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Chemico-Biological Interactions 177 (2009) 181–189

Contents lists available at ScienceDirect

Chemico-Biological Interactions

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re-clinical antitumour evaluation of Biphosphinic Palladacycle Complexn human leukaemia cells

arlos R. Oliveiraa, Christiano M.V. Barbosab, Fábio D. Nascimentob, Camilla S. Lanetzki c,arília B. Meneghinc, Flávia E.G. Pereirac, Edgar J. Paredes-Gamerob, Alice T. Ferreirab,

iago Rodriguesc, Mary L.S. Queirozd, Antonio C.F. Cairesc,varne L.S. Tersariol c, Claudia Bincolettoa,∗

Departamento de Farmacologia, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, SP, BrazilDepartamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, SP, BrazilCentro Interdisciplinar de Investigacão Bioquímica, Universidade de Mogi das Cruzes, Mogi das Cruzes, SP, BrazilDepartamento de Farmacologia e Hemocentro, Faculdade de Ciências Médicas, FCM, Universidade Estadual de Campinas, UNICAMP, Campinas, SP, Brazil

r t i c l e i n f o

rticle history:eceived 31 July 2008eceived in revised form 17 October 2008ccepted 20 October 2008vailable online 5 November 2008

eywords:PC

a b s t r a c t

Previous studies reported by our group have introduced a new antitumoural drug called BiphosphinicPalladacycle Complex (BPC). In this paper we show that BPC causes apoptosis in leukaemia cells (HL60and Jurkat), but not in normal human lymphocytes. IC50 values obtained for both cell lines using the MTTand trypan blue exclusion assays 5 h after BPC treatment were lower than 8.0 �M. Using metachromaticfluorophore, acridine orange, we observed that BPC elicited lysosomal rupture of leukaemic cells. Further-more, BPC triggered caspase-3 and caspase-6 activation and apoptosis in cell lines, inducing chromatincondensation, apoptotic bodies, and DNA fragmentation. Interestingly, the lysosomal cathepsin B inhibitor

uman leukaemia cellspoptosisysosome

CA074 markedly decreased BPC-induced caspase-3 and caspase-6 activation as well as cell death. Lyso-somal BPC-induced membrane destabilisation was not dependent on reactive oxygen species generation,which was consistent with the absence of cellular HL60 and Jurkat membrane lipid peroxidation. We con-clude that, following BPC treatment, lysosomal membrane rupture precedes cell death and the apoptoticsignalling pathway is initiated by the release of cathepsin B in the cytoplasm of leukaemia cells. As no

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. Introduction

Cancer cells characteristically provide their own growth signals,gnore growth-inhibitory signals, replicate without limit, sustainngiogenesis, invade through basal membranes and capillary walls,roliferate in unnatural locations, and avoid cell death [1]. Althoughesistance to cell death is attributed to the inhibition of clas-ic apoptosis and its hallmarks, particularly caspase activationnd mitochondrial outer-membrane permeabilisation, it is possi-

le that alternative, non-apoptotic cell death pathways are alsoelevant to carcinogenesis [2,3]. Therefore, changes in lysosomalrafficking and content that support invasion and angiogenesisould account for disorders in the regulation of apoptotic and

∗ Corresponding author at: Departamento de Farmacologia, Escola Paulista deedicina, Universidade Federal de São Paulo (UNIFESP), Rua três de maio, 100 Térreo,

EP 04044-020 São Paulo, SP, Brazil.E-mail address: claudia.bincoletto@pq.cnpq.br (C. Bincoletto).

ccctpttta[

009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2008.10.034

ytes were observed, we suggest that BPC is more selective for transformedrbated lysosome expression.

© 2008 Elsevier Ireland Ltd. All rights reserved.

on-apoptotic cell death, particularly through the aberrant cellu-ar release of a class of proteases, cathepsins, which are usuallyequestered within the lysosomal lumen [4]. Lysosomes containatabolic hydrolases that participate in the digestion of autophagicaterial, tissue invasion or acute cell death after lysosomal mem-

rane permeabilisation (LMP). LMP can be induced by classicalpoptotic stimuli, such as intracellular second messengers, reactivexygen species (ROS), as well as lysosomotropic agents [4].

The role of the lysosomal cell death pathway in transformedells remains largely unexplored. Tumour cell death is oftenaspase-independent [5,6] due to acquired defects in the classicaspase-dependent pathways of apoptosis during the malignantransformation, and these alternative pathways may representotential targets for cancer therapy [7]. For instance, enhancing

he lysosomal cell death pathway may be a therapeutic strategyo overcome blocks in caspase-dependent cell death. Indeed, theopoisomerase inhibitor, camptothecin, induces apoptosis in hep-tocellular carcinoma cells via a cathepsin D/B-mediated pathway8]. Moreover, cathepsin B has been shown to play a dominant role

1 gical Interactions 177 (2009) 181–189

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Fig. 1. Chemical structure of BPC and cytotoxicity for leukaemia cell lines (Jurkat andHL60) and human PBMC (with or without PHA). Chemical structure of BPC (A). Cellswere treated with different BPC concentrations for 5 h (B and C). In the absence of thecompound, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide)]reduction or trypan blue dye were considered as 100%. Results represent the meansaHu

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82 C.R. Oliveira et al. / Chemico-Biolo

n executing the apoptotic program in several tumour cell lines [5].s a result, it seems that cathepsin B may play two opposing roles inalignancy: as an executioner of apoptosis in cytotoxic signalling

ascades and as a mediator of tumour invasion.Transformation of cells strongly affects processing, trafficking,

nd subcellular localization of lysosomal enzymes [9,10] and it isonceivable that cathepsins might take part in the transformation-nduced death of tumour cells. The overexpression of cathepsins,ften observed in tumours, renders cancer cells more susceptible toell death via the lysosomal pathway [11]. Thus, it should be possi-le to exploit this pathway to differentially modulate susceptibilityf cancer cells to death.

A previous study reported by our group introduced a newrganometallic class of drugs called Biphosphinic Palladacycleomplexes (BPC), which presents important biological properties12,13]. One isomer of this organometallic class prolonged the sur-ival of Walker tumour-bearing rats and inhibited extracellularathepsin B activity [12]. Most recently, a study showed that BPClso caused lysosomal membrane destabilisation that precededther apoptotic manifestations in K562 leukaemia cells [13]. Welso demonstrated, through acute toxicological studies, that BPCresented absence of detectable toxic effects on kidney and liver13].

Taking all these findings into consideration, the elucida-ion of the biochemical and molecular mechanisms mediatingntileukaemic properties of BPC, in various human leukaemic cellines, is likely to provide additional relevant information to betternderstand its effects on transformed cells, as well as new insights

nto possible benefits of this compound compared to currently usedherapeutic agents. To further investigate the pharma-toxicologicalotential of this new drug, we analysed the molecular mech-nisms involved in the effectiveness of BPC to produce lethalffects on both human promyelocytic leukaemia cells (HL60) and-lymphoma cells (Jurkat). Cytotoxic effects produced by BPC onuman peripheral blood mononuclear cells were also evaluated.he results obtained showed that BPC presented lethal effects onoth leukaemia cells studied, but was ineffective to induce apopto-is in normal human lymphocytes.

. Materials and methods

.1. Biphosphinic Palladacycle Complex [Pd(C2,N-(S(−)mpa)(dppf)] Cl

Ionic compounds having chelating biphosphinic ligand with theeneral structure Pd(C2,N-dmpa)(L)] Cl (L = bidentate dppf ligand)ere synthesised from the reactions of the starting cyclopalladated

omplexes with the biphosphinic ligand (L). From the describedtarting compounds, cyclopalladated complexes were synthesisedy reactions with the 1,1′-bis(diphenylphosphine)ferrocene (dppf)Fig. 1A) [14].

.2. Cell culture

HL60 and Jurkat cell lines, obtained from Rio de Janeiroell Bank/RJ, Brazil, were grown in suspensions in RPMI 1640edium containing l-glutamine (0.2 g/L), antibiotics (penicillin

00 �g/mL; streptomycin 100 �g/mL) and supplemented with 10%eat-inactivated fetal bovine serum, in 5% CO2 humidified atmo-

phere at 37 ◦C. In all experiments, 3 × 105 cells/mL were seedednd, after 48 h, the cells were treated with different BPC concen-rations for 5 h. The BPC complex was first dissolved in dimethylulfoxide (DMSO) and then in supplemented medium. The finaloncentration of DMSO in the test medium and controls was 0.1%.

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nd standard errors of three experiments run in triplicate. IC50% values found forL60 and Jurkat cells using the MTT assay were 6.0 and 7.6 �M, respectively, andsing the trypan blue exclusion assay, 5.1 and 5.0 �M, respectively.

.3. Cell viability

Viability of leukaemia cells was assessed by the trypan blue dyexclusion and MTT reduction assays, as previously reported [15,16].ach BPC concentration was tested in three different experimentsun in triplicate.

.4. Lymphocyte proliferation assay

Blood was collected from healthy donors, and human peripheral

lood mononuclear cells (PBMC) were isolated by Ficoll/Hypaqueradient. Mononuclear cells were cultured under the same con-itions described for leukaemia cell lines, except for the additionf 5 �g/mL phytohemagglutinin (PHA) in each well and incubatedor 5 h. Cells were plated at a density of 1 × 106 plating/mL in a

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idtDfi3(Cells were treated with BPC (5.0, 10.0 or 25.0 �M) in duplicateand incubated at 37 ◦C for 5 h. Tert-butylhydroperoxide (t-BHP)(0.5 mM), a chemical compound commonly used to induce oxida-tive stress in biological systems, was employed as the positivecontrol. The fluorescence intensity of the cell suspension was

Table 1Effects of BPC on human peripheral blood mononuclear cells (PBMC) and toxicitytowards lymphocytes.

BPC concentrations (�M) Viability (%)

BPC BPC-PHA

5.0 98 ± 2.1 >131 ± 5

C.R. Oliveira et al. / Chemico-Biolo

6-well plate in the presence of BPC for 5 h. MTT, dissolved in PBSt 5 �g/mL, was added to all wells (10 �L per 100 �L medium), andlates were incubated at 37 ◦C for 4 h. Acid–isopropanol (100 �L.04N HCl in isopropanol) was added to all wells to dissolve theark blue crystals [15,16]. The plates were read on a Bio-Teckniversal microplate reader (ELx800 Instruments Inc.) using a testavelength of 570 nm. Cellular viability was evaluated by trypanlue exclusion assay.

.5. Lysosomal destabilisation after BPC exposure

To observe the lysosomotropic properties of BPC molecule, wesed the acridine orange (AO) uptake and relocation methods, asescribed previously [17–19]. AO, a metachromatic fluorophore,ccumulates mainly in the acidic vacuolar apparatus, preferentiallyn secondary lysosomes. When excited by blue light (relocation

ethod) it shows red and green fluorescence at high (lysosomal) orow (nuclear and cytosolic) concentrations, respectively. However,f green excitation light is used (uptake method), only concentratedysosomal AO is demonstrated by its red or orange fluorescence.upture of initially AO-loaded lysosomes may be monitored as an

ncrease in cytoplasmic diffuse green, or a decrease in granular reduorescence [20,21]. For imaging, HL60 and Jurkat cells were grownn cover glasses and labelled in vivo with 5 �g/mL of AO in RPMI640 medium without serum for 15 min at 37 ◦C in 5% CO2. There-fter, HL60 and Jurkat cells were washed with RPMI 1640 mediumnd then exposed to 10.0 and 7.5 �M of BPC, respectively, for 2.5r 5 h at 37 ◦C in 5% CO2. The fluorescent signals of AO were takenith a Zeiss LSM 510 confocal microscope (Jena, Germany). Frac-

ions of isolated lysosomes were also prepared from HL60 andurkat cells homogenised in 9 vol. of 0.33 M sucrose containingmM Hepes and 5 mM MgCl2 (pH 7.4) and centrifuged at 450 × g

or 2 min. The supernatant was centrifuged (3500 × g for 10 min)nd the resulting supernatant was centrifuged again (10,000 × gor 10 min), yielding a lysosome-rich fraction [22]. The lysosomesolated fraction was also incubated with BPC for 5 min and theapacity of BPC to directly destabilise the membrane of isolatedysosome was evaluated by measuring cysteine activity using fluo-ogenic substrate Z-FR-MCA [23]. Supernatant cathepsin B activityn BPC-treated lysosomes was expressed as a percentage of totalctivity in relation to lysosomes exposed to Trinton X-100 (0.1%)positive control).

.6. Assessment of apoptosis

DNA fragmentation evaluation was performed in both condi-ions: HL60 and Jurkat (106/mL) cells treated with BPC for 5 h andith cathepsin B inhibitor CA-074 [N-(l-3-trans-propylcarbamoyl-

xirane-2-carbonyl)-l-isoleucyl-l-proline] for 2 h before BPC treat-ent. For the experiments, cells were centrifuged at 300 × g formin, washed twice with ice-cold PBS, incubated in 200 �L of lysisuffer (10 mM Tris–HCl, 10 mM EDTA, pH 8.0, 0.5% sodium sar-osinate and 1 mg/mL proteinase K) for 3 h at 56 ◦C, and treatedith 0.5 mg/mL RNase for 1 h at 56 ◦C. DNA was extracted withhenol/chloroform/isoamyl alcohol (25:24:1, v/v) before loading.amples were mixed with loading buffer (50 mM Tris, 10 mM EDTA,% (w/w) low-melting-point agarose, and 0.025% (w/w) bromophe-ol blue) and loaded onto a pre-solidified 2% agarose gel containing.1 �g/mL ethidium bromide. The agarose gels were run at 50 V for0 min in TBE buffer, and then observed and photographed under

V light.

We also used AO combining fluorescence microscopy to deter-ine the morphological characteristic of apoptotic cells [24,25].

or the experiments, 1 �L of stock solution containing 100 �g/mLf AO was added to 25 �L of HL60 and Jurkat cell suspensions and

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Interactions 177 (2009) 181–189 183

iewed using fluorescent microscopy with a band fluorescein filter520–560 nm).

.7. Caspase activity

After HL60 and Jurkat cells incubation with BCP for 5 h (6.0 �M),irect measurements of caspase activity were performed usingolorimetric protease kits (R&D Systems, USA), according to theanufacturer’s recommendations. This experiment was performed

n both conditions: treated with BPC for 5 h and with cathepsin Bnhibitor CA-074 for 2 h before BPC treatment (6.0 �M). The cas-ase activity assay is based on the spectrophotometer detectionf the chromophore p-nitroanilide (pNA) after cleavage from theubstrates X-pNA, where X stands for the amino acid sequencesecognised by the specific caspase-3 and caspase-6. Accordingo the procedure, 2 × 106 cells were pelleted by centrifugationnd lysed on ice. The protein concentration in the lysed sampleas measured using a Bio-Rad Protein assay (Bio-Rad Laborato-

ies, Inc., USA). The optic density of samples was measured at05 nm.

.8. Lysosomal membrane lipid peroxidation

Damage to cell membrane by ROS can result in peroxidationf lipid components, which can be studied by the generation ofalondialdehyde (MDA). BPC-treated Jurkat cell suspension (6.0

r 15.0 �M for 5 h) was centrifuged at 50 × g for 5 h at 4 ◦C andhe pellet was treated with 1 mL 1% of 2-thiobarbituric acid (TBA)dissolved in 50 mM NaOH), 0.1 mL of 10 M NaOH, and 0.5 mLf 20% H3PO4, followed by incubation at 85 ◦C for 20 min. Afterooling the reaction mixture, the MDA-TBA complex formed wasxtracted with 2 mL of n-butanol and its absorbance was measuredt 535 nm with a DU-70 spectrophotometer (Beckman Coulter Inc.).DA concentration was calculated from C = 150,000 M−1 cm−1

26].

.9. Reactive oxygen species (ROS) generation

ROS production was studied by measuring the fluorescencentensity of dichlorofluorescein (DCF). Non-fluorescent 2,7-ichlorofluorescein-diacetate (DCFH-DA) diffuses into the cellhrough the plasma membrane and is hydrolysed within the cell toCFH. Intracellular oxidation converts DCFH into the fluorescent

orm, DCF [27]. Briefly, Jurkat cells were cultured until confluencen 25-cm2 flasks and incubated with 1 �M DCFH-DA at 37 ◦C for0 min. Cells were washed twice in Hank’s balanced salt solutionBSS), counted and seeded (5 × 105 cells/well) into 6-well plates.

10.0 97 ± 1.4 >127 ± 415.0 94 ± 4.0 >101 ± 5

BMC was treated with BPC and stimulated with PHA (5.0 �g/ml) for 5 h. Viabil-ty was determined by staining with trypan blue dye. The results are expressed asercentage of the control group (BPC non-treated cells).

184 C.R. Oliveira et al. / Chemico-Biological Interactions 177 (2009) 181–189

Fig. 2. Induction of LMP. A decrease in lysosomal (red) and an increase in cytosolic (green) fluorescence of AO-stained cells reflecting lysosomal rupture. HL60 and Jurkatcells were incubated with AO, washed, and treated with BPC for 2.5 or 5 h. BPC-non-treated HL60 and Jurkat cells (A–C). (A) Intact lysosomes within most cells. (B) Low greenfluorescence due to well-defined lysosomal membrane. (C) Absence of co-localization between red and green fluorescence. HL60 and Jurkat cells treated with BPC for 2.5 h(D–F) and 5 h (G–I). (D) and (G) A few intact lysosomes expressed by an intense red fluorescence. (E) and (H) Increasing green fluorescence due to AO release from lysosomesinto the cytosol. (F) and (I) A clear co-localization between green and red fluorescence, which strongly suggests lysosomal membrane rupture. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of the article.)

gical Interactions 177 (2009) 181–189 185

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Fig. 3. Analysis of lysosomal stability in a lysosomal fraction. A leukaemia lysoso-mal fraction HL60 or Jurkat cells (0.6 mg protein/mL) in buffered 300 mM sucrosewas exposed to BPC at the concentration indicated in the picture for 5 min at 37 ◦C.After centrifugation (10,000 × g for 10 min), supernatant cathepsin B activity in BPC-treated lysosomes (9.0, 18 or 36 �M) was expressed as a percentage of total activityitw+

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etermined using a fluorescence spectrophotometer (F-2500,ITACHI) with excitation at 503 nm and emission at 529 nm.

.10. Statistical analysis

Values are the mean of three independent experiments run inriplicate. Data for each assay are expressed as mean ± S.D. and weretatistically analysed using ANOVA. Multiple comparisons amongroup mean differences were checked using the Tukey post-test.ifferences were considered significant when P-value was lower

han 0.05. Results were expressed as a percentage of the controlsnd the computer software package “Origin” was used to determineC50 values (concentration required to inhibit 50% of the evaluatedarameter).

. Results

.1. BPC-dependent cytotoxicity for leukaemia cells (HL60 andurkat)

To establish the specificity of BPC for leukaemia cells, as com-ared with untransformed cells, the effects of BPC on the viabilityf HL60, Jurkat, and peripheral blood mononuclear cells (PBMC)nd lymphocytes were determined in parallel using the MTT assay.he results demonstrated that BPC presented cytotoxicity for botheukaemia cell lines studied (Fig. 1B), exhibiting an IC50% lowerhan 8.0 �M (Fig. 1B). Effects of BPC on PBMC were much less pro-ounced (Fig. 1C), since no toxic effects were observed in the rangef concentration studied (5–20 �M). Based on visual aspect of theells using trypan blue dye we also found that BPC produced anypparent harm to the PBMC and lymphocytes at 5.0, 10.0 or 15 �MTable 1).

.2. BPC-induced lysosomal permeability

HL60 and Jurkat cells exposed to 10 �M and 7.5 �M BPC,espectively, were examined for fluorescence staining by theysosomotropic weak base AO to evaluate whether BPC inducedncreasing lysosomal permeability. HL60 BPC-non-treated cells areresented in Fig. 2A–C. As early as 2.5 h following BPC exposure,ells decreased red fluorescence, indicating translocation of AOrom acidic lysosomes to the cytoplasm (Fig. 2D–F). After 5 h incu-ation, BPC-treated HL60 and Jurkat cells diffused cytoplasmaticnd nuclear staining that varied in intensity (Fig. 2G–I). This translo-ation of lysosomal contents to the cytosol and nucleus indicateshat BPC caused increasing lysosomal permeability for both cellines. Similar results were observed using isolated fractions of HL60ysosomes exposed to BPC for 5 min (Fig. 3). It is possible to see inig. 3 that BPC really leads to lysosomal leakage during the firstmin after incubation, as reflected by the increase in cathepsinctivity in a dose-dependent manner.

.3. BPC-induced apoptosis

We also observed in this study that HL60 and Jurkat cells under-ent apoptosis after in vitro treatment with BPC. As depicted in

ig. 4A and B, exposure of HL60 and Jurkat cells to BPC resultedn a classical characteristic DNA fragmentation regardless of the

oncentration used. Leukaemia BPC-treated cells also exhibitedhromatin condensation, expressed by dense green areas and apop-otic bodies, which are membrane-enclosed vesicles that haveudded from the cytoplasmatic extension (Fig. 4C). Taken together,hese results suggest that the apoptotic process also mediates BPCytotoxicity for HL60 and Jurkat cells.

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n relation to lysosomes exposed to Trinton X-100 (0.1%) (positive control). Notehe dose-dependent increase in cathepsin B activity as a result of BPC exposure,hich suggests lysosomal rupture. *Significant in relation to BPC-non-treated cells;

significant in relation to BPC-treated leukaemia cells.

.4. Cathepsin B contribution to BPC-induced apoptosis

Using HL60 cell line, which is a useful model for the studyf factors affecting proteinase synthesis by human mononuclearhagocytes and Jurkat cells, we verified that pre-treating bothell lines with cathepsin B inhibitor CA074 prior to BPC treat-ent (6.0 �M) reduced activation of both caspase-3 and caspase-6

Fig. 5). In addition to that, CA074 also significantly increased HL60nd Jurkat cell viability (Fig. 6A) and prevented leukaemia cell DNAragmentation (Fig. 6B). These data indicate that cathepsin B con-ributed to the downstream initiation of caspase activation leadingo apoptosis.

.5. Effects of BPC on free radical metabolism

ROS and membrane lipid peroxidation were also evaluated inhis study after BPC leukaemia cell treatment to verify whetherhey were involved in BPC-induced cell death. ROS generation (DCFuorescence intensity) was studied in Jurkat cells after 5 h incuba-ion with BPC. DCF fluorescence intensity was not different between.0 or 15 �M BPC-treated Jurkat cells and control samples (Fig. 7A).imilar results were observed for HL60 cells. The absence of ROSeneration in BPC-treated cells was consistent with the results thatemonstrate absence of membrane lipid peroxidation in leukaemiaells (Fig. 7B).

. Discussion

Programmed cell death is a natural process for removingnwanted cells such as those with potentially harmful mutations,berrant substratum attachment, or alterations in cell-cycle con-rol [27]. Therefore, drugs designed to restore programmed celleath might be effective against many cancers [28]. Selective killingf tumour cells might be achievable with such drugs, because

nlike normal cells, cancer cells are under stress, destined to die,nd highly dependent on aberration of the apoptosis signallingathways to stay alive [27]. However, cancer cells that possesslterations in proteins involved in cell death signalling are oftenesistant to chemotherapy and are more difficult to treat using

186 C.R. Oliveira et al. / Chemico-Biological Interactions 177 (2009) 181–189

Fig. 4. DNA fragmentation of Jurkat (A) and HL60 (B) cells after 5 h incubation with BPC alines. The pictures are representative of similar results obtained from two independent expexpressed by dense green areas and apoptotic bodies (C). (For interpretation of the referearticle.)

Fig. 5. Alterations in caspase-3 and caspase-6 activity in HL60 and Jurkat cells after5 h incubation with BPC (6.0 �M). Control experiments with the same BPC amountwere carried out after leukaemia cell cathepsin B inhibitor CA074 pre-incubation for2 h. Caspase activity in BPC-treated cells was calculated as the percentage of caspase-3 and caspase-6 activity in the control (basal caspase-3 and caspase-6 activity innon-treated leukaemia samples). Each column represents the mean ± S.D. of twoexperiments run in triplicate. *P < 0.01 compared to control; +P < 0.05 compared toHL60 BPC-treated cells; oP < 0.05 compared to Jurkat BPC-treated cells.

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t different concentrations. Note that the apoptosis process is presented in both celleriments. Leukaemia BPC-treated cells (6.0 �M) exhibited chromatin condensationnces to color in this figure legend, the reader is referred to the web version of the

hemotherapeutic agents that primarily work by inducing classicalignals of apoptosis. Thus, the development of anticancer agentsith different modes of action is of key importance to overcome

linical therapy resistance [29]. Several lines of evidence indicatehat apoptosis also plays an important role in the response of acuteeukaemia patients to chemotherapy [30], an aggressive neopla-ic disease with poor prognosis. The primary cause of treatmentailures in adult patients with leukaemia is the emergence of drugesistance, which involves multiple molecular mechanisms, suchs defects in inherent programmed cell death pathways.

Recently, the development of neoplasic diseases has been asso-iated with altered lysosomal trafficking and increased expressionf lysosomal proteases termed cathepsins. Emerging experimen-al evidence suggests that such alterations in lysosomes may behe Achilles’ heel of cancer cells by sensitising them to death path-ays involving LMP [31]. Based on these findings, one of the goalsf this study was to determine whether possible changes in the

ysosomal membrane stabilisation could account for BPC-inducedell death. For this purpose, using HL60 and Jurkat cell lines, we

nalysed the capacity of a new organometallic compound, hereinalled BPC, to induce apoptosis. The results obtained demonstratedhat BPC was cytotoxic for both cell lines and presented an IC50alue below 8.0 �M using the trypan blue exclusion assay after 5 hf incubation. The same compound might also have reduced the

C.R. Oliveira et al. / Chemico-Biological Interactions 177 (2009) 181–189 187

Fig. 6. Cytotoxic effects of BPC on Jurkat and HL60 cells after pre-incubation withcathepsin B inhibitor CA074 for 2 h. Cells were treated with several concentrations ofBPC for 5 h and leukaemia cell survival was studied (A). IC50 values obtained for HL60and Jurkat cells using the MTT assay after pre-incubation with CA074 were 10.0 and1fc

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fitclclsBm(llsorganelles (e.g. mitochondria). Mitochondria undergo alterations

1.5 �M, respectively. The same condition of leukaemia cell treatment with CA074or 2 h prevented DNA fragmentation induced by BPC (6.0 �M) treatment in bothell lines (B).

itochondrial activity 5 h post-treatment, as measured by the MTTeduction assay. Both tests used in this evaluation provide an impor-ant tool for evaluating cytotoxicity of compounds with potentialherapeutic activity [32]. The trypan blue exclusion test allows thevaluation of the structural integrity of the cell membrane, whereashe reduction of 3-(4-5-dimethylthiazole-2yl)-2,5-diphenyl tetra-olium bromide (MTT) allows the assessment of mitochondrialunction through succinate dehydrogenase activity [16]. Usually theC50% values obtained for trypan blue exclusion assay are lower thanhose obtained for MTT because the membrane integrity is moreusceptible to chemical insult than the enzyme succinate dehydro-enase, which is required for formazan formation by reduction ofTT.Programmed cell death was observed in both cell lines using

wo methods to predict the hallmarks of apoptosis, such as DNAragmentation and morphological changes (chromatin condensa-ion). Interestingly, the same concentrations that were lethal toeukaemia cells (HL60 and Jurkat) were ineffective against nor-

al human lymphocytes, even in the presence of PHA, whichuggests that, at the concentrations of 5.0 and 10.0 �M, BPC isot immunosuppressive. Since transformed cells presented alteredxpression of cysteine cathepsins, altered trafficking of lysosomes,

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ells 5 h after BPC exposure. Positive controls tert-butylhydroperoxide (t-BHP) (A)nd Fe2+ (B). These cells did not show significant changes in DCF fluorescence inten-ity or MDA formation post-BPC treatment. The results are expressed as a percentagef the control (leukaemia BPC-non-treated cells).

nd enhanced secretion of cathepsins, leading to the appearance ofsp70 on the lysosomal membrane and increased volume of acidicompartments [32], we suggest that BPC, due to its lysosomotropicroperties, is more selective for malignant cells, which probablyresent increased sensitivity to lysosomal cell death pathway.

The present data allowed the proposal of a coherent pathwayor BPC-induced apoptosis in HL60 and Jurkat cells as illustratedn Fig. 8. Firstly, BPC targets lysosomes and induces leakage or rup-ure since this is a hydrophobic (lipid-solvable) compound (Fig. 1A)apable of penetrating and partitioning into lipid membrane of cel-ular organelles. The proton trapping amino group of BPC becomesharged at acidic lysosomal pH and accumulates heavily insideysosomes (Fig. 1A). Thus, it is possible for BPC to disturb lyso-omal membrane stability. This is supported by our finding thatPC could induce relocation or release of cathepsin B, a lysosomalarker enzyme, to the cytosol, as well as AO, a lysosomotropic dye

Figs. 2 and 3), events that took place as early as 5 min after isolatedysosome BPC exposure. Secondly, once cathepsin B is released fromysosomes to the cytosol, it may convert certain (unknown) sub-tances to bioactive molecules, which directly attack other cellular

s a result of the attack of cathepsin B and/or other molecules,nd in this study, using the MTT assay we observed a possibleitochondrial reduction activity 5 h after BPC treatment. Finally,

aspase-3 and caspase-6 are activated and execute apoptosis. The

188 C.R. Oliveira et al. / Chemico-Biological

Fig. 8. Proposed pathway for BPC-induced apoptosis in leukaemia cells. BPC maydirectly target lysosomes and induce lysosomes leakage or partial rupture leadingto the release of cathepsin B from lysosomes to the cytosol. Cathepsin B may con-vert certain unknown substances to bioactive molecules, which may attack othercellular organelles (e.g. mitochondria, nucleus). Moreover, cathepsin B may directlyattack the organelles. As a result of the attack of cathepsin B and/or other bioactivemsSa

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olecules, mitochondria may undergo apoptotic alteration. Thus, apoptotic factors,uch as cytochrome c and caspases, are released or activated and execute apoptosis.ince CA-074 inhibits BPC-induced cell death, we propose that cathepsin B servess a death mediator.

mportance of cathepsin B release from lysosomes to cytosol forpoptosis induction in HL60 and Jurkat cells was further demon-trated when cathepsin B inhibitor CA074 prevented caspase-3 andaspase-6 activation as well as apoptosis induction. These resultsre in accordance with data found in the literature demonstratinghat lysosomal permeabilisation appears to be an early event in thepoptotic cascade, preceding other hallmarks of apoptosis such asestabilisation of mitochondria and caspase activation [33,34].

Lysosomes are the major site for cellular catabolism and containwide spectrum of hydrolytic enzymes that can degrade nearly

ll cellular components. Among the most powerful hydrolyticnzymes are the cathepsins, particularly cathepsin B, which haveeen associated with apoptosis [35,36]. From this point of view,n uncontrolled leakage of these enzymes from lysosomes to theytosol may be lethal to cells.

Besides extralysosomal signals, it is clear that many eventsccurring within the lysosome, i.e. iron-catalysed oxidative reac-ions, are also very important to promote LMP. Indeed, oxidativetress, together with intra-lysosomal iron, generates oxygen rad-cals via the Fenton reaction, which can promote oxidation of

embrane lipids and LMP [37]. Since the BPC complex presentsron and palladium as part of its structure, we have recently sug-ested a relationship between ROS generation, membrane lipideroxidation, and BCP cytotoxicity. However, when we analysedhese events in HL60 and Jurkat cells through MDA formation andCP fluorescence intensity, respectively, 5 h after BPC incubation,e observed that BPC-induced insufficient amounts of ROS and

ow levels of lipid peroxidation compared with leukaemia BPC-on-treated cells. From this standpoint, it is important to mention

hat rapidly growing tumour cells present a marked reduction of

itochondrial content and low levels of O2− [38–40]. Moreover, a

educed rate of lipid peroxidation has been observed in Yoshidaepatomas [41] and in colon carcinoma. These observations are

n accordance with the results presented here, which demonstrate

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hat leukaemia BPC-treated cells produce lower levels of ROS andecreased lipid peroxidation capacity, suggesting that the alter-tion in the cellular redox status is not essential for BPC-inducedell death.

Taken together, these results suggest that lysosomal enzymectivity and lysosome acidic compartment contribute to BPC-nduced apoptosis preceded by a loss of lysosomal membranentegrity. However, additional research is needed to elucidate theellular mechanism underlying the contributions of lysosomes tohe intrinsic pathway of BPC-induced apoptosis.

onflict of interest

There is no conflict of interest.

cknowledgements

This study was supported by grants from Fundacão de AmparoPesquisa do Estado de São Paulo (Process 99/00639-2), Conselhoacional de Desenvolvimento Científico e Tecnológico (CNPq) andAEP/Universidade de Mogi das Cruzes.

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