Differential induction of apoptosis by tumor necrosis factor-related apoptosis-inducing ligand in...

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Gynecologic Oncology 93 (2004) 594–604

Differential induction of apoptosis by tumor necrosis factor-related

apoptosis-inducing ligand in human ovarian carcinoma cells

Denis Lane, Andreanne Cartier, Sylvain L’Esperance, Marceline Cote,Claudine Rancourt, and Alain Piche*

Departement de Microbiologie et Infectiologie, Faculte de Medecine, Universite de Sherbrooke, 3001, Sherbrooke, Canada, J1H 5N1

Received 26 August 2003

Available online 10 May 2004

Abstract

Objectives. In this study, we examine the sensitivity of a panel of ovarian carcinoma cells, which includes four primary ovarian cancer cell

samples, and four normal ovarian epithelium samples to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We also examine

the intracellular regulation of TRAIL-mediated apoptosis.

Methods. The sensitivity to TRAIL was determined by short-term survival assays on seven ovarian carcinoma cell lines, four primary

samples of ovarian cancer, and four normal ovarian epithelium samples. We assessed the activation of the apoptotic pathway in TRAIL-

resistant and -sensitive tumor cells. The expression of TRAIL receptors was determined by flow cytometry. The protein expression of FADD,

XIAP, caspase-8, caspase-3, BAX, and c-FLIP were determined by immunoblot analyses.

Results. We show that ovarian cancer cells display variable sensitivity to TRAIL-induced apoptosis although most cell lines have similar

sensitivity to cisplatin. Normal ovarian epithelium samples were mostly sensitive to TRAIL. In sensitive cells, TRAIL induced caspase-8-

dependent apoptosis, which subsequently led to activation of caspase-3. Both sensitive and resistant cells expressed caspase-8, caspase-3,

FADD, XIAP, and c-FLIP at similar levels. A significant enhancement in cell death was observed in TRAIL-resistant cells when c-FLIPLlevels were downregulated by RNA interference.

Conclusions. These data suggest that sensitivity to TRAIL and chemotherapy does not necessarily correlate in human ovarian cancer cells.

Cancerous cells isolated from patients with ovarian cancer show variable sensitivity to TRAIL but most normal ovarian epithelial cells are

sensitive. In human ovarian cancer cells, c-FLIPL may participate to the regulation of the TRAIL signaling cascade.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Apoptosis; TRAIL; Caspase-8; Caspase-3; Ovarian carcinoma

Introduction cancer is less than 5% mainly because of the development of

Ovarian carcinoma is the leading cause of death from

gynecologic cancer in North America [1]. The vast majority

of the patients present with late stage disease [2]. Primary

cytoreductive surgery in combination with chemotherapy

(cisplatin/carboplatin and taxol) has produced higher initial

response rates in the range of 70% but this has not

consistently resulted in prolonged survival [3]. The long-

term survival for patients with advanced high-grade ovarian

0090-8258/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygyno.2004.03.029

* Corresponding author. Departement de Microbiologie et Infectiolo-

gie, Universite de Sherbrooke, 3001, 12ieme Avenue Nord, Sherbrooke,

Quebec, Canada, J1H 5N4. Fax: +1-819-564-5392.

E-mail address: [email protected] (A. Piche).

drug resistance [2].

Apoptosis plays a critical role in cellular homeostasis and

it prevents the development of tumor cells. Apoptosis is

defined by distinct morphological and biochemical changes

mediated by a family of caspases, which become activated

following apoptotic stimuli. There are two alternative path-

ways that initiate apoptosis: the intrinsic pathway is medi-

ated by the mitochondria and the extrinsic pathway is

mediated by death receptors on the cell surface. In both

pathways, activated effector caspases can cleave different

cellular substrates, leading to the biochemical and morpho-

logical hallmarks of apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand

(TRAIL) is a member of the TNF family that triggers rapid

D. Lane et al. / Gynecologic Oncology 93 (2004) 594–604 595

apoptosis in vitro and in vivo in various tumor cells without

marked toxicity on normal cells [4–8]. TRAIL binds to its

death receptors, TRAIL-R1 and -R2, which are expressed

widely in most human tissues [9,10]. In addition to R1 and

R2, TRAIL interacts with two decoy receptors (TRAIL-R3

and -R4) [11–13]. TRAIL-R1 and -R2 have a cytoplasmic

death domain (DD), but the decoy receptors have a

truncated intracellular domain or no intracellular domain

and thus are unable to transduce death signals. Upon

binding to TRAIL, activated TRAIL receptors (R1 and

R2) recruit the adaptor molecule FADD (Fas-associated

death domain) [14]. Then, FADD via its death effector

domain (DED) recruits pro-caspase-8 to form the DISC

(death-inducing signaling complex) [15,16]. When

recruited to the DISC, pro-caspases-8 is activated through

a series of proteolytic cleavage steps. The mechanism of

activation of pro-caspases generally involves the cleavage

within the proteolytic caspase domain resulting in active

caspase comprised of large (a) and small (h) subunits as

well as removal of the N-terminal domain, subsequently

activating downstream effector caspases such as caspase-3

leading to apoptosis.

Preclinical studies of recombinant TRAIL in animal

models have demonstrated potent antitumor effect [17].

Based on these data, TRAIL represents an interesting

potential therapeutic molecule for ovarian cancer. However,

not all tumor cell lines respond to TRAIL. The lack of

response has been associated with several factors, including

overexpression of decoy receptors [21], overexpression of

intracellular protein c-FLIP [6,22], loss of caspase-8 expres-

sion [23,24], activation of transcription factor NF-nB [25],

and alteration in gene expression of Bcl-2 family members

[27–29]. Previous studies on ovarian cancer cell lines have

focused on the enhancement of TRAIL-mediated apoptosis

by chemotherapeutic agents [18–20]. In these studies,

ovarian cancer cell lines displayed variable sensitivity to

TRAIL-induced cytotoxicity that was enhanced by the

combination with chemotherapy. However, the sensitivity

of primary ovarian carcinoma cells and normal ovarian

epithelial cells has not been described. Furthermore, major

determinants of TRAIL resistance in ovarian cancer cells

have not been clearly identified.

In this study, we demonstrate that TRAIL induces

apoptosis in only a limited number of ovarian cancer cell

lines, whereas primary ovarian carcinoma cells display

variable sensitivity to TRAIL. The lack of response to

TRAIL was associated with a lack of activation of the

apoptotic cascade although the downstream common path-

way remains functional in resistant cells. Our data also

demonstrate that the ratios of death and decoy receptors

do not correlate with TRAIL resistance. Most interestingly,

we show that in the TRAIL-sensitive cell lines, TRAIL

enhances pro-caspase-8 and pro-caspase-3 activation and

promote apoptosis, while the expression of the full-length

55 kDa c-FLIPL protein may regulate TRAIL-induced

apoptosis.

Materials and methods

Reagents

Recombinant human TRAIL was purchased from Pepro-

Tech, inc. (Rocky Hill, NJ). The tetrapeptide caspase

inhibitors, z-DEVD-fmk and z-IETD-fmk, were obtained

from R&D Systems (Minneapolis, MN) and prepared as 20

mM stocks in DMSO and stored in aliquots at � 20jC until

further use. cis-Diamminedichloroplatinum (cisplatin) and

staurosporine were purchased from Sigma Canada Ltd

(Oakville, Ontario, Canada). Anti-human caspase-8 antibod-

ies were purchased from Cell Signalling (Beverly, MA).

Caspase-3 antibodies were obtained from BD Biosciences

(Mississauga, Ontario, Canada). Anti-TRAIL-R1, -R2, -R3,

R4, and anti-XIAP were purchased from R&D Systems.

Anti-c-FLIPL and - c-FLIPS were purchased from Calbio-

chem (La Jolla, CA). FADD antibodies were purchased

from Chemicon International (Temecula, CA). Anti-Bcl-2

antibody was obtained from DAKO, antitubulin from Sig-

ma, and anti-Bax from Santa Cruz Biotechnology Inc (Santa

Cruz, CA). HRP-conjugated anti-mouse, rabbit, or goat

antibodies were purchased from Jackson Immuno Research

Laboratories (West Grove, PA).

Cell culture

The SKOV3 and OVCAR3 cell lines were obtained

from the American Type Culture Collection (Manassas,

VA). The SKOV3.ip1 human epithelial ovarian cancer cell

line was kindly provided by J. Price (MD Anderson Cancer

Center, Houston, TX). The SKOV3.ip1 cell line was

established from ascites that developed in a nu/nu mouse

given an intraperitoneal injection of SKOV3 cells and

showed to be more aggressive [26]. The human ovarian

carcinoma cell lines UCI-101, CAOV3, PA-1, and OV-4

were obtained from D.T. Curiel (Gene Therapy Center,

University of Alabama at Birmingham, AL). Primary cul-

tures were established from ovarian tumors. COV2 and

COV17 were derived from ascitic fluids obtained at the

time of paracenthesis from patients with either primary or

recurrent stage III serous ovarian carcinoma diseases.

OVC116 and OVC118 were isolated from ascites of

patients with stage III Mullerian carcinosarcoma and mu-

cinous adenocarcinoma, respectively. All samples were

supplied by Dr. Paul Bessette at Centre Hospitalier Uni-

versitaire de Sherbrooke. Ascites fluids were aliquoted into

10 ml of samples and red blood cells were lysed. Cells were

centrifuged though a Ficoll–Hypaque gradient. Viable cells

were removed from the interface and plated in RPMI-1640

containing low serum concentrations (FBS, 2%) and insulin

(10 Ag/ml) (Sigma Canada Ltd). All the cell lines, except

OVCAR3 and cancerous cells isolated from ascetic fluids,

were maintained at 37jC in a humidified incubator con-

taining 5% CO2 in DMEM/F12 (BioMedia, Drummond-

ville, Quebec, Canada) supplemented with 10% heat-

Fig. 1. Sensitivity of human ovarian carcinoma cells and normal ovarian

epithelium samples to the cytotoxic action of TRAIL as determined by XTT

assay. Seven ovarian carcinoma cell lines (A), four primary samples of

ovarian cancer (B), and four normal ovarian epithelium samples (C) were

seeded (confluence 60–70%) into 96-well plates and treated for 48 h with

recombinant human TRAIL or growth medium as control. Experiments

were repeated three times and data are expressed as the mean of triplicate

samples: bars, F SE.

D. Lane et al. / Gynecologic Oncology 93 (2004) 594–604596

inactivated fetal bovine serum (FBS) (BioMedia) and anti-

biotics. The OVCAR3 cell line was maintained in RPMI-

1640 (BioMedia) with 20% FBS and insulin. COV2 were

maintained in DMEM/F12 with 20% FBS while COV17,

OVC116, and OVC118 were maintained in Medium 199/

MCDB 105 (1:1) (Wysent, St. Bruno, Quebec, Canada)

supplemented with 10% FBS. The normal ovarian epithelial

cells were generated from patients undergoing oophorecto-

mies from various reasons (other than cancer) and were

provided by Dr. Paul Bessette. Cells were scraped from the

outer surface of the ovaries and plated into Medium 199/

MCDB 105 (1:1) containing 10% FBS and maintained in

the same medium. Immunohistochemical analysis was

performed on normal ovarian cells, and positive staining

for cytokeratins 8 and 18 confirmed the epithelial origin.

Normal nonimmortalized ovarian epithelial cells were used

between passages 4 and 8.

Apoptosis assays

Nuclear staining obtained with Hoechst 33258 (10 Ag/ml) (Sigma) was viewed and photographed using an Olym-

pus fluorescence microscope. Cells with typical apoptotic

nuclear features were identified and counted in 10 randomly

selected fields on numbered slides. Caspase-3 fluorogenic

protease assay was performed according the manufacturer’s

protocol (R&D Systems). In brief, 3 � 106 cells were lysed

in 250 Al of cell lysis buffer, and total cell lysates were

incubated with 50 AM of DEVD-AFC substrate for 1 h.

Caspase-3 activity was measured on a Versa Fluor fluorom-

eter (BioRad, Hercules, CA). Protein concentration of the

lysates was measured with BioRad protein assay kit accord-

ing to the manufacturer’s recommendations.

Immunoblot analysis and immunoprecipitation

Whole-cell extracts, obtained at various times after the

addition of TRAIL (200 ng/ml), were separated by 12%

SDS-PAGE gels. Proteins were transferred to PVDF mem-

branes (Amersham Pharmacia Biotech Inc., Baie d’Urfe,

Quebec, Canada) by electroblotting, and immunoblot anal-

ysis was performed as previously described [30]. All pri-

mary antibodies were incubated overnight at 4jC. Proteinswere visualized by enhanced chemiluminescence (Amer-

sham Pharmacia Biotech Inc.).

Cytotoxicity assays

Cytotoxicity and cell survival were determined by the

XTT assay. Briefly, cells were plated at 15,000–20,000

cells/well in 96-well plates. The next day, cells (confluence,

60–70%) were treated with increasing concentrations of

human TRAIL as indicated and incubated for 48 h. In some

experiments, synthetic caspase inhibitors (25 AM z-DEDV-

fmk, or z-IETD-fmk) were added 1 h before the addition of

200 ng/ml of TRAIL. At the termination of the experiment,


the culture media were removed and a mixture of PBS and

fresh media (without phenol red) containing phenazine

methosulfate and XTT (Sigma) was added for 30 m. The

absorbance of each well was determined using a microplate

reader at 450 nm (TecanSunrise, Research Triangle Park,

NC). The percentage of cell survival was defined as the

relative absorbance of untreated versus treated cells. All

assays were performed in triplicate and repeated three

times. For these assays, primary cultures of ovarian cancer

cells were used at passage 10–30 depending on the sample,

and surface epithelial ovarian cells were used at passage

V 8.

Flow cytometry for TRAIL receptor expression

Each ovarian cancer cell lines were incubated with the

following unlabeled primary antibodies for 1 h at 4jC:human anti-TRAIL-R1, -R2, -R3, and -R4 (R&D Sys-

tems). The isotypic control antibody was a normal goat

IgG (R&D Systems). After three washes with PBS, cells

were incubated with FITC-conjugated donkey anti-goat

antibody (Jackson ImmunoResearch) for 45 min at 4jC.Cells were analyzed immediately using a FAC-scan (Beck-

ton Dickinson).

Fig. 2. Activation of caspase-8 and caspase-3 in TRAIL-treated human ovarian c

TRAIL treatment. TRAIL-resistant cell lines, SKOV3.ip1 and COV2, and the TRA

indicated times. Caspase-8 and caspase-3 activations were determined by Western

55,000 zymogen. Cleavage of caspase-3 is detected by decrease of the inactive M

fluorometric assay. Two TRAIL-sensitive (CAOV3, OVCAR3) and two TRAIL-r

lysates were analyzed for active caspase-3 using a fluorogenic substrate. The r

expressed as percentage of untreated controls.

Transfection with siRNA oligonucleotides

The siRNA oligonucleotide duplexes were synthesized

with the Silencerk siRNA construction kit from Ambion

according to the manufacturer’s protocol. The antisense

strand of the siRNAs corresponded to AA(N)19 sequences

in the coding region of the c-FLIPL mRNA. The GC

content of the duplexes was kept within the 40–55%.

The siRNA oligonucleotides were specific for c-FLIPL:

(5V-AAGACACATACAAGATGAAGACCTGTCTC-3V).The control GAPDH siRNA was synthesized using the

primers supplied by Ambion. The fluorescein-labeled lu-

ciferase GL2 duplex, which was used to assess the efficacy

of transfection, was purchased from Dharmacon Research,

Inc. The SKOV3.ip1 cells (6 � 104) were seeded in 6-

well plates and allowed to adhere for 24 h. The cells were

transfected with a mixture containing Oligofectamine

(Invitrogen Life Technologies), optiMEM (Gibco), and of

siRNA oligonucleotides (50 AM) according to the protocol

suggested by the manufacturer. The RNA complex was

then added to the media covering 6-well plates containing

SKOV3.ip1 cells. The cells were incubated for 4 h at 37jCin a CO2 incubator and medium containing FBS was then

added. For Western blotting, the cells were lysed 8–120

arcinoma cell lines. (A) Kinetic of caspase-8 and caspase-3 cleavage after

IL-sensitive cell line CAOV3 were treated with TRAIL (200 ng/ml) for the

blot. Caspase-8 produces an Mr 18,000 active submit from an inactive Mr

r 32,000 form. (B) Activation of caspase-3 after TRAIL treatment using a

esistant cell lines were treated with 200 ng/ml TRAIL for various time, and

elative fluorescent units were normalized for protein content. Results are


h post-transfection depending on the experiment, and total

cell lysates were analyzed by immunoblot.

Fig. 3. Cisplatin and staurosporine induce apoptosis in both TRAIL-

sensitive (CAOV3, OVAR3) and -resistant (SKOV3.ip1, COV2) ovarian

cancer cell lines. (A) The cells were left untreated or were treated for 24

h with equitoxic concentrations of cisplatin (10 Ag/ml) or staurosporine (50

ng/ml). Cells were stained with Hoechst 33258 and quantification of the

percentage of apoptotic nuclei was established by counting apoptotic nuclei

in 10 independent fields (mean F SE; n = 2). (B) Caspase-3 cleavage in

TRAIL-sensitive and -resistant cell lines treated with cisplatin was

measured in a fluorometric analysis using DEVD-AFC as substrate as

suggested by the manufacturer’s recommendations. (C) Various ovarian

cancer cells were treated with increasing concentration of cisplatin, as

indicated, and cell viability was measured by XTT assay 72 h later. The data

represent cell survival as a percentage of untreated cells. Data points show

the mean F SE for three independent experiments.

Results

Human ovarian cancer cells and normal ovarian cells

display variable sensitivity to TRAIL

We first examined the cytotoxic effects of TRAIL on a

panel of human ovarian cancer cells and normal primary

ovarian cells using a standard XTT cell survival assay. Only

two (OVCAR3, CAOV3) out of seven ovarian cancer cell

lines were efficiently killed by TRAIL in a dose-dependent

manner (Fig. 1A). In contrast, the remaining cell lines were

either partially resistant (40–80% cell death; PA-1, SKOV3,

SKOV3.ip1) or resistant ( < 20% cell death; UCI-101, OV-4)

to TRAIL at concentrations up to 500 ng/ml (Fig. 1A).

Analysis of TRAIL-induced cytotoxicity in primary ovarian

carcinoma cells showed variable sensitivity. Two primary

samples of ovarian cancer isolated from ascites of women

with stage III ovarian cancer (COV2 and COV17) were

highly resistant to TRAIL-induced cell death (Fig. 1B),

whereas the remaining two were sensitive (OVC116 and

OVC118). Of the four normal ovarian epithelium samples

tested, all but one displayed > 50% cell death in response to

TRAIL (Fig. 1C). These results demonstrated that most of

the ovarian cancer cells were resistant ( < 50% cell death) to

TRAIL and raised the possibility that molecular alterations

in the TRAIL signaling cascade leading to resistance may be

a common finding in human ovarian cancer cells. In

contrast, however, normal ovarian epithelium samples were,

for the most part, sensitive to TRAIL-induced cytotoxicity.

Activation of caspase-8 and caspase-3 in TRAIL-induced

cell death

To determine whether TRAIL-mediated cell death in

ovarian cancer cells occurred by activation of the apoptotic

cascade, TRAIL-sensitive (CAOV3) and -resistant cell lines

(SKOV3 ip1, COV2) were treated with 200 ng/ml of

TRAIL (little additional killing was seen with higher doses

for most cell lines) for various periods of time, and cell

lysates were analyzed by immunoblot assay for evidence of

caspase-8 and -3 activation. In TRAIL-sensitive CAOV3

cells, activation of caspase-8 and -3 was detected within 2

h after TRAIL addition and increased with time for up to 8 h

(Fig. 2A). Activation of caspase-8 and -3 was also detected

in the TRAIL-sensitive OVCAR3 cell line (data not shown).

Furthermore, caspase-8 and caspase-3 activation in CAOV3

and OVCAR3 was readily blocked in the presence of z-

IETD-fmk (25 AM) and z-DEVD-fmk (25 AM), two inhib-

itors specific for caspase-8 and caspase-3, respectively

(results not shown). In contrast, no significant activation

of caspase-8 and caspase-3 was observed, even after 8 h, in

TRAIL-resistant SKOV3 ip1 and COV2 cells (Fig. 2A).

Similar results were obtained in other resistant cell lines

such as UCI-101, PA-1, and OV-4 (results not shown). To

further demonstrate the differential activation of caspase-3

between TRAIL-sensitive and -resistant cells, caspase-3

activity was measured from cell extracts by analyzing the

cleavage of the synthetic substrate DEVD-AFC. Release of


AFC fluorochrome from the peptide substrate was mea-

sured, allowing quantification of the amount of caspase-3

activity in the extracts (Fig. 2B). Consistent with our

previous results, caspase-3 activation was significantly

higher in TRAIL-sensitive cell lines CAOV3 and OVCAR3

than in resistant cell lines. These results demonstrate that

TRAIL induces apoptosis through activation of the caspase

cascade in sensitive but not in resistant ovarian cancer cells.

Furthermore, the fact that activation of caspase-3 is blocked

by the addition of inhibitors caspase-8 suggests that resis-

tance may be associated with a defect located upstream of

caspase-3 in the TRAIL signaling cascade.

The final common pathway of apoptosis is functional in

TRAIL-resistant cell lines

Two distinct pathways (the death receptor pathway and

the mitochondrial pathway) can lead to effector caspase

activation and ultimately to apoptotic cell death (final

common pathway). Therefore, alterations that disrupt apo-

ptosis downstream of the mitochondria could lead to resis-

tance to agents that activate either one the upstream

pathways. To determine whether the lack of response to

TRAIL was associated with a nonfunctional downstream

pathway, TRAIL-sensitive and -resistant ovarian carcinoma

Fig. 4. Surface analysis of death and decoy TRAIL receptors. Flow cytometric anal

and COV2. Solid line histograms represent staining by specific antibodies for TR

control antibody.

cell lines were treated with cisplatin or staurosporine, which

primarily activate the mitochondrial pathway. Hoechst

33222 staining was used to visualize the extent of nuclear

fragmentation before and following treatment. Quantifica-

tion of nuclear changes showed that after a 24-h drug

treatment, cisplatin- or staurosporine-mediated apoptosis

were observed in all cell line (ranging from 10% to 20%

of apoptotic nuclei) when compared to untreated cells (mean

apoptotic nuclei < 5%) (Fig. 3A). More importantly, the

extent of apoptosis was similar between TRAIL-sensitive

(CAOV3, OVCAR3) and TRAIL-resistant cell lines (SKO-

V3.ip1) or TRAIL-resistant cancerous cells derived from

ascetic fluids (COV2). We also assessed caspase-3 activity

in these cell lines with or without exposure to cisplatin. As

shown in Fig. 3B, caspase-3 activity was enhanced by

cisplatin treatment in both TRAIL-sensitive and -resistant

cell lines.

To further characterize the general response to chemo-

therapy of cancerous cells isolated from ascitic fluids, the

cells were exposed to different doses of cisplatin. All cells

tested displayed similar responses to cisplatin (Fig. 3C)

whether or not they were resistant to TRAIL. Taken togeth-

er, these results suggest that the downstream common

pathway of apoptosis is functional in both TRAIL-resistant

and TRAIL-sensitive cell types.

ysis of TRAIL-R1, -R2, -R3, and -R4 expression on OVCAR3, SKOV3.ip1,

AIL receptors, and dotted line histograms represent staining with isotypic

Fig. 5. Endogenous expression of TRAIL signaling molecules in human

ovarian carcinoma cell lines. Expression of caspase-8, caspase-3, c-FLIPL,

c-FLIPS, FADD, XIAP, and BAX in TRAIL-sensitive CAOV3 and

OVCAR3, and TRAIL-resistant SKOV3.ip1, UCI-101, and COV2 ovarian

cancer cell lines. Lysates were separated on SDS-PAGE and subjected to

Western blotting. Tubulin was used as control to ensure equal loading.


TRAIL sensitivity does not correlate with surface expression

of TRAIL receptors R1 and R2 or with decoy receptors R3

and R4

TRAIL can potentially interact with four distinct recep-

tors at the surface of the cell: death receptors TRAIL-R1, -

R2, and decoy receptors TRAIL-R3, -R4. Because TRAIL-

R3 and -R4 bind to TRAIL without directly signaling for

cell death, potential mechanisms of resistance include the

overexpression of these decoy receptors. Also, cell-surface

downregulation of TRAIL-signaling receptors R1 and R2

may contribute to resistance. To test these hypotheses, we

performed flow cytometry analysis for cell-surface expres-

sion of TRAIL receptors in both sensitive and resistant cell

lines. As shown in Fig. 4, death and decoy receptors were

expressed in all cell lines, albeit at various degrees, but

more importantly no consistent differences in receptor

expression were observed between TRAIL-sensitive and

-resistant cells. There results demonstrated that TRAIL

resistance in ovarian cancer cells cannot be explained by

alterations in TRAIL-receptor expression but suggest that

resistance is more likely to be associated with alterations

of intracellular TRAIL-signaling molecules located up-

stream of caspase-3.

Intracellular levels of TRAIL-signaling molecules in

sensitive and resistant cell lines

Several intracellular proteins are capable of modulating

the cellular response to TRAIL, including caspase-8 [23,24],

c-FLIP [31–35], FADD [36], and XIAP [37]. We thus

examined the expression of intracellular levels of various

TRAIL-signaling molecules by immunoblotting to deter-

mine whether low levels or absence of expression of these

proteins was associated with resistance to TRAIL. As shown

in Fig. 5, TRAIL-sensitive and -resistant cells displayed

similar intracellular levels of the various molecules tested

suggesting that TRAIL resistance in human ovarian carci-

noma cells is not associated with altered expression of any

of the of the proteins tested. Interestingly, untreated CaOV3

cells expressed a truncated 43 kDa c-FLIPL form in addition

to the full-length 55 kDa form suggesting that c-FLIPL may

be processed in the absence of TRAIL in these cells. In

OVCAR3 cells, which are also TRAIL-sensitive, only the

full-length c-FLIPL protein was detected by immunoblot in

untreated cells.

Expression of XIAP and c-FLIP in TRAIL-treated ovarian

cancer cells

The X-linked IAP (XIAP) is an intracellular anti-apopto-

tic protein believed to be an important determinant of

chemosensitivity in ovarian cancer [6]. XIAP is a direct

inhibitor of caspase-3, caspase-7, and caspase-9 [38].

TRAIL-sensitive and -resistant cell lines were treated with

TRAIL for various period of time, and cell lysates were

analyzed by immunoblot assay for expression of XIAP. In

TRAIL-sensitive CAOV3 cells, there was a time-dependent

endogenous cleavage of XIAP as demonstrated by depletion

of the full-length 53 kDa XIAP protein over time and the

concomitant appearance of a 30 kDa fragment that reacts

with an anti-XIAP antibody specific for an epitope found in

the BIR2 region (Fig. 6). This phenomenon was suppressed

by the presence of caspase-8 (z-IETD-fmk, 25 AM) and

caspase-3 (z-DEVD-fmk, 25 AM) inhibitors (data not

shown). In contrast, the expression of XIAP did not vary

over time in the TRAIL-resistant cell lines SKOV3 ip1 and

COV2 upon treatment will TRAIL, suggesting that XIAP

cleavage is a direct result of the caspase cascade activation

in sensitive cells.

Using a similar approach, we evaluated whether the

expression of cFLIPL and c-FLIPS differed in TRAIL-sensi-

tive and -resistant cells upon treatment with TRAIL. C-

FLIPL and c-FLIPS expression remained unchanged over

time in all cell lines tested (Fig. 6). In contrast, c-FLIPL was

partially processed in CAOV3-sensitive cells as demonstrat-

ed by the detection of full-length 55 and 43 kDa forms in the

absence of TRAIL. Upon treatment with TRAIL (200 ng/ml)

in these cells, the expression of the full-length, uncleaved 55

kDa c-FLIPL protein increased in a time-dependent manner,

whereas the 43/41 kDa c-FLIPL cleavage products were

Fig. 6. Effects of TRAIL treatment on expression of XIAP and c-FLIP in ovarian carcinoma cell lines. The TRAIL-sensitive cell line CAOV3 and TRAIL-

resistant SKOV3.ip1 and COV2 cell lines were treated with TRAIL (200 ng/ml) for the times indicated. Equal amounts of protein prepared from each time-

point were loaded and analyzed by Western blotting for expression of XIAP, c-FLIPL, and c-FLIPS.


depleted correspondingly. After 24 h of TRAIL treatment,

the p43/41 c-FLIPL became completely undetectable by

Western blot in CAOV3 (data not shown). This phenomenon

is probably specific to CAOV3 as we did not observe c-

FLIPL processing in the three other sensitive cell lines

(OVCAR3, OVC116, and OVC118). The full-length c-

FLIPL remained unchanged over time in the presence of

Fig. 7. Effects of c-FLIPL downregulation on TRAIL-induced cell death.

SKOV3.ip1 cells were cultured for 24 h in the presence of absence of

siRNAs and then for an additional 48 h with or without TRAIL (200 ng/

ml). Cells were collected for Western blotting of c-FLIPL (A) and cell death

assessments by XTT assay (B). Mean F SE (n = 3).

TRAIL (200 ng/ml). Similar results were seen in TRAIL-

resistant SKOV3 ip1 and COV2 cells even after 24 h.

To further assess the role of c-FLIPL in ovarian cancer

cells, the influence of c-FLIPL downregulation by RNA

interference before TRAIL treatment was assessed. Whereas

transfection of the c-FLIPL siRNAs in the TRAIL-resistant

cell line SKOV3.ip1 alone had no detectable effect (data not

shown), a marked increase in cell death was evident when

TRAIL was subsequently added (Fig. 7B). Transfection of

SKOV3.ip1 with control GAPDH siRNAs had no signifi-

cant effect on TRAIL cytotoxicity. These findings support

the fact that c-FLIPL may be involved, at least in some

ovarian cancer cell lines, in regulating TRAIL-induced

apoptosis.

Discussion

TRAIL has been shown to be a potent inducer of

apoptosis in different cellular system; however, not all

cancer cells undergo apoptosis when treated with TRAIL.

Understanding the molecular signaling events that control

the response to TRAIL in tumor cells is important given the

therapeutic potential of this molecule [7,8,39–41]. In this

study, we examined a panel of human ovarian cancer cells,

which included cancerous cells isolated from ascetic fluids

of women with ovarian cancer, for their susceptibility to

TRAIL-induced apoptosis. We showed that TRAIL effi-

ciently (>80% cell death) killed only two out of seven

human ovarian cancer cell lines tested, which is consistent

with previous reports [5,18,19,43,45]. Cancerous cells iso-

lated from woman with ovarian cancer also displayed

variable sensitivity to TRAIL. In contrast, most of the

normal ovarian epithelial cells were killed (>50% cell death)

by TRAIL. Although initial reports on TRAIL suggested a

specificity for tumor cells [9,10], the TRAIL cytotoxicity


observed in normal primary ovarian epithelial cells is

consistent with recent data demonstrating that TRAIL is

capable of inducing efficient apoptosis in normal human

keratinocytes [42], hepatocytes [43], and prostate epithelial

cells [44].

Altered expression of the death receptors TRAIL-R1

and -R2 has been previously associated with resistance to

TRAIL in some tumor cells. Several studies have demon-

strated high expression levels of death receptors in TRAIL-

sensitive malignant gliomas [5,46,47]. In contrast, low or

undetectable levels of TRAIL-R1 have been found in

some, but not all, resistant cancer cells [21]. Our results,

however, demonstrated a lack of correlation between

expression levels of death and decoy receptors, and the

sensitivity of human ovarian cancer cells to TRAIL. We

found that ovarian cancer cells primarily express TRAIL-

R2 whereas TRAIL-R1 was expressed at much lower

levels at the cell surface, suggesting that TRAIL-induced

apoptosis may occur primarily through TRAIL-R2 activa-

tion. In addition, expression of TRAIL-2 was similar in

TRAIL-sensitive and -resistant cell lines as demonstrated

by FACS analysis. We cannot rule out, however, that

mutations in the death domain of TRAIL-R2, which would

result in loss of apoptotic function, were present in

resistant cells. This would have account for their lack of

sensitivity to TRAIL. Naturally occurring mutants of DD-

containing receptors have been described, but it appears to

be an infrequent phenomenon [48]. Another alternative that

may have accounted for resistance to TRAIL is the auto-

crine production of a soluble decoy receptor by resistant

cells. Osteoprotegerin (OPG) is a member of the TNF

receptor family, which can inhibit TRAIL-induced apopto-

sis in prostate cancer and myeloma cell lines when TRAIL

is used at relatively low dose (50 ng/ml) [54,55]. In

addition, condition media only produced a modest inhibi-

tion of TRAIL-induced apoptosis [54]. Although we can-

not rule out the possibility that resistant ovarian cancer

cells release OPG in the medium, the fact that concen-

trations of TRAIL up to 500 ng/ml did not overcome

resistance makes it unlikely that the release of OPG, if any

in these ovarian cancer cells, play a significant role in

TRAIL resistance.

We have also demonstrated that TRAIL-induced cell

death is characterized by the activation of caspase-8 and

caspase-3 in sensitive cells whereas TRAIL-resistant cells

showed little or no evidence of caspases activation. Inhibi-

tion of caspase-8 or caspase-3 in sensitive cells completely

abrogated TRAIL killing, indicating that the caspase path-

way is primarily activated in TRAIL-induced apoptosis.

Furthermore, treatment of TRAIL-resistant cells with anti-

cancer drugs resulted in activation of caspase-3 and mor-

phological features of cellular apoptosis suggesting that the

mitochondrial pathway of apoptosis is functional in ovarian

carcinoma cells. It would also suggest that regulation of

TRAIL-induced apoptosis in these cells occurs upstream of

caspase-3 in the death receptor pathway.

When ectopically overexpressed, XIAP has been shown

to effectively inhibit cellular apoptosis by direct inhibition

of caspase-3 [49]. Downregulation of XIAP by antisenses

also results in activation of the apoptotic cascade in chemo-

resistant ovarian cancer cells [50]. Here, we provide evi-

dence that during TRAIL-induced apoptosis, XIAP is

cleaved into at least one fragment of 30 kDa (Fig. 6). In

support of this observation, a recent study also showed that

XIAP cleavage occurs in Jurkat anti-FAS antibody-treated

cells [38]. Cleavage of XIAP produces two 30 kDa frag-

ments with reduced ability to inhibit caspase-3. XIAP is

cleaved in vitro by various caspases, including caspase-8 and

caspase-3 [38]. It is thus not surprising that TRAIL-induced

activation of the death receptor pathway of apoptosis results

in cleavage of XIAP. In fact, significant cleavage of XIAP

occurs after the activation of caspase-8 and caspase-3, a

phenomenon suppressed by caspase-8 and -3 inhibitors.

This finding is consistent with the fact that caspase-3

directly cleaves XIAP. The lack of XIAP cleavage in the

absence of caspase-8 and caspase-3 activation in TRAIL-

resistant cells is consistent with these data. However,

whether the cleavage of XIAP seen in sensitive cells is

simply the results of the TRAIL-induced activation of

caspases or whether XIAP cleavage contributes directly to

the sensitivity to TRAIL cannot be determined based on our

data.

c-FLIP, a protease-deficient caspase-8 homolog, is

expressed mainly in a long (c-FLIPL) and a short (c-FLIPS)

splice form. The latter contains only two tandems of DEDs

and inhibits procaspase-8 activation in the DISC [51]. In

contrast, c-FLIPL shares extensive homology with procas-

pase-8, with a C-terminal domain that is highly homolo-

gous to the procaspase-8 protease domain yet enzy-

matically inactive due to the lack of key active site

residues. The role of c-FLIPL in TRAIL-induced apoptosis

is controversial. Gene transfer-mediated overexpression of

c-FLIPL has been associated with inhibition of TRAIL-

induced apoptosis [51]. However, in cell lines that have

been examined quantitatively, the level of endogenous c-

FLIPL is merely 1% that of endogenous procaspase-

8 [52,53]. It is unclear what role c-FLIPL plays in these

cells. Furthermore, pro-apoptotic activities of c-FLIPL have

also been described. Overexpression of c-FLIPL in HEK

293T cells causes efficient cell death [32–35,51]. Our

results demonstrated that expression levels of endogenous

c-FLIPL were very similar in all cell lines tested with the

exception perhaps of COV2, which expressed lower levels

(Fig. 5). These results indicate that c-FLIPL levels do not

correlate with TRAIL sensitivity. Processing of c-FLIPLoccurs very early during TRAIL-induced apoptosis [6].

Cleavage of c-FLIPL, which depends on caspase-8, gen-

erates a fragment of 43 kDa. Interestingly, we found that

the TRAIL-sensitive CAOV3 cell line expressed, in the

absence of TRAIL, the cleaved c-FLIPL fragment as well

as the full-length form (Figs. 5 and 6), suggesting that c-

FLIPL can be processed in the absence of activated


caspase-8 in these cells. During TRAIL-mediated apoptosis

in CaOV3, the expression of the full-length 55 kDa c-

FLIPL protein increased in a time-dependent manner

whereas the 43 kDa c-FLIPL cleavage products were

depleted correspondingly. A comparison of the cleavage

kinetics among pro-caspase-8, pro-caspase-3, and c-FLIPL(Figs. 2 and 6) showed that in sensitive cell lines, process-

ing of pro-caspase-8 and pro-caspase-3 preceded that of

accumulation of full-length c-FLIPL. Accumulation of the

full-length c-FLIPL protein is somehow surprising in the

presence of activated caspase-8 and caspase-3 and the

reason is unknown but may be related to inhibition of

ubiquitination and subsequent degradation of c-FLIPL. The

accumulation of full-length c-FLIPL in the TRAIL-sensi-

tive cell line CAOV3 is consistent with recent data

suggesting that the full-length protein may promote cas-

pase-8/10 activation when part of an heterocomplex with

these caspases [35,56]. Although interesting, this observa-

tion was limited to CAOV3. The three other TRAIL-

sensitive cell lines tested showed no evidence of c-FLIPLprocessing/accumulation (data not shown) suggesting that

c-FLIPL cleavage does not necessarily correlate with

TRAIL sensitivity. In contrast to these results, downregu-

lation of c-FLIPL levels by RNA interference in TRAIL-

resistant cells increased the toxicity of that cytokine

supporting the idea that c-FLIPL may participate to the

regulation of the TRAIL signaling cascade, at least in some

ovarian cancer cell lines. Thus, the role of c-FLIPL in

TRAIL-sensitive and resistant cells appears distinct.

In contrast to c-FLIPL, c-FLIPS has been shown to be a

dedicated apoptosis inhibitor preventing the first cleavage of

procaspase-8 in the DISC [51]. Expression levels of endog-

enous c-FLIPS were comparable in CAOV3, SKOV3.ip1,

and COV2 (Fig. 5). Similarly, the levels of c-FLIPS protein

remained unchanged during treatment with TRAIL not

cleaved during TRAIL-mediated apoptosis.

In conclusion, our results indicate that human ovarian

carcinoma cells display a variable sensitivity to TRAIL.

Normal ovarian epithelium samples however are mostly

sensitive to TRAIL, a finding that may be important from

a therapeutic standpoint. In ovarian cancer cells, c-FLIPLappears to be a determinant for TRAIL sensitivity. Further

studies are needed to elucidate the molecular mechanisms

leading to TRAIL resistance in human ovarian carcinoma

cells.

Acknowledgments

This work was supported by the National Cancer

Institute of Canada with funds from the Terry Fox Run

and a grant from Valorisation Recherche Quebec through

the Montreal Centre for Experimental Therapeutics in

Cancer. C.R. is supported by the National Cancer Institute

of Canada with funds from the Canadian Cancer Society and

the Reseau Cancer FRSQ Axe Sein/Ovaire.

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