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Cisplatin-mediated sensitivity to TRAIL-induced cell death in human granulosa tumor cells
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Gynecologic Oncology 108 (2008) 632 – 640 www.elsevier.com/locate/ygyno
Cisplatin-mediated sensitivity to TRAIL-induced cell death in human granulosa tumor cells Dori C. Woods, Claudia Alvarez, A.L. Johnson ⁎ Department of Biological Sciences and the Walther Cancer Research Center, The University of Notre Dame, Notre Dame, IN 46556, USA Received 8 October 2007 Available online 14 January 2008
Abstract Objectives. The goal of the present study was to determine the efficacy of combinatorial treatment using cisplatin and tumor necrosis factorrelated apoptosis including ligand (TRAIL) to promote apoptosis in granulosa cell tumor (GCT) lines, in vitro. Methods. Two human GCT lines (COV434 and KGN) were treated with cisplatin or TRAIL, alone or in combination. The cytotoxic effects of each treatment were evaluated using a methyl tetrazolium salt (MTS) assay. Initiation of TRAIL-induced apoptosis was verified by PARP- and FLIP-cleavage. Overexpression and knockdown studies were conducted to evaluate the role of p53 in TRAIL-induced cell death. Real-time PCR was used for gene expression analysis of the TRAIL receptor dr5 and the pro-apoptotic bax following treatment with cisplatin. Results. Treatment with TRAIL (100–200 ng/ml) led to a slight, but significant, loss of cell viability following an 18-h culture. This effect was enhanced following pre-treatment with cisplatin (25 μM) for 2 or 18 h. Moreover, pre-treatment with cisplatin decreased the maximal effective dose of TRAIL from 100 ng/ml to as low as 3 ng/ml in both cell lines. GCT lines overexpressing or deficient in p53 were used to determine the requirement for p53 on TRAIL-induced apoptosis. While the level of p53 expression enhanced both the death-inducing and TRAIL-sensitizing effects of cisplatin, TRAIL-induced cell death was found to occur independent of p53. Conclusions. These data suggest that the efficacy of cisplatin in GCT cells can be enhanced through combinatorial treatment with TRAIL. This result is due to both p53-dependent (cisplatin) and -independent (TRAIL) mechanisms. Combinatorial treatment of GCTs with cisplatin and TRAIL may provide an efficacious addition to cisplatin-based regimens. © 2007 Elsevier Inc. All rights reserved. Keywords: Granulosa cell tumors; TRAIL; Cisplatin; p53; Apoptosis
Introduction Granulosa cell tumors (GCTs) are the most common malignant sex-chord neoplasm, yet the overall occurrence of GCTs is relatively rare. Current estimates indicate that GCTs represent approximately 7% of all ovarian cancers [1]. Nevertheless, the etiology, genetics, and pharmacologic management of GCTs have been comparatively understudied compared to ovarian surface epithelium (OSE)-derived cancers. As such, the basis for targeted granulosa cell therapy has been largely derived from cancers originating from human (h) OSE, with the pre-
⁎ Corresponding author. Fax: +1 574 631 7413. E-mail address: [email protected] (A.L. Johnson). 0090-8258/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2007.11.034
vailing treatment consisting of a platinum-based regimen [1]. Although the overall GCT response rate to platinum-based therapy is estimated at 63% to 80%, the established propensity for eventual relapse is indicative of the need for more effective combinatorial regimes [1]. Although considerable information exists regarding the efficacy of the endogenously produced cytokine, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), to enhance cisplatin-induced cell death in a variety of cancers (including those of hOSE origin) [2], the efficacy of this combinatorial treatment in targeting specifically GCTs has not been reported. Interest in the potential therapeutic application of TRAIL originates from studies demonstrating its cytotoxicity in tumor cells, while most normal cells are resistant to TRAIL-induced apoptosis [3]. The anti-tumorigenic effects of TRAIL are further
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illustrated by its ability to induce regression of cancer xenografts, while healthy tissue remains unaffected [4,5]. Importantly, it has recently been reported that primary cultured normal ovarian granulosa cells are resistant to TRAIL-induced apoptosis, while treatment with cisplatin renders them TRAILsensitive [6]. TRAIL is reported to mediate its biological effects on cancerous cells primarily through two Tumor Necrosis Factor Receptor (TNFSRF) superfamily death receptors, DR4 (TNFSRF10A) and KILLER/DR5 (TNFSRF10B) [7–9]. TRAIL binding induces receptor homotrimerization, which modifies the conformation of the cysteine-rich cytoplasmic domain. This conformational change enables the well-characterized intracellular death domain (DD) motif to associate with the cytoplasmic adaptor protein, Fas-associated death domain protein (FADD), and promote formation of the deathinducing signaling complex [10,11]. In turn, the death effector domain of FADD interacts with initiator caspases (e.g., caspase8) [12]. Consequent activation of caspase-8 cleaves the BH3interacting domain death agonist (Bid), and initiates mitochondrial release of cytochrome c (cytC). Re-localization of cytC to the cytosol initiates the intrinsic cell death pathway, including eventual downstream activation of the executioner caspase, caspase-3. Ultimately, this cascade of signaling events leads to self-amplification of caspase-3 activation and results in cell death (reviewed in [13,14]). Significantly, the efficacy of TRAIL-mediated cytotoxicity is enhanced in wild-type p53 expressing cells following treatment with DNA-damaging chemotherapeutics, such as cisplatin [15]. It has been shown that this increase in TRAIL sensitivity is due, at least in part, to p53-dependent up-regulation of DR5 subsequent to DNA damage. Additionally, it has been shown that treatment with cisplatin sensitizes cells to TRAIL-induced cell death downstream of receptor activation by perturbing mitochondrial function and initiating intrinsic apoptotic mechanisms, including up-regulation and activation of the p53-responsive, pro-apoptotic protein, Bax [16]. Due to the increased efficacy of TRAIL-induced cell death via p53 activity, it has been postulated that activation of p53 by DNA damaging agents (e.g., cisplatin) may provide therapeutic utility in the treatment of some cancers [15]. The potential for cisplatin to modulate cell death in GCTs via extrinsic (e.g., DR5) or intrinsic (bax, mitochondrial perturbation) mechanisms in a p53-dependent manner has yet to be evaluated. In general, the lack of information regarding selective treatment for GCTs, as opposed to hOSE derived cancers, is due not only to the comparatively rare incidence of GCTs, but also to the paucity of appropriate in vitro model systems, and in particular, established human GCT lines. The recent description of adulttype GCT lines, COV434 [17] and KGN [18], has provided new opportunities for developing more targeted GCT therapies. Accordingly, in the present report, we provide evidence for: (1) TRAIL plus dr5 mRNA expression and TRAIL-induced cell death in GCT (COV434 and KGN) cell lines; (2) cisplatininduced cell death and enhanced TRAIL sensitivity in GCT cell lines; and (3) the facilitation of cisplatin-induced death and TRAIL sensitivity in a p53-dependent manner.
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Materials and methods Cell culture and reagents The adult-type human ovarian GCT lines, COV434 and KGN, were generously provided by Dr. De Geyter (University of Basel, Switzerland), and Drs. Nishi and Yanase (Kyushu University, Japan), respectively. Both cell lines were maintained in a subconfluent state during all experiments. COV434 and KGN were cultured in cell culture medium (COV434, DMEM; KGN, DMEM/ Ham's F12; Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 1% nonessential amino acids, and 1% antibiotic/antimycotic. All cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2. For cell culture experiments, adherent cells were trypsinized, washed with an equal volume of cell culture medium, and plated in either 6- or 96-well Corning polystyrene cell culture dishes. Recombinant human TRAIL was purchased from Peprotech (Rocky Hill, NJ), and cisplatin (cis-platinum(II)-diammine dichloride) was from Sigma–Aldrich (St. Louis, MO). The selective caspase inhibitor, Z-IETD-FMK (IETD), was from R&D Systems (Minneapolis, MN). Optimally effective doses for TRAIL, cisplatin, and Z-IETD-FMK were empirically determined for each cell line.
Nested PCR and p53 sequencing In lieu of amplification and sequence analysis of the entire p53 gene, mutational analysis was performed on the five highly conserved domains, where most functional mutations are reported to occur [19]. To identify potential p53 mutations in COV434 and KGN cell lines, nested PCR was used followed by automated sequencing. cDNA was synthesized from freshly isolated total RNA and random hexamers using the Promega Reverse Transcription System (Promega, Madison, WI). Samples were incubated for 15 min at room temperature, then 15 min at 45 °C, followed by 5 min at 95 °C for enzyme inactivation. PCR reactions were subsequently performed using High Fidelity Taq polymerase, with a proof-reading polymerase designed for mutational analysis (ClonTech, Palo Alto, CA). Nested PCR reactions for mutational analysis, including the primers and reaction conditions, were performed essentially as described by Khan et al. [20]. Briefly, primers specific for the coding region between exons 3 and 10 were used to amplify an initial product (outer primers). The resulting product was then used in a subsequent PCR reaction using two sets of primers designed to amplify the coding region between exons 4 and 9 (inner primers). The two sets of inner primers consisted of an overlapping region to ensure total amplification of the conserved region of p53. The products were then sequenced using the ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA).
Amplification of TRAIL mRNA by RT–PCR Primers for TRAIL PCR amplification from COV434, KGN, and granulosa cells collected from IVF patients (provided by Dr. B.R. Rueda, The Vincent Center for Reproductive Biology, Boston, MA) were designed using MacVector (v.6.5.3; Cary, NC), and were specific for exons 1 and 5 (TRAIL sense: 5′ACCTGCGTGCTGATCGTG-3′ [exon 1]; antisense: 5′-ATTTTGCGGCCCAGAGCC-3′[exon 5]). RNA was isolated and cDNA synthesized as described above. Thermocycling conditions were: 94 °C for 3 min, then 94 °C for 45 s, 55 °C for 45 s, 72 °C for 90 s for 35 cycles, followed by 72 °C for 10 min. Amplified products were subcloned and sequenced to verify nucleic acid identity.
Cell density analysis for granulosa tumor cell viability Adherent KGN and COV434 cells were removed from culture flasks and seeded in a 96-well cell culture plate at a density of 2 × 104 (COV434) or 1 × 104 (KGN). After an overnight culture, the medium was removed and replaced, then cells were cultured in the absence or presence of cisplatin (1–50 μM), TRAIL (3–100 ng/ml), or cisplatin plus TRAIL. Following the time period specified for each experiment, cell density (a measure of metabolic activity and viability) was determined as previously described [6,21] using the colorimetric methyl tetrazolium salt (MTS) Cell Titer 96 Aqueous One Solution cell proliferation assay (Promega). The colorimetric reagent was added to each well of the plate,
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incubated for 1–4 h, and the absorbance values read at 490 nm. Activation of apoptotic pathways was further verified through detection of poly(ADP-ribose) polymerase (PARP) and FLICE inhibitory protein (FLIP) cleavage, as described below.
Western blot analysis Cells were harvested and lysed in ice-cold lysis buffer (150 mM NaCl, 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM pervanidate, 1 mM EDTA, 1% Igepal, 0.25% deoxycholic acid, 1 mM NaF, and 50 mM Tris–HCl, pH 7.4), and cleared through centrifugation at 13,000×g. Between 10 and 40 μg of total cellular protein was loaded on an SDS polyacrylamide gel for electrophoresis, followed by a transfer to nitrocellulose
membranes. The membranes were blocked for 1 h at room temperature in 5% milk in TTBS (Tris-buffered saline–0.1% Tween 20), then incubated with primary antibody overnight at 4 °C. Rabbit anti-PARP serum (Cell Signaling, Danvers, MA) was used at a dilution of 1:1000. The anti-FLIP serum (Santa Cruz; used a 1:1000 dilution) was raised in rabbits against the amino terminus. Rabbit anti-DR5 (Sigma–Aldrich), recognizing both the unprocessed and mature proteins, was used at a 1:500 dilution, while rabbit anti-bax serum was from Santa Cruz (Santa Cruz, CA) and diluted 1:1000. Following incubation with primary antibody, membranes were washed for 5 min in TTBS then incubated with horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG; Pierce Endogen, Rockford, IL) for 1 h at room temperature. The membranes were incubated with ECL Western blotting detection reagent (Pierce) for 1 min, then wrapped and exposed to X-ray film for 3 to 10 min. Membranes
Fig. 1. Expression of TRAIL mRNA in COV434 and KGN cell lines plus non-neoplastic (IVF-derived) granulosa cells. (A) PCR co-amplification of TRAIL (413 bp) and an alternatively spliced variant (TRAILδ; 265 bp) from COV434 (COV), KGN and IVF-derived cells. Negative (no RT) control (Neg.). (B) Clustal alignment depicting full-length TRAIL (GenBank accession: U37518) and a novel TRAIL splice variant (TRAIL-δ; EU183231) cloned from COV434 and KGN cells. (C) Top: The 5 exons from the full-length TRAIL pre-mRNA results in an 846 total nt product (middle). Bottom: mRNA encoding TRAIL-δ, lacking exons 3 and 4, results in a predicted 698 nt product.
D.C. Woods et al. / Gynecologic Oncology 108 (2008) 632–640 were subsequently blotted for α-tubulin (Pierce) to demonstrate equal loading of protein.
Real-time quantitative PCR Forward and reverse primers specific for DR5, bax, and 18s rRNA have been described previously [21], and were subsequently validated for use with real-time PCR by determining the optimal amplification efficiency and primer concentrations as described by the system manufacturer (Applied Biosystems). Gene expression analysis was performed as previously described [21], using ABgene Absolute QPCR SYBR Green Mix (ABgene, Rochester, NY). Briefly, random-primed, reverse transcribed cDNA synthesis reactions were performed utilizing the Promega Reverse Transcription System (Promega, Madison, WI), as described by the manufacturer. For real-time PCR, primers and were added to 25 μl total reaction volume using reagents provided in the kit. Final concentrations of the sense and antisense primers were determined for each primer pair based on optimal amplification efficiency. Reactions were completed on an ABI 7700 Thermocycler (Applied Biosystems). Thermocycling conditions were as follows: 95 °C for 15 min followed by a program of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s for a total of 40 cycles. The Ct (defined as the cycle number at which the fluorescence exceeds a threshold level) was determined for each reaction (run in triplicate) using the Sequence Detection software (v.1.6.3; Applied Biosystems), while quantification was accomplished using the ΔΔCt method [22]. The target Ct was determined for each sample then normalized to the 18s rRNA Ct from the same sample (18s rRNA Ct subtracted from the target Ct yields the ΔCt). These values were expressed as fold-difference compared to an appropriate control sample using the 2−ΔΔCt equation.
Cell transfection for overexpression of p53 and siRNA
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Means were designated as statistically significant when p values b0.05 were obtained.
Results Amplification and sequencing of TRAIL in COV434 and KGN granulosa tumor cell lines PCR amplification revealed the presence of TRAIL mRNA in both KGN and COV434 cell lines, as well as in granulosa cells obtained from IVF patients. Interestingly, in addition to the anticipated 413 bp TRAIL PCR product, a smaller 265 bp product was also detected (Fig. 1A). Sequencing revealed a novel truncated variant form of TRAIL with a deletion of nucleotides 272–419, corresponding to deletions of both exon 3 (43 nt) and exon 4 (105 nt) (Figs. 1B, C; GenBank accession EU183231). Treatment with cisplatin enhances TRAIL-induced cell death Treatment of COV434 and KGN cells with TRAIL at 100 and 200 ng/ml for 18 h resulted in a slight (∼10%), but statistically significant, induction of cell death compared to control cells (Fig. 2A; p b 0.05). By comparison, treatment with cisplatin (1 to 50 μM) induced cell death in a dose-dependent fashion following 18 h in COV434 cells, while the magnitude of
The GFP-expressing wild-type and nuclear localization p53 constructs were provided by Dr. Tyler Jacks (Massachusetts Institute of Technology, Center for Cancer Research), and have been described previously [23]. The p53-specific and control siRNAs were purchased from Santa Cruz. Cells were seeded in 6well cell-culture plates for 1 or 2 days at a density of 5 × 105 or 2 × 105 (COV434 and KGN, respectively) to allow adequate time for cell adherence. Transfections were performed using the Lipofectamine 2000 transfection reagent using conditions recommended by the manufacturer (Invitrogen). For each transfection, 6 μl of Lipofectamine was added to 100 μl of cell culture medium in the absence of antibiotics and serum. Additionally, 6 μg of DNA or siRNA was added to 100 μl of serum/antibiotic free cell culture medium. The Lipofectamine and DNA or RNA solutions were then combined and incubated for 45 min at room temperature. Following this incubation, 800 μl of serum- and antibioticfree cell culture medium was added to each Lipofectamine complex, and the entire 1 ml solution added to the cells. The cells were cultured for 5 h, followed by the addition of 1 ml of appropriate cell culture medium containing 20% FBS. Cells were incubated overnight, then the medium was removed and replaced with normal growth medium. Cells were harvested 24 h later for analysis or seeded in a 96-well plate to assay cell viability. Control (mock) transfections with Lipofectamine reagent were performed with each transfection. For p53 overexpression studies, data are compared to cells transfected with the pEGFPN1 vector alone. For p53 siRNA studies, data were compared to cells transfected with non-silencing (control) siRNA. Knockdown efficiency was monitored by PCR.
Confocal microscopy Immunolocalization studies were performed by confocal analysis as previously described [21] using a mouse monoclonal antibody directed against cytochrome c (PharMingen International; used at 1:100 dilution).
Statistical analysis Data are expressed as mean ± SEM and compared by one-way ANOVA followed by LSD post-hoc analysis or by t test using SPSS statistical software. Each experiment was independently replicated a minimum of three times.
Fig. 2. TRAIL and cisplatin effects on cell viability in COV434 and KGN cell lines. (A) Treatment with TRAIL (50–200 ng/ml) for 18 h results in a slight, but significant, decrease in cell viability in both cell lines at the two highest doses. (A, B; a, b) p b 0.05; n = 4 replicate experiments. (B) Treatment with cisplatin (1–50 μM) for 18 h results in a loss of cell viability in both cell lines. Different letters denote p b 0.05; n = 4 replicate experiments.
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Fig. 3. Combinatorial effects of cisplatin plus TRAIL treatment on GCT viability and apoptosis. (A) Pre-treatment with cisplatin (25 μM for 2 h) enhances the effectiveness of TRAIL (100 ng/ml added during the last 18 h of culture) to decrease cell viability. Data are expressed relative to control cultured cells (not shown). (A, B, C; a, b, c) p b 0.05; n = 5 replicate experiments. (B) TRAIL (100 ng/ml) induces PARP cleavage following treatment for 4 h (top panel) and 18 h (bottom panel), while treatment with cisplatin (25 μM) increases PARP cleavage by 18 h. (C, D) Pre-treatment with cisplatin for 18 h sensitizes GCT lines to a subsequent 4 h (C) or 18 h (D) challenge with TRAIL. Different letters denote p b 0.05 from 5 to 7 replicate experiments. (E) In addition, cisplatin pre-treatment effectively reduces the maximal effective dose of TRAIL to as low as 3 ng/ml in COV434 and KGN cell lines. *,+ denote p b 0.05 compared to 0 ng TRAIL/ml by t test; n = 5 replicate experiments.
this response in KGN cells was less pronounced (Fig. 2B; p b 0.05). In both COV434 and KGN cell lines, the effect of cisplatin (25 μM) plus TRAIL (100 ng/ml) was enhanced when cells were pre-treated with cisplatin for 2 h (Fig. 3A). An 89-kDa PARP cleavage product (a marker of apoptosis) was induced by TRAIL (100 ng/ml) following as little as 4 h in culture, whereas cisplatin (25 μM)-induced PARP cleavage was visible following 18 h of culture (Fig. 3B). Following a more prolonged pretreatment with cisplatin (for 18 h), TRAIL treatment for 4 h significantly reduced the number of viable cells compared to cells treated with cisplatin alone (Fig. 3C). This reduction in cell viability was further enhanced following an extended 18 h incubation with TRAIL (Fig. 3D). Not only was cisplatin effective in sensitizing the COV434 and KGN cell lines to TRAIL after
4 h, pre-treatment with cisplatin for 18 h significantly reduced the effective dose of TRAIL to as low as 3 ng/ml (compare Fig. 3E to Fig. 2A). Effects of cisplatin occur independent of caspase-8 activity Treatment with TRAIL (100 ng/ml), but not cisplatin (25 μM), for 4 h resulted in detection of a p43 N-terminal FLIP-cleavage product as determined by Western blot analysis (Fig. 4A), indicative of caspase-8 activation [24]. To evaluate the role of caspase-8 in cisplatin-induced TRAIL sensitivity, COV434 and KGN cells were pretreated for 2 h without or with the caspase-8 inhibitor, IETD (20 μM). Inhibition of caspase-8 activity significantly attenuated the cytotoxic effects of TRAIL, both in the absence and presence of
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Notably, there was no detectable rescue of viability in sip53 transfected cells treated with cisplatin plus TRAIL versus those treated with cisplatin alone. These findings provide evidence that the effects of TRAIL, but not those of cisplatin, occur independent from p53 activity. Cisplatin increases dr5 and bax mRNA and protein levels in GCT cell lines Treatment with cisplatin for 18 h resulted in significantly elevated levels of bax and dr5 mRNA (Fig. 7A) plus protein (Fig. 7B) in both KGN and COV434 cell lines. Up-regulation of functional Bax is corroborated by the re-localization of cytC from the mitochondria to the cytoplasm (Fig. 7C). To further investigate whether the effects of cisplatin on bax and dr5 transcription in KGN and COV434 are mediated by p53, each cell line was transfected with non-silencing siRNA (siC) or
Fig. 4. (A) TRAIL treatment (100 ng/ml) for 4 h results in FLIP cleavage, as indicated by the presence of the 43-kDa N-terminal FLIP cleavage product. (B) The selective pharmacologic caspase-8 inhibitor Z-IETD-FMK (IETD; 20 μM) attenuates the effects of TRAIL treatment for 18 h alone or in combination with cisplatin (25 μM) pretreatment for 18 h in both COV434 and KGN cell lines. ⁎p b 0.05 versus absence of IETD, by t test, n = 5 replicate experiments.
cisplatin (Fig. 4B). By contrast, IETD failed to alter cisplatininduced cytotoxicity. Cisplatin-mediated cell death and sensitivity to TRAIL is p53-dependent A nested PCR technique described by Khan et al. [20] was used to determine the mutational status of p53 (see Materials and methods). No nucleotide substitutions, additions, or deletions were detected in either transformed cell line compared to wild-type p53 (p53-wt; data not shown). Transfection of COV434 and KGN cells with p53-wt or nuclear localized p53 (p53-NL) (Fig. 5A) facilitated a significant increase in cisplatininduced cytotoxicity compared to cells expressing the plasmid vector pEGFP-N1 (Fig. 5B). Moreover, cells transfected with p53 were significantly more sensitive to treatment with TRAIL following pre-treatment with cisplatin. This sensitizing effect was specific to cells pre-treated with cisplatin, as treatment with TRAIL treatment alone did not result in greater cytotoxicity compared to pEGFP-N1 expressing cells. Furthermore, targeted knockdown of p53 using siRNA (sip53; Fig 6A) did not enhance cell viability following treatment with TRAIL when compared to cells transfected with the non-silencing siRNA (Fig. 6B). By comparison, transfection with sip53 increased the percentage of viable cells following treatment with cisplatin.
Fig. 5. Overexpression of p53 leads to an increase in sensitivity to cisplatin, but not TRAIL, in COV434 and KGN cell lines. (A) Localization of GFP-tagged wild-type p53 (wt-p53; top panels) within the cytoplasm, and nuclear-localized p53 (p53-NL; bottom panels) in KGN cells. Nucleus in marked by arrow. (B) Overexpression of p53-NL enhances the cell death-inducing effects of cisplatin compared to cells treated with the pEGFP-N1 vector alone. Additionally, overexpression of p53-NL enhanced cisplatin-induced TRAIL sensitivity, while having no effect on TRAIL treatment alone. ns: non-significant; ⁎p b 0.05 by t test compared to vector alone. n = 5 replicate experiments.
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expression of TRAIL mRNA in both GCT lines, as well as in non-neoplastic granulosa cells (Fig. 1A). Interestingly, sequence analysis demonstrated the presence of a novel TRAIL mRNA splice variant lacking exons 3 and 4 (TRAIL-δ). As previously described for the human TRAIL variants, TRAIL-β and TRAIL-γ [26], deletion of these exons results in a predicted truncated TRAIL-δ protein consisting of 101 amino acids (aa). Notably, this variant is similar to the 98 aa TRAIL-β variant, which lacks apoptotic activity presumably due to a truncated extracellular binding domain. Unlike TRAIL-γ, which also lacks this binding domain, TRAIL-β does not strongly associate with the cell surface and nuclear membranes. Although the biological function(s) of any TRAIL variant has not been unequivocally established, it has been speculated that such variants may play a passive regulatory role in TRAIL actions by reducing the amount of biologically active TRAIL [26].
Fig. 6. Knockdown of p53 leads to a partial rescue of cisplatin-induced cell death. (A) PCR amplification following transfection with siRNA specifically targeting p53 (sip53) in COV434 and KGN cell lines. Non-silencing control siRNA (siCon) was used to control for non-specific effects. Neg., no RT control. (B) Percent rescue of cell viability compared to cells transfected with siCon (not shown). Treatment with sip53 resulted in a partial reversal of the effects of cisplatin (∼60% in COV, ~30% in KGN), however, there was no further increase in cell viability in cells treated with cisplatin plus TRAIL. Down-regulation of p53 mRNA expression did not provide a significant rescue effect on TRAILreduced cell viability. ns: p N 0.05, n = 5 replicate experiments.
sip53. In both GCT lines, transfection with sip53 resulted in a significant decrease in levels of cisplatin-induced bax and dr5 mRNA (Fig. 8). Discussion Published clinical data suggest that while platinum-based therapy is up to 80% effective in treating advanced stage GCTs, the potential for increasing the efficacy of chemotherapeutic therapy plus the opportunity to minimize tumor reoccurrence following initial therapy emphasize the need for the development of more targeted combinatorial regimens [1]. Due to the relative infrequency of GCTs and scarcity of in vitro model systems, approaches to addressing these issues have only recently been initiated. The results presented herein demonstrate the effects of TRAIL enhanced apoptotic cell death via combinatorial treatment with the conventional chemotherapeutic, cisplatin, in GCT cell lines, in vitro. The expression of TRAIL has been reported in normal granulosa cells from various animal models [6,25], yet it is not known whether the human GCT lines express this naturally occurring ligand. PCR amplification revealed the endogenous
Fig. 7. Cisplatin-induced bax and dr5 mRNA plus protein expression in COV434 and KGN cell lines. (A) Real-time PCR data indicate that cisplatin (Cis; 25 μM) treatment for 18 h increases levels of bax and dr5 mRNA in both cell lines. ⁎p b 0.05 compared to control (Con) cultured cells; n = 3 replicate experiments. (B) Cisplatin, but not TRAIL (100 ng/ml for 18 h), also induced bax and DR5 protein in both COV434 and KGN cells. (C) Immunofluorescence depicting localization of cytochrome c (cytC) in KGN. Left: Punctate staining (arrows) indicative of mitochondrial localized cytC in control cultured cells. Right: Induced release of cytC into the cytoplasm following treatment with cisplatin for 8 h. Dotted circle depicts outline of the nucleus.
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Fig. 8. Results of real-time PCR analyses indicate that transfection with siRNA targeting p53 (sip53) decreases cisplatin (25 μM)-induced bax and dr5 mRNA expression in COV434 and KGN cell lines when compared to cells transfected with non-silencing siRNA (siCon). * Indicates significance by t test, n = 3.
Nevertheless, further experimental work is required to determine what, if any, role TRAIL splice variants have in modulating the apoptotic activity of TRAIL in human granulosa cells. Treatment of GCT cell lines with TRAIL resulted in a slight, but statistically significant, increase in cell death, indicated by both a reduction in cell viability (Fig. 2A) and the rapid (within 4 h) initiation of PARP (Fig. 3B) and FLIP (Fig. 4A) cleavage. The cytotoxic effects of TRAIL were enhanced following pretreatment with cisplatin for 2 or 18 h (Figs. 3A, C, D). This finding is consistent with earlier reports in hOSE tumor cell lines, in which cisplatin increased the ability of TRAIL to initiate apoptosis as indicated by an increase in PARP cleavage and caspase-3 activation [2]. The additive effect of cisplatin on TRAIL-induced cell death in GCT cells is attributed to the effects of the extrinsic (e.g., death receptor-induced) pathway, as suggested by the ability of the selective caspase-8 inhibitor, Z-IETD-FMK to prevent this combinatorial effect (Fig. 4). This finding is similar those reported from hOSE tumor cell lines where a pan-caspase inhibitor, Z-VAD-FMK, inhibited the cytotoxic effects of TRAIL [2]. Additionally, the intrinsic pathway may be of critical importance in this additive effect, since a recent study in thoracic cancer cells indicates that mitochondrial-mediated amplification of caspase-8 are critical components for enhancing the cytotoxic effects of TRAIL [27]. One established mechanism by which cells are sensitized to TRAIL is through the DNA-damage-inducible p53 pathway, which modulates cell death through both extrinsic (e.g., DR5) and intrinsic (e.g., bax) activity. Data presented herein indicate that p53-wt expression is not sufficient to significantly enhance the death-inducing effects of TRAIL (Fig. 5B). By comparison, both cisplatin-induced cell death and cisplatin-enhanced sen-
sitivity to TRAIL were evidenced in p53 overexpressing cells. It has been shown that in addition to amplifying transcription of pro-apoptotic genes, cytosolic p53 can inhibit the activity of the anti-apoptotic Bcl-x by forming a p53/Bcl-x inhibitory complex [28]. In addition to an apoptogenic role at the mitochondria, nuclear-localized p53 can affect mitochondrial function through the up-regulation of the pro-apoptotic Bax protein [30–32]. To investigate the potential for p53-mediated events at the level of the nucleus, nuclear-localized p53 (p53-NL) was overexpressed in COV434 and KGN cells. Similar to results obtained with cytosolic-localized p53-wt, p53-NL significantly enhanced both the death-inducing and TRAIL-sensitizing effects of cisplatin. This result is not unexpected, as treatment of renal tubule cells with cisplatin results in the nuclear localization wild-type p53, independent from caspase activation [29]. Results from the present studies indicate that knockdown of p53 does not rescue the death-inducing effects of TRAIL, but does lead to at least a partial reversal of cisplatin-induced death in both GCT cell lines (Fig. 6). Significantly, both cisplatin-induced dr5 and bax mRNA expression were blocked in p53-deficient cells (Fig. 8). Taken together, these data suggest that the effects of cisplatin on TRAIL-mediated cell death are, at least in part, p53 dependent. Nevertheless, it has been shown that cells with mutated p53 are also susceptible to combinatorial treatment with cisplatin, when neither TRAIL nor cisplatin alone is sufficient to induce cell death [2]. The present findings provide further supportive evidence for this in GCT tumor cells, as downregulation of p53 resulted only in a partial reversal of cisplatininduced cell death, and no significant reversal of TRAILinduced death, either alone or when combined with cisplatin treatment.
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In summary, GCTs exhibit a unique phenotype when compared to cancers originating from hOSE cells. Most notably, GCTs retain gonadotropin sensitivity and steroidogenic capacity, placing GCTs within the category of endocrine tumor. Despite these unique characteristics, the clinical chemotherapeutic treatment of advanced GCTs is currently derived from conventional therapies utilized to treat ovarian cancers of surface epithelial origin. The results presented herein are among the first to describe the potential for chemotherapeutic utility specifically on GCT cell lines, in vitro. The finding that the efficacy of cisplatin treatment is partially dependent on p53, yet the death-inducing effects of TRAIL are p53-independent, is significant because it implicates multiple mechanisms and sites of action for synergistic activity. Further studies are warranted to evaluate the potential for cisplatin to enhance TRAIL activity in GCTs in animal models.
[12]
[13] [14] [15] [16]
[17]
[18]
Acknowledgments [19]
The authors thank Martin Vonau and Morgan Haugen for PCR amplifications, Dr. Han-Ken Liu for contributions to early stages of this study, and Dr. Neil Lobo for the automated nucleic acid sequencing. Research was supported by grants from the United States Department of Defense (DAMD17-03-1-0206) and The University of Notre Dame Walther Cancer Research Center. C. Alvarez was supported by a National Science Foundation REU fellowship (DBI-0453325).
[20]
[21]
[22]
References
[23]
[1] Colombo N, Parma G, Zanagnolo V, Insinga A. Management of ovarian stromal cell tumors. J Clin Oncol 2007;25:2944–51. [2] Cuello M, Ettenberg SA, Nau MM, Lipkowitz S. Synergistic induction of apoptosis by the combination of TRAIL and chemotherapy in chemoresistant ovarian cancer cells. Gynecol Oncol 2001;81:380–90. [3] Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995;3:673–82. [4] Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 1999;5:157–63. [5] Roth W, Isenmann S, Naumann U, Kugler S, Bahr M, Dichgans J, et al. Locoregional Apo2L/TRAIL eradicates intracranial human malignant glioma xenografts in athymic mice in the absence of neurotoxicity. Biochem Biophys 1999;265:479–83. [6] Johnson AL, Ratajczak C, Haugen MJ, Liu HK, Woods DC. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) expression and activity in hen granulosa cells. Reproduction 2007;133:609–13. [7] Pan G, O'Rourke K, Chinnaiyan AM, Gentz R, Ebner R, Ni J, et al. The receptor for the cytotoxic ligand TRAIL. Science 1997;276:111–3. [8] Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 1997;277:815–8. [9] Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, et al. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 1997;277:818–21. [10] Schneider P, Thome M, Burns K, Bodmer JL, Hofmann K, Kataoka T, et al. TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD-dependent apoptosis and activate NF-kappaB. Immunity 1997;7:831–6. [11] Chaudhary PM, Eby M, Jasmin A, Bookwalter A, Murray J, Hood L.
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31] [32]
Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NF-kappaB pathway. Immunity 1997;7:821–30. Bodmer JL, Holler N, Reynard S, Vinciguerra P, Schneider P, Juo P, et al. TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat Cell Biol 2000;2:241–3. Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 2001;104:487–501. Johnson AL, Bridgham JT. Caspase-mediated apoptosis in the vertebrate ovary. Reproduction 2002;124:19–27. El-Diery WS. Insights into cancer therapeutic design based on p53 and TRAIL receptor signaling. Cell Death Differ 2001;8:1066–75. Tsai WS, Yeow WS, Chua A, Reddy RM, Nguyen DM, Schrump DS, et al. Enhancement of Apo2L/TRAIL-mediated cytotoxicity in esophageal cancer cells by cisplatin. Mol Cancer Ther 2006;5:2977–90. Zhang H, Vollmer M, De Geyter M, Litzistorf Y, Ladewig A, Durrenberger M, et al. Characterization of an immortalized human granulosa cell line. Mol Hum Reprod 2000;6:146–53. Nishi Y, Yanase T, Mu Y, Oba K, Ichino I, Saito M, et al. Establishment and characterization of a steroidogenic human granulosa-like tumor cell line, KGN, that expresses functional follicle-stimulating hormone receptor. Endocrinology 2001;142:437–45. Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science 1991;253:49–53. Khan ZAJ, Jonas SK, Le-Marer N, Patel H, Wharton RQ, Tarragona A, et al. p53 mutations in primary and metastatic tumors and circulating tumor cells from colorectal carcinoma patients. Clin Cancer Res 2000;6: 3499–504. Woods DC, Liu H-K, Nishi Y, Yanase T, Johnson AL. Inhibition of proteosome activity sensitizes human granulosa tumor cells to TRAILinduced cell death. Cancer Lett in press. doi:10.1016/j.canlet.2007.10.016. Livak KJ, Schmittgen TG. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 2001;25: 402–8. Boyd SD, Tsai KY, Jacks T. An intact HDM2 RING-finger domain is required for nuclear exclusion of p53. Nat Cell Biol 2000;9:563–8. Niikura Y, Nonaka T, Imajoh-Ohmi S. Monitoring of caspase-8/FLICE processing and activation upon Fas stimulation with novel antibodies directed against a cleavage site for caspase-8 and its substrate, FLICE-Like Inhibitory Protein (FLIP). J Biochem (Tokyo) 2002;132:53–62. Inoue N, Manabe N, Matsui T, Maeda A, Nakagawa S, Wada S, et al. Roles of tumor necrosis factor-related apoptosis-inducing ligand signaling pathway in granulosa cell apoptosis during atresia in pig ovaries. J Reprod Dev 2003;49:313–21. Krieg A, Krieg T, Wenzel M, Schmitt M, Ramp U, Fang B, et al. TRAILbeta and TRAIL-gamma: two novel splice variants of the human TNFrelated apoptosis-inducing ligand (TRAIL) without apoptotic potential. Br J Cancer 2003;88:918–27. Nguyen DM, Yeow WS, Ziauddin MF, Baras A, Tsai W, Reddy RM, et al. The essential role of the mitochondria-dependent death-signaling cascade in chemotherapy-induced potentiation of Apo2L/TRAIL cytotoxicity in cultured thoracic cancer cells: amplified caspase 8 is indispensable for combination-mediated massive cell death. Cancer J 2006;12:257–73. Petros AM, Gunasekera A, Xu N, Olejniczak ET, Fesik SW. Defining the p53 DNA-binding domain/Bclx-(L)-binding surface interface using NMR. FEBS Lett 2004;559:171–4. Cummings BS, Schnellmann RG. Cisplatin-induced renal cell apoptosis: caspase-3 dependent and -independent pathways. J Pharmacol Exp Ther 2002;302:8–17. Henry H, Thomas A, Shen Y, White E. Regulation of the mitochondrial checkpoint in p53-mediated apoptosis confers resistance to cell death. Oncogene 2002;21:748–60. Miyashita T, Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 1995;80:293–9. Liu J, Yin S, Reddy N, Spencer C, Sheng S. Bax mediates the apoptosissensitizing effect of maspin. Cancer Res 2004;64:1703–11.
Gynecologic Oncology 108 (2008) 632 – 640 www.elsevier.com/locate/ygyno
Cisplatin-mediated sensitivity to TRAIL-induced cell death in human granulosa tumor cells Dori C. Woods, Claudia Alvarez, A.L. Johnson ⁎ Department of Biological Sciences and the Walther Cancer Research Center, The University of Notre Dame, Notre Dame, IN 46556, USA Received 8 October 2007 Available online 14 January 2008
Abstract Objectives. The goal of the present study was to determine the efficacy of combinatorial treatment using cisplatin and tumor necrosis factorrelated apoptosis including ligand (TRAIL) to promote apoptosis in granulosa cell tumor (GCT) lines, in vitro. Methods. Two human GCT lines (COV434 and KGN) were treated with cisplatin or TRAIL, alone or in combination. The cytotoxic effects of each treatment were evaluated using a methyl tetrazolium salt (MTS) assay. Initiation of TRAIL-induced apoptosis was verified by PARP- and FLIP-cleavage. Overexpression and knockdown studies were conducted to evaluate the role of p53 in TRAIL-induced cell death. Real-time PCR was used for gene expression analysis of the TRAIL receptor dr5 and the pro-apoptotic bax following treatment with cisplatin. Results. Treatment with TRAIL (100–200 ng/ml) led to a slight, but significant, loss of cell viability following an 18-h culture. This effect was enhanced following pre-treatment with cisplatin (25 μM) for 2 or 18 h. Moreover, pre-treatment with cisplatin decreased the maximal effective dose of TRAIL from 100 ng/ml to as low as 3 ng/ml in both cell lines. GCT lines overexpressing or deficient in p53 were used to determine the requirement for p53 on TRAIL-induced apoptosis. While the level of p53 expression enhanced both the death-inducing and TRAIL-sensitizing effects of cisplatin, TRAIL-induced cell death was found to occur independent of p53. Conclusions. These data suggest that the efficacy of cisplatin in GCT cells can be enhanced through combinatorial treatment with TRAIL. This result is due to both p53-dependent (cisplatin) and -independent (TRAIL) mechanisms. Combinatorial treatment of GCTs with cisplatin and TRAIL may provide an efficacious addition to cisplatin-based regimens. © 2007 Elsevier Inc. All rights reserved. Keywords: Granulosa cell tumors; TRAIL; Cisplatin; p53; Apoptosis
Introduction Granulosa cell tumors (GCTs) are the most common malignant sex-chord neoplasm, yet the overall occurrence of GCTs is relatively rare. Current estimates indicate that GCTs represent approximately 7% of all ovarian cancers [1]. Nevertheless, the etiology, genetics, and pharmacologic management of GCTs have been comparatively understudied compared to ovarian surface epithelium (OSE)-derived cancers. As such, the basis for targeted granulosa cell therapy has been largely derived from cancers originating from human (h) OSE, with the pre-
⁎ Corresponding author. Fax: +1 574 631 7413. E-mail address: [email protected] (A.L. Johnson). 0090-8258/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2007.11.034
vailing treatment consisting of a platinum-based regimen [1]. Although the overall GCT response rate to platinum-based therapy is estimated at 63% to 80%, the established propensity for eventual relapse is indicative of the need for more effective combinatorial regimes [1]. Although considerable information exists regarding the efficacy of the endogenously produced cytokine, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), to enhance cisplatin-induced cell death in a variety of cancers (including those of hOSE origin) [2], the efficacy of this combinatorial treatment in targeting specifically GCTs has not been reported. Interest in the potential therapeutic application of TRAIL originates from studies demonstrating its cytotoxicity in tumor cells, while most normal cells are resistant to TRAIL-induced apoptosis [3]. The anti-tumorigenic effects of TRAIL are further
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illustrated by its ability to induce regression of cancer xenografts, while healthy tissue remains unaffected [4,5]. Importantly, it has recently been reported that primary cultured normal ovarian granulosa cells are resistant to TRAIL-induced apoptosis, while treatment with cisplatin renders them TRAILsensitive [6]. TRAIL is reported to mediate its biological effects on cancerous cells primarily through two Tumor Necrosis Factor Receptor (TNFSRF) superfamily death receptors, DR4 (TNFSRF10A) and KILLER/DR5 (TNFSRF10B) [7–9]. TRAIL binding induces receptor homotrimerization, which modifies the conformation of the cysteine-rich cytoplasmic domain. This conformational change enables the well-characterized intracellular death domain (DD) motif to associate with the cytoplasmic adaptor protein, Fas-associated death domain protein (FADD), and promote formation of the deathinducing signaling complex [10,11]. In turn, the death effector domain of FADD interacts with initiator caspases (e.g., caspase8) [12]. Consequent activation of caspase-8 cleaves the BH3interacting domain death agonist (Bid), and initiates mitochondrial release of cytochrome c (cytC). Re-localization of cytC to the cytosol initiates the intrinsic cell death pathway, including eventual downstream activation of the executioner caspase, caspase-3. Ultimately, this cascade of signaling events leads to self-amplification of caspase-3 activation and results in cell death (reviewed in [13,14]). Significantly, the efficacy of TRAIL-mediated cytotoxicity is enhanced in wild-type p53 expressing cells following treatment with DNA-damaging chemotherapeutics, such as cisplatin [15]. It has been shown that this increase in TRAIL sensitivity is due, at least in part, to p53-dependent up-regulation of DR5 subsequent to DNA damage. Additionally, it has been shown that treatment with cisplatin sensitizes cells to TRAIL-induced cell death downstream of receptor activation by perturbing mitochondrial function and initiating intrinsic apoptotic mechanisms, including up-regulation and activation of the p53-responsive, pro-apoptotic protein, Bax [16]. Due to the increased efficacy of TRAIL-induced cell death via p53 activity, it has been postulated that activation of p53 by DNA damaging agents (e.g., cisplatin) may provide therapeutic utility in the treatment of some cancers [15]. The potential for cisplatin to modulate cell death in GCTs via extrinsic (e.g., DR5) or intrinsic (bax, mitochondrial perturbation) mechanisms in a p53-dependent manner has yet to be evaluated. In general, the lack of information regarding selective treatment for GCTs, as opposed to hOSE derived cancers, is due not only to the comparatively rare incidence of GCTs, but also to the paucity of appropriate in vitro model systems, and in particular, established human GCT lines. The recent description of adulttype GCT lines, COV434 [17] and KGN [18], has provided new opportunities for developing more targeted GCT therapies. Accordingly, in the present report, we provide evidence for: (1) TRAIL plus dr5 mRNA expression and TRAIL-induced cell death in GCT (COV434 and KGN) cell lines; (2) cisplatininduced cell death and enhanced TRAIL sensitivity in GCT cell lines; and (3) the facilitation of cisplatin-induced death and TRAIL sensitivity in a p53-dependent manner.
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Materials and methods Cell culture and reagents The adult-type human ovarian GCT lines, COV434 and KGN, were generously provided by Dr. De Geyter (University of Basel, Switzerland), and Drs. Nishi and Yanase (Kyushu University, Japan), respectively. Both cell lines were maintained in a subconfluent state during all experiments. COV434 and KGN were cultured in cell culture medium (COV434, DMEM; KGN, DMEM/ Ham's F12; Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 1% nonessential amino acids, and 1% antibiotic/antimycotic. All cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2. For cell culture experiments, adherent cells were trypsinized, washed with an equal volume of cell culture medium, and plated in either 6- or 96-well Corning polystyrene cell culture dishes. Recombinant human TRAIL was purchased from Peprotech (Rocky Hill, NJ), and cisplatin (cis-platinum(II)-diammine dichloride) was from Sigma–Aldrich (St. Louis, MO). The selective caspase inhibitor, Z-IETD-FMK (IETD), was from R&D Systems (Minneapolis, MN). Optimally effective doses for TRAIL, cisplatin, and Z-IETD-FMK were empirically determined for each cell line.
Nested PCR and p53 sequencing In lieu of amplification and sequence analysis of the entire p53 gene, mutational analysis was performed on the five highly conserved domains, where most functional mutations are reported to occur [19]. To identify potential p53 mutations in COV434 and KGN cell lines, nested PCR was used followed by automated sequencing. cDNA was synthesized from freshly isolated total RNA and random hexamers using the Promega Reverse Transcription System (Promega, Madison, WI). Samples were incubated for 15 min at room temperature, then 15 min at 45 °C, followed by 5 min at 95 °C for enzyme inactivation. PCR reactions were subsequently performed using High Fidelity Taq polymerase, with a proof-reading polymerase designed for mutational analysis (ClonTech, Palo Alto, CA). Nested PCR reactions for mutational analysis, including the primers and reaction conditions, were performed essentially as described by Khan et al. [20]. Briefly, primers specific for the coding region between exons 3 and 10 were used to amplify an initial product (outer primers). The resulting product was then used in a subsequent PCR reaction using two sets of primers designed to amplify the coding region between exons 4 and 9 (inner primers). The two sets of inner primers consisted of an overlapping region to ensure total amplification of the conserved region of p53. The products were then sequenced using the ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA).
Amplification of TRAIL mRNA by RT–PCR Primers for TRAIL PCR amplification from COV434, KGN, and granulosa cells collected from IVF patients (provided by Dr. B.R. Rueda, The Vincent Center for Reproductive Biology, Boston, MA) were designed using MacVector (v.6.5.3; Cary, NC), and were specific for exons 1 and 5 (TRAIL sense: 5′ACCTGCGTGCTGATCGTG-3′ [exon 1]; antisense: 5′-ATTTTGCGGCCCAGAGCC-3′[exon 5]). RNA was isolated and cDNA synthesized as described above. Thermocycling conditions were: 94 °C for 3 min, then 94 °C for 45 s, 55 °C for 45 s, 72 °C for 90 s for 35 cycles, followed by 72 °C for 10 min. Amplified products were subcloned and sequenced to verify nucleic acid identity.
Cell density analysis for granulosa tumor cell viability Adherent KGN and COV434 cells were removed from culture flasks and seeded in a 96-well cell culture plate at a density of 2 × 104 (COV434) or 1 × 104 (KGN). After an overnight culture, the medium was removed and replaced, then cells were cultured in the absence or presence of cisplatin (1–50 μM), TRAIL (3–100 ng/ml), or cisplatin plus TRAIL. Following the time period specified for each experiment, cell density (a measure of metabolic activity and viability) was determined as previously described [6,21] using the colorimetric methyl tetrazolium salt (MTS) Cell Titer 96 Aqueous One Solution cell proliferation assay (Promega). The colorimetric reagent was added to each well of the plate,
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incubated for 1–4 h, and the absorbance values read at 490 nm. Activation of apoptotic pathways was further verified through detection of poly(ADP-ribose) polymerase (PARP) and FLICE inhibitory protein (FLIP) cleavage, as described below.
Western blot analysis Cells were harvested and lysed in ice-cold lysis buffer (150 mM NaCl, 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM pervanidate, 1 mM EDTA, 1% Igepal, 0.25% deoxycholic acid, 1 mM NaF, and 50 mM Tris–HCl, pH 7.4), and cleared through centrifugation at 13,000×g. Between 10 and 40 μg of total cellular protein was loaded on an SDS polyacrylamide gel for electrophoresis, followed by a transfer to nitrocellulose
membranes. The membranes were blocked for 1 h at room temperature in 5% milk in TTBS (Tris-buffered saline–0.1% Tween 20), then incubated with primary antibody overnight at 4 °C. Rabbit anti-PARP serum (Cell Signaling, Danvers, MA) was used at a dilution of 1:1000. The anti-FLIP serum (Santa Cruz; used a 1:1000 dilution) was raised in rabbits against the amino terminus. Rabbit anti-DR5 (Sigma–Aldrich), recognizing both the unprocessed and mature proteins, was used at a 1:500 dilution, while rabbit anti-bax serum was from Santa Cruz (Santa Cruz, CA) and diluted 1:1000. Following incubation with primary antibody, membranes were washed for 5 min in TTBS then incubated with horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG; Pierce Endogen, Rockford, IL) for 1 h at room temperature. The membranes were incubated with ECL Western blotting detection reagent (Pierce) for 1 min, then wrapped and exposed to X-ray film for 3 to 10 min. Membranes
Fig. 1. Expression of TRAIL mRNA in COV434 and KGN cell lines plus non-neoplastic (IVF-derived) granulosa cells. (A) PCR co-amplification of TRAIL (413 bp) and an alternatively spliced variant (TRAILδ; 265 bp) from COV434 (COV), KGN and IVF-derived cells. Negative (no RT) control (Neg.). (B) Clustal alignment depicting full-length TRAIL (GenBank accession: U37518) and a novel TRAIL splice variant (TRAIL-δ; EU183231) cloned from COV434 and KGN cells. (C) Top: The 5 exons from the full-length TRAIL pre-mRNA results in an 846 total nt product (middle). Bottom: mRNA encoding TRAIL-δ, lacking exons 3 and 4, results in a predicted 698 nt product.
D.C. Woods et al. / Gynecologic Oncology 108 (2008) 632–640 were subsequently blotted for α-tubulin (Pierce) to demonstrate equal loading of protein.
Real-time quantitative PCR Forward and reverse primers specific for DR5, bax, and 18s rRNA have been described previously [21], and were subsequently validated for use with real-time PCR by determining the optimal amplification efficiency and primer concentrations as described by the system manufacturer (Applied Biosystems). Gene expression analysis was performed as previously described [21], using ABgene Absolute QPCR SYBR Green Mix (ABgene, Rochester, NY). Briefly, random-primed, reverse transcribed cDNA synthesis reactions were performed utilizing the Promega Reverse Transcription System (Promega, Madison, WI), as described by the manufacturer. For real-time PCR, primers and were added to 25 μl total reaction volume using reagents provided in the kit. Final concentrations of the sense and antisense primers were determined for each primer pair based on optimal amplification efficiency. Reactions were completed on an ABI 7700 Thermocycler (Applied Biosystems). Thermocycling conditions were as follows: 95 °C for 15 min followed by a program of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s for a total of 40 cycles. The Ct (defined as the cycle number at which the fluorescence exceeds a threshold level) was determined for each reaction (run in triplicate) using the Sequence Detection software (v.1.6.3; Applied Biosystems), while quantification was accomplished using the ΔΔCt method [22]. The target Ct was determined for each sample then normalized to the 18s rRNA Ct from the same sample (18s rRNA Ct subtracted from the target Ct yields the ΔCt). These values were expressed as fold-difference compared to an appropriate control sample using the 2−ΔΔCt equation.
Cell transfection for overexpression of p53 and siRNA
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Means were designated as statistically significant when p values b0.05 were obtained.
Results Amplification and sequencing of TRAIL in COV434 and KGN granulosa tumor cell lines PCR amplification revealed the presence of TRAIL mRNA in both KGN and COV434 cell lines, as well as in granulosa cells obtained from IVF patients. Interestingly, in addition to the anticipated 413 bp TRAIL PCR product, a smaller 265 bp product was also detected (Fig. 1A). Sequencing revealed a novel truncated variant form of TRAIL with a deletion of nucleotides 272–419, corresponding to deletions of both exon 3 (43 nt) and exon 4 (105 nt) (Figs. 1B, C; GenBank accession EU183231). Treatment with cisplatin enhances TRAIL-induced cell death Treatment of COV434 and KGN cells with TRAIL at 100 and 200 ng/ml for 18 h resulted in a slight (∼10%), but statistically significant, induction of cell death compared to control cells (Fig. 2A; p b 0.05). By comparison, treatment with cisplatin (1 to 50 μM) induced cell death in a dose-dependent fashion following 18 h in COV434 cells, while the magnitude of
The GFP-expressing wild-type and nuclear localization p53 constructs were provided by Dr. Tyler Jacks (Massachusetts Institute of Technology, Center for Cancer Research), and have been described previously [23]. The p53-specific and control siRNAs were purchased from Santa Cruz. Cells were seeded in 6well cell-culture plates for 1 or 2 days at a density of 5 × 105 or 2 × 105 (COV434 and KGN, respectively) to allow adequate time for cell adherence. Transfections were performed using the Lipofectamine 2000 transfection reagent using conditions recommended by the manufacturer (Invitrogen). For each transfection, 6 μl of Lipofectamine was added to 100 μl of cell culture medium in the absence of antibiotics and serum. Additionally, 6 μg of DNA or siRNA was added to 100 μl of serum/antibiotic free cell culture medium. The Lipofectamine and DNA or RNA solutions were then combined and incubated for 45 min at room temperature. Following this incubation, 800 μl of serum- and antibioticfree cell culture medium was added to each Lipofectamine complex, and the entire 1 ml solution added to the cells. The cells were cultured for 5 h, followed by the addition of 1 ml of appropriate cell culture medium containing 20% FBS. Cells were incubated overnight, then the medium was removed and replaced with normal growth medium. Cells were harvested 24 h later for analysis or seeded in a 96-well plate to assay cell viability. Control (mock) transfections with Lipofectamine reagent were performed with each transfection. For p53 overexpression studies, data are compared to cells transfected with the pEGFPN1 vector alone. For p53 siRNA studies, data were compared to cells transfected with non-silencing (control) siRNA. Knockdown efficiency was monitored by PCR.
Confocal microscopy Immunolocalization studies were performed by confocal analysis as previously described [21] using a mouse monoclonal antibody directed against cytochrome c (PharMingen International; used at 1:100 dilution).
Statistical analysis Data are expressed as mean ± SEM and compared by one-way ANOVA followed by LSD post-hoc analysis or by t test using SPSS statistical software. Each experiment was independently replicated a minimum of three times.
Fig. 2. TRAIL and cisplatin effects on cell viability in COV434 and KGN cell lines. (A) Treatment with TRAIL (50–200 ng/ml) for 18 h results in a slight, but significant, decrease in cell viability in both cell lines at the two highest doses. (A, B; a, b) p b 0.05; n = 4 replicate experiments. (B) Treatment with cisplatin (1–50 μM) for 18 h results in a loss of cell viability in both cell lines. Different letters denote p b 0.05; n = 4 replicate experiments.
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Fig. 3. Combinatorial effects of cisplatin plus TRAIL treatment on GCT viability and apoptosis. (A) Pre-treatment with cisplatin (25 μM for 2 h) enhances the effectiveness of TRAIL (100 ng/ml added during the last 18 h of culture) to decrease cell viability. Data are expressed relative to control cultured cells (not shown). (A, B, C; a, b, c) p b 0.05; n = 5 replicate experiments. (B) TRAIL (100 ng/ml) induces PARP cleavage following treatment for 4 h (top panel) and 18 h (bottom panel), while treatment with cisplatin (25 μM) increases PARP cleavage by 18 h. (C, D) Pre-treatment with cisplatin for 18 h sensitizes GCT lines to a subsequent 4 h (C) or 18 h (D) challenge with TRAIL. Different letters denote p b 0.05 from 5 to 7 replicate experiments. (E) In addition, cisplatin pre-treatment effectively reduces the maximal effective dose of TRAIL to as low as 3 ng/ml in COV434 and KGN cell lines. *,+ denote p b 0.05 compared to 0 ng TRAIL/ml by t test; n = 5 replicate experiments.
this response in KGN cells was less pronounced (Fig. 2B; p b 0.05). In both COV434 and KGN cell lines, the effect of cisplatin (25 μM) plus TRAIL (100 ng/ml) was enhanced when cells were pre-treated with cisplatin for 2 h (Fig. 3A). An 89-kDa PARP cleavage product (a marker of apoptosis) was induced by TRAIL (100 ng/ml) following as little as 4 h in culture, whereas cisplatin (25 μM)-induced PARP cleavage was visible following 18 h of culture (Fig. 3B). Following a more prolonged pretreatment with cisplatin (for 18 h), TRAIL treatment for 4 h significantly reduced the number of viable cells compared to cells treated with cisplatin alone (Fig. 3C). This reduction in cell viability was further enhanced following an extended 18 h incubation with TRAIL (Fig. 3D). Not only was cisplatin effective in sensitizing the COV434 and KGN cell lines to TRAIL after
4 h, pre-treatment with cisplatin for 18 h significantly reduced the effective dose of TRAIL to as low as 3 ng/ml (compare Fig. 3E to Fig. 2A). Effects of cisplatin occur independent of caspase-8 activity Treatment with TRAIL (100 ng/ml), but not cisplatin (25 μM), for 4 h resulted in detection of a p43 N-terminal FLIP-cleavage product as determined by Western blot analysis (Fig. 4A), indicative of caspase-8 activation [24]. To evaluate the role of caspase-8 in cisplatin-induced TRAIL sensitivity, COV434 and KGN cells were pretreated for 2 h without or with the caspase-8 inhibitor, IETD (20 μM). Inhibition of caspase-8 activity significantly attenuated the cytotoxic effects of TRAIL, both in the absence and presence of
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Notably, there was no detectable rescue of viability in sip53 transfected cells treated with cisplatin plus TRAIL versus those treated with cisplatin alone. These findings provide evidence that the effects of TRAIL, but not those of cisplatin, occur independent from p53 activity. Cisplatin increases dr5 and bax mRNA and protein levels in GCT cell lines Treatment with cisplatin for 18 h resulted in significantly elevated levels of bax and dr5 mRNA (Fig. 7A) plus protein (Fig. 7B) in both KGN and COV434 cell lines. Up-regulation of functional Bax is corroborated by the re-localization of cytC from the mitochondria to the cytoplasm (Fig. 7C). To further investigate whether the effects of cisplatin on bax and dr5 transcription in KGN and COV434 are mediated by p53, each cell line was transfected with non-silencing siRNA (siC) or
Fig. 4. (A) TRAIL treatment (100 ng/ml) for 4 h results in FLIP cleavage, as indicated by the presence of the 43-kDa N-terminal FLIP cleavage product. (B) The selective pharmacologic caspase-8 inhibitor Z-IETD-FMK (IETD; 20 μM) attenuates the effects of TRAIL treatment for 18 h alone or in combination with cisplatin (25 μM) pretreatment for 18 h in both COV434 and KGN cell lines. ⁎p b 0.05 versus absence of IETD, by t test, n = 5 replicate experiments.
cisplatin (Fig. 4B). By contrast, IETD failed to alter cisplatininduced cytotoxicity. Cisplatin-mediated cell death and sensitivity to TRAIL is p53-dependent A nested PCR technique described by Khan et al. [20] was used to determine the mutational status of p53 (see Materials and methods). No nucleotide substitutions, additions, or deletions were detected in either transformed cell line compared to wild-type p53 (p53-wt; data not shown). Transfection of COV434 and KGN cells with p53-wt or nuclear localized p53 (p53-NL) (Fig. 5A) facilitated a significant increase in cisplatininduced cytotoxicity compared to cells expressing the plasmid vector pEGFP-N1 (Fig. 5B). Moreover, cells transfected with p53 were significantly more sensitive to treatment with TRAIL following pre-treatment with cisplatin. This sensitizing effect was specific to cells pre-treated with cisplatin, as treatment with TRAIL treatment alone did not result in greater cytotoxicity compared to pEGFP-N1 expressing cells. Furthermore, targeted knockdown of p53 using siRNA (sip53; Fig 6A) did not enhance cell viability following treatment with TRAIL when compared to cells transfected with the non-silencing siRNA (Fig. 6B). By comparison, transfection with sip53 increased the percentage of viable cells following treatment with cisplatin.
Fig. 5. Overexpression of p53 leads to an increase in sensitivity to cisplatin, but not TRAIL, in COV434 and KGN cell lines. (A) Localization of GFP-tagged wild-type p53 (wt-p53; top panels) within the cytoplasm, and nuclear-localized p53 (p53-NL; bottom panels) in KGN cells. Nucleus in marked by arrow. (B) Overexpression of p53-NL enhances the cell death-inducing effects of cisplatin compared to cells treated with the pEGFP-N1 vector alone. Additionally, overexpression of p53-NL enhanced cisplatin-induced TRAIL sensitivity, while having no effect on TRAIL treatment alone. ns: non-significant; ⁎p b 0.05 by t test compared to vector alone. n = 5 replicate experiments.
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expression of TRAIL mRNA in both GCT lines, as well as in non-neoplastic granulosa cells (Fig. 1A). Interestingly, sequence analysis demonstrated the presence of a novel TRAIL mRNA splice variant lacking exons 3 and 4 (TRAIL-δ). As previously described for the human TRAIL variants, TRAIL-β and TRAIL-γ [26], deletion of these exons results in a predicted truncated TRAIL-δ protein consisting of 101 amino acids (aa). Notably, this variant is similar to the 98 aa TRAIL-β variant, which lacks apoptotic activity presumably due to a truncated extracellular binding domain. Unlike TRAIL-γ, which also lacks this binding domain, TRAIL-β does not strongly associate with the cell surface and nuclear membranes. Although the biological function(s) of any TRAIL variant has not been unequivocally established, it has been speculated that such variants may play a passive regulatory role in TRAIL actions by reducing the amount of biologically active TRAIL [26].
Fig. 6. Knockdown of p53 leads to a partial rescue of cisplatin-induced cell death. (A) PCR amplification following transfection with siRNA specifically targeting p53 (sip53) in COV434 and KGN cell lines. Non-silencing control siRNA (siCon) was used to control for non-specific effects. Neg., no RT control. (B) Percent rescue of cell viability compared to cells transfected with siCon (not shown). Treatment with sip53 resulted in a partial reversal of the effects of cisplatin (∼60% in COV, ~30% in KGN), however, there was no further increase in cell viability in cells treated with cisplatin plus TRAIL. Down-regulation of p53 mRNA expression did not provide a significant rescue effect on TRAILreduced cell viability. ns: p N 0.05, n = 5 replicate experiments.
sip53. In both GCT lines, transfection with sip53 resulted in a significant decrease in levels of cisplatin-induced bax and dr5 mRNA (Fig. 8). Discussion Published clinical data suggest that while platinum-based therapy is up to 80% effective in treating advanced stage GCTs, the potential for increasing the efficacy of chemotherapeutic therapy plus the opportunity to minimize tumor reoccurrence following initial therapy emphasize the need for the development of more targeted combinatorial regimens [1]. Due to the relative infrequency of GCTs and scarcity of in vitro model systems, approaches to addressing these issues have only recently been initiated. The results presented herein demonstrate the effects of TRAIL enhanced apoptotic cell death via combinatorial treatment with the conventional chemotherapeutic, cisplatin, in GCT cell lines, in vitro. The expression of TRAIL has been reported in normal granulosa cells from various animal models [6,25], yet it is not known whether the human GCT lines express this naturally occurring ligand. PCR amplification revealed the endogenous
Fig. 7. Cisplatin-induced bax and dr5 mRNA plus protein expression in COV434 and KGN cell lines. (A) Real-time PCR data indicate that cisplatin (Cis; 25 μM) treatment for 18 h increases levels of bax and dr5 mRNA in both cell lines. ⁎p b 0.05 compared to control (Con) cultured cells; n = 3 replicate experiments. (B) Cisplatin, but not TRAIL (100 ng/ml for 18 h), also induced bax and DR5 protein in both COV434 and KGN cells. (C) Immunofluorescence depicting localization of cytochrome c (cytC) in KGN. Left: Punctate staining (arrows) indicative of mitochondrial localized cytC in control cultured cells. Right: Induced release of cytC into the cytoplasm following treatment with cisplatin for 8 h. Dotted circle depicts outline of the nucleus.
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Fig. 8. Results of real-time PCR analyses indicate that transfection with siRNA targeting p53 (sip53) decreases cisplatin (25 μM)-induced bax and dr5 mRNA expression in COV434 and KGN cell lines when compared to cells transfected with non-silencing siRNA (siCon). * Indicates significance by t test, n = 3.
Nevertheless, further experimental work is required to determine what, if any, role TRAIL splice variants have in modulating the apoptotic activity of TRAIL in human granulosa cells. Treatment of GCT cell lines with TRAIL resulted in a slight, but statistically significant, increase in cell death, indicated by both a reduction in cell viability (Fig. 2A) and the rapid (within 4 h) initiation of PARP (Fig. 3B) and FLIP (Fig. 4A) cleavage. The cytotoxic effects of TRAIL were enhanced following pretreatment with cisplatin for 2 or 18 h (Figs. 3A, C, D). This finding is consistent with earlier reports in hOSE tumor cell lines, in which cisplatin increased the ability of TRAIL to initiate apoptosis as indicated by an increase in PARP cleavage and caspase-3 activation [2]. The additive effect of cisplatin on TRAIL-induced cell death in GCT cells is attributed to the effects of the extrinsic (e.g., death receptor-induced) pathway, as suggested by the ability of the selective caspase-8 inhibitor, Z-IETD-FMK to prevent this combinatorial effect (Fig. 4). This finding is similar those reported from hOSE tumor cell lines where a pan-caspase inhibitor, Z-VAD-FMK, inhibited the cytotoxic effects of TRAIL [2]. Additionally, the intrinsic pathway may be of critical importance in this additive effect, since a recent study in thoracic cancer cells indicates that mitochondrial-mediated amplification of caspase-8 are critical components for enhancing the cytotoxic effects of TRAIL [27]. One established mechanism by which cells are sensitized to TRAIL is through the DNA-damage-inducible p53 pathway, which modulates cell death through both extrinsic (e.g., DR5) and intrinsic (e.g., bax) activity. Data presented herein indicate that p53-wt expression is not sufficient to significantly enhance the death-inducing effects of TRAIL (Fig. 5B). By comparison, both cisplatin-induced cell death and cisplatin-enhanced sen-
sitivity to TRAIL were evidenced in p53 overexpressing cells. It has been shown that in addition to amplifying transcription of pro-apoptotic genes, cytosolic p53 can inhibit the activity of the anti-apoptotic Bcl-x by forming a p53/Bcl-x inhibitory complex [28]. In addition to an apoptogenic role at the mitochondria, nuclear-localized p53 can affect mitochondrial function through the up-regulation of the pro-apoptotic Bax protein [30–32]. To investigate the potential for p53-mediated events at the level of the nucleus, nuclear-localized p53 (p53-NL) was overexpressed in COV434 and KGN cells. Similar to results obtained with cytosolic-localized p53-wt, p53-NL significantly enhanced both the death-inducing and TRAIL-sensitizing effects of cisplatin. This result is not unexpected, as treatment of renal tubule cells with cisplatin results in the nuclear localization wild-type p53, independent from caspase activation [29]. Results from the present studies indicate that knockdown of p53 does not rescue the death-inducing effects of TRAIL, but does lead to at least a partial reversal of cisplatin-induced death in both GCT cell lines (Fig. 6). Significantly, both cisplatin-induced dr5 and bax mRNA expression were blocked in p53-deficient cells (Fig. 8). Taken together, these data suggest that the effects of cisplatin on TRAIL-mediated cell death are, at least in part, p53 dependent. Nevertheless, it has been shown that cells with mutated p53 are also susceptible to combinatorial treatment with cisplatin, when neither TRAIL nor cisplatin alone is sufficient to induce cell death [2]. The present findings provide further supportive evidence for this in GCT tumor cells, as downregulation of p53 resulted only in a partial reversal of cisplatininduced cell death, and no significant reversal of TRAILinduced death, either alone or when combined with cisplatin treatment.
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In summary, GCTs exhibit a unique phenotype when compared to cancers originating from hOSE cells. Most notably, GCTs retain gonadotropin sensitivity and steroidogenic capacity, placing GCTs within the category of endocrine tumor. Despite these unique characteristics, the clinical chemotherapeutic treatment of advanced GCTs is currently derived from conventional therapies utilized to treat ovarian cancers of surface epithelial origin. The results presented herein are among the first to describe the potential for chemotherapeutic utility specifically on GCT cell lines, in vitro. The finding that the efficacy of cisplatin treatment is partially dependent on p53, yet the death-inducing effects of TRAIL are p53-independent, is significant because it implicates multiple mechanisms and sites of action for synergistic activity. Further studies are warranted to evaluate the potential for cisplatin to enhance TRAIL activity in GCTs in animal models.
[12]
[13] [14] [15] [16]
[17]
[18]
Acknowledgments [19]
The authors thank Martin Vonau and Morgan Haugen for PCR amplifications, Dr. Han-Ken Liu for contributions to early stages of this study, and Dr. Neil Lobo for the automated nucleic acid sequencing. Research was supported by grants from the United States Department of Defense (DAMD17-03-1-0206) and The University of Notre Dame Walther Cancer Research Center. C. Alvarez was supported by a National Science Foundation REU fellowship (DBI-0453325).
[20]
[21]
[22]
References
[23]
[1] Colombo N, Parma G, Zanagnolo V, Insinga A. Management of ovarian stromal cell tumors. J Clin Oncol 2007;25:2944–51. [2] Cuello M, Ettenberg SA, Nau MM, Lipkowitz S. Synergistic induction of apoptosis by the combination of TRAIL and chemotherapy in chemoresistant ovarian cancer cells. Gynecol Oncol 2001;81:380–90. [3] Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995;3:673–82. [4] Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 1999;5:157–63. [5] Roth W, Isenmann S, Naumann U, Kugler S, Bahr M, Dichgans J, et al. Locoregional Apo2L/TRAIL eradicates intracranial human malignant glioma xenografts in athymic mice in the absence of neurotoxicity. Biochem Biophys 1999;265:479–83. [6] Johnson AL, Ratajczak C, Haugen MJ, Liu HK, Woods DC. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) expression and activity in hen granulosa cells. Reproduction 2007;133:609–13. [7] Pan G, O'Rourke K, Chinnaiyan AM, Gentz R, Ebner R, Ni J, et al. The receptor for the cytotoxic ligand TRAIL. Science 1997;276:111–3. [8] Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 1997;277:815–8. [9] Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, et al. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 1997;277:818–21. [10] Schneider P, Thome M, Burns K, Bodmer JL, Hofmann K, Kataoka T, et al. TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD-dependent apoptosis and activate NF-kappaB. Immunity 1997;7:831–6. [11] Chaudhary PM, Eby M, Jasmin A, Bookwalter A, Murray J, Hood L.
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31] [32]
Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NF-kappaB pathway. Immunity 1997;7:821–30. Bodmer JL, Holler N, Reynard S, Vinciguerra P, Schneider P, Juo P, et al. TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat Cell Biol 2000;2:241–3. Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 2001;104:487–501. Johnson AL, Bridgham JT. Caspase-mediated apoptosis in the vertebrate ovary. Reproduction 2002;124:19–27. El-Diery WS. Insights into cancer therapeutic design based on p53 and TRAIL receptor signaling. Cell Death Differ 2001;8:1066–75. Tsai WS, Yeow WS, Chua A, Reddy RM, Nguyen DM, Schrump DS, et al. Enhancement of Apo2L/TRAIL-mediated cytotoxicity in esophageal cancer cells by cisplatin. Mol Cancer Ther 2006;5:2977–90. Zhang H, Vollmer M, De Geyter M, Litzistorf Y, Ladewig A, Durrenberger M, et al. Characterization of an immortalized human granulosa cell line. Mol Hum Reprod 2000;6:146–53. Nishi Y, Yanase T, Mu Y, Oba K, Ichino I, Saito M, et al. Establishment and characterization of a steroidogenic human granulosa-like tumor cell line, KGN, that expresses functional follicle-stimulating hormone receptor. Endocrinology 2001;142:437–45. Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science 1991;253:49–53. Khan ZAJ, Jonas SK, Le-Marer N, Patel H, Wharton RQ, Tarragona A, et al. p53 mutations in primary and metastatic tumors and circulating tumor cells from colorectal carcinoma patients. Clin Cancer Res 2000;6: 3499–504. Woods DC, Liu H-K, Nishi Y, Yanase T, Johnson AL. Inhibition of proteosome activity sensitizes human granulosa tumor cells to TRAILinduced cell death. Cancer Lett in press. doi:10.1016/j.canlet.2007.10.016. Livak KJ, Schmittgen TG. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 2001;25: 402–8. Boyd SD, Tsai KY, Jacks T. An intact HDM2 RING-finger domain is required for nuclear exclusion of p53. Nat Cell Biol 2000;9:563–8. Niikura Y, Nonaka T, Imajoh-Ohmi S. Monitoring of caspase-8/FLICE processing and activation upon Fas stimulation with novel antibodies directed against a cleavage site for caspase-8 and its substrate, FLICE-Like Inhibitory Protein (FLIP). J Biochem (Tokyo) 2002;132:53–62. Inoue N, Manabe N, Matsui T, Maeda A, Nakagawa S, Wada S, et al. Roles of tumor necrosis factor-related apoptosis-inducing ligand signaling pathway in granulosa cell apoptosis during atresia in pig ovaries. J Reprod Dev 2003;49:313–21. Krieg A, Krieg T, Wenzel M, Schmitt M, Ramp U, Fang B, et al. TRAILbeta and TRAIL-gamma: two novel splice variants of the human TNFrelated apoptosis-inducing ligand (TRAIL) without apoptotic potential. Br J Cancer 2003;88:918–27. Nguyen DM, Yeow WS, Ziauddin MF, Baras A, Tsai W, Reddy RM, et al. The essential role of the mitochondria-dependent death-signaling cascade in chemotherapy-induced potentiation of Apo2L/TRAIL cytotoxicity in cultured thoracic cancer cells: amplified caspase 8 is indispensable for combination-mediated massive cell death. Cancer J 2006;12:257–73. Petros AM, Gunasekera A, Xu N, Olejniczak ET, Fesik SW. Defining the p53 DNA-binding domain/Bclx-(L)-binding surface interface using NMR. FEBS Lett 2004;559:171–4. Cummings BS, Schnellmann RG. Cisplatin-induced renal cell apoptosis: caspase-3 dependent and -independent pathways. J Pharmacol Exp Ther 2002;302:8–17. Henry H, Thomas A, Shen Y, White E. Regulation of the mitochondrial checkpoint in p53-mediated apoptosis confers resistance to cell death. Oncogene 2002;21:748–60. Miyashita T, Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 1995;80:293–9. Liu J, Yin S, Reddy N, Spencer C, Sheng S. Bax mediates the apoptosissensitizing effect of maspin. Cancer Res 2004;64:1703–11.