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Endocrinology Vol. 145, No. 1 49-58
Copyright © 2004 by The Endocrine Society

Estrogen Inhibits Paclitaxel-Induced Apoptosis via the Phosphorylation of Apoptosis Signal-Regulating Kinase 1 in Human Ovarian Cancer Cell Lines

Seiji Mabuchi, Masahide Ohmichi, Akiko Kimura, Yukihiro Nishio, Emi Arimoto-Ishida, Namiko Yada-Hashimoto, Keiichi Tasaka and Yuji Murata

Department of Obstetrics and Gynecology, Osaka University Medical School, Osaka 565-0871, Japan

Address all correspondence and requests for reprints to: Dr. Masahide Ohmichi, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: masa{at}gyne.med.osaka-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The influence of postoperative estrogen replacement therapy on the sensitivity of ovarian cancer to paclitaxel remains elusive. We examined whether estrogen affects paclitaxel-induced apoptosis in the Caov-3 human ovarian cancer cell line, which expresses estrogen receptor. 17ß-Estradiol (E2) significantly reversed the paclitaxel-induced apoptosis and reduction of cell viability, and a highly selective estrogen receptor antagonist, ICI182,780, and a phosphatidylinositol 3-kinase inhibitor, LY294002, attenuated the reversal effect of E2 on paclitaxel-induced apoptosis and reduction of cell viability. E2 significantly induced the phosphorylation of Akt. Akt and apoptosis signal-regulating kinase 1 (ASK1) were physically associated, and E2 induced the phosphorylation of ASK1 at serine-83, which is a consensus Akt phosphorylation site. We confirmed a previous report showing that paclitaxel induces cell damage via the ASK1-c-Jun N-terminal protein kinase (JNK) cascade. E2 inhibited the paclitaxel-induced JNK activation, and the E2-induced inhibition of the paclitaxel-induced JNK activation was attenuated in cells treated with either ICI182,780 or LY294002 or transfected with ASK1S83A, in which a consensus Akt phosphorylation site at serine-83 was converted to alanine. The inhibitory effect of E2 on the paclitaxel-induced reduction of cell viability and apoptosis was diminished in cells transfected with ASK1S83A. These results indicate that E2 inhibits paclitaxel-induced cell damage by inhibiting JNK activity via phosphorylation of Akt-ASK1. Thus, treatment of ovarian cancer with paclitaxel might be less effective in the setting of postoperative estrogen replacement therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVARIAN CARCINOMA RESULTS in more deaths than any other gynecological malignancy. In the vast majority of cases, it presents as advanced disease that is widespread in the abdominal cavity. Therefore, the majority of patients with ovarian cancer require treatment with cytotoxic chemotherapy after surgical management. Even though the incidence of ovarian carcinoma increases with age, a significant proportion of cases occur in premenopausal and perimenopausal women. Surgical management for premenopausal and perimenopausal patients with epithelial ovarian carcinoma will, by necessity, induce significant menopausal symptoms. Estrogen receptor (ER) is present in about 60% of cases of ovarian cancer (1). Although it was previously reported that estrogen replacement therapy (ERT) does not have a pronounced effect on the survival of patients with invasive ovarian carcinoma (2), it was recently reported that ERT induces ovarian cancer (3). It was reported that postoperative estrogen replacement therapy did not have a negative influence on the disease-free interval or overall survival of ovarian carcinoma survivors in a small study (4). Based on in vitro and in vivo data, estrogen probably acts as both a growth factor and a survival factor for breast cancer (5, 6). In vitro, breast cancer treatment with chemotherapy is markedly less effective in the setting of estrogen treatment (7, 8). In addition, it was reported that estrogen inhibits the paclitaxel-induced apoptosis of breast cancer cells (9). Paclitaxel, which is a microtubule-interfering agent, is also widely used for ovarian cancer treatment (10). However, the effect of postoperative ERT on the sensitivity of ovarian cancer to chemotherapy remains unknown.

Among the stress-activated kinases, apoptosis signal-regulating kinase 1 (ASK1) is a MAPK kinase kinase family member that acts upstream of c-Jun N-terminal protein kinase (JNK) and p38 kinases (11, 12). ASK1 plays a causal role in cell death induced by a number of stimuli, including microtubule-interfering agents (13), genotoxic stress (14), and TNF{alpha} (15). It was reported that the induction of apoptosis by paclitaxel is mediated by the activation of JNK through ASK1 (13) in breast cancer cells. The ability of estrogen to act as a survival factor for breast cancer is not well understood. It was reported that ER{alpha} binds to the p85{alpha} subunit of phosphatidylinositol 3-kinase (PI3K) in breast cancer cells (16) and vascular endothelial cells (17). PI3K phosphorylates the D-3 position of the phosphatidylinositol ring, catalyzing the synthesis of lipid mediators that act as second messengers transferring the signaling cascade to intracellular protein kinases. One of the principal targets of this cascade is the serine (Ser)-threonine protein kinase Akt. The activation of Akt mediates many of the downstream cellular effects of PI3K, including activation of cell survival pathways (18). Estrogen is known to stimulate the activity of Akt, which plays a central role in promoting the survival of a wide range of cell types (19, 20, 21). It was reported that ASK1 is a substrate for phosphorylation by Akt (22) and that this phosphorylation is associated with a decrease in the stimulation of ASK1 kinase activity (22, 23). Indeed, the inhibition of cisplatin-induced apoptosis via phosphorylation of ASK1 by Akt is reported more recently to play an important role in chemoresistance of ovarian cancer cells (23). Moreover, estrogen is known to prevent chemotherapy or radiation-induced apoptosis of breast cancer cells via a novel and rapid nongenomic mechanism (9).

These findings led us to examine whether estrogen inhibits paclitaxel-induced apoptosis in Caov-3 human ovarian cancer cell lines, which express ER and whether estrogen negatively regulates the paclitaxel-induced activation of JNK via phosphorylation of ASK1 by Akt.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
17ß-Estradiol (E2), anti-ß-actin antibody, and dimethylsulfoxide were purchased from Sigma (St. Louis, MO). ICI182780 was obtained from Tocris (Ballwin, MO). Paclitaxel was a gift from Bristol-Myers Squibb (Tokyo, Japan). LY294002 and a JNK inhibitor were purchased from Calbiochem (La Jolla, CA). The anticleaved poly (ADP-ribose) polymerase (PARP), anti-phospho-Akt (Ser-473), anti-Akt, anti-phospho-ASK1 (Ser-83), anti-ASK1, and anti-JNK antibodies and the stress-activated protein kinase (SAPK)/JNK assay kit were obtained from Cell Signaling Technology (Beverly, MA). The anti-hemagglutinin (HA), anti-ER{alpha}, and anti-ERß antibodies was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The Cell Titer 96-well proliferation assay was obtained from Promega (Madison, WI).

Cell culture
Human ovarian papillary adenocarcinoma cell line (Caov-3) (20, 21, 22, 24) cells were obtained from the American Type Culture Collection (Manassas, VA). Human ovarian cancer A2780 cell line derived from a patient before treatment was kindly provided by Dr. T. Tsuruo (Institute of Molecular and Cellular Biosciences, Tokyo, Japan) and Drs. R. F. Ozols and T. C. Hamilton (National Cancer Institute, Bethesda, MD) (24). The OVCAR-3 epithelial ovarian cancer cell line and MCF-7 human breast cancer cells were obtained from the American Type Culture Collection. The cells were grown in DMEM with 10% fetal calf serum, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate in the presence of 5% CO2 at 37 C. For estrogen induction assays, cells were cultured in phenol red-free DMEM containing 10% dextran-coated, charcoal-treated fetal calf serum for 48 h and then incubated with E2, ICI182780, or LY294002 alone or in combination.

Constructs
The plasmids encoding the wild-type ASK1 (ASK1-HA), dominant-negative ASK1 (DN-ASK1-HA), and mutant ASK1 (ASK1S83A-HA) were a kind gift from Dr. M. V. Chao (New York University, New York, NY) (22). The plasmid encoding the dominant-negative SAPK/JNK (pcDL-SR{alpha}-SAPK-VPF) (25) was a kind gift from Dr. E. Nishida (Kyoto University, Kyoto, Japan). The human ER{alpha} expression vector, pSG5-HEGO, was a kind gift from Dr. P. Chambon (Institut de Chimie Biologique, Strasbourg, France) (26).

Clone selection
Caov-3 cells were transfected for 12 h in 6-well tissue culture plates with 2 µg of the empty vector, pcDL-SR{alpha}-SAPK-VPF, ASK1-HA, or ASK1S83A-HA and the neomycin resistance gene using Lipofectamine plus (Life Technologies, Gaithersburg, MD). A2780 cells were transfected for 12 h in 6-well tissue culture plates with 2 µg of pSG5-HEGO and the neomycin resistance gene using Lipofectamine plus. Clonal selection was performed by adding geneticin to the medium at 200 µg/ml final concentration 2 d after the transfection. After 3 wk, several clones were isolated using cloning rings as described previously (27, 28, 29). Selected clones were then maintained in medium supplemented with geneticin (100 µg/ml), and only low-passage cells (P < 10) were used for the experiments described here.

Cytotoxicity
Cell viability was assessed by the addition of paclitaxel at the indicated concentrations for 24 h 1 d after seeding test cells into 96-well plates. The number of surviving cells was determined 24 h later by determination of A590 nm of the dissolved formazan product after the addition of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) for 1 h as described previously (27, 28, 29, 30). All experiments were carried out in quadruplicate and the viability was expressed as the ratio of the number of viable cells with paclitaxel treatment to that without treatment. The values shown are the means ± SE of three independent experiments performed in quadruplicate at three different passages of the cell lines.

Assay of JNK activity
Lysates were incubated with the N-terminal c-Jun (1–89)-glutathione-S-transferase fusion protein bound to glutathione-Sepharose beads. After JNK was selectively precipitated using the c-Jun fusion protein beads, the kinase reaction was carried out in the presence of cold ATP. The samples were then resolved by 12% SDS-PAGE and subjected to Western blotting with a phospho-specific c-Jun antibody as described previously (27, 30).

Western blot analysis
Cells were incubated in phenol red-free DMEM without serum for 16 h and then treated with various agents. They were then washed twice with PBS and lysed in ice-cold HNTG buffer (50 mM HEPES, pH 7.5; 150 mM NaCl; 10% glycerol; 1% Triton X-100; 1.5 mM MgCl2; 1 mM EDTA; 10 mM sodium pyrophosphate; 100 µM sodium orthovanadate; 100 mM NaF; 10 µg/ml aprotinin; 10 µg/ml leupeptin; and 1 mM phenylmethylsulfonyl fluoride) (27, 28, 29, 30). The lysates were centrifuged at 12,000 x g at 4 C for 15 min, and the protein concentrations of the supernatants were determined using the protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blocking was done in 10% BSA in 1 x Tris-buffered saline (TBS). Western blot analyses were performed with various specific primary antibodies. For Western blot analysis using immunoprecipitated proteins, cell lysates were prepared using HNTG buffer. Lysates were incubated with the indicated antiserum overnight and then immunoprecipitated for 2 h with protein A-Sepharose. Immune complexes were washed with ice-cold HNTG buffer, electrophoresed, and analyzed by immunoblotting with the indicated antiserum. The immunoblots were visualized with horseradish peroxidase-coupled goat antirabbit or antimouse immunoglobulin by using the enhanced chemiluminescence Western blotting system.

Statistics
Statistical analysis was performed by Student’s t test, and P < 0.01 was considered significant. Data are expressed as the mean ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of ER
To clarify whether ER is expressed in Caov-3 and OVCAR-3 cells, both of which are known to express ER{alpha} (31), and A2780 cells, which are known not to express ER{alpha} (32), Western blotting for ER{alpha} was performed. We first confirmed the equal level of ß-actin expression in each lane (Fig. 1Go, lower panel). The MCF-7 human breast cancer cell line was shown to express ER{alpha} as a positive control. We confirmed that both Caov-3 and OVCAR-3 cells expressed ER{alpha}, whereas A2780 cells did not (Fig. 1Go, lanes 2–4). The level of expression of ER{alpha} in Caov-3 was similar to that in MCF-7 cells (Fig. 1Go, lanes 1 and 2). A clonal line of A2780 expressing ER{alpha} (A2780-ER) was made, and its expression of ER{alpha} was confirmed by Western blotting (Fig. 1Go, lane 5). There was no detectable expression of ERß in Caov-3, OVCAR-3, or A2780 cells by Western blotting with anti-ERß antibody (data not shown).



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  		FIG. 1. Expression of ER. Lysates (250 µg protein) of MCF-7 (lane 1), Caov-3 (lane 2), OVCAR-3 (lane 3), A2780 (lane 4), and A2780-ER (lane 5) cells were resolved by 8% SDS-PAGE and subjected to Western blotting with anti-ER{alpha} (upper panel) or anti-ß-actin (lower panel) antibody. Experiments were repeated three times with essentially similar results.

 
Effect of E2 on paclitaxel-induced apoptosis
To examine whether E2 protects cells against paclitaxel-induced cell damage, the effect of pretreatment with E2 on the sensitivity to paclitaxel was compared among Caov-3, OVCAR-3, A2780, and A2780-ER cells. Treatment with paclitaxel for 24 h caused a dose-dependent reduction of cell viability in Caov-3 (Fig. 2AGo, panel i), OVCAR-3 (Fig. 2BGo, panel i), A2780 (Fig. 2CGo, panel i), and A2780-ER (Fig. 2DGo, panel i) cells. Although the expression of ß-actin detected in each lane was equal (Fig. 2Go, panel ii), treatment with paclitaxel also induced the cleavage of PARP in Caov-3 (Fig. 2AGo, panel ii, lane 2), OVCAR-3 (Fig. 2BGo, panel ii, lane 2), A2780 (Fig. 2CGo, panel ii, lane 2), and A2780-ER (Fig. 2DGo, panel ii, lane 2) cells, indicating that paclitaxel induced apoptosis in these cells. Whereas treatment with 10 nM E2 had no effect on the paclitaxel-induced reduction of cell viability (Fig. 2CGo, panel i) or apoptosis (Fig. 2CGo, panel ii, lane 3) in A2780 cells, it significantly reversed the paclitaxel-induced reduction of cell viability (panel i) and apoptosis (panel ii, lane 3) in Caov-3 (Fig. 2AGo), OVCAR-3 (Fig. 2BGo), and A2780-ER (Fig. 2DGo) cells. Moreover, 1 µM ICI182,780 (a highly selective ER antagonist) attenuated the reversal effect of E2 on the paclitaxel-induced reduction of cell viability (Fig. 2AGo, panel i) and apoptosis (Fig. 2AGo, panel ii, lane 5) in Caov-3 cells, indicating that the reversal effect of E2 is mediated by ER. In addition, 20 µM LY294002 (a PI3K inhibitor) attenuated the reversal effect of E2 on the paclitaxel-induced reduction of cell viability (Fig. 2AGo, panel i) and apoptosis (Fig. 2AGo, panel ii, lane 4) in Caov-3 cells. Because the level of expression of ER{alpha} in Caov-3 cells appeared to be higher than that in OVCAR-3 cells (Fig. 1Go, lanes 2 and 3), Caov-3 cells was used in most of the following experiments.



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  		FIG. 2. Effect of E2 on paclitaxel-induced apoptosis. Caov-3 cells (A, panel i) were treated with the indicated concentrations of paclitaxel with or without 10 nM E2, 10 nM E2 + 20 µM LY294002, or 10 nM E2 + 1 µM ICI182,780. OVCAR-3 (B), A2780 (C), and A2780-ER (D) were treated with the indicated concentrations of paclitaxel with or without 10 nM E2. Twenty-four hours later, cell viability was assessed by the MTS assay as described in Materials and Methods. Significant differences are indicated by asterisks. **, P < 0.01. Caov-3 cells (A, panel ii) were treated with 100 nM paclitaxel (lane 2) or 100 nM paclitaxel +10 nM E2 (lane 3), 100 nM paclitaxel +10 nM E2 + 20 µM LY294002 (lane 4), or 100 nM paclitaxel + 10 nM E2 + 1 µM ICI182,780 (lane 5) for 24 h. OVCAR-3 (B), A2780 (C), and A2780-ER (D) were treated with 100 nM paclitaxel with (lane 3) or without (lane 2) 10 nM E2 for 24 h. Lysates (250 µg protein) were subjected to Western blotting using anticleaved PARP (middle panel) or anti-ß-actin (lower panel) antibody. Relative densitometric units of the cleaved PARP bands are shown in the top panel, with the density of the control (1% fetal bovine serum) bands set arbitrarily at 1.0. Values shown represent the mean ± SE from at least three separate experiments. Significant differences are indicated by asterisks. **, P < 0.01. N.S., Not significant.

 
E2-induced Akt phosphorylation
Because the Akt cascade is known to mediate the survival function of estrogen in breast cancer cells (23), we first examined whether E2 induced the phosphorylation of Akt in Caov-3 cells. The cells were treated with E2 for various times and then used to prepare lysates that were subjected to Western blotting with anti-phospho-Akt or -Akt antibody. Although E2 did not affect the expression of Akt (Fig. 3AGo, lower panel), it induced phosphorylation of Akt (Fig. 3AGo, middle and top panels). The strongest responses were detected 1 h after E2 exposure. We then examined the mechanism by which E2 induces Akt phosphorylation. Although the expression of Akt was not affected by ICI182,780 or LY294002 (Fig. 3BGo, lower panel), E2-induced Akt phosphorylation was attenuated by these agents (Fig. 3BGo, middle and top panels). Taken together, the results shown in Figs. 2Go and 3Go suggest that an Akt cascade is involved in the survival function of E2 against paclitaxel-induced cell damage.



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  		FIG. 3. E2 induces Akt phosphorylation. A, Caov-3 cells were incubated with 10 nM E2 for various times and then harvested and used to prepare cell lysates. The lysates were subjected to SDS-PAGE and blotted with anti-phospho-Akt antibody (middle panel) or anti-Akt antibody (lower panel). B, Caov-3 cells were incubated with 1 µM ICI182780 or 10 µM LY294002 for 15 min before E2 stimulation at 10 nM for 1 h. Cell lysates were subjected to SDS-PAGE and blotted with anti-phospho-Akt antibody (middle panel) or anti-Akt antibody (lower panel). Relative densitometric units of the phospho-Akt bands are shown in the top panel, with the density of the control bands set arbitrarily at 1.0. Values shown represent the mean ± SE from at least three separate experiments. Significant differences are indicated by asterisks. **, P < 0.01.

 
E2-induced ASK1 phosphorylation
It was reported that Akt directly phosphorylates ASK1 on Ser-83 and that this phosphorylation is associated with a decrease in the induction of ASK1 kinase activity (22). Other substrates of Akt, such as Raf-1 (29, 33) and BAD (34), have been shown to associate with Akt. To examine whether Akt and ASK1 were physically associated, endogenous ASK1 and Akt were immunoprecipitated in Caov-3 cells and found to coimmunoprecipitate (Fig. 4AGo).



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  		FIG. 4. E2 induces ASK1 phosphorylation on Ser-83. A, Association between Akt and ASK1. Endogenous ASK1 and Akt were immunoprecipitated (IP) with their cognate antibodies. Rabbit IgG was used for immunoprecipitation as a negative control. The resulting immunoprecipitates were then subjected to Western blotting analysis with the same antibodies. The positions of molecular weight markers are noted on the left. B, Caov-3 cells were incubated with 10 nM E2 for various times and then harvested and used to prepare cell lysates. The lysates were subjected to SDS-PAGE and blotted with anti-phospho-ASK1 (Ser-83) antibody (middle panel) or anti-ASK1 antibody (lower panel). C, Caov-3 cells were incubated with 1 µM 182,780 or 10 µM LY294002 for 15 min before E2 stimulation at 10 nM for 1 h. Cell lysates were subjected to SDS-PAGE and blotted with anti-phospho-ASK1 antibody (middle panel) or anti-ASK1 antibody (lower panel). Relative densitometric units of the phospho-ASK1 bands are shown in the top panel, with the density of the control bands set arbitrarily at 1.0. Values shown represent the mean ± SE from at least three separate experiments. Significant differences are indicated by asterisks. **, P < 0.01.

 
Next we examined whether E2 induced the phosphorylation of ASK1 at Ser-83 in Caov-3 cells. The cells were treated with E2 for various times and then used to prepare lysates that were subjected to Western blotting with anti-phospho-ASK1 (Ser-83), which specifically recognizes ASK1 phosphorylated on Ser-83, or anti-ASK1 antibody. Although E2 did not affect the expression of ASK1 (Fig. 4BGo, lower panel), it induced phosphorylation of ASK1 at Ser-83 (Fig. 4BGo, middle and top panels). The strongest responses were detected 1 h after E2 exposure, like E2-induced Akt phosphorylation (Fig. 3AGo). We then examined the mechanism by which E2 induces ASK1 phosphorylation. Although the expression of ASK1 was not affected by ICI182,780 or LY294002 (Fig. 4CGo, lower panel), E2-induced ASK1 phosphorylation at Ser-83 was attenuated by these agents (Fig. 4CGo, middle and top panels), suggesting that E2 induces ASK1 phosphorylation at Ser-83 via ER-Akt cascade.

Paclitaxel-induced cell damage via ASK1-JNK cascade
We confirmed the previously reported finding (13) that paclitaxel induces cell damage via the ASK1-JNK cascade in Caov-3 cells. We developed clonal lines of Caov-3 cells that stably expressed HA epitope-tagged wild-type ASK1 (WT-ASK1) or dominant-negative ASK1 (DN-ASK1). We first confirmed that ASK1 protein products were ectopically expressed (Fig. 5AGo, panel i, middle panel) and overexpressed relative to the endogenous expression (Fig. 5AGo, panel i, top panel), despite the equal level of ß-actin expression detected in each lane (Fig. 5AGo, panel i, lower panel). To evaluate whether JNK is activated by paclitaxel in WT-ASK1- or DN-ASK1-expressing cells, the cultured cells were exposed to 100 nM paclitaxel for 1 h. Cell lysates were incubated with glutathione-S-transferase-c-Jun fusion protein, followed by precipitation and Western blotting using anti-phospho-c-Jun antibody. Paclitaxel induced JNK activation in WT-ASK1-expressing cells, but not in DN-ASK1-expressing cells (Fig. 5AGo, panel ii, middle and top panels), suggesting the involvement of ASK1 in the paclitaxel-induced JNK activation. We confirmed this result with other clonal derivatives of WT-ASK1 and DN-ASK1 (data not shown).



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  		FIG. 5. Paclitaxel-induced cell damage via ASK1-JNK cascade. A, Lysate samples of untransfected parental cells (panel i, lane 1), DN-ASK1 construct (panel i, lane 2), or WT-ASK1 (panel i, lane 3) expressing Caov-3 cells grown in 100-mm dishes were analyzed by electrophoresis on SDS-PAGE, followed by Western blotting with anti-ß-actin (lower panel), anti-HA (middle panel), or anti-ASK1 (top panel) antibody. For analysis of the effects of the expressed DN-ASK1 on paclitaxel-induced JNK activation (panel ii), DN-ASK1- or WT-ASK1-expressing Caov-3 cells were treated with paclitaxel at 100 nM for 1 h. The lysates were subsequently precipitated with c-Jun fusion protein bound to glutathione-Sepharose beads, and the kinase reaction was carried out in the presence of cold ATP as described in Materials and Methods. After the reaction was stopped by the addition of Laemmli sample buffer, samples were resolved by 12% SDS-PAGE and subjected to Western analysis using a phospho-specific c-Jun antibody. B, Caov-3 cells were incubated with 10 µM JNK inhibitor for 15 min before paclitaxel stimulation at 100 nM for 1 h. Cell lysates were subsequently assayed for JNK activity as described above. C, Lysate samples of untransfected parental cells (panel i, lane 1), DN-JNK construct (pcDL-SR{alpha}-SAPK-VPF) (panel i, lane 2), or empty vector (pcDL-SR{alpha})- (panel i, lane 3) expressing Caov-3 cells grown in 100-mm dishes were analyzed by electrophoresis on SDS-PAGE, followed by Western blotting with anti-ß-actin (lower panel) or anti-JNK1 (upper panel) antibody. For analysis of the effects of the expressed DN-JNK on paclitaxel-induced JNK activation (panel ii), cell lysates were subsequently assayed for JNK activity as described above. Relative densitometric units of the phospho-c-Jun bands are shown in A (panel ii), the upper panels of B, and C (panel ii), with the density of the control bands set arbitrarily at 1.0. Values shown represent the mean ± SE from at least three separate experiments. Significant differences are indicated by asterisks. **, P < 0.01. D, Cell viability in DN-ASK1- or DN-JNK-expressing, or JNK inhibitor-treated, cells after treatment with the indicated concentrations of paclitaxel was assessed as described in Materials and Methods. All experiments were carried out in quadruplicate, and the viability was expressed as the ratio of the number of viable cells with paclitaxel treatment to that without treatment. The values shown are the means ± SE of three independent experiments performed in quadruplicate at three different passages of the cell lines.

 
JNK inhibitor or an expression plasmid (pcDL-SR{alpha}-SAPK-VPF) encoding a dominant-negative JNK (DN-JNK) (25) was used to inhibit the JNK cascade. We first confirmed that JNK protein products were overexpressed relative to the level of endogenous expression (Fig. 5CGo, panel i, upper panel), despite the equal level of ß-actin expression detected in each lane (Fig. 5CGo, panel i, lower panel). Paclitaxel-induced JNK activation was clearly attenuated by JNK inhibitor (Fig. 5BGo) and paclitaxel-induced JNK activation in cells transfected with DN-JNK was clearly attenuated, compared with that in cells transfected with pcDL-SR{alpha} (Fig. 5CGo, panel ii), confirming the negative effects of both JNK inhibitor and the expression of DN-JNK on the paclitaxel-induced JNK activation. We confirmed this result with other clonal derivatives of DN-JNK (data not shown).

We then examined the effect of the expression of DN-ASK1 or DN-JNK, or the presence of JNK inhibitor, on the paclitaxel-induced cell damage. The expression of DN-ASK1 or DN-JNK or treatment with JNK inhibitor attenuated significantly the paclitaxel-induced reduction of cell viability (Fig. 5DGo), suggesting the involvement of the ASK1-JNK cascade in the paclitaxel-induced cell damage. We confirmed this result with other clonal derivatives of DN-ASK1 and DN-JNK (data not shown).

Effect of E2 on the paclitaxel-induced JNK activation
We next examined the effect of E2 on paclitaxel-induced JNK activation. E2 inhibited the paclitaxel-induced JNK activation (Fig. 6AGo, lane 3), and this effect of E2 was blocked by ICI182,780 or LY294002 (Fig. 6AGo, lanes 4 and 5). Moreover, we examined the involvement of Akt-dependent phosphorylation of ASK1 at Ser-83 in the E2-induced inhibition of the paclitaxel-induced JNK activation (Fig. 6BGo). An expression plasmid encoding ASK1S83A (22) was used to inhibit the phosphorylation of ASK1 on Ser-83. We first confirmed the overexpression of ectopically expressed ASK1 protein products (Fig. 6BGo, panel i, lower panel) and the negative effects of the expression of ASK1S83A on E2-induced phosphorylation of ASK1 at Ser-83 (Fig. 6BGo, panel i, upper panel). The E2-induced inhibition of the paclitaxel-induced JNK activation in cells transfected with ASK1S83A was clearly reversed, compared with that in cells transfected with WT-ASK1 and parental cells (Fig. 6BGo, panel ii). We confirmed this result with other clonal derivatives of WT-ASK1 and ASK1S83A (data not shown). These data suggest that E2 inhibited the paclitaxel-induced JNK activation through ER via the Akt-dependent phosphorylation of ASK1 at Ser-83.



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  		FIG. 6. Effect of E2 on the paclitaxel-induced JNK activation. A, Caov-3 cells were treated with 100 nM paclitaxel (lane 2) with 10 nM E2 (lane 3), 10 nM E2 + 1 µM ICI182,780 (lane 4), or 10 nM E2 + 20 µM LY294002 (lane 5) for 1 h. Cell lysates were subsequently assayed for JNK activity as described in Materials and Methods (middle panel) or subjected to Western blotting analysis with anti-Akt antibody (lower panel). B, Caov-3 cells expressing WT-ASK1, a mutant ASK1 construct (ASK1Ser83A) in which the consensus Akt phosphorylation site at Ser-83 was converted to alanine, or untransfected parental Caov-3 cells grown in 100-mm dishes were treated with 10 nM E2 for 1 h (panel i, lane 2), or 100 nM paclitaxel (panel ii, lane 2) or 100 nM paclitaxel +10 nM E2 (panel ii, lane 3) for 1 h. Cell lysates were subsequently subjected to Western blotting analysis with anti-phospho-ASK1 (Ser-83) (panel i, upper panel) or anti-ASK1 antibody (panel i, lower panel) or assayed for JNK activity (panel ii) as described in Materials and Methods. Relative densitometric units of the phospho-c-Jun bands are shown (A, top panel, and B, upper panel of panel ii), with the density of the control bands set arbitrarily at 1.0. Values shown represent the mean ± SE from at least three separate experiments. Significant differences are indicated by asterisks. **, P < 0.01.

 
E2 inhibits the paclitaxel-induced cell damage via the phosphorylation of ASK1 at Ser-83
To examine whether E2 inhibits the paclitaxel-induced cell damage via the Akt-dependent phosphorylation of ASK1 at Ser-83, we compared the effect of E2 on the paclitaxel-induced cell damage between WT-ASK1- and ASK1S83A-expressing cells. E2 strongly reversed the paclitaxel-induced reduction of cell viability (Fig. 7AGo) and apoptosis (Fig. 7BGo) in WT-ASK1-expressing cells but not in ASK1S83A-expressing cells, suggesting that E2 inhibits the paclitaxel-induced cell damage via the Akt-dependent phosphorylation of ASK1 at Ser-83. We confirmed this result with other clonal derivatives of WT-ASK1 and ASK1S83A (data not shown).



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  		FIG. 7. E2 inhibits the paclitaxel-induced cell damage via the phosphorylation of ASK1. A, WT-ASK1- (left panel) or ASK1Ser83A (right panel)-expressing Caov-3 cells were treated with the indicated concentrations of paclitaxel with or without 10 nM E2. Twenty-four hours later, cell viability was assessed by the MTS assay as described in Materials and Methods. Significant differences are indicated by asterisks. **, P < 0.01. B, WT-ASK1- (left panel) or ASK1Ser83A (right panel)-expressing Caov-3 cells were treated with 100 nM paclitaxel (lane 2) or 1 µM paclitaxel+10 nM E2 (lane 3) for 24 h. Lysates (250 µg protein) were subjected to Western blotting with anti-cleaved PARP antibody.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although postoperative ERT did not have a negative effect on the disease-free interval or overall survival of ovarian carcinoma survivors in a small study (4), the majority of patients with ovarian cancer require treatment with cytotoxic chemotherapy after surgical management. We showed here for the first time that estrogen inhibits paclitaxel-induced cell damage by inhibiting JNK activity via phosphorylation of Akt-ASK1 (Fig. 8Go). Although clinical trials would be necessary to determine the effect of postoperative ERT on the sensitivity to paclitaxel, the present data suggest that ovarian cancer treatment with paclitaxel would be markedly less effective in the setting of postoperative ERT.



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  		FIG. 8. Molecular mechanism of the inhibitory effect of E2 on paclitaxel-induced apoptosis. E2 inhibits paclitaxel-induced apoptosis by inhibiting JNK activity via the phosphorylation of Akt-ASK1.

 
Estrogen has an antiapoptotic effect on vascular endothelial cells (35, 36, 37) and breast cancer cells (9). On the other hand, estrogen has an apoptotic effect on vascular smooth muscle cells (37) and an erythroid cell line (38). Thus, the effects of estrogen differ among cells expressing ER. The signaling molecules involved in apoptosis and antiapoptosis might determine the function of estrogen in cells expressing ER. For example, estrogen-induced Akt activation (39) or ERK activation (40) or estrogen-induced inhibition of JNK activation (9) is involved in estrogen-induced antiapoptosis in breast cancer cells. On the other hand, estrogen-induced p38 activation is involved in estrogen-induced apoptosis in vascular smooth muscle cells (41). In the present report, we demonstrated that the estrogen-induced inhibition of JNK activity via the phosphorylation of ASK1 at Ser-83 is involved in the antiapoptotic effect of estrogen in paclitaxel-treated human ovarian cancer cell lines that express ER.

The antiapoptotic effect of estrogen in paclitaxel-treated human ovarian cancer cell lines is probably mediated by ER in the plasma membrane, as indicated in Fig. 8Go. Rapid, presumably nongenomic effects of estrogen have been reported in a variety of tissues (42, 43, 44, 45, 46); these effects often originate from the cell membrane. Although it is clear that membrane-based responses exist, the isolation, cloning, and convincing demonstration of the in vivo localization of an endogenous membrane-based ER distinct from the classic ERs remain to be shown. On the other hand, it was reported that estrogen up-regulates antiapoptotic Bcl-2 mRNA and protein in tumorigenic ovarian surface epithelium cells (47). Thus, both genomic and nongenomic effects of estrogen might be involved in the antiapoptotic effect of estrogen.

Which ER is involved in the antiapoptotic effect of estrogen in paclitaxel-treated human ovarian cancer cell lines? In normal ovaries, ERß mRNA was the predominant ER form, whereas in ovarian cancer cell lines ER{alpha} mRNA was markedly increased as compared with ERß mRNA (48). We have shown that ER{alpha} is expressed in Caov-3, OVCAR-3, and A2780-ER cells (Fig. 1Go) in which estrogen has an antiapoptotic effect. Although it was shown by RT-PCR that ERß mRNA is expressed in Caov-3 cells (49), we found that there is no detectable expression of ERß in Caov-3, OVCAR-3, and A2780 cells by Western blotting (data not shown). Therefore, the possibility that ERß is also involved in the antiapoptotic effect of estrogen still remains. However, all membrane forms described to date are related to ER{alpha} and not ERß (50, 51, 52).

The ASK1-JNK cascade (13, 53) and Raf-1 (54, 55, 56) are involved in paclitaxel-induced apoptosis. It was reported that phosphorylation by Akt inactivates the function of Akt substrates such as BAD (34), FKHR1 (57), Raf-1 (29, 33), and ASK1 (23). The regions surrounding Ser-136 in BAD (34) and Ser-259 in Raf-1 (33) conform to a consensus sequence for phosphorylation by Akt. We previously reported that paclitaxel induced the phosphorylation of BAD at Ser-136 and Raf-1 at Ser-259 via an Akt-dependent mechanism and that inhibition of these cascades sensitizes ovarian cancer cells to paclitaxel (29). Thus, because we demonstrated previously that in paclitaxel-resistant cells paclitaxel induces Akt phosphorylation (29), it is possible that paclitaxel induces the phosphorylation of ASK1 at Ser-83, whose surrounding regions conform to a consensus sequence for phosphorylation by Akt (22), resulting in inhibition of JNK and thus leading to antiapoptosis. In addition, it was reported that the inhibition of cisplatin-induced apoptosis via phosphorylation of ASK1 by Akt plays an important role in chemoresistance of ovarian cancer cells (21). Thus, ASK1 might be an important molecule for determining the balance between apoptosis and antiapoptosis in cells treated with paclitaxel.

The majority of patients with ovarian cancer require treatment with cytotoxic chemotherapy. It is now well established that the combination of taxanes (paclitaxel or docetaxel) and platinum agents (cisplatin or carboplatin) is the most effective drug combination in first-line regimens. The signaling cascade of paclitaxel is not always the same as that of cisplatin. For example, although the expression of Raf-1 has no relationship with cisplatin (58), the level of paclitaxel-induced apoptosis seems to have some dependency on Raf-1 kinase activity (54, 55, 56). In addition, whereas ASK1-mediated JNK activity is involved in paclitaxel-induced apoptosis (13), we showed previously that JNK activity is involved in the repair of cisplatin-induced DNA damage (27). On the other hand, we previously reported that inducible Akt activity by cisplatin (28) and paclitaxel (29) promotes resistance to these drugs as constitutive and inducible Akt activity promotes resistance to chemotherapy, trastuzumab, and tamoxifen in breast cancer cells (59). Thus, activation of Akt in ovarian cancer might predict a worse outcome among patients treated with cytotoxic chemotherapy, as in breast cancer (60). We demonstrated that estrogen induces the activation of Akt in human ovarian cancer cell lines that express ER. This suggests that postoperative ERT for ovarian cancer patients treated with cytotoxic chemotherapy might lead to a worse outcome.


   Acknowledgments
 
We are grateful to Dr. M. V. Chao (New York University, New York, NY) for providing ASK1-HA, DN-ASK1-HA, and ASK1S83A-HA constructs, and Dr. E. Nishida (Kyoto University, Kyoto, Japan) for providing pcDL-SR{alpha}-SAPK-VPF, and Dr. P. Chambon (Institut de Chimic Biologique, Strasbourg, France) for providing pSG5-HEGO.


   Footnotes
 
Abbreviations: ASK1, Apoptosis signal-regulating kinase 1; E2, 17ß-estradiol; ER, estrogen receptor; JNK, c-Jun N-terminal protein kinase; MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PI3K, phosphatidylinositol 3-kinase; Ser, serine.

Received June 25, 2003.

Accepted for publication September 12, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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