Pomolic acid triggers mitochondria-dependent apoptotic cell death in leukemia cell line

Pomolic acid triggers mitochondria-dependent apoptotic

cell death in leukemia cell line

Janaina Fernandesa,1, Ricardo Weinlichb,1, Rachel Oliveira Castilhoc,Maria Auxiliadora Coelho Kaplanc, Gustavo Pessini Amarante-Mendesb,

Cerli Rocha Gattassa,*

aInstituto de Biofısica Carlos Chagas Filho, UFRJ, Ilha do Fundao, IBCCF, CCS Bl.G, Rio de Janeiro 21949-900, BrazilbDepartamento de Imunologia, Instituto de Ciencias Biomedicas, USP, Sao Paulo, e Instituto de Investigacao em Imunologia,

Instituto do Milenio, BrazilcNucleo de Pesquisas em Produtos Naturais, UFRJ, Rio de Janeiro, Brazil

Received 2 July 2004; received in revised form 28 August 2004; accepted 1 September 2004

Abstract

One of the major goals in chemotherapy is to circumvent anti-apoptotic strategies developed by tumor cells. In a previous

paper, we showed that pomolic acid (PA) is able to kill the leukemia cell line K562 and its MDR derivative, Lucena 1. Here, we

demonstrated that PA-induced apoptosis of HL-60 cells is dependent on the activation of caspases-3 and -9 and dissipation of

the mitochondrial transmembrane potential (Djm). Disruption of Djm precedes caspase activation and is not inhibited by

zVAD-fmk indicating mitochondria as the main target of PA. Our data pointed to the potential use of PA to overcome apoptosis

resistance.

q 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Pomolic acid; Apoptosis; Mitochondria membrane potential; Cancer therapy; Leukemia; MDR

0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights re

doi:10.1016/j.canlet.2004.09.001

Abbreviations: Djm, mitochondrial transmembrane potential;

ANT, adenine nucleotide translocator; Apaf-1, apoptotic protease

activating factor-1; CD95-L, CD95-ligand; CsA, cyclosporin;

DiOC6(3), 3,3 0-dihexyloxacarbocyanide iodide; MTT, 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PI, propi-

dium iodide; PTP, permeability transition pore; zVAD-fmk,

benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone.

* Corresponding author. Tel.: C55 21 2562 6564; fax: C55 21

2280 8193.

E-mail address: [email protected] (C.R. Gattass).1 Janaina Fernandes and Ricardo Weinlich contributed equally to

this paper.

1. Introduction

One of the major causes of chemotherapeutic

failures in cancer treatment is the development of

different kinds of resistance. The search for new

drugs able to overcome the resistance mechanisms

and leading to tumor cell death is of utmost

importance for cancer therapy. In a previous study

[1] we showed that pomolic acid (PA), a pentacyclic

triterpene isolated from Chrysobalanus icaco, was

highly effective in inhibiting the growth of both

Cancer Letters 219 (2005) 49–55

www.elsevier.com/locate/canlet

served.

J. Fernandes et al. / Cancer Letters 219 (2005) 49–5550

K562, a leukemia cell line and Lucena-1, a MDR

resistant leukemia line that overexpresses P-glyco-

protein (Pgp 170) [2]. However, apart from its

capacity to induce apoptosis [1], the mechanisms of

PA activity are presently unclear. As part of the

evaluation of the potential of new anti-cancer

compounds, this paper investigated the molecular

pathways of PA-induced apoptosis.

Several compounds used in anticancer chemother-

apy are believed to induce cell death via activation of

key elements of the apoptotic program. Apoptosis is

characterized by distinct morphological features such

as plasma membrane blebbing, cell shrinkage, chro-

matin condensation, DNA fragmentation, and break-

down of the cell into apoptotic bodies [3–5]. Recently,

it has been shown that mitochondria play an essential

role in the so-called intrinsic pathway of apoptosis [6],

the major route used by several chemotherapeutic

drugs [7,8]. Activation of the mitochondrial per-

meability transition pore (PTP) leads to dissipation of

mitochondria membrane potential (Djm) [9–11]. In

the cytosol, the complex formed by cytochrome c,

Apaf-1 and pro-caspase-9 (apoptosome) triggers the

activation of caspase-9 [12]. This caspase promotes

the activation of a cascade of caspases including

caspase-3, with the consequent cleavage of specific

caspase substrates and cell death [7,10]. Because

caspase activities can be detected in all cells under-

going apoptosis, regardless of their origin or death

stimuli, and inhibitors of caspases prevent the hall-

marks of apoptosis, these proteases are considered the

main executioners of apoptosis [13,14].

Due to the relevance of the potential anti MDR-

activity of PA and considering that characterization of

their mechanisms of action is a preliminary and

fundamental step to future clinical application/use of

new drugs, the aim of this paper is to clarify the

mechanism of action of this triterpene. Investigation

of PA-induced death pathways demonstrated that

induction of apoptosis by this triterpene involves

activation of caspases-9 and -3 in a process mediated

through loss of Djm, an unusual mechanism for drug-

induced apoptosis in HL-60 cells. Indeed, treatment of

cells with CSA an inhibitor of mitochondria PTP,

completely abrogates apoptosis suggesting that mito-

chondria is the main target for PA. Since it has been

proposed that drugs that act on mitochondria

may bypass MDR resistance mechanisms, our data

reinforce the potential of pomolic acid as a new anti-

MDR drug.

2. Materials and methods

2.1. Chemicals and cell culture

Pomolic acid isolated from Chrysobalanus

icacoL. as described previously [1] was dissolved

in dimethyl sulfoxide (DMSO, SIGMA, St Louis)

and diluted in RPMI 1640 for use. DiOC6(3) was

from Molecular Probes, Inc. (Eugene, OR), cyclos-

porin A was from Calbiochem (San Diego, CA,

USA), caspase inhibitor zVAD-fmk was from

Enzyme Systems (Livermore, USA), mAbs for

caspases-3 and -9 were from Pharmingen (San

Diego, CA, USA), anti-actin mAb was from ICN

(Costa Mesa, USA) and anti-mouse IgG-HRP from

Amersham Biosciences (Arlington, IL). The human

leukemia cell line HL-60 was cultured at 37 8C and

5% CO2 in RPMI 1640 (Life Technologies, Inc.,

USA) supplemented with heat inactivated 10% fetal

calf serum (FCS; Life Technologies, Inc., USA),

2 mM L-glutamine, 100 U/ml of penicillin and

100 mg/ml of streptomycin (Life Technologies,

Inc., USA).

2.2. Cell viability assay

Cell viability was assessed by MTT [15]. Cells

(104/well) were plated in 96 well tissue culture plates

for 24 h and then treated with different concentrations

of PA (1, 10, 25, 100 mg/ml) or DMSO (in the same

concentrations carried by the drug). After another

48 h, 20 ml (5 mg/ml) MTT was added to each well

and the plate was incubated at 37 8C in the dark for at

least 4 h. The formazan crystals were solubilized in

DMSO (200 ml/well) and the reduction of MTT was

quantified by absorbance (A570 nm, with 630 nm-

reference filter). Effects of the drug on cell viability

were calculated using cells treated with DMSO as

control.

2.3. Detection of apoptosis

Apoptosis was evaluated by cell morphology and

cell cycle analysis [16]. After 24 h resting, plated cells

J. Fernandes et al. / Cancer Letters 219 (2005) 49–55 51

(2!105/well) were treated with medium or different

concentrations (1, 10, 25, 100 mg/ml) of PA and

incubated for another 24 h. After this time, cells were

harvested and alternatively pelleted onto glass slides

for Giemsa staining or suspended in 300 ml of HFS-

Hypotonic Fluorescent Solution (50 mg/ml propidium

iodide (PI) and 0.1% Triton X-100 in 0.1% Na citrate

buffer). After 1 h incubation in the dark at 4 8C the

DNA content was measured by flow cytometry (FL-2)

(FACSCalibur, Becton Dickinson, San Jose, CA).

Data acquisition and analysis were controlled by

Cellquest software version 3.1f. Subdiploid popu-

lations were considered apoptotic. In some exper-

iments, cells were pre-incubated during 1 h with

zVAD-fmk (100 mM) or CSA (5 mM) before the

addition of PA to the cultures.

2.4. Measurement of mitochondrial transmembrane

potential (Djm)

Variations of mitochondrial membrane potential

(MMP) was assessed in cells plated and treated with

PA under the same conditions described for apoptosis

detection. Then cells were stained with the fluor-

ochrome DiOC6(3) (40 nM) as described previously

[17] and analyzed by flow cytometry (FL-1), accord-

ing to Ormerod [18].

2.5. Western blot analysis

SDS-PAGE and western-blot analysis were per-

formed as described elsewhere [19]. Briefly, plated

cells (2!105/well), treated with 25 mg/ml PA for 4, 8,

and 12 h, were collected by centrifugation, washed

once in ice-cold PBS, lysed directly in SDS-sample

buffer (50 mM Tris–HCl, pH 6.8, 2% SDS, 10%

glycerol, 2.5% 2-ME) and boiled for 5 min. Equal

amounts of protein were separated by SDS-PAGE and

transferred to polyvinylidene difluoride membranes at

150 mA for 5 h. Blots were blocked for 2 h in TBST

(10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.05%

Tween) containing 5% nonfat dried milk, probed with

anti inactive caspases-3 or -9 for 1 h at RT and then

with anti-mouse IgG-HRP. Blots were developed

using the enhanced chemiluminescence system (ECL,

Amersham, Arlington, IL).

2.6. Statistical analysis

All of the experiments were performed in triplicate

and repeated at least three times. Results are

expressed as meanGSE. Statistical comparisons

were made by Turkey test. P%0.05 was considered

significant.

3. Results

3.1. Pomolic acid induces apoptosis of HL-60

In a previous paper, we showed that pomolic acid

inhibited the proliferation and induced apoptosis of

leukemia cell lines [1]. In this paper, HL-60 cells were

used to investigate the intracellular pathways involved

in PA cytotoxic activity.

Use of the MTT assay revealed that PA inhibited

the viability of HL-60 cells in a dose-dependent

manner, reaching a plateau at 10 mg/ml after 48 h of

treatment (Fig. 1A). To further evaluate the nature of

this inhibition, we followed the kinetics of PA action

on cell morphology. Cells incubated with 25 mg/ml

PA and analyzed 4 and 8 h later by optical

microscopy, showed characteristic apoptotic mor-

phology with cell shrinkage, DNA fragmentation and

the presence of apoptotic bodies (results not shown).

Cell-cycle analysis was used to quantify this effect.

Appearance of the sub-diploid nuclei population,

indicative of DNA fragmentation, demonstrated

that PA-induced DNA fragmentation was dose- and

time-dependent (Fig. 1B). Although maximal induc-

tion of apoptosis was observed after 48 treatment with

25 mg/ml PA, at this concentration of triterpene, a

significant DNA fragmentation was already seen

around 12 h of treatment (Fig. 1C).

3.2. Pomolic acid induces caspase activation

To investigate the biochemical features that lead to

apoptosis induced by PA, we examined the effect of

this triterpene on activation of caspases-3 and -9. HL-

60 cells were treated with pomolic acid (25 mg/ml)

and samples collected at 4, 8 and 12 h were blotted

with mAbs that recognize the inactive form of the

caspases. Compatible with the DNA fragmentation

shown in Fig. 1c insert, after 8 h a reduction of

Fig. 1. Cytotoxic activity of pomolic acid (PA) is mediated by

apoptosis. (A) PA-induced decrease in cell viability is dose-

dependent. Cells were incubated with medium, PA (1, 5, 10, 25,

50, 100 mg/ml) or DMSO at the same concentrations carried by the

drug. After 48 h viability was assessed by MTT (5 mg/ml). (B) PA-

induced apoptosis is time and dose-dependent. Cells were treated

with different concentrations of pomolic acid (1, 10, 25 mg/ml) and

DNA-fragmentation was measured by FACS at several time points

(12, 24, 36 and 48 h) following incubation with PI (50 mg/ml). (C)

Short kinetics of DNA fragmentation. DNA fragmentation was

measured at 0, 2, 4, 8 and 12 h after treatment of cells with

25 mg/ml PA.

Fig. 2. PA-induced apoptosis is mediated by caspases activation.

(A) Kinetics of caspases activation. Western blot showing the

kinetics of pro-caspases-3 and -9 processing in cells treated with

25 mg/ml PA. Experiment representative of three others. (B) z-

VAD.fmk inhibited PA-induced apoptosis. Cells pre-treated with

100 mM z-VAD.fmk were incubated with 25 mg/ml PA for different

time intervals, stained with PI (50 mg/ml) and the sub-G1 peak was

measured by FACS. Results represent mean of 3 different

experiments.

J. Fernandes et al. / Cancer Letters 219 (2005) 49–5552

the inactive form of caspases -3 and -9 can already be

observed while after 12 h of treatment the bands

disappeared completely, indicating their full acti-

vation (Fig. 2A). Pre-treatment of cells for 1 h

with 100 mM z-VAD.fmk, a pan-caspase inhibitor

completely abrogated DNA fragmentation (Fig. 2B),

indicating the dependence of PA apoptotic activity on

caspase activation.

3.3. Pomolic acid induced a caspase-independent

loss of Djm

To evaluate the role of mitochondria in pomolic

acid-induced apoptosis, we investigated its ability to

induce alterations in mitochondrial potential. Cells

were treated with different concentrations of PA and

stained with DiOC6(3) at 0, 12, 24, 36 and 48 h after

treatment. The drop in fluorescence, indicative of

mitochondrial membrane potential disruption, was

measured by flow cytometry. The results demon-

strated that PA induced a dose- and time-dependent

loss of mitochondrial membrane potential (Fig. 3A).

Fig. 3. Pomolic acid (PA) causes loss of mitochondrial membrane

potential (Djm). (A) Kinetics of PA-triggered dissipation of Djm.

Cells treated with different concentrations of PA (1, 10, 25 mg/ml)

were incubated with 40 nM DiOC6(3) and the loss of the

fluorescence was measured at several time points (12, 24, 36 and

48 h). (B) Short kinetics of loss of Djm. Loss of Djm was measured

at 0, 2, 4, 8 and 12 h after treatment of cells with 25 mg/ml PA. (C)

Effect of z-VAD.fmk in PA-induced loss of Djm. Cells were pre-

treated with 100 mM z-VAD.fmk and Djm was measured by FACS

8 h after incubation with 25 mg/ml PA.

Fig. 4. CsA blocks PA-induced disruption of Djm and apoptosis.

(A) Effect of CsA on Djm. Cells were pre-treated for 1 h with 5 mM

CsA and Djm was measured by FACS after 8 h incubation with

25 mg/ml PA. (B) Effect of CsA on DNA fragmentation. Cells were

pre-treated for 1 h with 5 mM with CsA and after 8 h incubation with

25 mg/ml PA, were incubated with PI (50 mg/ml) and the sub-G1

peak was measured by FACS. (C) PA-induced Djm dissipation

precedes DNA fragmentation. After treatment with 25 mg/ml of PA

(0, 2, 4, 8 h) cells were stained with 50 mg/ml PI (DNA-

fragmentation) or 40 nM DiOC6(3) (Djm) and analyzed by FACS.

J. Fernandes et al. / Cancer Letters 219 (2005) 49–55 53

At 25 mg/ml of PA, uncoupling of mitochondria

membrane potential can already be observed after

4 h treatment (Fig. 3B).

To investigate whether the effect of PA on

mitochondrial potential was dependent on caspase

activation, we measured the disruption of MMP in

presence of zVAD-fmk (100 mM). No significant

alteration of Djm was observed (Fig. 3C), indicating

that the effect of PA on disruption of mitochondrial

membrane potential occurs in the absence of caspase

activation. Indeed, treatment of cells with 5 mM CsA,

a cyclophilin D ligand that inhibits mitochondria

potential uncoupling [20,21], blocked both the

PA-induced disruption of mitochondrial membrane

potential (Fig. 4A) and DNA fragmentation (Fig. 4B).

These data indicated mitochondria as the main target

of PA-triggered apoptosis. This hypothesis was

reinforced by kinetic studies (Fig. 4C) showing that

the uncoupling event preceded DNA fragmentation.

J. Fernandes et al. / Cancer Letters 219 (2005) 49–5554

Indeed, 4 h after pomolic treatment when no DNA

fragmentation was observed, the loss of Djm is

already 20% (Fig. 4C).

4. Discussion

Pomolic acid has recently been shown to inhibit the

growth and to induce apoptosis of leukemia cell lines,

including one that overexpresses Pgp, a multidrug

resistance protein [1]. Using HL-60 cells to character-

ize the molecular pathways involved in this effect, this

paper showed that apoptosis induced by PA is triggered

by alteration of mitochondria membrane potential.

In recent years, several studies performed in vitro

have shown that the cytotoxic effects of several types

of drugs on human tumor cell lines are mediated by

apoptosis [7,22]. In agreement with these obser-

vations, DNA-fragmentation analysis presented here

showed that PA-induced reduction of HL-60 cell

viability is also due to apoptosis.

Caspases represent the core to which most of the

death signals converge to induce apoptosis. Thus,

independent of which signal initiated the events that

lead to apoptosis, caspases play a fundamental role in

the apoptotic process [14,23]. Induction of apoptosis

by PA is also associated with activation of caspases,

as both initiator (caspase-9) and effector (caspase-3)

caspases are activated during this process. The

dependence of caspases activation on PA-triggered

apoptosis was evidenced by the complete abrogation

of apoptosis in cells treated with the broad-range

peptide inhibitor of caspases, zVAD-fmk.

Although apoptosis can be activated by different

signaling pathways, opening of the permeability

transition pore (PTP) and a subsequent drop in

mitochondrial membrane potential have been

suggested as the main mechanisms involved in

cytochrome c release and apoptosome formation

during apoptosis induced by a series of chemo-

therapeutic agents [7,8]. Assembly of the apoptosome,

a complex formed by cytochrome c, Apaf-1, procas-

pase-9 and dATP, triggers activation of caspases-9, -3

and others, leading to cleavage of specific substrates

and cell death [24]. Our data showed that loss of Djm

is an early event in PA-induced apoptosis. In fact,

disruption of mitochondrial membrane potential

started 4 h after drug exposition while DNA-

fragmentation was only observed 8 h after PA

treatment, indicating the involvement of the mito-

chondrial pathway on PA-induced death. A depen-

dency of the loss of mitochondrial potential on

caspase activity has also been reported [25]. However,

our data, showing that blockage of caspases activation

by zVAD-fmk, which completely abrogated PA-

triggered apoptosis, did not impair the Djm disrup-

tion, indicated that PA-induced loss of mitochondrial

potential is independent of caspase activation.

Investigation on the mechanism of PTP opening has

helped to elucidate its role on apoptosis. It has been

proposed that the basic structure of PTP is composed by

a voltage-dependent anion channel (VDAC), a matrix

isomerase cyclophilin-D (CyP-D) and an adenine

nucleotide translocator (ANT) [21,26]. Herein we

showed that inhibition of mitochondrial pore opening

by the ANT inhibitor, CsA, a ligand of cyclophilin-D,

prevented both mitochondrial membrane potential loss

and DNA fragmentation induced by PA. Thus, in HL-60

treated with PA, disruption of Djm occurs upstream of

apoptosome formation and effector caspases activation,

as already shown by studies involving other cytotoxic

drugs in different cell lines [27,28].

Due to the modulatory role of pre-mitochondrial

transduction signals on PTP opening and mitochon-

dria depolarization, substances whose activity is

mediated through mitochondria have been proposed

as important tools to overcome resistance mediated by

pre-mitochondrial signals [11]. Data presented in this

paper showing that (1) blockage of caspase activation

abrogates DNA-fragmentation but had no effect on

PA-induced loss of Djm and (2) treatment with CSA,

which prevented loss of MMP also blocked PA-

induced DNA-fragmentation, pointed to mitochondria

as the main target of PA. Thus, our data emphasizes

PA as a promising experimental cytotoxic agent to

develop new strategies directed to overcome apopto-

sis resistance due to defects in the intrinsic death

receptor-signaling pathway.

Acknowledgements

J.F is a recipient of a PhD fellowship from CNPq

and R.W. was a recipient of a MSc fellowship from

FAPESP. This research was supported by grants from

CNPq, FAPERJ, PRONEX (MCT), and FAPESP.

J. Fernandes et al. / Cancer Letters 219 (2005) 49–55 55

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