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Endocrinology Vol. 145, No. 3 1302-1313
Copyright © 2004 by The Endocrine Society

Tamoxifen Inhibits Cell Proliferation via Mitogen-Activated Protein Kinase Cascades in Human Ovarian Cancer Cell Lines in a Manner Not Dependent on the Expression of Estrogen Receptor or the Sensitivity to Cisplatin

Seiji Mabuchi, Masahide Ohmichi, Akiko Kimura, Yoshihide Ikebuchi, Koji Hisamoto, Emi Arimoto-Ishida, Yukihiro Nishio, Kazuhiro Takahashi, 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
 
Although tamoxifen (TAM), which is widely used in the treatment of breast cancer, also has a beneficial effect on cisplatin-refractory ovarian cancer, the biological mechanism of this effect has remained obscure. TAM, besides its action as an antiestrogen, also inhibits cell proliferation of estrogen receptor (ER)-negative cells by an unknown mechanism. Therefore, we examined the roles of the MAPK family in the antiproliferative effect of TAM on cisplatin-resistant Caov-3, which expresses ER and cisplatin-sensitive A2780, which does not express ER. The number of viable cells was reduced by TAM dose-dependently. TAM induced the activation of ERK, c-Jun N-terminal protein kinase (JNK), and p38 with different time courses. PD98059 canceled the reduction of the number of viable cells by 1 µM TAM and inhibited the TAM-induced cell-cycle arrest at the G1 phase and dephosphorylation of the retinoblastoma protein. Either expression of dominant-negative JNK or pretreatment with SB203580 canceled the reduction of the number of viable cells by 5 µM TAM and inhibited the apoptotic nuclear changes and the cleavage of poly (ADP-ribose) polymerase induced by TAM. These results provide evidence that whereas the ERK cascade is involved in the induction of cell-cycle arrest at the G1 phase by lower concentrations of TAM, the JNK or p38 cascade is involved in the induction of apoptosis by higher concentrations of TAM in both types of cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TAMOXIFEN (TAM), A NONSTEROIDAL triphenylethylene derivative, has been used extensively for the treatment of breast cancer (1). TAM is a potent estrogen receptor (ER) antagonist, and its pharmacology has been reviewed extensively (2). TAM is thought to exert its antiproliferative action by binding competitively to the ER, thereby blocking the mitogenic effect of estradiol (3). An important additional feature of TAM is its effectiveness in the treatment of estrogen-independent neoplasia, such as ER-negative breast cancer (4) and ovarian cancer (5). There is some evidence from observational studies that TAM may produce a response in a modest proportion of women with relapsed ovarian cancer (6). ER is present in about 60% of cases of ovarian cancer (7). TAM has also been shown to be cytotoxic to both ER-positive and ER-negative cells (8, 9), and this cytotoxic effect is believed to be mediated by the induction of apoptosis (10, 11). However, the mechanism underlying the antiproliferative action of TAM in tumors cells has not been completely clarified.

Many studies focusing on the effect of TAM have indicated that this compound acts in both a cytostatic (by causing G0/G1 arrest) and cytotoxic (by inducing apoptosis) manner (12, 13, 14). Whereas 1 µM TAM induced cell-cycle arrest at the G1 phase (15), between 5 and 50 µM TAM induced apoptosis (14). This dual effect of TAM suggests that this drug targets the checkpoint between cell cycle progression and apoptosis. It is speculated that TAM, by virtue of its high lipophilicity and partioning in the cell membrane, generates a transmembrane signal transduction cascade. Intracellular transmission of extracellular signals is mediated by several groups of sequentially activated protein kinases, which are collectively known as the MAPK cascades. The MAPK family has been classified into three subfamilies: ERKs; stress-activated protein kinases (SAPKs), also termed c-Jun N-terminal protein kinases (JNKs); and p38 kinase (16). Whereas ERK has been shown to be involved in growth and proliferation-related events, JNK and p38 have been implicated in apoptosis. TAM activates both ERK2 and JNK1 activities in HeLa cells (17), whereas in ER-negative MDA-MB-231 breast cancer cells TAM also induces JNK1 activity and expression of dominant-negative JNK prevents TAM-induced apoptosis (18). It was more recently reported that 4-hydroxytamoxifen activated p38 in MCF-7 cells and pretreatment with SB203580 largely protected the cells against 4-hydroxytamoxifen-induced apoptosis (19). Moreover, it was reported that TAM induced accumulation of hypophosphorylated retinoblastoma (Rb) protein (20) and 4-hydroxytamoxifen blocked the estradiol (E2)-induced phosphorylation of pRb, retinoblastoma protein (pRb) (21), suggesting that TAM inhibits breast cancer growth by mediating cell-cycle arrest at the G1 phase. Previously, we and other groups showed that ERK is involved in cell-cycle arrest at the G1 phase of human breast cancer cells (22), human ovarian cancer cells (23), NIH 3T3 murine fibroblasts (24), and human myeloblastic leukemia cells (25).

These findings led us to examine whether TAM has an antiproliferative effect and stimulates the ERK, JNK, and/or p38 cascade in two human ovarian cancer cell lines: cisplatin-resistant Caov-3 (26), which expresses ER (27), and cisplatin-sensitive A2780, which does not express ER (28). In addition, we examined the roles of the ERK, JNK, and p38 cascades in the TAM-induced phosphorylation of pRb, apoptotic nuclear changes, or cleavage of poly (ADP-ribose) polymerase (PARP) as a marker for apoptosis. Moreover, because the cytotoxicity of TAM has been attributed to the generation of oxidative stress (29), we examined the role of oxidative stress in the TAM-induced ERK, JNK, and p38 activation in both types of cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
TAM, {alpha}-tocopherol (vitamin E), myelin basic protein (MBP), anti-ß-actin antibody, and propidium iodide were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-ER{alpha} antibody and anti-ERK1 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-cleaved PARP antibody, PD98059, anti-SAPK/JNK antibody, the SAPK/JNK assay kit, a p38 activity assay kit, were obtained from Cell Signaling Technology (Beverly, MA). SB203580 was obtained from Calbiochem (La Jolla, CA). Hoechst 33258 was obtained from Molecular Probes (Eugene, OR). Anti-Rb antibody was obtained from PharMingen (San Diego, CA). The cell titer 96-well proliferation assay was obtained from Promega (Madison, WI).

Cell cultures
Human ovarian papillary adenocarcinoma cell line Caov-3 was obtained from 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) (30). The cells were cultured at 37 C in DMEM with 10% fetal bovine serum in a water-saturated atmosphere of 95% air and 5% CO2.

Constructs
The human ER{alpha} expression vector, pSG5-HEGO, was a kind gift from Dr. P. Chambon (Institut de Chimie Biologique, Strasbourg, France) (31). The plasmid encoding ERE-tk109-luc was a kind gift from Drs. B. D. Gehm and J. L. Jameson (Northwestern University, Chicago, IL). The plasmid encoding the dominant-negative SAPK/JNK (pcDL-SR{alpha}-SAPK-VPF) (32) was a kind gift from Dr. E. Nishida (Kyoto University, Kyoto, Japan).

Clone selection
Caov-3 and A2780 cells were transfected for 12 h in 6-well tissue culture plates with 2 µg of the empty vector, pcDL-SR{alpha}-SAPK-VPF, or pSG5-HEGO and the neomycin resistance gene using Lipofectamine plus (Life Technologies, Gaithersburg, MD). 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 (33). 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.

Luciferase assays
Cells cultured in 24-well plates were transfected with ERE-tk109-luc and pCMV-ß-galactosidase plasmid using Lipofectamine plus. At 48 h after transfection, serum-deprived cells were incubated with 10-8 M E2 for 24 h. Cells were harvested and luciferase assays were performed as described previously (34).

Rate of cell proliferation
The number of viable cells was assessed 24 h later of the addition of TAM at the indicated concentrations 1 d after seeding test cells into 96-well plates in phenol red-free DMEM with 10% charcoal stripped serum (CSS) by determination of A590 nm of the dissolved formazan product after the addition of 3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium (MTS) for 1 h as described previously (23, 35). All experiments were carried out in quadruplicate, and the number of viable cells is expressed as the ratio of the number of viable cells with TAM 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. For the trypan blue exclusion test, test cells were plated at a density of 5 x 103 cells per well in 24-well plate and allowed to attach overnights. The cells were growth-arrested by incubation in phenol red-free DMEM with 10% CSS for 24 h, which was ascertained by cell cycle analysis of the viable cells using fluorescence-activated cell sorter (FACS), and were then treated with TAM at the indicated concentrations for 24 h. The Neubauer chamber was used to count the cell numbers and the trypan blue exclusion test was carried out to determine the viable cells. All experiments were carried out in quadruplicate, and the number of viable cells is expressed as the ratio of the number of viable cells with TAM 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.

Flow cytometric analysis
Caov-3 and A2780 cells were seeded onto 100-mm dishes and grown in phenol red-free DMEM with 10% CSS. After the cells had grown to subconfluence, they were rendered quiescent and challenged without 1% CSS or with 1% CSS, 1% CSS+1 µM TAM, or 1% CSS+1 µM TAM+100 nM PD98059 for 24 h. Then after release using trypsin-EDTA, they were harvested, washed twice with PBS/0.1% dextrose, and fixed in 70% ethanol at 4 C. The cells were stained at room temperature for 2–3 h in 50 µg/ml propidium iodide and 100 Kunitz U/ml Rnase A in PBS. The cells were then counted on a FACS (Becton Dickinson, Mountain View, CA), and the percentages of cells in the G1, S, and G2/M phases of the cell cycle were determined using ModFit LT software (Verity Software House, Topsham, ME).

Assay of ERK activity
Cells were lysed in HNTG buffer (36) and lysates were immunoprecipitated with anti-ERK1 rabbit polyclonal antibody. The immunoprecipitated products were resuspended in 30 µl of kinase assay buffer containing 10 µg of MBP and 40 µM [{gamma}-32P] ATP (1 µCi). The kinase reaction was allowed to proceed at room temperature for 5 min and stopped by the addition of Laemmli sodium dodecyl sulfate sample buffer (37). Reaction products were resolved by 15% SDS-PAGE as described previously (38).

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 (26).

Assay of p38 activity
Lysates were incubated with immobilized phospho-p38 monoclonal antibody. After phospho-p38 was selectively immunoprecipitated from the cell lysates, the immunoprecipitated products were resuspended in 50 µl of kinase buffer containing 200 µM ATP and 2 µg activating transcription factor (ATF)-2 fusion protein. The kinase reaction was allowed to proceed at 30 C for 30 min and stopped by the addition of sodium dodecyl sulfate sample buffer. Reaction products were resolved by 10% SDS-PAGE and then subjected to Western blotting with anti-phospho-ATF-2 antibody.

Western blotting
Immunoprecipitates with the indicated antiserum or whole-cell lysates were resolved on gels containing the indicated percentage of SDS-PAGE and subjected to Western blotting using the indicated antibody as described previously (26, 33, 35).

Morphological study of apoptosis
Cells were plated on culture chamber slides. After incubation for 24 h, the cells were treated with TAM at the indicated concentrations for 24 h. Cells were washed once with PBS and fixed with PBS containing 1% glutaraldehyde for 30 min at room temperature. The cells were incubated with PBS containing 10 mg/liter Hoechst 33258 for 15 min in the dark and examined by fluorescence confocal microscopy.

Statistics
Statistical analysis was performed using one-way ANOVA followed by Fisher’s least significant difference test, and P < 0.05 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 cells, which are cisplatin resistant, and A2780 cells, which are cisplatin sensitive, Western blotting for ER{alpha} was performed. Caov-3 cells expressed ER and A2780 cells did not (Fig. 1AGo, left panel), as reported previously (27, 28). A clonal line of A2780-expressing ER{alpha} (A2780-ER) was made, and its expression of ER{alpha} was confirmed by Western blotting (Fig. 1AGo, right panel). Moreover, to confirm that A2780-ER expresses functional ER, estrogen-induced transcriptional transactivation was assessed by transfection of the estrogen-responsive reporter plasmid ERE-tk-luc. Treatment with 10-8 M E2 for 24 h significantly induced ERE-tk-luc reporter activity in A2780-ER but not A2780 cells (Fig. 1BGo). Moreover, treatment with 10-6 M ICI 182,780 (pure antiestrogen) for 24 h significantly inhibited the E2-induced ERE-tk-luc reporter activity in A2780-ER cells, confirming that A2780-ER cells express functional ER.



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  		FIG. 1. Effect of TAM on the proliferation of Caov-3, A2780, and A2780-ER cells. A, Lysates (250 µg protein) were resolved by 8% SDS-PAGE and subjected to Western blotting with anti-ER{alpha} antibody. Experiments were repeated three times with essentially similar results. B, A2780 or A2780-ER cells were transiently cotransfected with 0.4 µg of the reporter construct ERE-tk-luc and 0.04 µg of an internal control, pCMV-ß-gal. After transfection, cells were treated with 10-8 M E2 for 24 h or 10-6 M ICI 182,780 + 10-8 M E2 for 24 h. Luciferase activity was normalized relative to ß-galactosidase activity, and the control value was set at 1.0. Data are expressed as the mean fold activation ± SE of six transfections. Significant differences are indicated by asterisks. **, P < 0.01. C and D, A2780, A2780-ER, or Caov-3 cells were growth arrested by incubation in phenol red-free DMEM with 10% CSS for 24 h, which was ascertained by cell cycle analysis of the viable cells using FACS, and were then treated with the indicated concentrations of TAM for 24 h. The number of viable cells was assessed by the trypan blue exclusion test (C) or MTS assay (D) as described in Materials and Methods.

 
Effect of TAM on the number of viable cells
To examine whether TAM has any effect on the number of viable cells of Caov-3, A2780, and A2780-ER cells, the cells were treated with the indicated concentrations of TAM for 24 h, and the number of viable cells was assessed by the trypan blue exclusion test (Fig. 1CGo) or MTS assay (Fig. 1DGo). Treatment with TAM caused a dose-dependent reduction of the number of viable cells. Regardless of their ER status, the three cell lines showed a similar IC50 (~3–4 µM). These results suggest that an ER-independent mechanism also functions in the TAM-induced reduction of the number of viable ovarian cancer cells.

Differential activation of ERK, JNK, and p38 cascades by TAM
The MAPK family is known to be involved in the regulation of cell proliferation. We therefore investigated whether TAM induces the activation of ERK, JNK, and p38. Activation of ERK by TAM occurred within 30 min, was maximal from 1–3 h, and was sustained until 6 h in both types of cells (Fig. 2AGo, upper panel). We confirmed that the total amount of ERK in each lane was the same (Fig. 2AGo, lower panel). Activation of JNK by TAM occurred at 5 min, reached a plateau at 30 min, and declined thereafter in both types of cells (Fig. 2BGo, upper panel). We confirmed that the total amount of JNK in each lane was the same (Fig. 2BGo, lower panel). We also examined whether p38 activity is affected by TAM. The activation of p38 occurred at 30 min, reached a plateau at 1 h, and declined thereafter in both types of cells (Fig. 2CGo, upper panel). We confirmed that the total amount of p38 in each lane was the same (Fig. 2CGo, lower panel). These results demonstrate that TAM induces the activation of ERK, JNK, and p38 with differing time courses.



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  		FIG. 2. The effect of TAM on the activity of ERK (A), JNK (B), and p38 (C). A, A2780 and Caov-3 cells were treated with 1 µM TAM for the indicated times (lanes 1–6). ERK activity was analyzed as described in Materials and Methods. Equal amounts of protein were analyzed by Western blotting using a specific antibody against total ERK. Relative densitometric units of MBP bands are shown in the upper panel, with the density of the vehicle bands (0 min) 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. B, A2780 and Caov-3 cells were treated with 5 µM TAM for the indicated times (lanes 1–6). JNK activity was analyzed as described in Materials and Methods. Equal amounts of protein were analyzed by Western blotting using a specific antibody against total JNK. Relative densitometric units of phospho-c-Jun bands are shown in the upper panel, with the density of the vehicle bands (0 min) 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 (C) A2780 and Caov-3 cells were treated with 5 µM TAM for the indicated times (lanes 1–6). p38 activity was analyzed as described in Materials and Methods. Equal amounts of protein were analyzed by Western blotting using a specific antibody against total p38. Relative densitometric units of phospho-ATF2 bands are shown in the upper panel, with the density of the vehicle bands (0 min) 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.

 
Prevention of TAM-induced cell-cycle arrest at the G1 phase by PD98059
To examine the hypothesis that the reduction of the number of viable cells by TAM is the result of activation of the ERK cascade, PD98059, an inhibitor of MAPKs or ERKs (39, 40, 41), was used in an MTS assay. PD98059 at 100 nM completely prevented the TAM-induced ERK activation (Fig. 3AGo). In the presence of 100 nM PD98059, the ability of 1 µM TAM to induce a reduction of the number of viable cells was significantly decreased in both types of cells (Fig. 3BGo). Figure 3CGo shows the FACS analyses of DNA content at 24 h after release from quiescence by incubation in culture medium supplemented without 1% CSS or with 1% CSS, 1% CSS+1 µM TAM, or 1% CSS+1 µM TAM+100 nM PD98059. The data reveal that TAM induced cell-cycle arrest at the G1 phase. In the presence of 100 nM PD98059, the ability of 1 µM TAM to induce the cell-cycle arrest at the G1 phase was reduced in both types of cells (Fig. 3CGo).



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  		FIG. 3. The effect of PD98059 on the TAM-induced activation of ERK (A), growth inhibition (B), and reduction of pRb phosphorylation (C). A, Cells were preincubated with (lanes 3 and 4) or without (lanes 1 and 2) 100 nM PD98059 for 30 min and then treated with 1 µM TAM for 1 h (lanes 2 and 4). ERK activity was analyzed as described in Materials and Methods. Relative densitometric units of MBP bands are shown in the upper panel, with the density of the vehicle 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 B, Assays of the number of viable cells using untreated cells and cells pretreated with or without 100 nM PD98059 for 30 min and then treated with or without 1 µM TAM for 24 h were carried out by the MTS assay as described in Materials and Methods. Significant differences are indicated by asterisks. **, P < 0.01. C, Caov-3 and A2780 cells were rendered quiescent and challenged without 1% CSS or with 1% CSS, 1% CSS+1 µM TAM, or 1% CSS+1 µM TAM+100 nM PD98059 for 24 h before being harvested, fixed with ethanol, and stained with propidium as described in Materials and Methods. The stained cells of each sample were analyzed by FACS. The percentage of cells at the G1, S, or G2/M phase of the cell cycle was determined using ModFit LT software. The data shown are from one of at least three independent experiments performed with similar results. D, Cells were preincubated with (lanes 3 and 4) or without (lanes 1, 2, and 5) 100 nM PD98059 for 30 min and then treated with 1% CSS for 24 h (lanes 2 and 3) or with 1% CSS plus 1 µM TAM for 24 h (lanes 4 and 5). Lysates were subsequently immunoprecipitated with anti-pRb antibody, and the immunoprecipitates were subjected to Western blotting with anti-pRb antibody (upper panel). Lysates were subjected to Western blotting with anti-ß-actin antibody (lower panel).

 
To confirm that 1 µM TAM induced the cell-cycle arrest at the late G1 phase, we examined the effect of TAM on the phosphorylation of pRb because this phenomenon is a crucial milestone for the cell to advance into the S phase. Stimulation of the cells for 24 h with 1% CSS caused reduced mobility, reflecting hyperphosphorylation of pRb (Fig. 3DGo, upper panel, lane 2). The addition of TAM to the 1% CSS led to a repression of pRb phosphorylation (Fig. 3DGo, upper panel, lane 4). In the presence of PD98059, however, TAM was not able to inhibit the induction of pRb phosphorylation by 1% CSS (Fig. 3DGo, upper panel, lane 3). We confirmed that the expression of ß-actin detected in each lane was equal (Fig. 3DGo, lower panel). These results demonstrate that in both types of cells, the ERK cascade is involved in the signaling leading to hypophosphorylation of pRb and cell-cycle arrest at the G1 phase in cells stimulated with TAM.

Prevention of TAM-induced apoptosis by either dominant-negative JNK or SB203580
We next examined the involvement of the JNK cascade or p38 cascade in the reduction of the number of viable cells by TAM. An expression plasmid (pcDL-SR{alpha}-SAPK-VPF) that encodes a dominant-negative JNK (DN-JNK) was used to inhibit the JNK cascade (32). TAM-induced JNK activation in cells transfected with DN-JNK was clearly attenuated, compared with that in cells transfected with pcDL-SR{alpha} (Fig. 4AGo). TAM (5 µM) had significantly less ability to reduce the number of viable cells in both types of cells transfected with DN-JNK than in the cells transfected with pcDL-SR{alpha} (Fig. 4BGo). SB203580 was then used to inhibit the p38 cascade. SB203580 at 1 µM completely abolished the TAM-induced p38 activation (Fig. 5AGo). In the presence of 1 µM SB203580, 5 µM TAM had significantly lower ability to reduce the number of viable cells in both types of cells (Fig. 5BGo).



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  		FIG. 4. The effect of DN-JNK on TAM-induced reduction of viable cell number. A, Empty vector (pcDL-SR{alpha})- (lanes 1 and 2) or DN-JNK construct- (lanes 3 and 4) expressing A2780 or Caov-3 cells were treated with 5 µM TAM for 30 min (lanes 2 and 4). JNK activity was analyzed as described in Materials and Methods. Relative densitometric units of phospho-c-Jun bands are shown in the upper panel, with the density of the vehicle 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. B, Assays of the number of viable pcDL-SR{alpha}- or DN-JNK-expressing A2780 or Caov-3 cells after treatment with or without 5 µM TAM for 24 h were carried out by the MTS assay as described in Materials and Methods. Significant differences are indicated by asterisks. **, P < 0.01. N.S., not significant.

 


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  		FIG. 5. The effect of SB203580 on TAM-induced reduction of viable cell number. A, Cells were preincubated with (lanes 3 and 4) or without (lanes 1 and 2) 1 µM SB203580 for 30 min and then treated with 5 µM TAM for 1 h (lanes 2 and 4). p38 activity and total p38 were analyzed as described in legend of Fig. 2Go. Relative densitometric units of phospho-ATF2 bands are shown in the upper panel, with the density of the vehicle 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 B, Assays of the number of viable A2780 or Caov-3 cells pretreated with or without 1 µM SB203580 for 30 min and then treated with or without 5 µM TAM for 24 h were carried out by MTS assay as described in Materials and Methods. Significant differences are indicated by asterisks. **, P < 0.01.

 
Because the reduction of the number of viable cells by the trypan blue exclusion test (Fig. 1CGo) or MTS assay (Figs. 1DGo, 4BGo, and 5CGo) do not always indicate the apoptosis, we examined the involvement of the JNK cascade or p38 cascade in the apoptotic nuclear changes or the cleaved PARP induced by TAM (Fig. 6Go). TAM (5 µM) induced the aggregation of chromatin with apoptotic nuclear changes and the cleaved PARP in both parental cells (Fig. 6BGo, left panels, and Fig. 6DGo, lane 2) and cells transfected with empty vector pcDL-SR{alpha} (Fig. 6AGo, left panels, and Fig. 6CGo, lane 2). TAM-induced apoptotic nuclear changes and cleaved PARP were attenuated in cells transfected with DN-JNK (Fig. 6AGo, right panels, and Fig. 6CGo, lane 4) or treated with SB203580 (Fig. 6BGo, right panels, and Fig. 6DGo, lane 3).



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  		FIG. 6. The effect of DN-JNK or SB203580 on TAM-induced the apoptosis. A, pcDL-SR{alpha}- or DN-JNK-expressing A2780 or Caov-3 cells were plated on culture chamber slides. After incubation for 24 h, the cells were treated with 5 µM TAM for 24 h. B, A2780 or Caov-3 cells plated on culture chamber slides were pretreated with 1 µM SB203580 for 30 min and then treated with 5 µM TAM for 24 h. Cells were stained with Hoechst 33258 and the apoptotic nuclear changes was examined by fluorescence confocal microscopy as described in Materials and Methods. C, pcDL-SR{alpha}- (lanes 1 and 2) or DN-JNK construct- (lanes 3 and 4) expressing A2780 or Caov-3 cells were treated with 5 µM TAM for 30 min (lanes 2 and 4). D, Cells were preincubated with (lane 3) or without (lanes 1 and 2) 1 µM SB203580 for 30 min and then treated with 5 µM TAM for 1 h (lanes 2 and 3). Lysates (250 µg protein) were subjected to Western blotting using anti-cleaved PARP (middle panel) or anti-ß-actin (lower panel) antibody. The positions of molecular-weight markers are noted on the left. Relative densitometric units of the cleaved PARP 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.

 
Lack of cross-talk among ERK, JNK, and p38 cascades
We examined whether cross-talk occurs among the ERK, JNK, and p38 cascades. TAM-induced ERK activation was not blocked in cells transfected with DN-JNK (Fig. 7AGo). In addition, TAM-induced JNK activation was not blocked in cells treated with PD98059 (Fig. 7BGo) or SB203580 (Fig. 7CGo). Moreover, TAM-induced p38 activation was not blocked in cells transfected with DN-JNK (Fig. 7DGo). These results suggest that there is no cross-talk among the ERK, JNK, and p38 cascades in either type of cell.



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  		FIG. 7. No cross-talk among ERK, JNK, p38 cascades. A, Empty vector (pcDL-SR{alpha})- (lanes 1 and 2) or DN-JNK construct (pcDL-SR{alpha}-SAPK-VPF)- (lane 3) expressing A2780 or Caov-3 cells were grown in 100-mm dishes, treated with 1 µM TAM for 1 h, and analyzed for ERK activity as described in the legend of Fig. 3AGo. Relative densitometric units of MBP bands are shown in the upper panel, with the density of the vehicle bands set arbitrarily at 1.0. Values shown represent the mean ± SE from at least three separate experiments. Significant differences from vehicle are indicated by asterisks. **, P < 0.01; N.S., not significant. B, Cells grown in 100-mm dishes were preincubated with (lane 3) or without (lanes 1 and 2) 100 nM PD98059 for 30 min and then treated with 5 µM TAM for 30 min, followed by analysis of JNK activity as described in the legend of Fig. 3BGo. Relative densitometric units of phospho-c-Jun bands are shown in the upper panel, with the density of the vehicle bands set arbitrarily at 1.0. Values shown represent the mean ± SE from at least three separate experiments. Significant differences from vehicle are indicated by asterisks. **, P < 0.01; N.S., not significant. C, Cells grown in 100-mm dishes were preincubated with (lane 3) or without (lanes 1 and 2) 1 µM SB203580 for 30 min and then treated with 5 µM TAM for 30 min, followed by analysis of JNK activity as described in the legend of Fig. 3BGo. Relative densitometric units of phospho-c-Jun bands are shown in the upper panel, with the density of the vehicle bands set arbitrarily at 1.0. Values shown represent the mean ± SE from at least three separate experiments. Significant differences from vehicle are indicated by asterisks. **, P < 0.01; N.S., not significant. D, Empty vector (pcDL-SR{alpha})- (lanes 1 and 2) or DN-JNK construct (pcDL-SR{alpha}-SAPK-VPF)- (lane 3) expressing A2780 or Caov-3 cells were grown in 100-mm dishes, treated with 5 µM TAM for 1 h, and then analyzed for p38 activity as described in the legend of Fig. 3CGo. Relative densitometric units of phospho-ATF2 bands are shown in the upper panel, with the density of the vehicle bands set arbitrarily at 1.0. Values shown represent the mean ± SE from at least three separate experiments. Significant differences from vehicle are indicated by asterisks. **, P < 0.01; N.S., not significant.

 
Effect of antioxidant on TAM-induced ERK, JNK, and p38 activation
The cytotoxicity of TAM has been attributed to the generation of oxidative stress (29). Therefore, we examined the role of oxidative stress in the TAM-induced ERK, JNK, and p38 activation in both types of cells. Pretreatment with the lipid-soluble antioxidant VE ({alpha}-tocopherol) prevented TAM-induced activation of ERK (Fig. 8AGo), JNK (Fig. 8BGo), and p38 (Fig. 8CGo).



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  		FIG. 8. Effect of antioxidant on TAM-induced ERK, JNK, and p38 activation. Cells were pretreated with 0.5 mM VE for 1 h (lane 3) and then treated with 1 µM TAM for 1 h (A), 5 µM TAM for 30 min (B), or 5 µM TAM for 1 h (C). ERK (A), JNK (B), and p38 (C) activities were determined as described in Materials and Methods. Relative densitometric units of MBP (A), phospho-c-Jun (B), and phospho-ATF2 (C) bands are shown in the upper panel, with the density of the vehicle 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

 
Moreover, we examined the effect of VE on the TAM-induced reduction of the number of viable cells (Fig. 9AGo) and apoptosis (Fig. 9Go, B and C). Pretreatment with VE significantly attenuated the reduction of the number of viable cells induced by TAM between 1 and 5 µM (Fig. 9AGo). In the presence of VE, TAM was not able to inhibit the induction of pRb phosphorylation by 1% serum (data not shown). TAM (5 µM)-induced apoptotic nuclear changes and cleaved PARP were attenuated in cells treated with VE (Fig. 9BGo, right panels, and Fig. 9CGo, lane 3).



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  		FIG. 9. Effect of antioxidant on TAM-induced growth inhibition and apoptotic nuclear change. A, Assays of the number of viable cells using untreated cells and cells pretreated with 0.5 mM VE for 1 h followed by treatment with the indicated concentration of TAM for 24 h were carried out by MTS assay as described in Materials and Methods. B, A2780 or Caov-3 cells plated on culture chamber slides were pretreated with 0.5 mM VE for 1 h and then treated with 5 µM TAM for 24 h. Cells were stained with Hoechst 33258, and the apoptotic nuclear changes was examined by fluorescence confocal microscopy as described in Materials and Methods. C, Cells were pretreated with 0.5 mM VE for 1 h (lane 3) and then treated with 5 µM TAM for 1 h (lanes 2 and 3). Lysates (250 µg protein) were subjected to Western blotting using anticleaved PARP (middle panel) or anti-ß-actin (lower panel) antibody. The positions of molecular-weight markers are noted on the left. Relative densitometric units of cleaved PARP 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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results demonstrate a reduction of the number of viable cells by TAM in three human ovarian cancer lines: cisplatin-resistant Caov-3, which expresses ER; cisplatin-sensitive A2780, which does not express ER; and A2780-ER, which expresses ER. TAM caused ERK, JNK, and p38 activation with different kinetics in these types of cells. Suppression of ERK activation by PD98059 blocked the reduction of the number of viable cells as well as cell-cycle arrest at the G1 phase by 1 µM TAM. Suppression of either JNK activation by exogenous expression of a dominant-negative JNK or p38 activation by treatment with SB203580 blocked the reduction of the number of viable cells as well as apoptotic nuclear changes and the cleavage of PARP induced by 5 µM TAM. These results provide evidence that whereas the ERK cascade is involved in the TAM-induced cell-cycle arrest at the G1 phase, both the JNK and p38 cascades are involved in TAM-induced apoptosis in both ER-positive and -negative, and both cisplatin-resistant and -sensitive ovarian cancer cells.

TAM is thought to exert its antiproliferative action by binding competitively to ER and thereby blocking the mitogenic effect of E2 (3). How does TAM induce cytotoxicity in both ER-positive and -negative cells? It is tempting to speculate that TAM, by virtue of its high lipophilicity and partioning in the cell membrane, generates oxidative stress and transmembrane signal transduction events. It has been reported that TAM, by initially partitioning into the membranes, induces MAPK family members ERK (17), JNK (17, 18), and p38 (19). It was reported that TAM at 10 µM induces oxidative stress and apoptosis in ER-negative A2780 human ovarian cancer cell lines (28). However, the mechanisms of these effects remain elusive. In this study, we showed that both JNK and p38 cascades are involved in the induction of apoptosis by 5 µM TAM in A2780 cells. It was also reported that TAM at a lower dose (1 µM) induces cell-cycle arrest at the G1 phase in breast cancer cell lines (15), and the mechanism of this effect also remains elusive. In this study, we also showed that the ERK cascade is involved in the induction of cell-cycle arrest at the G1 phase by 1 µM TAM. In addition, we demonstrated that the lipid-soluble antioxidant VE blocked TAM-induced ERK, JNK, and p38 activation and the TAM-induced reduction of the number of viable cells in both ER-positive and -negative cell lines, implicating oxidative stress in these effects, as reported previously (18). This is the first report showing the role of oxidative stress in mediating the effects of TAM on the ERK, JNK, and p38 cascades as well as the reduction of the number of viable cells in human ovarian cancer cell lines in a manner not dependent on the expression of ER.

Although JNK (17, 18) and p38 (19) are involved in TAM-induced apoptosis, as we showed in this study, the mechanism of the involvement of ERK remains elusive. The present results raise the question of how TAM converts the outcome of ERK signaling from mitogenesis to growth inhibition. The potential ability of signal duration to affect growth factor signal outcome has been suggested in different contexts (22). For example, sustained activation of ERK has been associated with regulation of cell differentiation by nerve growth factor in PC12 cells, whereas transient activation of ERK by epidermal growth factor leads to cell proliferation rather than differentiation. The duration of ERK activity seems to be important in determining cell cycle progression. We reported that sustained activation of ERK by GnRH agonist in Caov-3 cells is involved in GnRH agonist-induced cell-cycle arrest at the G1 phase (23), whereas transient activation of ERK by cisplatin (26, 33) and paclitaxel (35) in A2780 and Caov-3 cells is involved in the cellular repair of cisplatin- and paclitaxel-induced cell damage. TAM-induced ERK activation was sustained in both A2780 and Caov-3 cells. Thus, it is possible that prolonged activation of ERK by TAM might mediate the mechanism of promoting cell-cycle arrest or slowing the cell cycle. The transition of cells from G1 to S phase in the cell cycle is controlled by pRb activity (42, 43). In G1 phase, pRb is hypophosphorylated and exerts its antiproliferative function. Pretreatment with PD98059 prevented the dephosphorylation of pRb (Fig. 3DGo) and cell-cycle arrest at the G1 phase (Fig. 3CGo) induced by TAM. These results suggest that TAM activates the ERK cascade, which in turn leads to dephosphorylation of pRb, followed by cell-cycle arrest at the G1 phase in human ovarian cancer cells. On the other hand, although the ERK cascade has been reported to contribute to the induction of apoptosis (44, 45), pretreatment with PD98059 did not prevent the apoptotic nuclear changes or the cleavage of PARP induced by TAM (data not shown).

It is known that parallel cascades leading to the activation of ERK, JNK, or p38 exist (16, 26). It is notable that the kinetics of TAM-induced ERK activation are different from those of JNK activation and of p38 activation. The activation of JNK by TAM was not dependent on the ERK or p38 cascade because neither PD98059 (Fig. 7BGo) nor SB203580 (Fig. 7CGo) affected JNK activation. Activation of ERK (Fig. 7AGo) or p38 (Fig. 7DGo) by TAM was still detected in DN-JNK-expressing cells. Moreover, we previously reported that a GnRH agonist induced cell-cycle arrest at the G1 phase via the ERK cascade but not the JNK cascade in Caov-3 cells (23). These results provide further evidence that ERK, JNK, and p38 are independently activated in TAM-treated A2780 and Caov-3 cells and that the TAM-induced cell-cycle arrest at the G1 phase is independent of TAM-induced apoptosis.

The majority of patients with ovarian cancer require treatment with cytotoxic chemotherapy. It is now well established that platinum agents (cisplatin or carboplatin) are the most important drugs to be included in first-line regimens. More recently, randomized trials have confirmed the benefit of the addition of taxanes to platinum-containing regimens. Despite the high response rate to first line chemotherapy, the majority of patients with advanced ovarian cancer relapse and become candidates for further chemotherapy, which can palliate symptoms and improve survival even in recurrent disease. There is some evidence from observational studies (5, 6) that TAM may produce a response in a modest proportion of women with relapsed ovarian cancer. Our data showing that TAM induces a reduction of the number of viable cisplatin-resistant cells also indicate the possibility of the usefulness of TAM in cases of cisplatin-refractory ovarian cancer. Moreover, it was reported that TAM delays the development of resistance to cisplatin in human ovarian cancer cells (46). Our data show that TAM also induces a reduction of the number of viable cisplatin-sensitive cells. TAM may confer useful clinical benefits in combination therapy with cisplatin. Thus, TAM, which has relatively mild toxicity, might serve a useful role in the adjuvant and advanced settings of ovarian cancer.


   Acknowledgments
 
We are grateful to Dr. E. Nishida (Kyoto University, Kyoto, Japan) for providing pcDL-SR{alpha}-SAPK-VPF, Dr. P. Chambon (Institut de Chimie Biologique, Strasbourg, France) for providing pSG5-HEGO, and Drs. B. D. Gehm and J. L. Jameson (Northwestern University, Chicago, IL) for providing ERE-tk109-luc.


   Footnotes
 
Abbreviations: A2780-ER, A2780-expressing ER{alpha}; ATF, activating transcription factor; CSS, charcoal stripped serum; DN-JNK, dominant-negative JNK; E2, estradiol; ER, estrogen receptor; FACS, fluorescence-activated cell sorter; JNK, c-Jun N-terminal protein kinase; MBP, myelin basic protein; MTS, 3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt; PARP, poly (ADP-ribose) polymerase; pRb, retinoblastoma protein; SAPK, stress activated protein kinase; TAM, tamoxifen; VE, vitamin E.

Received June 6, 2003.

Accepted for publication November 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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