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Endocrinology Vol. 144, No. 2 623-630
Copyright © 2003 by The Endocrine Society

ARTICLE

Resistance of Human Ovarian Cancer Cells to Tumor Necrosis Factor {alpha} Is a Consequence of Nuclear Factor {kappa}B-Mediated Induction of Fas-Associated Death Domain-Like Interleukin-1ß-Converting Enzyme-Like Inhibitory Protein

Chao Wu Xiao, Xiaojuan Yan, Yulian Li, Shrikanth A. G. Reddy and Benjamin K. Tsang

Department of Obstetrics and Gynecology and Cellular and Molecular Medicine (C.W.X., X.Y., Y.L., B.K.T.), Reproductive Biology Unit and Division of Gynecologic Oncology, University of Ottawa, Ottawa Health Research Institute, The Ottawa Hospital, Ottawa, Ontario, Canada K1Y 4E9; Nutrition Research Division (C.W.X.), Food Directorate, Health Products and Food Branch, Health Canada, Banting Research Centre, Ottawa, Ontario, Canada K1A 0L2; Department of GI Oncology and Digestive Diseases (S.A.G.R.), Division of Medicine, M. D. Anderson Cancer Center, University of Texas, Houston, Texas 77030

Address all correspondence and requests for reprints to: Dr. Benjamin K. Tsang, Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), 725 Parkdale Avenue, Ottawa, Ontario, Canada K1Y 4E9. E-mail: btsang{at}ohri.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The purpose of the present studies was to examine the role and regulation of Fas-associated death domain-like IL-1- converting enzyme-like inhibitory protein [FLIP; long (FLIPL) and short (FLIPS) forms] in human ovarian epithelial cancer cells by TNF{alpha} and their significance in the resistance of the cells to the proapoptotic action of the cytokine. OV2008, A2780-s, and OVCAR-3 cells were cultured in serum-free media with or without cycloheximide (CHX, 10 µg/ml) ± TNF{alpha} (5, 10, 20 ng/ml) or transfected with a mammalian expression vector containing either a dominant negative inhibitor {kappa}B (I{kappa}B), FLIPS sense or antisense cDNA and cultured with or without TNF{alpha}. In the presence of CHX, TNF{alpha} increased caspase-8 and -3 cleavage and apoptosis. It also induced I{kappa}B phosphorylation, nuclear factor {kappa}B activation, and the expression of FLIPS but not of FLIPL. Overexpression of dominant negative I{kappa}B attenuated TNF{alpha}-induced FLIPS expression and enhanced TNF{alpha}-induced apoptosis. Apoptosis induced by TNF{alpha} and CHX was facilitated by FLIPS antisense expression but attenuated by sense transfection. This study demonstrates that TNF{alpha} up-regulates FLIPS expression, and this effect is mediated by the activation of nuclear factor {kappa}B. The induction of FLIPS expression by TNF{alpha} might contribute to the resistance of ovarian epithelial cancer cells to the proapoptotic action of the cytokine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HUMAN OVARIAN SURFACE epithelial cancer is the leading cause of death from gynecologic malignancy in the Western world. Although clinical and histological prognostic factors are well understood, the biologic process leading to uncontrolled cellular growth is unclear. It is well established that the control of cellular homeostasis involves a balance between cell proliferation and apoptosis and the uncontrolled cell proliferation and/or suppressed apoptosis lead to excessive tissue growth.

TNF{alpha} is a pleiotropic cytokine that can induce differentiation, proliferation, and apoptosis in many cell types (1, 2) and has been suggested to play an important role in the biology of ovarian cancer and tumorigenesis. Ovarian tumor cells produce a macrophage colony-stimulating factor, a potent chemoattractant for monocytes that secretes TNF{alpha}. TNF{alpha} concentrations are significantly increased in ovarian cancer patients (3), and the levels of TNF{alpha} expression are positively correlated with tumor grade (4). TNF{alpha} has selective cytolytic activity against some but not all tumor cells (5). The resistance of human epithelial tumor cells to TNF{alpha} appears to be associated with the expression of this cytokine (5, 6, 7, 8) and controlled by a protein synthesis-dependent mechanism (9). However, the intracellular mechanism(s) involved in the resistance of ovarian cancer cells to TNF{alpha} is not clear.

The actions of TNF{alpha} are mediated by its two receptors, TNFR1 and TNFR2 (10, 11, 12). TNFR1 contains an intracellular death domain required for induction of apoptosis and is coupled to a nuclear factor {kappa}B (NF{kappa}B) activation pathway. Binding of TNF{alpha} to its receptors activates caspase-8 and caspase-3 (13, 14, 15, 16) as well as induces I{kappa}B phosphorylation and degradation and activates NF{kappa}B (17, 18, 19, 20, 21, 22). NF{kappa}B activation regulates the expression of a number of genes involved in the modulation of TNF{alpha}-induced apoptosis, including zinc finger protein A20 (23, 24, 25), members of the Bcl-2 family (26), Bcl-2 homolog Bfl-1/A1 (27), inhibitor of apoptosis proteins (IAP) (28, 29), and Fas-associated death domain (FADD)-like IL-1ß-converting enzyme-like inhibitory protein (FLIP) (30, 31, 32).

FLIP is a FADD-binding suppressor of apoptosis. FLIP is present in two spliced isoforms, long (FLIPL) and short (FLIPS) (33). Both isoforms contain two death effector domains, a structure resembling the N-terminal half of caspase-8 (34, 35, 36). In addition, FLIPL isoform has an inactive caspase-like domain. FLIP is recruited to the death-inducing signaling complex through the adaptor molecule, FADD, thereby preventing the recruitment of caspase-8 into the complex and subsequent caspase-8 activation and then suppresses apoptosis (33, 35, 37). However, the role of FLIP is controversial in some cell types because its overexpression has been reported to induce apoptosis (36, 38, 39, 40). Although recent data have shown that FLIP plays an important role in TNF{alpha}-induced, NF{kappa}B-mediated antiapoptotic response, the expression and role of the two FLIP splice variants appears to be cell type specific (30, 31). Furthermore, whether FLIP overexpression is related to the resistance of human ovarian epithelial cancer cells to TNF{alpha} is unknown.

In the present studies, we examined the role and regulation of FLIP expression by TNF{alpha} in a human ovarian cancer cell lines in vitro and demonstrated that TNF{alpha} induces NF{kappa}B-mediated FLIPS expression, which protects the cells from cytotoxic action of the cytokine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and reagents
Agarose, Tris, phenylmethylsulfonyl fluoride (PMSF), and EGTA were from Sigma (St. Louis, MO). Enhanced chemiluminescence Western blotting detection kit and [{gamma}32P]-dATP (30 Ci/mmol) were obtained from Amersham (Arlington Heights, IL). RPMI 1640, DMEM/F-12 media, and fetal bovine serum (FBS) were from Life Technologies, Inc. (Burlington, Ontario, Canada). Nitrocellulose membrane, acrylamide (electrophoresis grade), N,N’-methylene-bis-acrylamide, ammonium persulfate, dithiothreitol (DTT), glycine, and a protein assay kit were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). X-ray films were from Eastman Kodak Co. (Rochester, NY). Recombinant human TNF{alpha} was from R&D Systems Inc. (Minneapolis, MN). CHX was from BDH Laboratory Supplies (Poole, UK). NF{kappa}B probe and T4 polynucleotide kinase were from Promega Corp. (Madison, WI). Polyclonal rabbit caspase-3 antibody was from PharMingen (Mississauga, Canada). Rabbit polyclonal antihuman phosphorylated and total I{kappa}B-{alpha} antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal antibody recognizing full-length caspase-8, the cleavage intermediates p43 and p41, and the p18 active subunits were generously provided by Dr. M. Peter (German Cancer Research Center, Heidelberg, Germany). Rabbit polyclonal antihuman X-linked IAP (XIAP) antibody was a generous gift from Dr. Eric LaCasse, ApoptoGen Inc. (Ottawa, Canada). Rabbit polyclonal antihuman FLIP antibody was from Alexis Biochemicals (San Diego, CA). The pCMV-I{kappa}B construct containing serine-to-alanine mutation at residue 32 on I{kappa}B{alpha} that cannot be degraded because of mutated phosphorylation sites was from Shrikanth Reddy (M. D. Anderson Cancer Center, Houston, TX).

Cell culture
Human ovarian epithelial cancer cells were cultured in RPMI 1640 (for OV2008 and OVCAR-3) or DMEM/F-12 (for A2780-s) supplemented with FBS (10%, vol/vol), nonessential amino acids (0.1 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37 C under 5% CO2 and 95% air. After a 24-h plating period, the culture medium was changed and the treatments were added as described hereafter. At the end of the culture period, cells were trypsinized and aliquoted for the assessment of nuclear morphology and protein extraction. Cell number in each treatment group was determined by hemocytometry. Cell viability was determined by the trypan blue dye exclusion test as previously described (41).

Preparation of plasmid DNA
The cDNA fragment encoding the open reading frame of human FLIPS (nucleotides 294–956) was prepared by RT-PCR using a set of primers: 5'-ATGTCTGCTGAAGTCATCCA-3' (294–313) and 5'-CATGGAACAATTTCCAAGAA-3' (937–956). The primers were designed based on the human FLIPS sequences (GenBank accession no. U97075) obtained from the GenBank database and the PCR products were subcloned into pcDNA3.1/CT-GFP-TOPO expression vector (Invitrogen, Carlsbad, CA). The sense and antisense hFLIPS-pcDNA3.1/CT-GFP constructs were verified by automated sequence analysis.

Transient transfection
OV2008, A2780-s, and OVCAR-3 cells were seeded in 6-well plates (1 x 106 cell/well) and transfected the following day with 4 µg of the vectors pcDNA3.1/CT-GFP, pCMV alone, or pcDNA3.1/CT-GFP containing hFLIPS and pCMV containing mutated I{kappa}B, using the Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, cells were treated with TNF{alpha} (20 ng/ml) for 6 h and then harvested for further analyses. The overall transfection efficiency assessed by the presence of green fluorescent protein (GFP) expression under a fluorescent microscope is about 40–50%.

Quantitation of FLIP mRNA by semiquantitative RT-PCR
Total RNA was isolated from cultured cells with TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s instructions. Two micrograms total RNA were reversely transcribed for cDNA synthesis, using oligo-deoxythymidine as primer. One tenth of the cDNA synthesized was then amplified with the following primers: human FLIPL [forward: 5'-GACAGCTGAGACAACAAGGACC-3' (58–79), reverse: 5'-GTCTCCACAGCTTTTCTGTCCA-3' (624–602)]; human FLIPS [forward: 5'-GAGACCACCCAGAAGGAAAGAG-3' (66–87), reverse: 5'-G GGTCTCCACAGCTTTTCTGTC-3' (537–516)]; ß-actin [forward: 5'-GAAACTACCTTCAACTCCATC-3', reverse: 5'-CGAGGCCAGGATGGAGCCGCC-3']. Human FLIPL and FLIPS PCR cycle conditions were 95 C for 15 min, 94 C for 45 sec, 60 C for 1 min, and 72 C for 1 min for 35 cycles, 72 C for 10 min. Human ß-actin conditions were 95 C for 15 min, 94 C for 45 sec, 55 C for 1 min, and 72 C for 1 min for 25 cycles, 72 C for 10 min. Samples were resolved on a 2% agarose gel and visualized with ethidium bromide. FLIPS mRNA levels were normalized with its respective ß-actin contents.

Protein extraction and Western blot analysis
Cells were sonicated in a lysis buffer (pH 7.4) containing NaCl (150 mM), sodium dodecyl sulfate (0.1%), sodium deoxycholate (0.5%), Nonidet P-40 (1%) in PBS and protease inhibitors [PMSF (1 mM), aprotinin (10 µg/ml), sodium orthovanadate (1 mM)]. The sonicates were pelleted (15,000 x g, 20 min) and supernatant was retained and stored at -20 C. Protein content of the extracts was determined with the DC protein assay reagent (Bio-Rad Laboratories, Inc.). Samples were mixed with loading buffer, resolved by 12% SDS-PAGE and electrotransferred (30 V, overnight) onto nitrocellulose membranes. The total protein on the nitrocellulose membranes was stained with ponceau S solution and scanned. After blocking for 1 h with nonfat milk powder (5%) in Tris-buffered saline [Tris (10 mM), NaCl (150 mM)] and Tween-20 (0.05%; TBS-T), membranes were incubated for 3 h with primary antibodies in TBS-T containing 5% nonfat milk powder, and subsequently with horseradish peroxidase-conjugated secondary antibody (1:5,000 to 10,000) in TBS-T with milk powder (reverse transcription, 45 min). Immunoreactivity was detected by chemiluminescence autoradiography (enhanced chemiluminescence kit) in accordance with the manufacturer’s instructions. The intensity of protein bands of interest was densitometrically determined and normalized by the respective stained total protein.

EMSA
Nuclear extracts of OV2008 cells were prepared as previously described (42) with minor modifications. Briefly, 3 x 106 cells were pelleted (200 x g, 5 min) and resuspended in 30 µl buffer A [HEPES (10 mM, pH 7.9), KCl (10 mM), MgCl (1.5 mM), DTT (0.5 mM), PMSF (0.5 mM), Nonidet P-40 (0.67%)]. Cells were allowed to swell (0 C, 15 min), and centrifuged (10,000 x g, 4 C). The supernatant was collected and stored at -80 C. The cell pellet (containing cell nuclei) was resuspended in 30 µl buffer B [HEPES (20 mM, pH 7.9), NaCl (0.4 M), EDTA (0.2 mM), MgCl (1.5 mM), DTT (0.5 mM), PMSF (0.5 mM)] and rocked vigorously (4 C, 15 min). The nuclear extract was centrifuged (10,000 x g, 30 min) and stored at -80 C. Double-stranded DNA oligonucleotides containing consensus sequences (5'-AGTTGAGGGGACTTTCCCAGGC-3') for NF{kappa}B was 32P-labeled with [32P]-ATP and T4 polynucleotide kinase. Nuclear proteins (8 µg) were incubated with radiolabeled DNA probes (reverse transcription, 20 min) in the binding buffer (20 mM HEPES, 0.2 mM EDTA, 0.2 mM EGTA, 100 mM KCl, 5% glycerol, 2 mM DTT, pH 7.9). Nuclear acid-protein complexes were resolved on a native 5% polyacrylamide gel in Tris-buffered EDTA (1x, pH 8.0) and detected by autoradiography.

Assessment of apoptosis
Cells were fixed in 4% neutral buffered formalin and then resuspended in Hoechst 33248 staining solution (0.1 µg/ml, overnight), as previously described (43, 44). Cells with typical apoptotic nuclear morphology were identified and counted.

Statistical analyses
Results are expressed as the mean ± SEM of three independent experiments. Statistical analyses were carried out by one- or two-way ANOVA. Significant differences between treatment groups were determined by the Tukey’s test. Statistical significance was inferred at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF{alpha} induced apoptosis in the presence of CHX
OV2008 cells were cultured in serum-free RPMI medium for 0, 3, and 6 h in the absence or presence of TNF{alpha} (20 ng/ml), the protein synthesis inhibitor CHX (10 µg/ml), or CHX (10 µg/ml) plus TNF{alpha} (20 ng/ml). Neither TNF{alpha} nor CHX alone could induce cell death, but in the presence of CHX, TNF{alpha} significantly increased the number of apoptotic cells in a time-dependent manner (P < 0.001; Fig. 1AGo). The proapoptotic effects of TNF{alpha} during a 6-h culture in the presence of CHX were concentration dependent (P < 0.001), and a significant increase in apoptosis was evident at concentrations as low as 5 ng/ml (P < 0.001; Fig. 1BGo). To examine whether the above proapoptotic effects of TNF{alpha} were specific to the OV2008 cell line, two additional ovarian epithelial cancer cell lines (A2780-s and OVCAR-3) were treated with TNF{alpha} in the absence or presence of CHX for 6 or 12 h. In the presence of CHX, TNF{alpha} significantly induced apoptosis in all three cell lines (Fig. 2Go, A–C), although the response of OVCAR-3 cells to TNF{alpha} was much lower than those of OV2008 and A2780-s. Nonetheless, these findings suggest that a cell survival factor(s) might have been induced by TNF{alpha} and suppressed the apoptotic process elicited by the cytokine.



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  		Figure 1. Time- and dose-dependent effect of TNF{alpha} and cycloheximide (CHX) on apoptosis in OV2008. Cells were cultured (A) in RPMI alone (CTL) or in the presence of TNF{alpha} (20 ng/ml), CHX (10 µg/ml), or CHX + TNF{alpha} for 0, 3, or 6 h or (B) in the presence of TNF{alpha} (5, 10, 20 ng/ml) or TNF{alpha}+CHX for 6 h. The cells were collected for apoptosis analysis. Mean ± SEM (n = 3).

 


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  		Figure 2. CHX decreased TNF{alpha}-induced FLIPS content and induced apoptosis in ovarian epithelial cancer cells. OV2008 (A and E), A2780s (B and F), and OVCAR-3 (C and G) cells were cultured in media alone (CTL) or in the presence of TNF{alpha} (20 ng/ml), CHX (10 µg/ml), or CHX + TNF{alpha} for 6 or 12 h. The cells were collected for apoptosis (A–C) and Western blot (D–G) analyses. D, Representative images of three replicate experiments of each cell line. The images for each experiment were scanned, quantified, and normalized by respective stained total protein (E–G). Mean ± SEM (n = 3).

 
TNF{alpha} induced the cleavages of caspase-8, caspase-3, and XIAP in the presence of CHX
To identify the potential survival factor(s) induced by TNF{alpha} in the ovarian cancer cells and determine where on the TNF{alpha} death pathway this factor could be acting, we examined by Western analysis the influence of the cytokine on the cleavage of procaspase-8 and procaspase-3 as well as XIAP in the absence and presence of CHX in vitro. Neither TNF{alpha} (20 ng/ml) nor CHX (10 µg/ml) alone had any apparent effects on the processing of these intracellular proteins. However, in the presence of CHX, TNF{alpha} induced cleavage of the caspases and XIAP, a phenomenon suppressed by the presence of caspase inhibitors [ZVAD (50 µM) and DEVD (20 µM); Fig. 3Go]. Moreover, TNF{alpha} alone had no effect on XIAP content in OV2008 cells. These observations suggest that, in the presence but not absence of the protein synthesis inhibitor, TNF{alpha} is capable of activating caspase-8 and -3 and inducing XIAP cleavage during the induction of apoptosis. Moreover, the putative survival factor is likely acting upstream of caspase-8.



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  		Figure 3. Effect of TNF{alpha} and CHX on caspase-8, -3, and XIAP cleavages in OV2008. Cells were pretreated with or without either ZVAD (50 µM) or DEVD (20 µM) for 2 h and then cultured in RPMI alone (CTL) or in the presence of TNF{alpha} (20 ng/ml), CHX (10 µg/ml), or CHX + TNF{alpha} for 6 h. The cells were collected for Western blot analysis of caspase-8, -3, and XIAP. The panels show representative images of three repeated experiments.

 
TNF{alpha} increased FLIPS mRNA steady-state levels and protein contents
To determine whether FLIP is the putative survival factor induced by the cytokine, OV2008 cells were incubated with TNF{alpha} (20 ng/ml) for different duration (1, 3, 6 h) and FLIPS and FLIPL mRNA abundance were measured by semiquantitative RT-PCR. Whereas TNF{alpha} has no apparent effect on steady-state FLIPL mRNA levels, it rapidly increased FLIPS mRNA abundance. A significant elevation in FLIPS mRNA levels was evident within 1 h of TNF{alpha} challenge and sustained for at least 6 h (P < 0.01; Fig. 4Go, A and B). The protein contents of FLIPS, but not FLIPL (data not shown) in all three cell lines were significantly increased at 6 h after TNF{alpha} treatment and maintained at a high level for at least 12 h (P < 0.05; Fig. 2Go, D–G). CHX significantly decreased basal and TNF{alpha}-induced FLIPS content in all three ovarian cancer cell lines (P < 0.01; Fig. 2Go, D–G).



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  		Figure 4. Effect of TNF{alpha} on FLIP expression in OV2008 cells. Cells were cultured in RPMI in the absence (open bar) or presence (filled bar) of TNF{alpha} (20 ng/ml) for 1, 3, or 6 h. FLIPL, FLIPS, and ß-actin mRNA steady-state levels were measured by semiquantitative RT-PCR. The images were scanned, quantified, and normalized by ß-actin. Mean ± SEM (n = 3).

 
NF{kappa}B activation is involved in the TNF{alpha}-modulated FLIPS expression
To assess whether NF{kappa}B activation is involved in TNF{alpha}-induced FLIPS expression, we examined the temporal changes in phosphorylated I{kappa}B and total I{kappa}B contents, p65 NF{kappa}B subunit translocation, and NF{kappa}B-binding ability in OV2008 cells in response to TNF{alpha}. Addition of TNF{alpha} (20 ng/ml) to the cancer cell cultures resulted in increased phosphorylated I{kappa}B level, which reached a maximum (5 min) and subsequently dropped to pretreatment level (Fig. 5AGo). Total I{kappa}B level remained constant until 5 min following the TNF{alpha} challenge, after which a biphasic response was observed: a marked decrease by 15 min and a gradual increase after 30 min (Fig. 5AGo). NF{kappa}B binding ability, as measured by EMSA, was markedly increased after TNF{alpha} challenge, reaching a maximum at 15 min, and decreased gradually thereafter (Fig. 5AGo). Immunocytochemical localization of the p65 subunit of NF{kappa}B was primarily restricted to the cytoplasm of the untreated OV2008 cells but was evident in the nucleus following TNF{alpha} stimulation (Fig. 5BGo).



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  		Figure 5. I{kappa}B phosphorylation and NF{kappa}B activation induced by TNF{alpha} in OV2008. A, Cells were cultured in RPMI in the presence of TNF{alpha} (20 ng/ml) for 0, 5, 15, 30, 60, and 120 min and collected for Western blotting of phosphorylated I{kappa}B (P-I{kappa}B) and total I{kappa}B (T-I{kappa}B). NF{kappa}B-binding ability was measured by EMSA. B, Cells were treated with or without TNF{alpha} (20 ng/ml) for 30 min and fixed. NF{kappa}B translocation induced by TNF{alpha} was detected immunocytochemically using an anti-p65 antibody. Images are the representatives of three repeated experiments.

 
To determine whether the TNF{alpha}-induced FLIPS expression is mediated via NF{kappa}B activation, influence of a dominant negative I{kappa}B (32-serine substituted with alanine) expression on NF{kappa}B activation and FLIPS abundance was examined. Transfection of the OV2008 cells with the mutated I{kappa}B construct decreased TNF{alpha}-induced phosphorylated I{kappa}B content, increased total I{kappa}B levels and suppressed NF{kappa}B activation induced by the cytokine (Fig. 6Go). These responses were coincidental to a marked decrease in TNF{alpha}-induced FLIPS mRNA expression. FLIPL mRNA abundance was not affected by any of the treatments (Fig. 6Go).



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  		Figure 6. Inhibition of TNF{alpha}-induced NF{kappa}B activation suppressed FLIPS expression. Cells were cultured for 48 h in RPMI+10% FBS in the presence of vector or vector containing a dominant negative I{kappa}B, and then TNF{alpha} (20 ng/ml) was added into cultures. Cells were collected after either 30 min for cytosolic and nuclear protein preparation to measure P-I{kappa}B, T-I{kappa}B, and NF{kappa}B or 6 h for total RNA extraction to determine FLIPL, FLIPS, and ß-actin mRNA levels by RT-PCR. FLIPS mRNA steady-state level was normalized by ß-actin. Mean ± SEM (n = 3).

 
Changes in FLIPS contents modulated TNF{alpha}-induced apoptosis in ovarian cancer cells
To confirm the role of FLIPS in the suppression of TNF{alpha}-induced apoptosis, the influence of FLIPS down-regulation by expression of either mutated I{kappa}B or FLIPS antisense before cytokine treatment was assessed. Whereas expression of mutated I{kappa}B or FLIPS antisense in OV2008 cells alone had no detectable effect on procaspase-3 cleavage, procaspase-3 cleavage was very evident when TNF{alpha} was subsequently added (P < 0.001 and P < 0.05, respectively; Fig. 7AGo). These treatments also resulted in a marked increase in apoptosis (P < 0.01; Fig. 7BGo). Transfection of the three ovarian cancer cell lines with FLIPS sense cDNA significantly increased the FLIPS protein contents in both the absence and presence of CHX, although to a lesser extent in the latter condition (Fig. 8AGo). In addition, overexpression of FLIPS markedly attenuated the apoptotic response of cells to TNF{alpha} plus CHX, compared with that of cells transfected only with the vector (P < 0.01; Fig. 8BGo). These findings support the contention that FLIPS expression in ovarian cancer cells in response to TNF{alpha} is involved in suppressing the proapoptotic action of the cytokine.



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  		Figure 7. Effects of FLIPS down-regulation on TNF{alpha}-induced apoptosis. OV2008 cells were cultured for 48 h in RPMI+10% FBS in the absence or presence of vectors or vectors containing either a dominant negative I{kappa}B or FLIPS antisense cDNA and then for another 6 h with (filled bar) or without (open bar) TNF{alpha} (20 ng/ml). Cells were collected for Western blotting of caspase-3 (A) and apoptosis assessment (B). Mean ± SEM (n = 3).

 


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  		Figure 8. Effect of FLIPS overexpression on TNF{alpha}-induced apoptosis in the presence of CHX. OV2008, A2780-s, and OVCAR-3 cells were transfected with either vector or vector containing sense FLIPS cDNA for 24 h and then incubated in the absence (open bar) or presence (filled bar) of TNF{alpha} (20 ng/ml) and CHX (10 µg/ml) for 6 h. Cells were collected for Western blotting of FLIPS (A) and apoptosis assessment (B). Mean ± SEM (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TNF{alpha} is a multifunctional cytokine and, depending on the cell type, can induce differentiation, proliferation, or apoptosis (1, 2). Although TNF{alpha} has been tested as a death inducer in various cell lineages, the ability of this cytokine to induce apoptosis is not universal (45, 46, 47, 48, 49). The effectiveness of TNF{alpha} is variable, and the cellular mechanism(s) responsible for the variability is poorly understood. In the present study, we have demonstrated for the first time that FLIPS plays a key role in conferring cellular resistance to the cytotoxic action of TNF{alpha} in human ovarian surface epithelial cancer cells. We have shown that TNF{alpha} alone is unable to induce apoptosis in human ovarian epithelial cancer cells but, in the presence of CHX, significantly increase the number of apoptotic cells in vitro. Although TNF{alpha} induces the expression of FLIPS but not of FLIPL in those cells in the concentration- and time-dependent manner, lowering FLIPS levels by antisense expression facilitated the proapoptotic action of the cytokine. These findings support our hypothesis that increased expression of FLIPS in response to TNF{alpha} challenge prevents downstream death signaling by the cytokine in this cell type. This contention is consistent with the current observations that whereas TNF{alpha} alone had no effect on caspase-8 cleavage in OV2008 cells, cotreatment of the cells with CHX and the cytokine resulted in significant cleavage of this caspase and increased apoptosis, which could be prevented by sense FLIPS cDNA expression.

It is of interest to note that the role of FLIP in conferring resistance to cell surface receptor-mediated apoptosis is not confined to the action of TNF{alpha}. In this context, the TNF-related apoptosis-inducing ligand (TRAIL or Apo2L) is a potent death inducer in primary and transformed keratinocytes and a marked difference in sensitivity observed between them could be accounted for by the differences in the expression of FLIPL (50). Whereas the expression of FLIP was highest in the TRAIL-resistant melanomas, and low or undetectable in the sensitive counterpart, addition of actinomycin D to TRAIL-resistant melanomas decreased intracellular concentrations of FLIP and increased TRAIL sensitivity (51). In addition, antigen receptor signaling in primary B cells is known to up-regulate FLIPL and suppress the Fas- and TRAIL-receptor mediated apoptosis (52) and Fas-mediated apoptosis associated with the pathophysiology of rheumatoid arthritis is regulated at the level of caspase-8 through increased FLIP expression (53). Moreover, c-FLIP-/- embryonic fibroblasts are highly sensitive to FasL- or TNF-induced apoptosis and show rapid induction of caspase activities. The c-FLIP-/- mouse embryos rarely survive past d 10.5 of embryogenesis, suggesting that c-FLIP mediates cytoprotection against death factor-induced apoptosis (54).

It has been previously demonstrated that the inability of TNF{alpha} to induce apoptosis was due to the induction of survival factors, including IAP (28, 29, 55) and the Bcl-2 family (27, 56). Although our recent studies (55) have shown that XIAP is important in determining the apoptotic responsiveness of rat ovarian granulosa cells to TNF{alpha}, the present findings indicate that this intracellular survival protein plays a minimal role, if any, in conferring resistance of the human ovarian cancer cells (OV2008, A2780-s, and OVCAR-3) to the cytotoxic action of the cytokine. In this latter context, TNF{alpha} failed to increase XIAP content in the ovarian cancer cells. However, it is of interest to note that, in the presence of protein synthesis inhibitor CHX, TNF{alpha} induced XIAP cleavage in OV2008 cells, a process sensitive to the presence of the caspase inhibitors ZVAD and DEVD. These findings, together with the observations that cleavage of XIAP produces an N-terminal BIR-2 fragment with reduced ability to inhibit caspase-3 and suppress apoptosis (57), support the contention that the caspase-3-mediated decrease in XIAP content may be involved in the execution of apoptosis in ovarian cancer cells in response to TNF{alpha}.

TNFR1 contains an intracellular death domain required for induction of apoptosis (58) and is coupled to a NF{kappa}B activation pathway (17, 18, 59, 60). NF{kappa}B activation regulates the expression of a number of genes involved in the prevention of TNF{alpha}-induced apoptosis (23, 24, 25, 27, 28, 29, 56). However, the mechanism(s) involved in the regulation of FLIP expression in human ovarian cancer cells is not completely understood. In the present study, we have observed that challenge of OV2008 cells with TNF{alpha} resulted in a rapid increase in phosphorylated I{kappa}B content, which was temporarily associated with NF{kappa}B translocation to the nucleus, increased nuclear NF{kappa}B-binding activity and FLIPS mRNA abundance. Overexpression of a dominant negative I{kappa}B attenuated TNF{alpha}-induced I{kappa}B phosphorylation and nuclear NF{kappa}B binding ability and suppressed FLIPS expression in response to this cytokine. These results are consistent with the recently published evidence showing that TNF{alpha}-induced FLIPS expression is mediated through the activation of the NF{kappa}B signaling pathway (30, 31).

The intracellular action of FLIP in the suppression of TNF{alpha}-induced apoptosis in human ovarian cancer cells is not known. Death receptors belonging to the TNF receptor family are characterized by an intracellular death domain that serves to recruit adapter proteins including TRADD and FADD and cysteine proteases, such as caspase-8 (15, 16). Activation of caspase-8 on the aggregated receptor leads to apoptosis (13, 14). Triggering of death receptors is mediated through the binding of specific ligands of the TNF family, which are homotrimeric type-2 membrane proteins displaying three receptor binding sites (58, 61, 62, 63). Intracellular proteins interacting with the apoptotic pathway are potential modulators of death receptors. FLIP resembles caspase-8 in structure and contains a FADD-binding domain. All isoforms of FLIP lack the active-center cysteine residue and function as dominant negatives for caspase-8 (33, 35, 37). They interact with both FADD and caspase-8 to inhibit the apoptotic signal of death receptors (64). Our current observation that, in the presence of CHX, TNF{alpha} increases caspase-8 cleavage (a process associated with activation) and induces apoptosis in human ovarian epithelial cancer cells and that overexpression of FLIPS prevented the apoptotic response are consistent with the above concept.

In conclusion, TNF{alpha} induces FLIPS expression in human ovarian cancer cells via I{kappa}B-mediated NF{kappa}B activation. FLIPS is a key determinant of the sensitivity of the cells to proapoptotic action of the cytokine.


   Acknowledgments
 
We thank Dr. Eric LaCasse (Apoptogen Inc., Ottawa, Canada) and Dr. M. Peter (German Cancer Research Center, Heidelberg, Germany) for generously providing rabbit polyclonal antihuman XIAP antibody and mouse monoclonal anticaspase-8 antibody used in the present studies.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research (MOP-15691) and the National Cancer Institute of Canada (with funds from the Canadian Cancer Society, Grant 013335). C.W.X. was a recipient of the Canadian Institute of Health Research Postdoctoral Fellowships.

Abbreviations: CHX, Cycloheximide; DTT, dithiothreitol; FADD, Fas-associated death domain; FBS, fetal bovine serum; FLIP, FADD-like IL-1-converting enzyme-like inhibitory protein; FLIPL, long isoform of FLIP; FLIPS, short isoform of FLIP; GFP, green fluorescent protein; IAP, inhibitor of apoptosis proteins; I{kappa}B, inhibitor {kappa}B; NF{kappa}B, nuclear factor {kappa}B; PMSF, phenylmethylsulfonyl fluoride; TBS-T, Tris-buffered saline and Tween 20; TRAIL, TNF-related apoptosis-inducing ligand; XIAP, X-linked IAP.

Received September 5, 2001.

Accepted for publication November 1, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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