding kinases (McCubrey et al., 2007; Lee and McCubrey, 2002;Steelman et al., 2008). This fact can explain the increased prolifer-ation rates and the resistance to cell death shown by tumour cellsin comparison with nontransformed cells.

The aim of the present work has been to approach the correla-tion of the toxicity and apoptosis induced by Ptx with MEK/ERKalterations on in a specific model of human acute promyelo-cytic leukaemia, the NB4 cell line. This cell line using a modelthat shows unique features with respect to cell progression, dif-ferentiation and resistance to antitumour drugs (Lanotte et al.,1991).

There are several kinds of leukaemia cells. Among them, Ptxhas proven to be and efficient antitumour drug on promyelo-cytic leukaemia U937 cells (Liao and Lieu, 2005), in acute myeloidleukaemia HL60 cells (Wan et al., 2004), in murine limphoblas-tic L1210 cells (Santos-Silva et al., 2001) and Jurkat T leukaemialymphocytes (Zhou et al., 2000).

Our results show a toxic action of Ptx on NB4 Cells produc-ing reduction of cell viability and increase of DNA fragmentationcompatible with an increment of cell death by apoptosis. Ptx at30 �M showed an increased time-dependent effect showing sig-nificant levels (24%) of early and late apoptosis after treatment for

19 h.

To elucidate the main factors involved in Ptx-induced apoptosis,we studied p53 expression changes in response to Ptx treatment.

1106 D. Morales-Cano et al. / Experimental and Toxicologic Pathology 65 (2013) 1101– 1108

Fig. 5. Level of caspase 3 in human leukaemia NB4. (A) Representative Westernblot of expression of caspase 3 in cells treated with 20 �M PD98050 for 90 min andsubsequently with 30 �M Ptx for 19 h. Anti-human �-tubulin monoclonal antibodywas used to demonstrate uniform protein load. (B) Percentage of relative intensityobtained from the corresponding Western-blots. Data are expressed as mean ± SEMof at least three different experiments. Significant differences between treated cellswith respect to control (untreated) cells (used as a negative control) are indicated as*p < 0.05. Non-significant differences between cells treated with PD98059 and Ptxwith respect to Ptx treated cells (positive control) were observed.

Fig. 6. Level of caspase 9 in human leukaemia NB4 cells. (A) Representative Westernblot of expression of caspases 9 in cells pretreated with 20 �M PD98059 for 90 minand subsequently with 30 �M Ptx for 19 h. Anti-human �-Tubulin monoclonal anti-body was used to demonstrate uniform protein load. (B) Percentage of relativeintensity obtained from the corresponding Western-blots. Data are expressed as themean ± SEM of at least three different experiments. Significant differences betweentreated cells with respect to control (untreated) cells (used as a negative control)are indicated as *p < 0.05. Non-significant differences between cells pretreated withPD98059 and treated with Ptx with respect to Ptx treated cells (positive control)were observed.

Fig. 7. Cell viability of NB4 cells treated with PD98059 and Paclitaxel. (A) Percent-age of cell viability in cells treated with PD98059 for 90 min and subsequently withPtx for 19 h. Cell viability was determined by flow cytometry, measuring the imper-meability to PI in non-permeabilized cells. Data are expressed as the mean ± SEMof at least three different experiments. Significant differences between treated cellswith respect to control (untreated) cells (used as a negative control) are indicatedas ***p < 0.001. Significant differences between cells treated with PD98059 and Ptxwith respect to Ptx treated cells (positive control) are indicated as ###p < 0.001.

Fig. 8. Apoptosis analyses of NB4 cells treated with different concentrations ofPD98059 and subsequently incubated with 30 �M Ptx. (A) Percentage of apoptoticcells in the upper-right and lower-right quadrants of the corresponding dot plotdiagrams. Cells were stained with FITC-conjugated annexin V-FITC and PI and ana-lyzed by flow cytometry. Data are expressed as the mean ± SEM of at least threedifferent experiments. Significant differences between treated cells with respect tocontrol (untreated) cells (used as a negative control) are indicated as **p < 0.01 and***p < 0.001. Non-significant differences between cells pretreated with PD98059 andtreated with Ptx with respect to Ptx treated cells (positive control) were also stud-ied. (B) Representative dot plot diagrams obtained with 20 �M PD98059, 30 �M Ptxor PD98059 plus Ptx.

D. Morales-Cano et al. / Experimental and Toxi

Fig. 9. Cell cycle analysis of NB4 cells treated with PD98059 and Ptx. Subdiploid DNAcontent was analyzed using flow cytometry by PI staining after cell permeabiliza-tion. Data are expressed as the mean ± SEM of at least three different experiments.Significant differences between treated cells with respect to control (untreated) cells(tw

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used as a negative control) are indicated as ***p < 0.001. Differences between cellsreated with PD98059 and Ptx with respect to Ptx treated cells (positive control)ere not significant.

tx significantly reduced p53 levels which might be correlated with possible role of this protein in the cytotoxic effect of Ptx in NB4uman leukaemia cells. This reduction of p53 seems to be related toNA fragmentation which also begins in the early hour of treatmentnd is fully effective (up to 50% of subdiploid DNA) at 24 h of Ptxreatment.

Variations in levels of apoptotic factors such as Bax and Bcl2ere observed after Ptx treatment. The increment in Bax expression

s clearly significant, what can be an indication of apoptosis via thentrinsic pathway. The reduction of Bcl-2 factor expression can leado a loss of survival signals. These results are similar to those foundn Jurkat leukaemia cells, where apoptosis is induced by lactoferrin

ithout variations in Bcl2 (Lee et al., 2009). They also match thosereviously described by Vrana et al. (2006) for myeloid leukaemiaells, where the antiapoptotic BCL2 family is rapidly upregulatedpon exposure to chemotherapeutic microtubule disrupting agentsith a transient maintenance of cell viability resulting eventually

n cell death (Vrana et al., 2006).Shortly after Ptx treatment, there was a clear reduction of

FkB factors which correlated with reduction of the expressionf p53, ERK, Bcl-2 and procaspases. Similar results were observedn lymphocytic leukaemia cells (L1210) (Santos-Silva et al., 2006,001).

Many tumour cell lines show an activation of the ERK path-ay with an overexpressed activity of the corresponding kinases

McCubrey et al., 2007; Steelman et al., 2008; Santos-Silva et al.,001; Zhou et al., 2000) what can explain the increased prolifer-tion rates and the resistance to cell death of tumour cells. Thenhibition or activation of MAP kinases in general and of ERK inarticular can respectively increase the effects of Ptx or produceesistance to this drug (Oh et al., 2006; Xu et al., 2009a,b). The lev-ls of pErk showed a reduction after 19 h with Ptx. This responseas also observed in myeloid leukaemia cells (ML-1) treated with

tx (Stadheim et al., 2001). Thus, Ptx toxic effect could be mediatedy Erk and the reduction of their phosphorylated forms also in NB4ells.

We evaluated the tentative feasibility of combined use of annhibitor of MEK/ERK pathway and the antimitotic drug Ptx tonduce programmed cell death in leukaemia cells. Our resultshow a slight decrease in ERK levels after the combined treat-

ent (PD98059 plus Ptx) with almost total dissapearance of p-ERK.own-regulation of the MEK/ERK pathway leads to changes in pro-

iferation and induction of apoptosis in NB4 cells similarly to thateen in AML cells (Liao and Lieu, 2005; Cagnol and Chambard,

cologic Pathology 65 (2013) 1101– 1108 1107

2010; Nishioka et al., 2010). These effects can be related to thereduction of expression of antiapoptotic proteins such as Bcl-2probably responsible for the increased apoptosis shown in thisstudy.

The MAPK pathway is activated by Ptx and inhibition of MAPKactivation can increase the sensitivity to this drug in different typesof cancer, breast, ovarian and colon and lung cancer cells (Xu et al.,2009a,b; Yu et al., 2001). In contrast, inhibition of MAPK activationby PD98059 markedly reduced the ability of Ptx to induce apoptosisin breast carcinoma cells and other cell lines (Bacus et al., 2001).These different effects may be due to the different cell genetic statusand to the drug concentrations used.

Several authors have shown that combination of MEK/ERKinhibitors and Ptx produced either increased or decreased toxiceffects over the antitumor action of the Ptx alone in HL-60, MCF7 orU937 cells, depending on the cell line (Bacus et al., 2001; Yu et al.,2001; Lee et al., 2009; Qiu et al., 2006).

In NB4 cells, the observed differences may rely on the high con-stitutive expression of ERK which has also been observed in otherkinds of tumours.

Caspase-3, caspase 8 and caspase 9 expression is correlatedto the activation of apoptosis cascade (Santos-Silva et al., 2006;Shimizu and Tsujimoto, 2000; Zou et al., 1997).

Ptx could induce apoptosis through a caspase-dependent way.Ptx activates caspases 3 and 9 in melanoma, carcinoma, glioblas-toma and in leukaemia HL60 cells (Selimovic et al., 2008; Pushkarevet al., 2004; Lu et al., 2005; George et al., 2008) as we have foundin NB4 leukaemia cells. On the contrary, caspase 9 does not playany observed role in Ptx mediated apoptosis in lymphoblasticleukaemia cells (CCRF-HSB-2) where caspases 3, 6, 8 and 10 arerequired (Park et al., 2004).

Combined sequential treatment with PD and Ptx did not affecteither the levels or the precursor forms of caspase 3 and caspase9 or those of the active forms of these caspases in comparisonto the effect produced by Ptx alone. These results could explainthe observations on subdiploid DNA values that are slightly lowerin cells treated with PD98059 plus Ptx than with Ptx alone. Any-way, induction of apoptosis by caspases activation precedes DNAdamage.

Ptx alone reduced NB4 cells viability down to 72%. Pretreat-ment of NB4 cells with PD98059 previous to incubation with Ptxrenders a reduction of cell viability, as compared with untreatedcontrol group, that is significant lower than that obtained with Ptxalone.

Some toxic effects induced by Ptx treatment such as DNA frag-mentation can be reduced by PD98059 pretreatment. This couldbe related to the observed inhibition of ERK phosphorylation thatcould reduce DNA damage. The sub-G1 phase accumulation can becorrelated with cell death. Anyhow, the time-course sequences ofthe events of apoptosis concerning membrane alteration, caspasesactivation, expression changes in apoptosis factors and fragmenta-tion of DNA are different. Thus, one could think that pre-treatmentwith PD98059 although reducing DNA fragmentation could bemediated via mitochondria by decreasing the levels of Bcl2 andincreasing the level of Bax factor.

In summary, Ptx induced DNA cleavage and cell death mainlyas apoptosis in NB4 leukaemia cells. In prolonged treatment, Ptxalso produced reductions of p53, changes in Bax and Bcl-2 andactivation of caspases 3 and caspase 9. Ptx produced NFkB factordepletion and decrease of pERK1/2. Pretreatment with PD98059neither activated caspases nor significantly enhanced the apoptoticeffect of Ptx. Thus, inhibition of ERK phosphorylation by PD98059

in human NB4 leukaemia cells does not seem to be an essentialpathway for bursting an increased induction of apoptosis by Ptx.Eventual applications of Ptx should consider particular effects ondifferent leukaemia cell types.

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cell lymphoma cell line and its molecular mechanisms. Zhonghua Xue Ye Xue

108 D. Morales-Cano et al. / Experimental an

cknowledgements

This work was supported in part by grants from CCG06-AH/SAL-0672, CCG10-UAH/SAL-5966, CAM-UAH 2011/060 and.I.S. PI060119. D. Morales-Cano was supported by a fellow-hip (Beca-Colaboración) from Ministerio de Educación (Spain). E.alvino was supported by a Miguel de Cervantes fellowship fromniversidad de Alcalá. We also want to thank Isabel Trabado forer technical assistance in cytometric analyses (C.A.I. Medicina-iología, Unidad de Cultivos, Universidad de Alcalá).

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